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https://openalex.org/W2587176644
https://europepmc.org/articles/pmc5372064?pdf=render
English
null
Pharmacist provided medicines reconciliation within 24 hours of admission and on discharge: a randomised controlled pilot study
BMJ open
2,017
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ABSTRACT To cite: Cadman B, Wright D, Bale A, et al. Pharmacist provided medicines reconciliation within 24 hours of admission and on discharge: a randomised controlled pilot study. BMJ Open 2017;7:e013647. doi:10.1136/bmjopen-2016- 013647 Strengths and limitations of this study Background: The UK government currently recommends that all patients receive medicines reconciliation (MR) from a member of the pharmacy team within 24 hours of admission and subsequent discharge. The cost-effectiveness of this intervention is unknown. A pilot study to inform the design of a future randomised controlled trial to determine effectiveness and cost-effectiveness of a pharmacist-delivered service was undertaken. ▪Pilot randomised controlled trial. ▪Intervention fidelity enhanced through compe- tency assessment of medicine reconciliation providers. ▪Robust process for identifying unintentional dis- crepancies at each stage developed to prevent results contamination. ▪Pragmatic design and therefore elements of the intervention found within the control arm. Method: Patients were recruited 7 days a week from 5 adult medical wards in 1 hospital over a 9 month period and randomised using an automated system to intervention (MR within 24 hours of admission and at discharge) or usual care which may include MR (control). Recruitment and retention rates were determined. Length of stay (LOS), quality of life (EQ-5D-3L), unintentional discrepancies (UDs) and emergency readmission (ER) within 3 months were tested as outcome measures. The feasibility of identifying and measuring intervention-associated resources was determined. ▸Prepublication history for this paper is available online. To view these files please visit the journal online (http://dx.doi.org/10.1136/ bmjopen-2016-013647). ▪Limited response to request for patient data 3 months postdischarge. BACKGROUND Medicines reconciliation (MR) is defined by the WHO as ‘the formal process in which healthcare professionals partner with patients to ensure accurate and complete medication information transfer at the inter- faces of care’.1 Researchers have identified unintentional error rates within hospital pre- scriptions on admission of between 30% and 70%2–5 and these can include omission of usual medicines, prescription of incorrect dosages and addition of medicines which have not been previously prescribed. The majority of these errors are believed to result from deficiencies in the MR process with such errors known to contribute to patient morbidity and mortality and increase the length of hospital stay (LOS).6–10 Received 1 August 2016 Revised 7 November 2016 Accepted 15 December 2016 Result: 200 patients were randomised to either intervention or control. Groups were comparable at baseline. 95 (99%) patients in the intervention received MR within 24 hours, while 62 (60.8%) control patients received MR at some point during admission. The intervention resolved 250 of the 255 UDs identified at admission. Only 2 UDs were identified in the intervention group at discharge compared with 268 in the control. The median LOS was 94 hours in the intervention arm and 118 hours in the control, with ER rates of 17.9% and 26.7%, respectively. Assuming 5% loss to follow-up 1120 patients (560 in each arm) are required to detect a 6% reduction in 3-month ER rates. Conclusions: The results suggest that changes in outcome measures resulting from MR within 24 hours were in the appropriate direction and readmission within 3 months is the most appropriate primary outcome measure. A future study to determine cost-effectiveness of the intervention is feasible and warranted. For numbered affiliations see end of article. Research Research Open Access Pharmacist provided medicines reconciliation within 24 hours of admission and on discharge: a randomised controlled pilot study Brit Cadman,1 David Wright,2 Amanda Bale,1 Garry Barton,3,4 James Desborough,2 Eman A Hammad,5 Richard Holland,3 Helen Howe,1 Ian Nunney,3 Lisa Irvine3,4 Pharmacist provided medicines reconciliation within 24 hours of admission and on discharge: a randomised controlled pilot study Brit Cadman,1 David Wright,2 Amanda Bale,1 Garry Barton,3,4 James Desborough,2 Eman A Hammad,5 Richard Holland,3 Helen Howe,1 Ian Nunney,3 Lisa Irvine3,4 Received 1 August 2016 Revised 7 November 2016 Accepted 15 December 2016 AIMS The aim of this study was therefore to pilot a RCT to inform the design of a future definitive study to deter- mine the clinical and cost-effectiveness of pharmacy pro- viding a full MR service on admission and at discharge. The objectives of the pilot study were to: The aim of this study was therefore to pilot a RCT to inform the design of a future definitive study to deter- mine the clinical and cost-effectiveness of pharmacy pro- viding a full MR service on admission and at discharge. The objectives of the pilot study were to: Intervention A standard operating procedure (SOP) based on hos- pital guidelines, outlined in figure 1, was used to deliver MR by a trained MR pharmacist (MRP) within 24 hours of admission (including weekends) and at the point of transfer of care out of hospital, or as soon as possible fol- lowing patient discharge from hospital to the next care provider. The five MRPs, all clinical pharmacists employed within the hospital, covered for each other’s holidays, sick leave and absences wherever possible. y p MRPs recorded all unintentional discrepancies (UDs), defined as differences between patient records with no identifiable rationale, they identified between the infor- mation they collated and the inpatient medication chart on admission and again any differences between the inpatient chart and discharge letter. MRPs followed up on all identified UDs to ensure that they were addressed prior to discharge. Despite national guidance few hospitals are providing MR as envisioned by NICE for all patient admissions.18 Thus, it would appear that further high-quality evidence which demonstrates cost-effectiveness is required to ensure that resources are appropriately allocated to this service in order to meet national recommendations. The most suitable approach to determine the cost- effectiveness of a complex intervention such as MR is to perform a RCT which collates data on cost from the appropriate perspective and outcomes which are most proximal to the intervention. Within the UK, it is add- itionally necessary to collect data on quality of life and mortality to enable the cost per quality-adjusted life year (QALY) to be estimated. Recent guidance, however, sug- gests that before an RCT of such nature is undertaken feasibility testing and piloting are required.19 O S Study design and location The trial was a randomised controlled pilot study under- taken at Cambridge University Hospitals NHS Foundation Trust (CUHFT) on five adult medical wards from a range of medical specialities where patients did not routinely receive MR from a pharmacist within 24 hours of admission. One similar ward was identified as a ‘backup’, in the eventuality that one of the study wards was closed for any reason (eg, norovirus outbreak) during the recruitment period. With the significant resources required for pharmacists to deliver MR services, it is not adequate to demonstrate reduction in errors (which may or may not result in adverse drug events), it is also important in resource- limited health systems to show that such investment con- stitutes value for money. There is currently a recognised lack of evidence supporting the cost-effectiveness of MR interventions provided by pharmacists.16 A model built in 2008 to estimate the likely cost-effectiveness of pre- venting medication error at hospital admission using MR suggested that a pharmacist-based intervention is likely to be cost-effective but was based solely on US error data and made assumptions concerning the severity of errors prevented.17 METHODS Recent systematic reviews of hospital-based MR prac- tices consistently demonstrate a reduction in medicine discrepancies; however, this has not been shown to result in reductions in postdischarge healthcare usage.13 14 When considering only pharmacist-led MR interventions a 19% reduction in the rate of all-cause readmissions was seen but similarly pooled data on mortality and com- posite readmission and emergency department (ED) visit did not find in favour of either pharmacist-led inter- vention or usual care.15 Recent systematic reviews of hospital-based MR prac- tices consistently demonstrate a reduction in medicine discrepancies; however, this has not been shown to result in reductions in postdischarge healthcare usage.13 14 Control Patients in the control arm received usual care which may or may not consist of MR and where it was provided it may not have occurred within 24 hours and could either be delivered by a pharmacist or pharmacy techni- cian. The MRPs within the intervention arm did not deliver MR to control patients and the SOP used for study intervention purposes was not automatically fol- lowed within the control arm. For the purposes of the study all MR details regarding interventions undertaken within the control arm were recorded and costed. Consequently, within the UK, patient safety guidance issued by the National Patient Safety Agency (NPSA) and the National Institute for Health and Care Excellence (NICE) recommended that policies for MR should be implemented in hospitals for all adult patient admissions, and that pharmacy should be involved in the MR process within 24 hours of admission.11 NICE guidance on For numbered affiliations see end of article. For numbered affiliations see end of article. Correspondence to Professor David Wright; D.J.Wright@uea.ac.uk Trial registration number: ISRCTN23949491. Trial registration number: ISRCTN23949491. 1 Cadman B, et al. BMJ Open 2017;7:e013647. doi:10.1136/bmjopen-2016-013647 Open Access response to the intervention and quality of data obtained; pharmacy involvement was based primarily on one ran- domised controlled trial (RCT) which demonstrated that the inclusion of the pharmacist in MR reduced the error rate from 44% to 19%.2 Data on cost-effectiveness which usually underpins recommendations made by NICE12 are not available and therefore whether this intervention represents an appropriate use of National Health Service (NHS) resources is unknown. ▸Determine potential recruitment and retention rates; ▸Determine potential recruitment and retention rates; ▸Determine potential recruitment and retention rates; ▸Develop and test the process for measuring resource usage associated with the intervention and use of other NHS. Outcome measures Inclusion criteria Although undertaken as a pilot study with study aims to identify the most suitable outcome measure, LOS was nominally selected as the primary outcome measure for this pilot trial. Secondary outcome measures were unplanned (emergency) readmission at 3 months, quality of life (EQ-5D-3L) and UDs. ▸Adult (≥18 years of age); ▸Admitted with at least one prescribed medicine to one of the five medical wards; ▸Patient had not already received MR from the phar- macy team as part of routine pharmaceutical input at the time of recruitment; ▸Identified from hospital computer system as having been admitted straight from the ED to one of the five participating wards within the previous 24 hours. Intervention fidelity To enhance intervention fidelity all MRPs were observed by the principal investigator on at least three occasions to confirm adherence to the SOP. All MRPs had pro- vided MR to more than 30 patients in the year previous to delivering the intervention for the trial. j p y ▸Identify the most suitable outcome measure for a future definitive trial with respect to proximity and Cadman B, et al. BMJ Open 2017;7:e013647. doi:10.1136/bmjopen-2016-013647 Open Access Figure 1 Outline of service standard operating procedure. GP, general practitioner. Open Access Recruitment Patients randomised to the control group received usual care; this may have included elements of MR by members of the pharmacy team and in some cases may have occurred within 24 hours of admission but postran- domisation. While this may have affected patients in the control arm an intention-to-treat analysis was performed and consequently for the purpose of the analysis patients remained in their allocated arm. A recruitment target of 200 patients was set for the 9-month pilot phase. Study wards were visited every morning by the research assistant (RA) during the study period to identify potential participants. The nurse in charge of the ward confirmed that it was appropriate for the patient to be approached to be consented to partici- pate in the study. Patients were recruited based on the following inclusion and exclusion criteria. Resource use The time taken by the pharmacy team to deliver the MR was monitored in the intervention and control group (if applicable). Additionally, the following items were requested in both groups: Recruitment took place between July 2012 and April 2013 (9 months and 2 weeks), resulting in a recruitment rate of 5.2 patients per 7 days. ▸Time in hospital (LOS); ▸Medication (in patient medication and GP medica- tion list at 3 months); There was one postrandomisation exclusion in each arm (they were subsequently found not to meet the inclusion criteria), and were accordingly excluded from all analyses. Table 1 summarises the characteristics of the remaining 198 participants (96 intervention 102 control). The groups were broadly comparable. ); ▸Rehospitalisations; ▸Rehospitalisations; ▸Other healthcare contacts. With the exception of the final item, which included all health professional contacts and was requested from the participant at 3 months postdischarge, these were extracted from medical records. One patient in the intervention arm did not receive the MR as he was not on the ward when the pharmacist visited. Data were erroneously not collected for this patient postrandomisation. The time taken by the pharmacist to deliver the intervention was recorded for the remaining 95 intervention participants, where the mean total time was 48.6 min (range 2–195 min). In the Data collection To enable comparison of intervention and control groups, age, gender, primary reason for admission, all comorbidities and the admission ward was recorded. Additionally consented patients or their third party con- sultees completed a quality of life score (EQ-5D-3L)20 on admission, including the related visual analogue scale (VAS). p p g p With the intervention required to be delivered within 24 hours of admission, patients were given a maximum of 2 hours to consider whether they wished to participate. Intervention patients received MR in accordance with the SOP. Information obtained by the RA for the purposes of the study was given to the MRP prior to the visit to prevent duplica- tion of effort and to ensure that the patient was not interviewed for the same information twice. LOS, reported in hours, was calculated as the differ- ence in time from arrival at the hospital to the time of discharge as recorded in the hospital information support system (HISS). Unplanned readmissions to the intervention hospital within the 3 months postdischarge were also obtained from HISS. EQ-5D-3L responses were also obtained via postal survey 3 months postdischarge, allowing the calculation of QALY21 in the subsequent economic evaluation. Randomisation was performed using the Norwich Clinical Trials Unit automated service with patients stratified by ward. When wards were later closed for infection control reasons, participants on the ‘backup’ ward were randomised and stratified as if they had entered the closed ward. Cadman B, et al. BMJ Open 2017;7:e013647. doi:10.1136/bmjopen-2016-013647 3 UD identification on LOS taken from a study undertaken at St James Hospital, Dublin22 gave quartiles for two groups as (3, 7, 5) and (2, 5, 12) days. To derive an estimate of variability from this, an underlying log-normal distribution was assumed, which is consistent with the position of the medians, and its SD estimated using the geometric mean of the ratios between the upper and lower quar- tiles. This produced an estimated SD of 1.26 which implied that a comparison between two groups of 100 patients would provide an expected half-width for the 95% CI of the difference between group means (on the natural log scale) of 0.35. Translated back to the CI for the ratio of average LOS between the intervention and control groups would extend by a factor of about 1.4 on either side of the point estimate. To enable the identification of UDs the following infor- mation was photocopied by the research assistant (RA) for all consented patients (both intervention and control) and stored securely: ▸All versions of the inpatient medication chart(s) and discharge letters; g ▸Medical notes during admission; ▸Medical notes during admission; ▸General practitioner (GP) medication list on admission; ▸GP medication list at 3-month postdischarge (when received from GP surgery); ▸Any additional medicines-related information brought in by the patient on admission, for example, copies of labels from patient medicines, handwritten or typed medicine lists. yp The RA was a trained nurse and consequently was experienced in medical data collation and extraction. RESULTS Nine hundred and nineteen patients were assessed for eligibility of which 224 did not meet inclusion criteria. Two hundred out of 310 patients who were subsequently approached by the RA consented to take part in the study (figure 2). Of those patients identified as poten- tially eligible but not approached this was primarily because either the nurse in charge of the ward advised that the patient was not suitable to be approached or that the end of the 24-hour window for intervention was due to expire. All potential discrepancies which were identified at the 3-month point as having translated into the patient notes were reported to the patient’s GP. Data analysis As a pilot study descriptive statistics were used to deter- mine the suitability of the different outcome measures and report the variation in the differences in order to determine a sample size for a future RCT. Similarly, com- pletion rates are reported for each source of resource use data and the EQ-5D-3L. Three months postdischarge the stored information was used to develop an ‘accurate medication list’ for the control arm patients by the research team on admission and at discharge. These were then compared with the inpatient chart on admission and discharge letter to identify any discrepancies. Medical notes were subse- quently reviewed, unblinded to group allocation, to enable differentiation between those which were UDs which could not be explained from the information available and those which were intentional. The primary outcome variable, LOS, was reported using median, arithmetic mean and geometric mean. The rate of Trust readmissions, Trust emergency read- missions and mortality are also reported for both arms, along with the mean change in the VAS from the EQ-5D-3L. The GP medication list obtained 3 months post- discharge (where available) was used to enable the iden- tification of discrepancies which still remained at 3 months. Without access to GP medical notes it was not possible to establish whether identified discrepancies were unintentional. Sample size calculation As a pilot study a formal power calculation was not per- formed. The consequences for the precision of the primary outcome variable (LOS) of the choice of sample size were however estimated. Summary statistics Cadman B, et al. BMJ Open 2017;7:e013647. doi:10.1136/bmjopen-2016-013647 4 Open Access Figure 2 Consort diagram. GP, general practitioner; MR, medicines reconciliation. p Figure 2 Consort diagram. GP, general practitioner; MR, medicines reconciliation. group. Neither of the UDs in the intervention arm iden- tified at discharge was present at 3 months. control group 62 (60.8%) participants received some form of MR, 31 (30.4%) from a pharmacist (mean reported time 15 min) and 31 (30.4%) from a pharmacy technician (mean reported time 12 min). Twenty-five (24.5%) control patients received MR within the 24-hour window. One hundred and fifty-four UDs identified in the control arm were potentially related to medicines to be prescribed postdischarge, that is, the remainder related to medicines prescribed during admission only. Owing to the limited number of GP records provided at 3 months data were only available for 82 (53.2%) UDs in the control arm at 3 months and 37 out of the original 154 (24%) were found from the medical records pro- vided to have been propagated into the patient notes. During the initial hospital admission 3 participants died, with a further 11 deaths during the 3 months follow-up period (see figure 2). In total six intervention and eight control patients died during the trial period (p=0.78, Fisher’s exact). Additionally, in the control arm, one participant was lost to follow-up (address not known) and one participant withdrew. After taking account of the 1 patient who did not receive the MR and a further loss to follow-up in the intervention arm, this left a total of 88 available cases in the intervention arm and 92 available cases in the control arm at final assessment point. In terms of outcomes, there were com- plete data available on LOS and readmission data. Table 3 provides a comparison of patient outcomes for the intervention and control group. The results suggest that LOS, mortality and rehospitalisation rates tend to be lower in the intervention arm although statistical sig- nificance was not demonstrated as 95% CIs overlapped. Based on those who responded, both groups had a higher mean quality of life (based on the VAS) at the 3-month follow-up with improvement higher in the control arm. Sample size calculation This difference between groups was not significant. p Table 2 provides a summary of the UDs which were identified at each stage. Sixteen (16%) intervention patients had no discrepancies at admission and dis- charge, compared with 12 (12%) control patients. Overall, two UDs were known to remain at discharge in the intervention arm compared with 268 in the control With regard to the response rates for the resource use data, there were complete data available (for those on whom it was requested) for LOS, medication data as part of the original admission, readmissions (to the 5 Cadman B, et al. BMJ Open 2017;7:e013647. doi:10.1136/bmjopen-2016-013647 Open Access Table 1 Comparison of demographics at baseline Demographic Measure Intervention (n=96) Control (n=102) Female N (%) 45 (46.9) 60 (58.8) Age (years) Mean (SD) 67.6 (19.0) 65.4 (20.2) Regular medicines Mean (SD) 5.84 (4.07) 6.67 (4.64) As required medicines Mean (SD) 0.85 (2.08) 0.95 (2.53) Ward 1 N (%) 26 (26.8) 27 (26.2) 2 N (%) 30 (30.9) 30 (29.1) 3 N (%) 10 (10.3) 9 (8.7) 4 N (%) 14 (14.4) 16 (15.5) 5 N (%) 16 (16.4) 14 (13.5) 6 N (%) 1 (1.03) 7 (6.8) EQ-5D quality of life (visual analogue scale) Mean (SD) 55.9 (23.2) 54.7 (23.5) Reason for admission Lower respiratory tract infection N (%) 9 (9.2) 11 (10.7) Troponin negative chest pain N (%) 7 (7.2) 4 (3.9) Heart failure N (%) 2 (2.1) 5 (4.9) Exacerbation chronic obstructive pulmonary disease N (%) 4 (4.1) 3 (2.9) Other N (%) 73 (75) 79 (76.7) We consider that emergency rehospitalisation within 3 months would be the most appropriate primary outcome measure for such a trial, as unlike the other outcome measures tested it reflects all of the MR activity which occurs in secondary and primary care. Furthermore, data collection was complete and this is a patient-orientated outcome, unlike medication errors which while representing a patient safety issue are a measure of the prescribing process. same hospital) and the intervention pharmacist times. Of the 62 controls for whom pharmacist/pharmacy tech- nician review occurred, the times were missing for N=27. At 3-month follow-up, medication data were retrieved from GPs for 86 participants (94.5% of those from whom it was requested) in each arm and 133 partici- pants completed and returned both the EQ-5D-3L and the health resource use questionnaires (66 intervention, 67 control; 73.5% of those requested from all living participants). Sample size calculation Owing to the need to recruit patients before they received MR as usual care, this reduced the generalis- ability of the sample with all of those who had already received MR being automatically excluded. This was further compounded by recruitment activities which were focussed towards mornings. Based on the pilot data, we calculated that for a full trial, 1120 patients would need to be recruited to detect a 6% reduction (conservative assumption, one SE below 9% reduction seen) in 3-month unplanned readmission rates from a starting point of 26% with 90% power, using a 5% significance level and assuming 5% loss to follow-up. With a hospital requirement that all patients receive MR within 24 hours of admission, it is unsurprising that two-thirds of the control arm received some form of MR during their hospitalisation, a quarter of whom received this within 24 hours. There is no specific requirement for MR on discharge, however, and this may explain some of the differences seen between the two groups. The majority of discrepancies identified in the control arm by the researchers were found not to have been resolved and therefore reasons for the relative DISCUSSION It has been suggested that organisations are probably unlikely to repeat the benefits from MR services reported in the literature if there are deficiencies in intervention inten- sity and breadth.24 The results from the control arm of this study support this assertion and suggest that if MR of a similar nature to that seen in the intervention arm was to be shown to be cost-effective, then this would require significantly more pharmacist time. Considering evidence published poststudy completion, limiting the study population to those over the age of 70 and including a postdischarge telephone call as part of the intervention may have further enhanced the intervention.13 In line with previous research, the intervention pre- vented a large number of unintentional medicines- related discrepancies both during admission and post- discharge.3–6 The data obtained suggest that just less than a quarter of UDs identified at discharge were found to actually translate into primary care records at 3 months. While reasons for non-translation were not elucidated, the research suggests that the use of number of UDs at discharge as an outcome measure may over- emphasise the problem. The proportion of patients screened for eligibility and eventually recruited to the study was 20% and this could have been improved by increasing the number of week- ends covered (from 80% to 100%). Although some patients were not suitable for inclusion in the study as they had already been reviewed by a member of the pharmacy team prior to being approached by the RA, there was a sufficient number of patients available for recruitment. Not all primary care medication lists were made avail- able to researchers and approaches to addressing this will require consideration for a future definitive study. Using data on ‘unplanned readmission at 3 months’ would seem to be the most appropriate primary outcome measure for a future definitive study as it is a patient-orientated outcome which is most likely to reflect the effect of errors which occur at all stages of the process. Similar to hospital readmission, LOS is a cost which would be captured in any cost-effectiveness analysis. LOS was however found to be largely skewed by a small number of individuals who were admitted for extended periods. DISCUSSION The results from this study which was performed to inform the design of a future RCT suggest that even though MR activities are taking place, such a trial is feas- ible with reasonable recruitment and retention rates and that both cost and outcome data can be effectively obtained. Table 2 Comparison of UDs Intervention Control Unintentional discrepancies No. UDs No. patients No. per patient No. UDs No. patients No. per patient Admission 255 95 2.80 309 102 3.0 Resolved during hospital stay 250 95 2.74 Unknown Remaining at discharge 2 91 0.02 268 99 2.71 UDs, unintentional discrepancies. 6 Cadman B, et al. BMJ Open 2017;7:e013647. doi:10.1136/bmjopen-2016-013647 Cadman B, et al. BMJ Open 2017;7:e013647. doi:10.1136/bmjopen-2016-013647 6 Open Access Table 3 Comparison of outcome measures Outcome Measure N Intervention N Control Mean difference (SE of the difference) Length of stay (hours) Geometric mean (95% CI) 95 99.6 (76.59 to 129.63) 102 109.3 (87.0 to 137.3) Median (range) 95 94.0 (12–1077) 102 117 (13–1546) Arithmetic mean (SD) 95 224.8 (293.1) 102 203.9 (246.8) 20.84 (38.75) Hospital readmissions N (%) 95 30 (31.6) 101 37 (36.6) Hospital readmissions (emergency) N (%) 95 17 (17.9) 101 27 (26.7) Mortality N (%) 95 6 (6.3) 95 8 (7.8) Quality of life visual analogue scale change from baseline (high score better) Mean (SD) 63 5.64 (23.6) 68 7.15 (26.2) 1.51 (4.36) Table 3 Comparison of outcome measures ineffectiveness of the control arm MR requires elucida- tion through a detailed process evaluation. The process of collating all medicines-related data at different time points and sealing it until 3 months post- discharge provides an opportunity to identify discrepan- cies without adversely affecting the intervention. While the identification of UDs should be undertaken blind of allocation, the resources required for this may not be warranted when considering that the intentional or unintentional nature of the discrepancy cannot always be accurately determined. The average time spent on the intervention found within this study was very similar to that reported in a MR time and motion study23 but three times greater than that in the control arm. The study SOP required pharmacists to undertake initial MR, follow-up on all interventions to ensure that discrepancies had been addressed, assess all discharge letters for accuracy and correct them. Patients in the control arm frequently did not always receive all four elements and this probably explains most of the difference in time provided. Author affiliations 1 Author affiliations 1Pharmacy Department, Cambridge University Hospitals, Cambridge, UK 2School of Pharmacy, University of East Anglia, Norwich, UK 3Norwich Medical School, University of East Anglia, Norwich, UK 4Norwich Clinical Trials Unit, University of East Anglia, Norwich, UK 5School of Pharmacy, University of Jordan, Jordan Author affiliations 1Pharmacy Department, Cambridge University Hospitals, Cambridge, UK 2School of Pharmacy, University of East Anglia, Norwich, UK 3Norwich Medical School, University of East Anglia, Norwich, UK 4Norwich Clinical Trials Unit, University of East Anglia, Norwich, UK 5School of Pharmacy, University of Jordan, Jordan Author affiliations 1Pharmacy Department, Cambridge University Hospitals, Cambridge, UK 2School of Pharmacy, University of East Anglia, Norwich, UK 3Norwich Medical School, University of East Anglia, Norwich, UK 4Norwich Clinical Trials Unit, University of East Anglia, Norwich, UK 5School of Pharmacy, University of Jordan, Jordan Twitter Follow David Wright @K-7833-2016 Acknowledgements The authors would like to thank Dr Tracey Sach for her contribution in the design of the health economics element of the project and in the development of the original grant proposal, Antony Warford for his contribution to the management, delivery and dissemination of the project as the patient and public involvement representative and Kellie Hempstead and the pharmacists volunteering to provide the intervention at weekends in particular Lynn Martin for their efforts in completing this study. The input by the Norwich Clinical Trials Unit throughout the study is also acknowledged. Quality of data collection with respect to LOS, mortal- ity and readmission rates was high as this information was available from hospital records. Resource usage data and quality of life scores were, however, only available for two-thirds of participants at follow-up. Consequently researchers powering a definitive study on readmission rate will need to consider the possibility of there being more missing data for the cost-effectiveness analysis and imputation of missing data30 may be necessary to address this. Contributors BC, EAH, DW, JD and RH participated in the conception and design of this study. AB, BC, DW,GB, IN and LI participated in the analysis of the data and interpretation of the results. AB, BC and DW drafted the article and all other authors performed critical review of the article. All authors approved the submission of this article. Funding This article presents independent research funded by the National Institute for Health Research (NIHR) under its Research for Patient Benefit (RfPB) Programme (Grant Reference Number PB-PG-0110-20116). Author affiliations 1 The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. This study was a pilot study and was not designed to obtain a definitive answer to whether MR provided to all patients within 24 hours of admission was cost-effective. However, it was conducted as an RCT, conforming to expected standards and consequently it can be assumed that it would be possible to perform a full-scale RCT in the future. An internal pilot would be warranted in such a trial as the trial should be multicentre in nature and consequently local variations in recruitment rates and service delivery would require identification and appro- priate local adaptation. Additionally, costs related to service delivery may differ between settings and conse- quently time for service delivery would require estimation. Disclaimer The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. Competing interests None declared. Patient consent Obtained. Patient consent Obtained. Ethics approval Ethical approval was received on 14 June 2012 from NRES Committee East of England—Essex Research Ethics Committee. Reference number: 12/44/0143. Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement No additional data are available. Data sharing statement No additional data are available. Open Access This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http:// creativecommons.org/licenses/by/4.0/ The pilot study has shown that it is feasible to perform an RCT to determine the effectiveness and cost- effectiveness of a pharmacist MR within 24 hours of admission and again at discharge. This study has demon- strated that this form of intervention does appear to reduce medication errors at discharge, and may reduce LOS and hospital readmissions. We consider that unplanned readmission at 3 months is the most suitable primary outcome measure but LOS, errors, mortality and quality of life should be captured. DISCUSSION Furthermore, it is not affected by errors which translate into primary care, that is, does When identifying UDs, we have assumed that the MRP gen- erated list in the intervention arm and the RA generated list in the control arm were accurate. Both are unrealistic assumptions and ideally within a definitive study all data should have been reviewed independent of the service and blinded to group alloca- tion. The unblinded identification of MRs and inability to confirm intentional or unintentional nature of errors in many instances also means that the data on UDs must be treated with further caution. 7 Cadman B, et al. BMJ Open 2017;7:e013647. doi:10.1136/bmjopen-2016-013647 Open Access not reflect the full impact of the service. Within the UK, hospitals are penalised for unplanned readmissions within 1 month of discharge and therefore collection of this data may also be warranted for a UK-based definitive study. understanding of the intervention. Pharmacists deliver- ing the intervention arm within a definitive study should follow an SOP and be trained to undertake the role to ensure standardisation in delivery. While the results of this pilot emulate other studies where prescribing errors were reduced in the intervention arm, there is an add- itional cost associated with providing the intervention and therefore high-quality evidence from a multicentre RCT is now needed to determine both its effectiveness and cost-effectiveness. Unplanned readmission has been used as a primary outcome measure within other similar MR trials.25–29 While differences in unplanned readmission at 3 months have been demonstrated,28 29 this has fre- quently not been the case at either 1 or 6 months.25–27 Within the first month, patients may still be using the medication they were discharged with and consequently this may lessen the impact of errors on the discharge letter resulting from incorrect translation into primary care. Kwan et al14 after systematically reviewing the litera- ture suggest that unplanned readmission data should probably be collected for more than 30 days post- discharge. Unplanned readmission at 6 months will be affected by more factors unrelated to the index admis- sion than at 3 months and therefore it may be more dif- ficult to identify the impact of MR intervention using this outcome measure. REFERENCES 1. World Health Organisation. High 5s Fact Sheet: The High 5s Assuring Medication Accuracy at Transitions of Care: Medicines Reconciliation Standard Operating Procedure. http://www.who.int/ patientsafety/implementation/solutions/high5s/h5s-fact-sheet.pdf? ua=1 Additionally, a thorough process evaluation is war- ranted in order to provide a more complete 2. The University of Sheffield SoHaRRS. A systematic review of the effectiveness and cost-effectiveness of interventions aimed at 8 Cadman B, et al. 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Performance on Indirect Measures of Race Evaluation Predicts Amygdala Activation
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https://orca.cardiff.ac.uk/101525/1/art%253A10.1186%252Fs13063-017-1902-y.pdf
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Causal inference with randomised clinical trials of chemotherapy : the importance of well-documented treatment side-effects
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MEETING ABSTRACTS Open Access Meeting abstracts from the 4th International Clinical Trials Methodology Conference (ICTMC) and the 38th Annual Meeting of the Society for Clinical Trials Liverpool, UK. 07–10 May 2017 Published: 8 May 2017 Published: 8 May 2017 Published: 8 May 2017 Background h l g The evaluation of innovation in surgery is a complex process challenged by evolution of technique, operator learning curves, inconsistent procedural quality, and strong treatment preferences among patients and clinicians [1]. Given these challenges, the development of early-stage novel surgical techniques has been criticized for poor-quality study methodology and data reporting [2, 3]. To address this, the IDEAL framework (Idea, Development, Exploration, Assessment, Long-term follow-up) proposes a five stage stepwise evaluation of innovative procedures to allow a more transparent and ethical introduction of new techniques [4]. The IDEAL framework was proposed in 2009 and there has been no systematic assessment of its use. We examine the uptake and utilization of IDEAL by surgical innovators by reviewing the pub- lished literature. Results We identified 311 papers citing one or more of the 11 key IDEAL papers. Of these, 30 described having followed the stage- appropriate IDEAL recommendations to conduct their innovation study. Interim analysis indicates considerable variation in uptake between clinical specialties and geographical regions. We are cur- rently undertaking more in-depth analysis on the studies of these Trials 2017, 18(Suppl 1):P2 Poster Presentations early users of IDEAL to examine how the framework and recom- mendations have been used. We also plan to conduct qualitative research with the Pis of these studies to learn more about how useful they found IDEAL as a tool for their research plan. Discussion P1 Ideal framework and recommendations: a literature review of its utilization by surgical innovators since 2009 Joshua Feinberg1, Claudia Ashton1, Allison Hirst1, Christopher Pennell2, Peter McCulloch1 1 2 Since its inception in 2009, surgical researchers worldwide are beginning to recognize and utilize the IDEAL recommendations. Early adopters have been concentrated within a few surgical specialties and focused on the pre-RCT developmental stages of IDEAL, where research guidance has previously been lacking. This review of the literature will help the IDEAL Collaboration to learn from the early adopters’ experiences and identify how to work with future surgical innovators to develop IDEAL as a practical framework in order to conduct the highest quality surgical research. 1University of Oxford; 2Maimonides Medical Center Correspondence: Joshua Feinberg Trials 2017, 18(Suppl 1):P1 Methods We searched Web of Science to identify all articles published be- tween 1st January 2009 and 30th September 2016 that cited any of the 11 key papers published by the IDEAL Collaboration. All abstracts were assessed by two independent researchers to identify papers ex- plicitly describing using IDEAL recommendations to conduct their primary research. Included papers were reviewed and categorized by characteristics including clinical specialty area, type of journal, coun- try of origin, publication date, and the IDEAL stage. Each paper was further critiqued on how well it met the specified IDEAL stage recom- mendations [1]. Development of a complex exercise intervention for prevention of shoulder dysfunction in high-risk women following treatment for breast cancer: prevention of shoulder problems trial (PROSPER) Helen Richmond1, Cynthia Srikesavan2, Esther Williamson2, Jane Moser3, Meredith Newman3, Sophie Rees1, Sarah E Lamb4, Julie Bruce1 1University of Warwick; 2University of Oxford; 3Oxford University Hospitals NHS Foundation Trust; 4University of Oxford/University of Warwick Correspondence: Helen Richmond Trials 2017 18(Suppl 1):P2 References 1. Pennell, C.P., et al., Practical guide to the Idea, Development and Exploration stages of the IDEAL Framework and Recommendations. British Journal of Surgery, 2016. 103(5): p. 607–615. 2. Angelos, P., The art of medicine The ethical challenges of surgical innovation for patient care. Lancet, 2010. 376(9746): p. 1046–1047. 3. Ergina, P.L., et al., Surgical Innovation and Evaluation 2 Challenges in evaluating surgical innovation. Lancet, 2009. 374(9695): p. 1097–1104. 4. Mcculloch, P., et al., Surgical Innovation and Evaluation 3 No surgical innovation without evaluation: the IDEAL recommendations. Lancet, 2009. 374(9695): p. 1105–1112. Trials 2017, 18(Suppl 1):200 DOI 10.1186/s13063-017-1902-y Trials 2017, 18(Suppl 1):200 DOI 10.1186/s13063-017-1902-y 1. Pennell, C.P., et al., Practical guide to the Idea, Development and Exploration stages of the IDEAL Framework and Recommendations. British Journal of Surgery, 2016. 103(5): p. 607–615. 2. Angelos, P., The art of medicine The ethical challenges of surgical innovation for patient care. Lancet, 2010. 376(9746): p. 1046–1047. 3. Ergina, P.L., et al., Surgical Innovation and Evaluation 2 Challenges in evaluating surgical innovation. Lancet, 2009. 374(9695): p. 1097–1104. 4. Mcculloch, P., et al., Surgical Innovation and Evaluation 3 No surgical innovation without evaluation: the IDEAL recommendations. Lancet, 2009. 374(9695): p. 1105–1112. Conclusions We followed the MRC theoretical framework to develop a multicompo- nent exercise programme for the prevention of shoulder problems fol- lowing breast cancer treatment. This complex intervention is currently being evaluated within a large UK pragmatic RCT [ISRCTN35358984]. To date, 105 women with newly diagnosed breast cancer have been recruited from 12 centres across England. P4 Randomised controlled trials and realist evaluation: in what contexts and how? Rebecca Randell1, Jon Hindmarsh2, Joanne Greenhalgh1, Natasha Alvarado3, Peter Gardner1, Dawn Dowding4, Alexandra Cope1, Julie Croft1, Andrew Long1, Alan Pearman1 1University of Leeds; 2King’s College London; 3Newcastle University; 4Columbia University Intervention development and treatment success in randomised controlled trials of rehabilitation Rebecca Randell1, Jon Hindmarsh2, Joanne Greenhalgh1, Natasha Alvarado3, Peter Gardner1, Dawn Dowding4, Alexandra Cope1, Julie Croft1, Andrew Long1, Alan Pearman1 1University of Leeds; 2King’s College London; 3Newcastle University; 4Columbia University Rebecca Randell1, Jon Hindmarsh2, Joanne Greenhalgh1, Natasha Alvarado3, Peter Gardner1, Dawn Dowding4, Alexandra Cope1, Julie Croft1, Andrew Long1, Alan Pearman1 1 f 2 3 Jacqueline Hill, Claire Pentecost, Katie Finning, Angelique Hilli, David A. Richards, Victoria A. Goodwin University of Exeter Correspondence: Jacqueline Hill Trials 2017, 18(Suppl 1):P3 y Correspondence: Rebecca Randell Trials 2017, 18(Suppl 1):P4 g Conclusions We have applied techniques for mixed methods analysis in innovative ways to explore the relationship between intervention development and treatment effects. This work may help us better understand the role of intervention development in explaining why so few interven- tions in rehabilitation that undergo experimental testing result in effect- ive new treatments. Other factors, including the design and conduct of fully-powered trials, may also help explain the relatively few number of treatment successes and require further research. Methods We included 15 superiority RCTs funded by NIHR-HTA from 1997 to July 2016, that evaluated a physical rehabilitation programme and re- ported their main findings in a peer-reviewed journal or NIHR-HTA monograph. We extracted data on intervention development in re- spect of five areas described by the credeci 2 reporting criteria for “development” and “feasibility & piloting”: (i) description of the inter- vention’s underlying theoretical basis; (ii) description of all of the intervention components; (iii) illustration of any intended interactions between different components; (iv) description of the pilot test and its impact on the definitive intervention; (v) consideration of the con- text’s characteristics in intervention modelling. We coded the ex- tracted data thematically. We classified primary outcome data into one of six categories developed by Djulbegovic et al. (2008) to differ- entiate studies where outcomes favour the intervention, the control, demonstrate no difference between trials arms and are conclusive or inconclusive. To examine the relationship between intervention de- velopment and primary outcome data, we are applying novel mixed methods analytical techniques. We are combining the narrative data on intervention development with the numeric data on treatment outcomes in a joint category/themes display: for each category de- fined by Djulbegovic et al. we will present a summary of the the- matic data on intervention development in each trial for whom the category applies. In this way, we will compare the intervention devel- opment work that has been undertaken for trials that result in differ- ent outcomes. Patient and public involvement (PPI) was central to the development of the PROSPER intervention from its inception. We engaged PPI members from the initial application phase and have had ongoing input throughout the project. In conjunction with PPI, development work began with a comprehensive literature review to identify sys- tematic reviews and RCTs of shoulder-specific exercises and general physical activity during and after breast cancer treatment. This pro- vided the broad theoretical basis for the content of a structured exer- cise programme which was further developed and refined in a workshop with clinical experts, researchers, and patient representa- tives. Individual face-to-face interviews were then conducted with seven women previously treated for breast cancer, providing feed- back on intervention content and patient-facing materials. The PROS- PER intervention was pilot tested with 18 women newly diagnosed with breast cancer, at three hospital sites, allowing further refinement to ensure feasibility for delivery within the UK NHS. Results We found that four trials were significantly in favour of the new treat- ment, one was significantly in favour of the control, eight had a true negative outcome and two were inconclusive. Our preliminary data ex- traction reveals that the amount of (reported) intervention development work undertaken prior to experimental testing differs considerably. We are now applying our mixed methods analytical procedures to investi- gate the relationship between outcomes and intervention development. Conclusions Background Shoulder dysfunction and pain following breast cancer treatment is common, impacting upon postoperative quality of life. Exercise may improve shoulder function and reduce the risk of postoperative © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Trials 2017, 18(Suppl 1):200 Page 2 of 235 Page 2 of 235 Trials 2017, 18(Suppl 1):200 found that very few new interventions achieved superior outcomes relative to controls. To date, no research has examined why so few rehabilitation interventions that undergo testing in RCTs result in ef- fective new treatments. complications. However, there is uncertainty around the optimal timing (commencement) and exercise dosage (frequency, intensity, length of time and type of exercise) required for optimal results. We considered Medical Research Council (MRC) guidance for the development of a complex intervention, which highlights the need for a planned, phased approach based on available evidence, appropriate theoretical princi- ples and thorough piloting. We developed a complex intervention for the prevention of shoulder dysfunction following breast cancer treat- ment for evaluation within the framework of a large pragmatic multi- centre randomised controlled trial in the UK NHS setting. M h d complications. However, there is uncertainty around the optimal timing (commencement) and exercise dosage (frequency, intensity, length of time and type of exercise) required for optimal results. We considered Medical Research Council (MRC) guidance for the development of a complex intervention, which highlights the need for a planned, phased approach based on available evidence, appropriate theoretical princi- ples and thorough piloting. We developed a complex intervention for the prevention of shoulder dysfunction following breast cancer treat- ment for evaluation within the framework of a large pragmatic multi- centre randomised controlled trial in the UK NHS setting. M th d (1) To establish work that has been undertaken to develop physical rehabilitation interventions prior to testing in an NIHR-HTA funded RCT. (2) To examine the relationship between intervention develop- ment and the primary outcome of experimental testing. Methods Results The literature review identified several systematic reviews and new clinical trials suggesting that early structured exercise, started within a few days of surgery, versus delayed exercise may improve shoulder range of movement (ROM) in the short and long term. Evidence sug- gested that shoulder flexion and abduction be restricted to 90 de- grees for the first postoperative week to reduce risk of increased wound drainage. There was also evidence to suggest that postopera- tive strength training was safe and that general physical activity can enhance physical and psychological recovery. The final PROSPER ex- ercise intervention, underpinned by evidence, comprised of three main components: specific exercises targeting shoulder range of motion and upper arm muscle strength, general physical activity, and behavioural strategies to improve adherence. The exercise programme is structured, individualised, supported by trained phys- iotherapists, and delivered over a 12-month period with a focus upon self-management at home. Women randomised to the exercise programme receive three face-to-face sessions with a physiotherap- ist, with the option of a further three appointments that can be deliv- ered either face-to-face or by telephone. Background g Randomised controlled trials (RCTs) of physical rehabilitation inter- ventions evaluate multi-faceted interventions that are delivered in complex healthcare systems. In a synthesis of the outcomes of trials of rehabilitation interventions funded by the UK National Institute for Health Research – Health Technology Programme (NIHR-HTA), we g It is widely agreed that, if the aim is to inform policy and practice, randomised controlled trials (RCTs) of complex interventions should be coupled with process evaluations. Realist evaluation provides a strong theoretical foundation to explore complex interventions, using a process of eliciting, testing, and refining stakeholders’ theories of Page 3 of 235 Trials 2017, 18(Suppl 1):200 Page 3 of 235 Cluster randomised controlled trials (CRCT) are used extensively in evaluations of healthcare interventions. However, cluster cross-over randomised trials are novel: a recent systematic review identified only 91 such studies [1]. While providing efficiency gains compared to crcts, the cross-over design adds complexity to the design and analyses. To date, the literature has been limited to the analysis of binary outcomes [2,3]. how an intervention works, for whom, and in what contexts. There is debate about the relationship between realist evaluation and RCTs. One concern is that RCTs take place in closely controlled contexts and so do not allow for exploration of how different contexts shape the outcomes of an intervention. In this presentation, we will draw on our experience of undertaking a three-phase realist process evalu- ation alongside an RCT comparing robotic and laparoscopic surgery to address two methodological questions: (1) To what types of trials can realist evaluation make a meaningful contribution?; and (2) How is that contribution best achieved? The headpost [4] study is an international multicentre randomised cross-over clinical trial comparing the effectiveness of the lying flat (0°) head position with the sitting up (> = 30°) head position, applied within the first 24 hours of admission to hospital for patients with acute stroke, on functional outcome according to the modified Rankin scale (MRS) by blind assessors at 90 days. A total of 114 sites were allocated either to (a) lying flat head position or (b) sitting up head position as the first intervention, to be applied to up to 70 con- secutive stroke patients before crossing over to the other head pos- ition. All eligible stroke patients presenting to the hospital from the start date were to be prospectively and consecutively enrolled. Methods In Phase 1, a literature review identified stakeholders’ theories concern- ing how robotic surgery becomes embedded into practice and its im- pacts on teamwork. These were refined through interviews with theatre teams across nine hospitals. In Phase 2, the theories were tested through a multi-site case study across four hospitals. Case sites were se- lected to ensure variation in the theatre teams’ experience of robotic surgery, an important contextual factor within the theories. Data were collected using multiple methods: structured and ethnographic obser- vation; video analysis; qualitative interviews; and questionnaires. In Phase 3, interviews were conducted at case sites with staff representing other surgical disciplines, to assess generalisability of the findings. Results This presentation outlines the statistical analysis planning and con- duct for the headpost study, taking account of its cluster cross-over nature. The primary efficacy analysis was conducted using a hierarch- ical cumulative logistic regression to allow direct modelling across all levels of clustering by including both random cluster and random cluster-period effects. The implications for the analysis of secondary and safety outcomes as well as strategies for sensitivity analyses and handling of missing data, are to be discussed. The focus will be on practicalities of analysis rather than mathematical aspects of cluster cross-over trials. While the RCT delivered important results on outcomes, the findings from the realist process evaluation further enhanced our understand- ing of the introduction of robotic surgery. The combination of methods deployed enabled us to identify and interrogate a range of perspectives on the differences between robotic and laparoscopic surgery and the ways in which robotic surgery is implemented in dif- ferent sites. Most strikingly, we were able to capture unanticipated consequences of robotic surgery in terms of impacts on teamwork, along with strategies used to counteract such unanticipated conse- quences. These issues relate to the introduction of robotic surgery as a surgeon-led process but which is dependent on support at differ- ent levels of the organisation. The process evaluation directed our at- tention to the importance of whole team training, experienced and dedicated teams, and suitably sized operating theatres. l Background The primary outcome was the modified Rankin score, a 7-level ordinal scale between 0 (completely independent) and 6 (dead), which is commonly used in stroke trials. References 1. Arnup SJ, Forbes AB, Kahan BC, et al. Appropriate statistical methods were infrequently used in cluster-randomized crossover trials. J Clin Epidemiol 2016;74:40–50. 2. Turner RM, White IR and Croudace T. Analysis of cluster randomized cross- over trial data: a comparison of methods. Stat Med 2007;26:274–289. Conclusions 3. Morgan KE, Forbes AB, Keogh RH, et al. Choosing appropriate analysis methods for cluster randomised cross-over trials with a binary outcome. Stat Med 2016 Sept 28. Realist evaluation provided a robust framework to identify and test stakeholders’ theories on deployment of robotic surgery. The results of this study move beyond the RCT to deliver clear guidance on how to deploy robotic surgery and how to ensure effective teamwork when undertaking robotic surgery. So, realist evaluation can play a valuable role alongside pragmatic rcts of complex interventions that seek to ex- plore effectiveness in a range of contexts, eliciting theories about how contexts shape outcomes and then collecting empirical data to test and refine them. Theory elicitation should happen before the RCT to ensure it secures relevant data to support testing of identified theories. 4. Muñoz-Venturelli P, Arima H, Lavados P, et al. Head Position in Stroke Trial (headpost) - Sitting up vs lying flat, positioning of patients with acute stroke: study protocol for a cluster randomised controlled trial. Trials 2015;16:256. P7 Implementing mobile electronic patient reported outcomes (EPRO) in a long-term trial with an aging and diverse population Ashley Hogan, Nicole Butler, Ashley N. Hogan, Alla Sapozhnikova, Ella Temprosa George Washington University Correspondence: Ashley Hogan Trials 2017, 18(Suppl 1):P7 Implementing mobile electronic patient reported outcomes (EPRO) in a long-term trial with an aging and diverse population Ashley Hogan, Nicole Butler, Ashley N. Hogan, Alla Sapozhnikova, Ella Temprosa George Washington University Correspondence: Ashley Hogan Trials 2017, 18(Suppl 1):P7 1. Arnup SJ, Forbes AB, Kahan BC, et al. Appropriate statistical methods were infrequently used in cluster-randomized crossover trials. J Clin Epidemiol 2016;74:40–50. p Monitoring the Primary Outcome Data In order to monitor the completeness of the primary outcome data, a monthly reporting system was developed by the trial statistician to allow close analysis of data completeness. The reports revealed 17.9% of missing primary outcome data from babies known to be alive at SD28. A large proportion of these did not have a reported SD28 CUSS. This was due to a variety of causes, including transfer out of neonates prior to SD28 from the recruiting site to smaller non- participating units. In recent years, a growing trend toward global research has led to randomized controlled trials (RCTs) becoming larger and increasingly more complex. More patients are being enrolled entailing greater use of multisite trials, case report forms (CRFs) are more complicated, and larger budgets are necessary to accommodate for the greater volume of participants and sites involved in a RCT. Centralized statis- tical monitoring (CSM) is commonly used for guaranteeing data qual- ity by detecting data issues early, such as errors, sloppiness, tampering, and fraud, before significant problems occur. Through off-site central monitoring, onsite monitoring can be more efficiently targeted. Equally important to ensuring data quality is assessing the adequacy of the trial design and performance. Design errors, if not discovered and addressed early, can largely bias study findings and make a trial difficult or impossible to interpret. Poor adherence to de- sign and a lack of oversight can result in an unsuccessful trial with drastic ramifications, including revoking one’s clinical and research privileges, funding, and leaving a tarnished reputation. Consequently, the purpose of this research was to apply CSM to the monitoring of various aspects related to the design and performance of a clinical trial. p p g Measures to optimise the data The monthly reports allow the TMG to take measures to maximise the completeness of the data obtained from the study. A transfer pack was developed to inform new sites of required information and an accompanying letter provided, to enable PIs to request primary outcome data from colleagues within the new units. The SD28 win- dow was extended from +/−3 days to −5/+10 days of SD28. This was considered by the neonatologists on the TMG, and the independent members of the TSC, to possess the same clinical validity. Evaluation of a state-wide chronic disease management program on health service utilisation using a propensity-matched control group Evaluation of a state-wide chronic disease management program on health service utilisation using a propensity-matched control group 2 Ashley Hogan, Nicole Butler, Ashley Ella Temprosa George Washington University Correspondence: Ashley Hogan Trials 2017, 18(Suppl 1):P7 Laurent Billot1, Kate Corcoran1, Alina Mcdonald1, Gawaine Powell-Davies2, Anne-Marie Feyer1 1 2 Laurent Billot1, Kate Corcoran1, Alina Mcdonald1, Gawaine Powell-Davies2, Anne-Marie Feyer1 y 1The George Institute for Global Health; 2Centre for Primary Health Care and Equity, University of New South Wales, University of Sydney) Correspondence: Laurent Billot Trials 2017, 18(Suppl 1):P5 y 1The George Institute for Global Health; 2Centre for Primary Health Care and Equity, University of New South Wales, University of Sydney) Correspondence: Laurent Billot Trials 2017, 18(Suppl 1):P5 y 1The George Institute for Global Health; 2Centre for Primary Health Care and Equity, University of New South Wales, University of Sydney) Correspondence: Laurent Billot Trials 2017, 18(Suppl 1):P5 1The George Institute for Global Health; 2Centre for Primary Health Care and Equity, University of New South Wales, University of Sydney) Correspondence: Laurent Billot Trials 2017, 18(Suppl 1):P5 The clinical research enterprise cannot escape the shift from paper case report forms (CRF) to direct data entry of case report forms. This daunting shift has the potential to reduce the workload of clinical sites and provide an environmentally friendly source of data collection while also improv- ing the quality and integrity of the data by removing the chance of tran- scription errors, minimizing missing data, allowing for real time logic and range checks, and leading to faster database locks. For our long-term multi-center clinical trial, the use of mobile electronic patient reported outcomes (EPRO) for self-administered questionnaires was a clear first step towards this shift implemented within our custom built web-based data collection and management system called MIDAS (Multi-modal Inte- grated Data Acquisition System). Mobile EPRO for self-administered ques- tionnaires increases flexibility of visit flow, and allows for the collection of sensitive information such as questions about sexual health. This abstract is not included here as it has already been published. P9 Data monitoring committee overseeing multiple international randomised controlled trials 1 2 3 Data collection via mobile data entry for six self-administered ques- tionnaires began in August 2016 and was accompanied by a survey to assess user acceptability. Of the 300 visits completed to date, 90% used the mobile EPRO version with nearly 100% survey response rate. This presentation will present the results of the survey as well as feedback from participants and staff including utilization rates, overall experience, font size, button size, view preference, ease of use, and whether paper or mobile entry is preferred. We will share results of the survey overall and by demographic subgroups using the ~1,000 visits expected by the time of the presentation. The COPE (Consortium for Organ Preservation in Europe) include three clinical trials to improve preservation and reconditioning strat- egies for kidneys and livers procured for transplantation aiming to in- crease the number and quality of grafts used. P8 Optimising primary outcome data collection in a neonatal trial Alison J. Deary1, Karen Willoughby2, Ana Mora1, Anna Curley3 1NHSBT Clinical Trials Unit, Long Road, Cambridge, CB2 0PT, UK; 2Department of Obstetrics and Gynaecology, Box 223, Level 2 Rosie Hospital Robinson Way, Cambridge CB2 OSW, UK; 3Neonatal Unit, National Maternity Hospital, Holles Street, Dublin 2, Eire Correspondence: Alison J. Deary Trials 2017, 18(Suppl 1):P8 y Despite the trials being led at different centres, they are centrally managed from one main centre where the Principal Investigator, the project’s governance and management are based. This is one of the reason why it was decided to set up a single ‘combined’ Data Moni- toring Committee to oversee the three trials with a single six- monthly meeting to review all three trials. This seems to be the most convenient approach in similar situations/ settings as it reduces the number of meetings to organise as well as expenses. However, it does not come without difficulties particularly when the same person is preparing the reports for all the studies and multiple sites and/or countries are involved. p Monitoring the Primary Outcome Data If no scan had been obtained within the new extended timeframe, our medical experts, or, in some cases, independent expert, were given approval to impute the primary outcome given sufficient supporting clinical evidence. Evaluation of a state-wide chronic disease management program on health service utilisation using a propensity-matched control group P6 Analysis of the head position in stroke trial (headpost): an international cluster randomised cross-over trial Laurent Billot, headpost Steering Committee The George Institute for Global Health Correspondence: Laurent Billot Trials 2017, 18(Suppl 1):P6 Trials 2017, 18(Suppl 1):200 Page 4 of 235 Page 4 of 235 Concern for EPRO implementation and anxiety flourished when mak- ing considerations for the aging and diverse population of the trial for whom the use of mobile devices may be less ubiquitous. The aging study population is majority female, and ethnically diverse. During the implementation of EPRO for self-administered question- naires, we kept in mind the needs of elderly participants with cogni- tive decline, dexterity problems, and visual impairments as well as the needs of participants who speak English as a second language or those with disabilities in reading and writing. This piece of work shows the value of the statisticians and data team on the trial management team working together to improve the sci- entific integrity of the study. Our missing primary outcome data cur- rently stands at approximately 1.4%. Liaising closely with research site teams and maintaining good relationships is crucial to trial success. This piece of work shows the value of the statisticians and data team on the trial management team working together to improve the sci- entific integrity of the study. Our missing primary outcome data cur- rently stands at approximately 1.4%. Liaising closely with research site teams and maintaining good relationships is crucial to trial success. P9 Data monitoring committee overseeing multiple international randomised controlled trials Virginia Chiocchia1, Susan Dutton2, Rutger Ploeg3 1Centre for Statistics in Medicine (CSM) and Surgical Intervention Trials Unit (SITU), University of Oxford; 2Oxford Clinical Trials Research Unit (OCTRU) and Centre for Statistics in Medicine (CSM), University of Oxford; 3Nuffield Department of Surgical Sciences, University of Oxford Correspondence: Virginia Chiocchia Trials 2017, 18(Suppl 1):P9 Surveillance of clinical trial performance using centralized statistical monitoring 1 2 2 2 p Monitoring the Primary Outcome Data Background planet-2 (ISRCTN87736839) is an international multicentre trial of platelet count thresholds for prophylactic platelet transfusions in pre- term neonates. The trial commenced recruitment in June 2011 and to date 573/660 neonates have been randomised to one of 2 arms. Depending on their allocated threshold, the baby receives a platelet transfusion when platelet counts drops to either below 50x10^9 or 25x10^9. The primary outcome measure for planet-2 is the propor- tion of patients who either die or experience a major bleed up to and including Study Day 28 (SD28). A cranial ultrasound scan (CUSS) at SD28 is the prime marker for major intracranial bleeds at this point. p The different benefits and challenges experienced will be illustrated in order to provide a helpful reference to anyone that may consider this option in similar situations. P10 Surveillance of clinical trial performance using centralized statistical monitoring Eileen Stock1, Zhibao Mi2, Kousick Biswas2, Ilana Belitskaya-Levy2 1Department of Veterans Affairs; 2Cooperative Studies Program Coordinating Center, Department of Veterans Affairs Correspondence: Eileen Stock Trials 2017, 18(Suppl 1):P10 Methods g g Four searches were run on the entire trial record. The following search terms were used: "feasibility trial", "feasibility study", "pilot trial" and "pilot study" and 3039 unique trial records were identified. The unique trial records ids were extracted from the search results and then the trial records were downloaded. Data such as the trial title, target sample size, recruitment start date, recruitment end date, the longitude and latitude of the recruiting centre were then extracted from the records, using regular expressions, and collated into an Excel where open- ended text fields (e.g., target sample size) were manually cleaned. Four searches were run on the entire trial record. The following search terms were used: "feasibility trial", "feasibility study", "pilot trial" and "pilot study" and 3039 unique trial records were identified. The unique trial records ids were extracted from the search results and then the trial records were downloaded. Data such as the trial title, target sample size, recruitment start date, recruitment end date, the longitude and latitude of the recruiting centre were then extracted from the records, using regular expressions, and collated into an Excel where open- ended text fields (e.g., target sample size) were manually cleaned. Shiny, a web application framework for R, was then used to create an online tool to interrogate the data. For various health conditions, specified by the researcher, the tool can be used to obtain descrip- tive summaries and graphical displays of pilot and feasibility studies for the following factors: period of recruitment, target sample size, target recruitment rate per month, location of trial centres. The Collaborative Studies Coordinating Center (CSCC) in the Department of Biostatistics at the University of North Carolina serves as the coordinat- ing center for ARIC and provides the infrastructure for the data collection using the CSCC-developed, web-based data management system, Caro- lina Data Acquisition and Reporting Tool (CDART). q p g Neurocognitive test data collection in ARIC began at Visit 2 (1990–92) and was repeated in Visits 3 (1993–95) and 4 (1996–98) using 3 neuro- cognitive tests. An ancillary study was conducted in 2004–06 on a subset of the ARIC cohort where the test battery was expanded. At Visit 5 (2011–13), the expanded neurocognitive test battery was collected on 6538 participants, enabling the investigators to examine cognitive function changes over 24 years, particularly in the areas of memory, language and executive function. Result and Conclusions This monthly report system provides the information to allow the trial manager to contact the site teams a few days before each ran- domised baby reaches SD28 to remind them of the need to perform a CUSS and this has proved very effective in optimising the data completeness. Study design quality metrics assessed for anomalies included adher- ence to inclusion and exclusion criteria, recruitment, administration of treatment, blinding, visit scheduling, patient follow-up, data Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 5 of 235 Page 5 of 235 immediately available for comparison to Visit 5 factor scores through an application called from CDART. The behind the scenes program- ming calculates the Visit 6 factor scores for the cognitive areas of interest allowing for immediate determination of cognitive domain failure and generalized cognitive decline compared to Visit 5 per- formance, despite the fact that not all participants completed the exact same battery at each visit. submission, and the reporting of safety measures. Each metric can be evaluated across sites as in a multisite RCT, or across clinicians to assist in identifying potential threats to a trial’s performance. A pro- gram was developed to apply CSM for monitoring the performance of a clinical trial. CSM was applied monthly, in conjunction with regu- larly scheduled risk-based monitoring. For continuous measures of trial performance, modified boxplots described distributions, differences in the proportion of outliers were assessed using chi-square analyses, dif- ferences by site were examined with analysis of variance (or the non- parametric equivalent) and further assessed using pairwise tests, and homogeneity of variance and sites with outlying or inlying variance were also determined. Confidence bands were used to provide add- itional monitoring of trial performance. For categorical measures, chi- square analyses and logistic regression were employed. The participants who show failure in at least 1 cognitive domain and significant global cognitive decline from Visit 5 will have additional data collected from a proxy or informant and will undergo a complete data review by members of the classification committee in order to deter- mine neurocognitive status as dementia, MCI, normal, or unclassifiable. y g g y CSM applied to study design elements can be used to assess trial performance over time throughout the duration of a study. P12 An online tool for exploring recruitment achievability for a feasibility and pilot studies in the UK y p Andrew Brand, Nicola Totton, Paul Brock Bangor University Correspondence: Andrew Brand Trials 2017, 18(Suppl 1):P12 The aim of our online tool, is to use openly available data to help in- form researchers, in the UK, whether a given target size, is broadly achievable for a feasibility or pilot study investigating a specific health condition. Information obtained from the online tool may also further help determine a suitable recruitment period for a feasibility or pilot study investigating a given health condition. P11 Operationalizing the use of latent variables in the process of determining an ARIC participant’s neurocognitive status at visit 6 Sheila Burgard1, James Bartow1, Sonia Davis1, Alden Gross2, Tom Mosley3, Richey Sharrett2 1University of North Carolina; 2Johns Hopkins University; 3University of Mississippi Medical Center Correspondence: Sheila Burgard Trials 2017, 18(Suppl 1):P11 Operationalizing the use of latent variables in the process of determining an ARIC participant’s neurocognitive status at visit 6 Sheila Burgard1, James Bartow1, Sonia Davis1, Alden Gross2, Tom Mosley3, Richey Sharrett2 1 2 3 Operationalizing the use of latent variables in the process of determining an ARIC participant’s neurocognitive status at visit 6 Sheila Burgard1, James Bartow1, Sonia Davis1, Alden Gross2, Tom Mosley3, Richey Sharrett2 1 2 3 p y g g g Ideally, data on the actual sample sizes obtain in the feasibility and pilot studies would have been more informative than the target size data. Unfortunately, we were unable to find an openly available source for this data. However we feel that the target sample size data, along with the recruitment period can provide a rough guide to the achievability of recruitment targets for feasibility and pilot studies. For instance, if a pilot study had a target sample size of 60 and a recruitment period of 18 months for a study investigating a health condition you are interested in, you might want to reconsider running a similar pilot study with a target size of 100 for 6 months. We therefore believe that this data has value in making informed de- cisions with regard to recruitment for a feasibility and pilot studies. We identified UK Clinical Trial Gateway (UKCTG) as providing the best source of UK based data to harvest. The UKCTG was set up by the National Institute for Health Research (NIHR) to essentially help re- cruit people to clinical trials in the UK. P12 An online tool for exploring recruitment achievability for a feasibility and pilot studies in the UK Because there were no facil- ities for downloading data from the UKCTG website, a web scraping methodological was adopted and implemented using R. y Ideally, data on the actual sample sizes obtain in the feasibility and pilot studies would have been more informative than the target size data. Unfortunately, we were unable to find an openly available source for this data. However we feel that the target sample size data, along with the recruitment period can provide a rough guide to the achievability of recruitment targets for feasibility and pilot studies. For instance, if a pilot study had a target sample size of 60 and a recruitment period of 18 months for a study investigating a health condition you are interested in, you might want to reconsider running a similar pilot study with a target size of 100 for 6 months. We therefore believe that this data has value in making informed de- cisions with regard to recruitment for a feasibility and pilot studies. 1University of North Carolina; 2Johns Hopkins University; 3University of Mississippi Medical Center Correspondence: Sheila Burgard Trials 2017, 18(Suppl 1):P11 Methods A challenge to these longitudinal analyses has been that the neurocognitive measures change over time due to scientific improvements in the instruments. A group of ARIC investigators employed factor analysis to level differing cognitive test batteries over visits to common, comparable measurements in the area of general cognition and the 3 cognitive domains of interest (Gross et.al., Epidemiology vol 26, no 6, 11/2015). g g p y Shiny, a web application framework for R, was then used to create an online tool to interrogate the data. For various health conditions, specified by the researcher, the tool can be used to obtain descrip- tive summaries and graphical displays of pilot and feasibility studies for the following factors: period of recruitment, target sample size, target recruitment rate per month, location of trial centres. P13 Is it possible to randomise patients to potentially not receive a dressing after surgery? Preliminary findings of the NIHR HTA Bluebelle pilot randomised controlled trial Leila Rooshenas1, The Bluebelle Study Group2 1University of Bristol; 2University of Bristol and University of Birmingham Correspondence: Leila Rooshenas Trials 2017, 18(Suppl 1):P13 Background g Dementia and mild cognitive impairment (MCI) pose a large and increasing health and societal burden on the aging US population. In 1987–1989 the NHLBI-supported prospective epidemiologic Atherosclerosis Risk in Communities (ARIC) study enrolled 15,792 participants from 4 distinct US geographical regions in order to investi- gate the causes of atherosclerosis and its clinical outcomes, including cognitive function. Since visit 1 in 1987–1989, there have been 4 follow-up visits for the cohort. ARIC is uniquely suited to contribute crit- ical information on the vascular, and potentially preventable, contribu- tions to MCI and dementia of different origins. g y p We identified UK Clinical Trial Gateway (UKCTG) as providing the best source of UK based data to harvest. The UKCTG was set up by the National Institute for Health Research (NIHR) to essentially help re- cruit people to clinical trials in the UK. Because there were no facil- ities for downloading data from the UKCTG website, a web scraping methodological was adopted and implemented using R. Result and Conclusions Monitor- ing trial performance helps to ensure the validity of a study and its design, consistency in reporting across sites and clinicians, and that a study's hypotheses are not being compromised. Continual monitor- ing of study design quality metrics through CSM enables corrective action to be taken early enough to address any potential threats to the design of a RCT, while also simultaneously improving data quality and the credibility of a study and its findings. p Objective Visit 6 is underway with continued neurocognitive emphasis that will allow quantification of cognitive decline, estimation of the incidence of mild cognitive impairment (MCI) and dementia, and tracking of progression from MCI at V5 to dementia. These measures will be Leila Rooshenas , The Bluebelle Study Group 1University of Bristol; 2University of Bristol and University of Birmingham Correspondence: Leila Rooshenas Trials 2017, 18(Suppl 1):P13 Trials 2017, 18(Suppl 1):200 Page 6 of 235 Page 6 of 235 y Results Recruitment: Achieving the required sample of n = 50 proved to be impossible over an 18 month recruitment period. For example, dur- ing a four-month screening period, coinciding with a relaxation of in- clusion criteria 434 patients were screened, 85 were found to be eligible for inclusion, with only 6 successfully recruited. Only 8 index cases were secured across the study duration. Support: Family support structures were found to be weak, with a number of eligible candidates reporting strained family relationships as a deterrent to participation. Family size: Many potential index cases did not have enough siblings and/or children required to participate. Motivation: Index cases lacked motivation, both in relation to their condition and willingness to participate. Age: The tight inclusion criteria for age of index cases (45–65 years) were found to be restrictive. Data analysis: This proved difficult due to the small sample size and the clustering of family data. p Design Cross-sectional feasibility study of index cases diagnosed with T2D and their first degree relatives (siblings and offspring). Index cases were recruited from the diabetes information database (DIAMOND) of a hospital in Northern Ireland. p Method Sample: The DIAMOND database was screened to identify adults with T2D, aged 45–65 years, with at least two siblings and two offspring, willing to participate in the study. For this feasibility study 50 partici- pants were sought (i.e.10 index cases each with 4 first degree rela- tives, spanning two generations). Measures: A range of lifestyle factors, biochemical and clinical markers were collected for all partici- pants. Location of the Study: The rationale underpinning the suitabil- ity of this location for the study was based on existing knowledge: 1. As Northern Ireland comprises the most homogenous population group in the UK, it was believed the majority of offspring would live locally; 2. The close family structure encountered in Northern Ireland would lead to strong support for research projects that involve a family member. Adults undergoing elective and emergency abdominal surgery were invited to take part in the pilot RCT. Recruitment took place between March-November 2016, across four NHS hospitals in England. Re- search nurses and surgeons provided information about the study in advance of surgery and obtained written consent. Patients who en- tered the RCT and health care professionals involved in their care were invited to take part in semi-structured interviews, to explore the acceptability of the dressing strategies under comparison. Results Recruitment figures met or exceeded targets across all centres. The numbers of patients approached and the proportion consenting indi- cated that a main trial would be feasible (446 approached, 363 con- sented, and 326 randomised, as of October 2016). Qualitative interviews provided further evidence to suggest that randomisation to the three dressing strategies was acceptable. Patients’ wound healing experiences were similar across all groups, with no notable clinical or practical concerns. Contrary to health care professionals’ prior assumptions, some patients reported practical advantages of not having a dressing, reflecting on the ‘low maintenance’ nature of wound care. Health care professionals did not report any particular difficulties in caring for patients in any of the groups, and did not perceive any changes to other aspects of their practice. The number of recorded protocol deviations and retention rates are currently undergoing analysis and will be available at the conference. Conclusion Scaling up: lessons from a feasibility study involving people with type 2 diabetes and their families yp Vivien Coates1, Karen McGuigan2, Alison Gallagher1, Brendan Bunting1, Maurice O'Kane3, Tracy Donaghy3, Geraldine Horigan1, Maranna Sweeney1 1Ulster University; 2North West Research, NI; 3Western Health & Social Care Trust Feasibility studies are routinely performed in a variety of clinical areas to help provide evidence prior to major monetary investment, hu- man resource and patient recruitment to a large randomised con- trolled trial (RCT). They can assess a variety of aspects including recruitment potential, multi-centre operational coordination and lo- gistical aspects of administering the intervention. As viability is their Correspondence: Vivien Coates Trials 2017, 18(Suppl 1):P14 Correspondence: Vivien Coates Trials 2017, 18(Suppl 1):P14 P14 P15 40 is the magic number Laura Pankhurst, Ana Mora, Alison J. Deary, Dave Collett NHS Blood and Transplant Correspondence: Laura Pankhurst Trials 2017, 18(Suppl 1):P15 P15 40 is the magic number Laura Pankhurst, Ana Mora, Alison J. Deary, Dave Collett NHS Blood and Transplant Correspondence: Laura Pankhurst Trials 2017, 18(Suppl 1):P15 Background (relating to diet and exercise) are recognised as important risk factors for the development of T2D, interventions at the level of the individ- ual to modify these are challenging. Evidence suggests that lifestyle behaviours are passed through families, from one generation to an- other. Therefore, when designing T2D interventions, it may be im- portant to consider behaviours developed within the shared family environment. g Recruiting to randomised controlled trials (RCTs) can be difficult, especially when habitual clinical practices are compared with lesser- known or novel approaches. Surgical RCTs can be particularly chal- lenging, due to ingrained clinician preferences and doctrine. Post- surgical wound care is an aspect of surgery in need of high quality evidence. It is common to apply dressings over closed wounds after most adult operations, despite there being limited evidence to sup- port or refute this practice. The NIHR-funded Bluebelle study aimed to determine the feasibility of an RCT that randomises patients to dif- ferent wound dressing strategies, including ‘no dressing’ (where the wound is exposed to air). The funder and health care professionals were sceptical about whether ‘no dressing’ would be acceptable to patients and clinical professionals, and questioned whether an RCT could successfully recruit participants. The Bluebelle study was thus funded to investigate these uncertainties. It consisted of two phases: a preliminary phase to explore current practice and select appropri- ate comparators (Phase A), and an external pilot RCT (phase B). In- formed by phase A findings, the pilot RCT sought to randomise patients to receiving either a ‘simple dressing’, 'glue-as-a-dressing’, or ‘no dressing’. The pilot addressed a number of objectives to deter- mine whether a full-scale RCT could be delivered. Two objectives were to investigate whether recruitment was feasible (target of 330 patients), and explore whether the comparison groups were accept- able to patients and health care professionals. Methods Aim To investigate the impact of the shared family environment on risk factors for T2D, and to determine the feasibility of conducting a fully powered study using this methodology. D i Conclusion The feasibility study provided key insights, impacting on scaling up deci- sions. We now know that identifying index cases for this study through a hospital data base is ineffective and would be better suited to a primary care setting. The data gathering methods and instruments worked effect- ively. In light of the difficulties encountered in the feasibility study, it was agreed that a fully powered study would not be developed. This pilot RCT demonstrated that it is feasible to recruit patients to an RCT of different wound dressing strategies, including ‘no dress- ing’. A full-scale trial will be designed on the basis of these findings, providing other aspects of trial conduct (e.g. Retention) are acceptable. Methods Like other research organisations, NHS Blood and Transplant (NHSBT) considers feasibility studies to be essential prior to significant invest- ment in a subsequent full scale RCT. As such, NHSBT have funded a number of feasibility studies, which have then improved the design and conduct of RCTs and larger research projects that have ultim- ately lead to changes in clinical practice. The NIHR Health Technology Assessment (HTA) and Research for Patient Benefit (rfpb) programme databases (as available from the NIHR website) were screened for pilot/feasibility studies of surgical interventions funded between 2005 and 2015. Pilot/feasibility work was defined as: Any research undertaken before a main study intended to inform the design and/or conduct of a future main study. A surgical intervention was defined as: A diagnostic, thera- peutic or adjunctive invasive intervention performed by a trained clinician, using hands, instruments and/or devices. Studies which were not pilot/feasibility work or where the surgical intervention was a co-intervention were excluded. It was rationalised that research funded by the NIHR programmes would embrace the higher quality methodological features necessary to identify the key design features of interest and will have been peer-reviewed as part of the funding process. Protocols for all included studies and the associated data sources were collated, including, where available, published papers from the pilot/feasibility work and the consequent main trial. A data extraction form was developed and piloted a priori enabling elicit- ation of the pilot/feasibility work rationale, and exploration of the as- sociations of key design features of pilot/feasibility work with the planning, conduct and outcome of any subsequent definitive main trial. The NHSBT Clinical Trials Unit has a growing portfolio of feasibility studies in transfusion medicine with five studies having a sample size of around the magic number of 40 patients: in set up REAL and DRIVE, currently recruiting REDDS (ISRCTN26088319) and EFIT (ISRCTN67540073); and completed CRYOSTAT (ISRCTN55509212). Al- though formal sample size calculations are not needed for feasibility studies, it is important that required patient numbers are properly justified. Although there is some guidance on this in the literature (for example Julious (2005), Billingham (2013), Teare (2014) and Whitehead (2016)) the background to the sample size for our feasibil- ity studies will be described and illustrated. Results 1341 studies funded by the HTA and rfpb NIHR programmes be- tween 2005 and 2015 were identified and screened, with 73 (5.4%) meeting the inclusion criteria. 30 (41%) were rcts with an internal pilot phase and 43 (59%) were other feasibility work. This included 28 (65%) randomised pilot studies, 3 (7%) non randomised pilot stud- ies and 12 (28%) other types of feasibility study, of which 8 (66%) were systematic reviews. Further findings, including the rationale for pilot/feasibility work and the associations of key design features with main trial design and/or conduct, will be presented. Conclusions Correspondence: Katherine Fairhurst Background Poor research design, conduct and analysis contribute to significant research waste. This is further compounded by the limited reporting and dissemination of results. Pilot and feasibility work has the poten- tial to contribute to the success of subsequent definitive main trials. It allows areas of methodological uncertainty in the main trial proto- col to be addressed and resolved before the main trial begins. Whilst it is particularly important to the design of trials of complex interven- tions such as surgery, little is known about how to optimally design pilot and feasibility work to inform surgical trials. Methods Our magic number of 40 is regarded as a compromise between the need for a short timescale in which feasibility can be assessed, suffi- cient data to allow recruitment rates to be determined in representa- tive centres, and whether the study interventions can be delivered successfully. Some general observations on the design of these stud- ies will also be included, concluding with a summary of the research which has resulted from our completed feasibility studies. Estimating the cost of prescribed medications in economic evaluation: does the current method reflect the true cost to the English NHS? Evidence from the comet feasibility study Kirsty Garfield, Matthew J. Ridd, Sandra P. Hollinghurst University of Bristol P16 Key design features of pilot and feasibility studies to inform successful surgical trials: a systematic analysis of funded studies Katherine Fairhurst1, Jane Blazeby2, Shelley Potter2, Amanda Blatch-Jones3, Ceri Rowlands2, Carrol Gamble2, Kerry Avery2 1University of Bristol; 2MRC conduct-II Hub for Trials Methodology Research & Centre for Surgical Research, University of Bristol; 3National Institute for Health Research Evaluation, Trials and Studies Coordinating Centre (NETSCC), University of Southampton Key design features of pilot and feasibility studies to inform successful surgical trials: a systematic analysis of funded studies Katherine Fairhurst1, Jane Blazeby2, Shelley Potter2, Amanda Blatch-Jones3, Ceri Rowlands2, Carrol Gamble2, Kerry Avery2 Key design features of pilot and feasibility studies to inform successful surgical trials: a systematic analysis of funded studies Katherine Fairhurst1, Jane Blazeby2, Shelley Potter2, Amanda Blatch-Jones3, Ceri Rowlands2, Carrol Gamble2, Kerry Avery2 y Correspondence: Kirsty Garfield Trials 2017, 18(Suppl 1):P17 Correspondence: Kirsty Garfield Trials 2017, 18(Suppl 1):P17 1University of Bristol; 2MRC conduct-II Hub for Trials Methodology Research & Centre for Surgical Research, University of Bristol; 3National Institute for Health Research Evaluation, Trials and Studies Coordinating Centre (NETSCC), University of Southampton Background g Economic evaluation guidance states that resource use should be valued using relevant unit costs. The most frequently used source for valuing prescribed medication use in the UK is the British National Formulary (BNF). However, from the perspective of the UK National Health Service (NHS), it is not clear whether this source reflects the true cost to the NHS. y Trials 2017, 18(Suppl 1):P16 References Julious SA (2005) Sample size of 12 per group rule of thumb for a pilot study. Pharmaceutical Statistics, 4: 287–291. Billingham SAM, Whitehead AL, Julious SA (2013) An audit of sample sizes for pilot and feasibility trials being undertaken in the United Kingdom registered in the United Kingdom Clinical Research Network database. BMC Medical Research Methodology, 13: 104. Conclusions Teare MD, Dimairo M, Shephard N, Hayman A, Whitehead A and Walters SJ (2014) Sample size requirements to estimate key design parameters from external pilot randomised controlled trials: a simulation study. Trials, 15: 264. The findings will inform a qualitative study comprising in depth semi-structured interviews and consensus methods to explore the perceptions and experiences of key stakeholders involved in pilot/ feasibility studies of surgical interventions. This work is important to develop future recommendations for the optimal design and con- duct of pilot/feasibility work of surgical interventions. Whitehead AL, Julious SA, Cooper CL, Campbell MJ (2016) Estimating the sample size for a pilot randomised trial to minimise the overall trial sample size for the external pilot and main trial for a continuous outcome variable. Statistical Methods in Medical Research, Background The rapid and recent global increase in prevalence of type 2 diabetes (T2D)[1,2] is of great concern. Although adverse lifestyle behaviours Page 7 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 7 of 235 Page 7 of 235 understand key design features associated with the optimal design and conduct of main surgical trials. Methods understand key design features associated with the optimal design and conduct of main surgical trials. Methods main aim, small sample sizes are used and so feasibility studies rarely have sufficient power to assess clinically important treatment differences. Background g Cost-effectiveness analysis (CEA) of randomised controlled trials pro- vide key evidence to inform health care decision making. Missing data is a particularly challenging issue in CEA because a large propor- tion of patients may not complete resource use or quality of life questionnaires. Multiple imputation (MI) is commonly used to impute the missing values by conditioning on the observed data, assuming the data are “missing at random” (MAR). However, a major concern is that the missing data are often related to the unobserved values, a mechanism also known as “missing not at random” (MNAR). For ex- ample, patients whose health is relatively poor may be less likely to complete quality of life questionnaires, even after conditioning on the observed data. Unless missing data are addressed appropriately under transparent assumptions, CEA studies may provide misleading estimates of effectiveness and cost-effectiveness, and potentially lead to wrong decisions. Iryna Schlackow1, Alastair Gray2, Linda Sharples3, Chris Jackson4, Nicky Welton5, Borislava Mihaylova2 g Conclusions Using this method may lead to more accurate estimates of the true cost to the NHS of prescribed medications, however assumptions around pharmacy discounts were required to estimate costs. Estimat- ing costs using this method is more time intensive when compared to applying published unit costs from the BNF or PCA. Whilst using this method for intervention medications can provide sensitivity ana- lyses around intervention costs, the value added of using this method to cost concomitant medications may be limited when con- sidering the researcher time required. Results We identified a method for estimating the NHS cost of prescribed medications dispensed by community pharmacists. This method in- corporates the basic price of the medication, pharmacy discounts, dispensing fees, payments for consumables and containers, and other associated costs. The unit cost of all intervention emollients es- timated using the alternative method were higher than costs listed in the BNF and PCA. The largest difference in unit costs was for Aveeno lotion, whereby the cost listed in the BNF and PCA was £5.33 and the cost estimated using the alternative method was £7.23. The smallest difference was for Doublebase gel at £6.09 in the PCA and £6.22 using the alternative method. g j Based on the identified manuscripts, and the reviewers’ comments, a taxonomy of methods will be suggested, with methods classification based on the main driver of survival (e.g. a single cause of death, cause-specific mortality, non-fatal disease events or other disease markers); underlying epidemiological disease model (e.g. Natural his- tory of the disease and competing risks); and assumptions about the treatment effect over time. Interdependence between these factors, with the appropriateness, advantages and limitations of each ap- proach and implications for performing cost-effectiveness analyses will be discussed. Methods and interim results A pearl growing strategy was applied to identify manuscripts that contained novel extrapolation methods, with the emphasis on methods largely based on a single randomised clinical trial. Firstly, a scoping search of the PubMed database was performed to identify recent methodological papers. Subsequently, reference lists of in- cluded manuscripts were checked, and finally, a panel of experts was asked to suggest further potentially relevant published methods. Method description was extracted using a pre-defined template. Ex- tracted information included the context that motivated method de- velopment (e.g. the need to incorporate cause-specific mortality); type of data used for the extrapolation (e.g. from an RCT, general population or a matched cohort); detailed statistical/modelling Methods The COMET study sought to determine the feasibility of conducting a randomised controlled trial in young children with eczema. Children were recruited from primary care and randomised to one of four commonly used emollients. The study also explored the feasibility of both collecting and costing the data required to perform an eco- nomic evaluation in this setting. As part of this we explored whether published prescribed medication costs from the BNF and Prescription Cost Analysis (PCA) represented the true cost to the NHS. In order to estimate the cost to the NHS we identified the method by which community pharmacies are reimbursed for the medications they To systematically analyse the protocols and published papers of funded pilot and feasibility studies of surgical interventions to Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 8 of 235 prescribe. Unit costs of the four intervention emollients were esti- mated using this method and compared to unit costs from the BNF and PCA. The total cost of study emollients prescribed over the trial period were also estimated and compared using the different methods. methodology; comments on generalisability and usability (e.g. neces- sary assumptions, incorporation of uncertainty and sensitivity analyses, compatibility with a cost-effectiveness framework, implementation in standard software); main strengths and comparison with other methods. A reviewers’ opinion, based on a consensus between at least two reviewers, was provided on whether the method accommodated aspects commonly of interest in cost-effectiveness analyses, such as heterogeneous population as well as the main driver behind the ex- trapolated survival (eg major nonfatal adverse events). P19 Cost-effectiveness analysis of clinical trials with missing data: using multiple imputation to address data missing not at random Baptiste Leurent, Manuel Gomes, James Carpenter London School of Hygiene & Tropical Medicine Correspondence: Baptiste Leurent Trials 2017, 18(Suppl 1):P19 P19 Cost-effectiveness analysis of clinical trials with missing data: using multiple imputation to address data missing not at random Baptiste Leurent, Manuel Gomes, James Carpenter London School of Hygiene & Tropical Medicine Correspondence: Baptiste Leurent Trials 2017, 18(Suppl 1):P19 P18 Methods for extrapolation from clinical trial data to inform economic evaluations: a taxonomy Iryna Schlackow1, Alastair Gray2, Linda Sharples3, Chris Jackson4, Nicky Welton5, Borislava Mihaylova2 1University of Oxford; 2Nuffield Department of Population Health, University of Oxford); 3Leeds Institute of Clinical Trials Research, University of Leeds; 4MRC Biostatistics Unit, Cambridge; 5School of Social and Community Medicine, University of Bristol Correspondence: Iryna Schlackow Trials 2017, 18(Suppl 1):P18 Methods for extrapolation from clinical trial data to inform economic evaluations: a taxonomy Iryna Schlackow1, Alastair Gray2, Linda Sharples3, Chris Jackson4, Nicky Welton5, Borislava Mihaylova2 1University of Oxford; 2Nuffield Department of Population Health, University of Oxford); 3Leeds Institute of Clinical Trials Research, University of Leeds; 4MRC Biostatistics Unit, Cambridge; 5School of Social and Community Medicine, University of Bristol Correspondence: Iryna Schlackow Trials 2017, 18(Suppl 1):P18 Background Lifetime economic evaluations are often performed alongside rando- mised clinical trials, to incorporate long-term effects of interventions. However, due to the limited duration of most randomised controlled trials, extrapolation of components such as survival, beyond study data is required. Aim To review extrapolation methods that are currently used in economic evaluations and to provide a taxonomy of these methods while dis- cussing motivation, advantages and limitations behind each ap- proach in the context of a cost-effectiveness framework. To provide an accessible framework to perform sensitivity analyses in CEA of clinical trials with data missing not at random. We first conducted a review of recently published CEA to assess the extent of missing data and approaches commonly used to address them. We also held discussions with various stakeholders (conduct- ing or using trial-based CEA) to identify the main barriers and strat- egies to wider use of these methods. Based on these findings, we proposed a practical framework to conduct sensitivity analyses when data are anticipated to be MNAR. We applied this framework to the Ten Top Tips trial, which evaluates an intervention for weight man- agement in primary care. This study illustrates a typical trial-based CEA, in which key endpoints such as self-reported QOL are likely to be MNAR. Conclusions The choice of an appropriate method depends on a range of factors, including presence of competing risks, specifics of the disease nat- ural history and assumptions on the treatment effect. Care must be taken in understanding the available options and their limitations prior to embarking on extrapolation. The increase in availability of relevant data is likely to contribute to emergence of novel ap- proaches to support extrapolation efforts. The COMET study was independent research funded by the National Institute for Health Research (Research for Patient Benefit Programme, PB-PG- 0712–28056). The views expressed in the publi- cation are those of the author(s) and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health. Conclusions Whilst there is variability in CRN resourcing nationally, the HIWG standardises the conduct of research in primary care settings, im- proving consistency and engagement with the primary care research infrastructure. Utilising GP clinical systems to embed research tools, results in simple, efficient and effective methods for primary care partners to conduct research. Scaling up of the HIWG over time will allow the group to provide a service for other clinical research teams conducting research in the primary care setting. Despite these useful features, the practical application of the EVSI in trial design has been restricted due to computational issues. How- ever, recently methods been developed to overcome these computa- tional barriers allowing researchers to use the EVSI when designing clinical trials. This will become more important as economic consider- ations come to the forefront of decision making for Clinical Trials. We will discuss the interpretation of the EVSI and how it can be used to aid trial design by finding economically viable designs. We will then discuss the recent computational advances for the EVSI that allow re- searchers to use this tool in practice to aid their decision making. Background Background The routine use of electronic patient records (EPRs) in primary care provides opportunities and challenges for researchers conducting clinical trials in this setting. Although the use of EPRs to search for eligible patient populations is well established they can also be used as a resource to improve trial conduct and quality. The Footprints in Primary Care study is a feasibility study and pilot cluster randomised trial exploring the acceptability of a GP practice level intervention for frequently attending patients. Two key components of the interven- tion are; increased continuity of care with a named GP, and delivery of a psychosocial consultation technique called BATHE. Development of a health informatics working group to enhance the conduct of clinical trials in primary care Sarah Lawton1 Simon Wathall2 Sarah Lawton , Simon Wathall 1Keele Clinical Trials Unit; 2Keele Clinical Trials Unit and NIHR Clinical Research Network: West Midlands Correspondence: Sarah Lawton Trials 2017, 18(Suppl 1):P21 p Methods A joint HI Working Group (HIWG) between Keele CTU and CRN WM has been established to oversee, develop, support, track and quality assure the HI operational activity for research. A range of innovative methods have been developed by the working group, which can be embedded into existing GP clinical systems, to include; eligibility and recruitment searches, data collection templates, pop-ups and elec- tronic tools to aid referrals and clinical assessments. These methods are tailored on a bespoke basis to the requirements of individual clinical research teams to perform feasibility, identification, eligibility, screening, recruitment, tagging and data collection functions and are provided together with instructions for use. Reference [1] Graffy J et al. Trials within trials - Researcher, funder and ethical perspectives on the practicality and acceptability of nesting trials of recruitment methods in existing primary care trials. 2010 Use of primary care electronic records to monitor and improve intervention delivery of a GP practice level intervention Clare Thomas1, Rebecca Barnes1, Helen Cramer1, Sandra Hollinghurst1, Sue Jackson2, Charlie Record3, Chris Metcalfe1, David Kessler1 1University of Bristol; 2University of Surrey; 3Frome Valley Medical Centre Correspondence: Clare Thomas Trials 2017, 18(Suppl 1):P22 Results 100% of Keele CTU supported research activity involving general practices has utilised the HIWG. The groups’ innovations assist to imple- ment a robust, standardised and automated method of performing re- search activity in primary care settings. Greater precision of sample identification, reduced paperwork and increased efficiencies can be achieved, assisting with the retention of research participants, resulting in accessible interrogation and interpretation of research data. Conclusions The Expected Value of Sample Information (EVSI) quantifies the ex- pected monetary value of a specific future trial. Theoretically, this could be an important tool for trial design for two reasons. Firstly, it would be possible to compare the monetary value of the trial directly with its cost to determine whether the trial is worthwhile. More im- portantly the EVSI could find the optimal trial design in terms of monetary benefit by comparing the trial value and cost for different trial designs. Results Our review provided further evidence that missing data was a com- mon issue in trial-based CEA (median complete cases was 63%), and Trials 2017, 18(Suppl 1):200 Page 9 of 235 Page 9 of 235 that sensitivity analyses under MNAR assumptions were rarely con- ducted (4%). During our discussions with stakeholders, the main bar- rier identified were the lack of practical guidance and software code to perform such analyses. We found that the pattern-mixture model was a desirable approach in CEA because it frames the sensitivity analysis in terms of differences between observed and missing data, which is readily understood by the different stakeholders. We illus- trated how this approach can be easily implemented with standard missing data methods such as MI, and provided a framework for con- ducting sensitivity analyses under a broad range of assumptions. This framework also addressed the elicitation of the plausible missing data mechanisms, and the reporting of results. Application to the Ten Top Tips trial showed that results can be very sensitive to the as- sumptions about the missing data. For example, the intervention was likely to be cost-effective under the MAR assumption, but appear not cost-effective for some of the MNAR scenarios. Background: Primary care infrastructure is complex and requires a number of different strategies which are innovative, efficient and transferable in order to successfully coordinate, recruit and retain both sites and participants in primary care research. Background: Primary care infrastructure is complex and requires a number of different strategies which are innovative, efficient and transferable in order to successfully coordinate, recruit and retain both sites and participants in primary care research. y Keele CTU is a registered UKCRC CTU, specialising in the develop- ment and delivery of both feasibility and definitive multicentre ran- domised clinical trials, an increasing portfolio of Clinical Trials of Investigational Medicinal Products (CTIMPs) and epidemiology stud- ies in both primary care settings and at the secondary care interface. Keele CTU has a strong HI function, with over 12 years’ experience in utilising primary care clinical systems and strong links with CRN WM. CRN WM is one of 15 clinical research delivery arms of the NHS. They are responsible for ensuring the effective delivery of research within the primary care infrastructure throughout the WM area. Methods Conclusions Missing data in CEA of clinical trials can result in misleading conclu- sions. This study proposes an accessible framework to perform CEA under a wide range of missing data assumptions, which will help fu- ture studies provide more transparent and robust evidence to inform decision-making. P20 Value of sample information as a tool in clinical trial design Anna Heath, Gianluca Baio University College London Correspondence: Anna Heath Trials 2017, 18(Suppl 1):P20 Biospecimen management system that streamlines processes and reduces inherent challenges Biospecimen management system that streamlines processes and reduces inherent challenges Ella Zadorozny, David E. Hallam, Tamara Haller, Sharon M. Lawlor University of Pittsburgh Correspondence: Ella Zadorozny Trials 2017, 18(Suppl 1):P23 g Ella Zadorozny, David E. Hallam, Tamara Haller, Sharon M. Lawlor University of Pittsburgh Correspondence: Ella Zadorozny Trials 2017, 18(Suppl 1):P23 We propose that a more targeted approach to drug re-supply will ad- dress these issues by both reducing the volumes of drugs delivered to sites and at the same time reducing the amount of drug wastage. Utilis- ing the central trial database allows us to identify exactly which post- randomisation visits are upcoming at each site and to assign deliveries to sites based upon this. So, if a site had a run of equal treatment allo- cations then our supply algorithm will dictate that drug supplies at this site six months later be weighted accordingly rather than issuing equal amounts of each drug to the site. Using this mechanism will help plan- ners more easily determine how many drugs will be needed for a trial and allow them to reduce the amount of contingency required and hence reduce the costs of running a drug trial. Correspondence: Ella Zado Trials 2017, 18(Suppl 1):P23 Developing procedures for biospecimen collection, processing, ship- ping, and storage that yield high quality research samples and data present many challenges in multi-center studies. Studies that require real-time and batch shipments from clinical sites to numerous central testing laboratories or biospecimen repositories increase the com- plexities required to assure integrity of the biospecimens and related data. The data management development team in the Epidemiology Data Center (EDC), Graduate School of Public Health, at the University of Pittsburgh has designed a web-based Sample Tracking System (STS) to streamline sample tracking and shipping from point of collection to testing laboratories and repositories. The system is flexible, scalable, and can be customized easily to meet the needs of individual studies. Modules included in the STS are: entry and editing via barcode scan- ner or keyboard, generation of shipping manifests, and receipt con- firmation at the batch and sample level, with database audit trails for all modules. Automated email notifications alert laboratory/repository personnel of incoming shipments and clinical site personnel of ship- ments received. Background Achieving and maintaining participant recruitment to clinical re- search, and specifically, clinical trials in primary care, is known to be challenging [1]. Experience gained from research supported by Keele Clinical Trials Unit (CTU), shows that targeted Health Informatic (HI) support early in the design phase of clinical trials may enhance the conduct of research and improve recruitment and retention rates. A collaborative approach involving Keele CTU and the NIHR Clinical Re- search Network: West Midlands (CRN WM) in the use of HI has been developed to embed clinical research within primary care settings. Page 10 of 235 Page 10 of 235 Page 10 of 235 Trials 2017, 18(Suppl 1):200 Methods and laboratory/repository personnel, facilitating smooth study startup. It was designed to accommodate unlimited clinical sites, la- boratory/repository destinations, sample types, and samples/aliquots with minimal setup time or expertise on the part of EDC data man- agement personnel. Data management personnel use administrative tools to define study name, site codes, sample types, sample names/ titles, sample states (e.g. Frozen, ambient), barcode formats, labora- tory/repository names, and protocol timepoints, and to define the re- lationships among samples, studies, sites, and laboratories/ repositories. Optional settings are provided for default volume, vol- ume/unit (ml, μg, slide, image), minimum and maximum volume/ unit, and earliest sample date. There are options to initialize barcodes in the database and then utilize initialized barcodes to provide vali- dations (e.g. Site, participant ID, sample type, timepoint) at the time of sample entry. At the time of receipt of shipments, the system al- lows receiving personnel to resolve issues and input comments at the batch or sample level. Automated searches were set up within the EPR system in the four intervention practices. These were designed to collect consultation data, such as the number and type of consultations and name of consulting GP, for patients eligible for the Footprints in Primary Care study. Information on study GP use of the BATHE technique, denoted by the GP adding a pre-specified read-code to the EPR when they had used the technique in consultations with study patients, were also collected. These automated searches were run in the practices every 6 weeks during the 12 month intervention period and anon- ymised data emailed to the research team. Consultations data were also collected for the same patients for the 12 months prior to the start of the study to provide a baseline comparison. Biospecimen management system that streamlines processes and reduces inherent challenges Claire Kerr, Mairi Warren University of Glasgow Correspondence: Claire Kerr Trials 2017, 18(Suppl 1):P25 Background The Robertson Centre for Biostatistics conducts and supports collab- orative research in clinical trials through the design, conduct, analysis Results The collection of data from EPRs at regular time points allowed the research team to monitor intervention delivery whilst the study was ongoing. This included assessment of the extent to which continuity of care had increased and the reach and dose of the BATHE consult- ation technique i.e. with how many patients had BATHE been used and on how many occasions. This made it possible for issues with intervention delivery, such as the low uptake of the BATHE technique amongst GPs or difficulty booking appointments with the named GP, to be followed up with study practice staff. Individualised feedback could also be provided to practices during top-up training sessions with the aim of improving intervention delivery. Furthermore the positive impact of these training sessions could be demonstrated by looking at subsequent EPR data. The STS is in use on several EDC projects and has facilitated biospecimen-related processes, reduced data management effort for system setup, maintenance, and monitoring, streamlined site and la- boratory/repository sample-related processes, and has improved real- time validations and the quality of sample-related data. P24 A targeted approach to drug-supply in RCTs limits wastage and can reduce costs. The experience of the MS-smart trial Allan Walker, Moira Ross University Of Edinburgh Clinical Trials Unit Correspondence: Allan Walker Trials 2017, 18(Suppl 1):P24 g Conclusions Allan Walker, Moira Ross University Of Edinburgh Clinical Trials Unit Correspondence: Allan Walker Trials 2017, 18(Suppl 1):P24 Within the Footprints in Primary Care study the use of data from eprs has been important for monitoring intervention delivery, reach and dose, in providing feedback to participating practice staff, and in helping to select a maximum-variation sample of staff and patients for interview. This information, alongside qualitative interview and observational data, has been instrumental to our understanding of the feasibility and acceptability of the intervention. This approach however is not without its challenges and further consideration is needed regarding how the process of data collection and the colla- tion of feedback would be delivered on a larger scale or in real-world implementation. The MS-Smart trial is a four-arm phase IIB randomised, double-blind placebo controlled clinical trial comparing the efficacy of neuropro- tective drugs in secondary progressive multiple sclerosis. g y p g p Treatment allocation is by minimisation without a site stratification element. Participant follow up is over two years and each participant has at least 6 post-randomisation clinic visits where trial drugs are provided. The cost of the trial drugs is significant so all reasonable steps should be taken to limit oversupply at site leading to drug wastage. pp y g g g Sending equal amounts of each of the four drugs to site pharmacies leads to wastage as the treatment allocation method does not guaran- tee a balance of allocated treatments among recruits at each site. In addition, site pharmacies often have limited storage space and find it difficult to accommodate deliveries of large volumes of trial drugs. Design Des g The VA NEPHRON-D study was a multi-center, double blind, ran- domized clinical trial to assess the effect of ACEI and ARB combined vs. ARB alone on the progression of kidney disease in individuals with diabetes and proteinuria. The safety endpoints of the trial in- cluded serious adverse events (SAE), acute kidney injury (AKI), hyperkalemia and mortality. A subset of the participants (~62%) who enrolled in a long-term follow-up substudy were consented for data collection via the EMR. For those participants with consent, data accumulated in their medical records during the study period were extracted from the VA Corporate Data Warehouse (CDW). We accessed the CDW centrally, captured the safety data and com- pared these records with those collected by the study personnel at VA Medical Centers participating in the VA NEPHRON-D trial. This assessment examines both general and study-specific safety end- points, and more importantly, provides evidence for how to use ex- tracted EMR data for documenting SAE and study outcomes in futures studies. Methods Over the past 6 years the Centre, in consultation with staff, has cus- tomised and developed an MS sharepoint site to manage key project information and activities relating to clinical studies including: Project planning and management; Change management; Document con- trol; Study communication; Management reporting Automated solution for tracking electronic case report form completion Elizabeth Hill, Joanna Illambas, Eddie Heath, Charlotte Friend, Hassan Nawrozzadeh, Emma Hall, Claire Snowdon, Judith Bliss, Rebecca Lewis The Institute of Cancer Research Correspondence: Elizabeth Hill Trials 2017, 18(Suppl 1):P26 Background The ICR-CTSU introduced electronic data capture (EDC) in 2012. This necessitated development of a solution to automatically monitor electronic case report form (ECRF) completion and track timely com- pletion of ECRFs. Background Electronic medical records (EMRs) are now frequently used for col- lecting patient-level data for clinical trials. With the Veterans Affairs (VA) Healthcare System, EMR data have been widely used in clinical trials to assess eligibility and facilitate referrals for recruitment, and to conduct follow-up and safety monitoring. More recently, the EMR is being used for point-of-care randomization trials and for conduct- ing trials from central location. Despite the great potential efficiency of using the EMR, it is of interest and importance to evaluate the in- tegrity of data captured from the EMR through a centralized monitor- ing algorithm without involvement of research personnel compared to that collected by local investigators or coordinators under protocol conditions. This investigation assesses the verification of safety data collection. D i The MS sharepoint site has been further developed to support: Func- tional areas; Archival; Audit Management; Centre Communication; Risk Management Result Hospital admission data were obtained from CDW's acute care, extended care, and observational care records. Study-collected SAEs were consolidated into a single hospital stay for comparison with EMR records. A high level of matching was found using the CDW to verify SAE reported during the active trial for hospital ad- missions within the VA healthcare system. Hospitalization records that were stored as scanned notes from non-VA admissions were not included as CDW records, which is an issue that still needs to be addressed for obtaining a more complete data collection. Also, identifying individual SAEs during the same hospitalization stay requires further investigation. AKI was a major safety endpoint in the study. Different definitions of AKI based on ICD-9 codes and change of creatinine during hospitalization were applied in the CDW data searches. The search results varied significantly de- pending on the AKI definition applied. Likewise, hyperkalemia identified by the CDW laboratory datasets had some discrepan- cies from the active trial setting where diagnosis of hyperkalemia was a combination of potassium level and other clinical factors. Details of the comparisons for each safety endpoint will be presented. A two part solution was developed: 1. Schedule forms were created within the EDC clinical database. These forms display details of ECRF expected and completed dates per trial participant for every visit and form (dependent on the par- ticipant’s treatment allocation and pathway within the trial). The ex- pected date of each ECRF can be calculated from any date field captured within the EDC system and is tailored as needed depending on requirements for each individual ECRF. The ECRF completed date uses a standard ECRF field “date form submitted”. As forms are com- pleted by site staff, ECRF completion progress can be viewed in real- time on the schedule forms. 2. An in-house C#.net Windows application was developed for use by ICR-CTSU to read the schedule form data from the EDC system and calculate ECRF compliance data as required. Compliance data can be provided per trial to produce outstanding ECRF reports for provision to site and to review ECRF response rates by form and participating site. g Conclusion h MS sharepoint has been a key tool in providing a consistent ap- proach to managing projects however, it has been recognised that the system should continue to evolve in order to meet changing regulatory and Centre requirements. The Centre continues to identify other areas where MS sharepoint could be used to aid process and quality improvement. p Challenges g Prior to the introduction of EDC, sites posted paper CRFs to the ICR- CTSU. Once received, CRFs were manually tracked onto an ICR-CTSU legacy system which also provided CRF compliance reports. With the introduction of EDC, a solution was required to record real-time com- pletion of ECRFs within the EDC system and to calculate ECRF com- pliance data for review and reporting purposes. S l i Ascertaining study participant safety using centralized electronic medical records in a clinical trial setting — lessons learned from the veterans affairs NEPHRON-D trial Ascertaining study participant safety using centralized electronic medical records in a clinical trial setting — lessons learned from the veterans affairs NEPHRON-D trial Yuan Huang, Gary Johnson, Tassos Kyriakides, Jane H. Zhang CSPCC, VA Connecticut Healthcare System West Haven; Yale University Correspondence: Yuan Huang Trials 2017, 18(Suppl 1):P27 Background The STS can be implemented as a stand-alone system or integrated with a data management system. It is efficient in regard to database setup and implementation and is user-friendly and intuitive for site Page 11 of 235 Trials 2017, 18(Suppl 1):200 assist trial staff at sites with monitoring expected ECRF completion time points for individual trial subjects. assist trial staff at sites with monitoring expected ECRF completion time points for individual trial subjects. and interpretation of clinical trials and other well conducted studies. The Centre’s staff consists of biostatisticians, database managers, soft- ware developers, technicians, health informaticians, health econom- ics, project managers and administrative staff contributing to some 120 clinical studies at present. Involvement in this volume of clinical studies has led the Centre to identify a software solution to more ef- fectively project manage our involvement in these studies whilst sup- porting the requirements of the Centre’s internal Standard Operating Procedures (sops) and Good Clinical Practice (GCP). and interpretation of clinical trials and other well conducted studies. The Centre’s staff consists of biostatisticians, database managers, soft- ware developers, technicians, health informaticians, health econom- ics, project managers and administrative staff contributing to some 120 clinical studies at present. Involvement in this volume of clinical studies has led the Centre to identify a software solution to more ef- fectively project manage our involvement in these studies whilst sup- porting the requirements of the Centre’s internal Standard Operating Procedures (sops) and Good Clinical Practice (GCP). P27 Ascertaining study participant safety using centralized electronic medical records in a clinical trial setting — lessons learned from the veterans affairs NEPHRON-D trial Yuan Huang, Gary Johnson, Tassos Kyriakides, Jane H. Zhang CSPCC, VA Connecticut Healthcare System West Haven; Yale University Correspondence: Yuan Huang Trials 2017, 18(Suppl 1):P27 P27 Ascertaining study participant safety using centralized electronic medical records in a clinical trial setting — lessons learned from the veterans affairs NEPHRON-D trial P28 Producing CDISC compliant data and metadata for regulatory submissions William Stevens, Karl Wallendszus, Martin Landray University of Oxford Correspondence: William Stevens Trials 2017, 18(Suppl 1):P28 Background There is numerous security measures that can be employed to safe- guard online systems, however due to the complex layered architec- ture of today’s applications there are various potential weak points. While following best practices should reduce the risk of malicious parties gaining access to systems, often there are financial or bureau- cratic obstacles to following best practices. Keeping all software and hardware components maintained with current patches represents a considerable amount of work and cost. Despite all this effort there is always the potential of previously unknown zero day exploits being discovered, new strains of malware being created and a dizzying array of new ways to trick computer users into disclosing their cre- dentials or granting access to third parties. An intrusion detection system (IDS) monitors a network or systems for malicious activity or policy violations. The use of an IDS, or combination of different IDS systems are generally considered best practice. There are very di- verse approaches to IDS implementation ranging from configurable rule based systems to machine learning adaptive systems therefore it can be advantageous to employ more than one IDS. An IDS is an im- portant security tool however they are of limited use if a malicious party compromises a system account and performs similar actions e.g. Accessing the trial database. Worse still an IDS is entirely blind to application level activity as most web applications utilise a single sys- tem account to perform all actions. g Steps h The main tasks involved in producing these items are: −Assess how collected data maps to SDTM datasets and outline this in annotated case report forms (CRFs). - Decide which ADAM datasets are needed for analysis, based on the Protocol and Data Analysis Plan. - Trans- form SDTM data into relevant ADAM datasets. - Generate ‘define.xml’ metadata documents. - Validate all datasets and metadata, correcting or documenting errors. - Produce guidance notes for the SDTM and ADAM datasets. Background The purpose of the data-related components of an FDA regulatory submission is to enable an FDA reviewer to understand the clinical trial data that was collected, check the consistency of the data, understand how analysis datasets were produced, and recreate se- lected analyses. Our experience is based on using the Clinical Data Interchange Stan- dards Consortium (CDISC) standards (http://www.cdisc.org/) for pre- paring regulatory submissions for 3 trials totalling 65,000 randomized participants. Study Data Tabulation Model (SDTM) datasets represent trial data in a standardised form. Analysis Data Model (ADAM) data- sets are derived from SDTM datasets, and represent data in a form that is easy to analyse and report on. ‘define.xml’ documents contain metadata for SDTM and ADAM datasets. Brief guidance notes accom- pany the datasets, explaining anything that cannot be understood using the metadata. How do you detect and deal with compromised EDC accounts? William Aitchison, Sharon Kean, Jonathan Gibb Correspondence: William Aitchison Trials 2017, 18(Suppl 1):P29 Correspondence: William Aitchison Trials 2017, 18(Suppl 1):P29 Objective The objective is to devise solution approaches given the scenario where, despite all best security practises being employed there exists Conclusion This investigation identifies several factors that affect the quality of EMR-mediated safety data collection compared to active study condi- tions and establishes the importance of an additional level of clinical review of EMR data. This solution provides a real-time automatic ECRF tracking system that allows central review of ECRF compliance data as required. The user-friendly schedule forms within the EDC clinical database also Trials 2017, 18(Suppl 1):200 Page 12 of 235 Page 12 of 235 P28 Producing CDISC compliant data and metadata for regulatory submissions William Stevens, Karl Wallendszus, Martin Landray University of Oxford Correspondence: William Stevens Trials 2017, 18(Suppl 1):P28 P28 Producing CDISC compliant data and metadata for regulatory submissions William Stevens, Karl Wallendszus, Martin Landray University of Oxford Correspondence: William Stevens Trials 2017, 18(Suppl 1):P28 the possibility that malicious parties could still gain access to some element of the system architecture, how can systems be designed to detect malicious activity by legitimate but compromised user, appli- cation or system accounts? Furthermore the question - when mali- cious activity is detected, what automated and external processes should occur must be explored. Conclusion Bespoke, modular and light-weight tools were useful during the de- velopment of our process for generating regulatory submission pack- ages because these tools are rapidly adaptable. The automated test suite helps prevent changes from having unanticipated conse- quences. From the perspective of programming and data modelling, the CDISC standards have some limitations which could be readily addressed in future versions of the standards. A review of the essentials and pitfalls of the Lugano classification in malignant lymphoma trials g y p Dewen Yang, David Raunig ICON Clinical Research Correspondence: Dewen Yang Trials 2017, 18(Suppl 1):P30 The core language has a small codebase (approx. 2000 lines of code) and few non-standard dependencies. Most of the functionality of the system is expressed in well-documented parameterized units (approx. 4000 lines of code). An automated test suite (approx. 4000 lines of code) verifies the functionality of each unit. Correspondence: Dewen Y Trials 2017, 18(Suppl 1):P30 Correspondence: Dewen Y Trials 2017, 18(Suppl 1):P30 To facilitate the comparison of patients and results by providing a standardized guidance on how data should be analyzed for therapy, response criteria for non-Hodgkin lymphoma (NHL) were published in 1999 by an international working group (IWG). The revision for both NHL and Hodgkin lymphoma (HL) was published in 2007 to in- corporate PET and bone marrow biopsy in response assessment. After years of experience with the 2007 criteria and recognizing the imaging technique progress, the 2nd revision called the Lugano clas- sification was published in 2014 to assess lymphoma therapeutic re- sponse in clinical trials. The Lugano guidelines have enhanced interpretation of CT assessment, imaging schedules, and PET scoring implications, rules for handling missing anatomy and challenging scenarios for the given therapeutic under investigation. The Lugano classification provides a renewed opportunity to guide lymphoma diagnosis and clinical management based on imaging findings. The new criteria also have been increasingly adopted in many lymphoma trials since its publication. Nevertheless, certain aspects of the new criteria lack sufficient detail for explicit interpretation and a few fea- tures open to potential pitfalls which need particular attention and further discussion. For instance, the five-point scale (5-PS) for FDG- PET assessment was incorporated to evaluate tumor metabolic re- sponse assessment in FDG-avid lymphoma types, but the 5-PS, Method We propose integrating simple IDS methods into the application and database layers. By identifying simple activity rules to identify un- usual usage the application and database can react in an appropriate manner based on the associated level of risk. Conclusion We use bespoke software tools for these steps (except validation, which is performed using industry standard software). SDTM and ADAM datasets are stored in a relational database. Data- sets are defined and produced using a domain-specific language that permits XML elements to be associated with parameterized units of software which generally perform SQL code generation (which can be executed to perform a data transformation), but which may also do other things, such as the generation of documents. Some exam- ples are: −Conversion of units for a defined set of lab results, while checking that there are no unexpected combinations of lab test and units. - Estimating dates from partial dates and upper and lower limits. - Generating CDISC define.xml documents. The authors will present an overview of IDS style methods suitable to clinical EDC systems, how to implement them and how to structure a framework for responding to them. P31 Will you walk a little faster? - joining the database development dance 1 2 2 2 Will you walk a little faster? - joining the database development dance 1 2 2 2 Writing of a manuscript, abstract, or other document in a research environment is a collaborative effort which oftentimes involves indi- viduals from academia, industry, and government agencies. Topics are proposed and must be managed according to study timelines and may require considerable time and resources to track over the course of a study. The data management development team in the Epidemiology Data Center (EDC), Graduate School of Public Health, at the University of Pittsburgh has designed a web-based Presenta- tions and Publications System (PNP) to streamline work flow, provide a repository for completed works, and facilitate tracking and report- ing of the presentation and publication process. Correspondence: Mary Rauchenberger Trials 2017, 18(Suppl 1):P31 Problem Trialists often feel that the release of a validated database is a limit- ing factor in the timeline of opening a clinical trial. There is an inher- ent tension between (i) the desire to be able to change requirements (such as Case Report Forms, eligibility and validation checks) as late as possible and (ii) the need for those requirements to be finalised early on so that development and testing can take place. We will de- scribe several approaches we have taken to address this dilemma. These focus initially on technical solutions, using our bespoke clinical data management system, developed using MS SQL Server and.NET. We will then expand to look at how these can be enhanced with process changes. This has led to a culture change resulting in much wider participation in the database development project, a livelier dance with more partners on the dancefloor. g p p p The system is comprised of a Pre-proposal module, which allows users to quickly enter potential topics, and a Proposal module, which begins when a more fully developed topic is submitted. g y The Pre-Proposal module facilitates sharing topic ideas and allows topics to be ranked and prioritized. Potential collaborators use this module to indicate an interest in participating in a writing group. Ap- proved topics are moved into the Proposal module for development into an abstract, manuscript or preliminary analysis proposal. The Proposal module is used to submit a more detailed description of the topic, set priorities for the proposal, and track and manage the activities and content. The main page of the Proposal module provides access to all abstracts, manuscripts, and grant proposals submitted for the project and contains key information such as the status of the proposal and the latest activity. Proposals are managed via a tracking page, which has tabs for the submitted proposal form, summary information (e.g. Stage and status of the proposal), detailed proposal tracking activities, and a reference library. Proposal activities are grouped into pre-defined categories (e.g. Writing group, re- viewers, scientific meetings, and journals); these categories are also available as individual tabs to allow reporting all manuscripts in a particular category. Objective Objective The objective is to devise solution approaches given the scenario where, despite all best security practises being employed there exists Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 13 of 235 Page 13 of 235 copied from Deauville criteria, relies on a vague description of quali- tatively assessing “Change of the hottest lesion” And no definitive guidance on “Significant change in FDG uptake”; moderately/mark- edly higher than liver or whether quantitative uptake measurements are allowed as the cut-off reference for the score 4 or 5; beside of the imaging scan window, imaging findings on CT and FDG PET-CT can be rarely conflicting. In addition, progressive disease with regard to splenomegaly assesses response with regard to both the baseline and to ‘prior increase’ which, if interpreted one way, can lead to ex- treme enlargement without progression. On the other hand, spleno- megaly can be caused by lymphoma-unrelated causes such as portal hypertension or use of hematoietic growth factors which make a question if splenomegaly alone can be used to define the progres- sive disease. To provide the most accurate assessment of response to therapeutic intervention, it is essential that trial oncologists and radi- ologists not only have a tangible understanding of the Lugano Classi- fication, but also proper insight into the practical limitations of the criteria. We will review the essential elements and provide few exam- ples to illustrate the limitations and ambiguity that can arise from dif- ferent interpretations of the Lugano classification. Furthermore, some suggestions will be made to stimulate further improvement in clinical trial settings. copied from Deauville criteria, relies on a vague description of quali- tatively assessing “Change of the hottest lesion” And no definitive guidance on “Significant change in FDG uptake”; moderately/mark- edly higher than liver or whether quantitative uptake measurements are allowed as the cut-off reference for the score 4 or 5; beside of the imaging scan window, imaging findings on CT and FDG PET-CT can be rarely conflicting. In addition, progressive disease with regard to splenomegaly assesses response with regard to both the baseline and to ‘prior increase’ which, if interpreted one way, can lead to ex- treme enlargement without progression. On the other hand, spleno- megaly can be caused by lymphoma-unrelated causes such as portal hypertension or use of hematoietic growth factors which make a question if splenomegaly alone can be used to define the progres- sive disease. Objective To provide the most accurate assessment of response to therapeutic intervention, it is essential that trial oncologists and radi- ologists not only have a tangible understanding of the Lugano Classi- fication, but also proper insight into the practical limitations of the criteria. We will review the essential elements and provide few exam- ples to illustrate the limitations and ambiguity that can arise from dif- ferent interpretations of the Lugano classification. Furthermore, some suggestions will be made to stimulate further improvement in clinical trial settings. development and encourage ownership. Rapid coding approaches, using group sessions and peer review, and group testing sessions, implementing fixes in real-time, have also been implemented to ac- celerate progress. The more agile dance is now perhaps a casual group samba, involving developers, data managers, data scientists, and business analysts. Di i Discussion Ultimately though, there is a limit to how fast you can dance. The culture change needed requires much earlier penetration into the trial project timeline, looking at team resourcing and key decision timepoints. Involving the database team at the earliest stages helps with understanding how the proposed trial flow can best be imple- mented, and with prioritisation of agreements needed for timely de- livery of the validated database. The dance becomes a unit-wide quadrille, with multiple partners and movements. Or, maybe more appropriately for this conference, a ceilidh! P32 Presentation and publication system (PNP): a tool to facilitate efficient tracking and reporting of the presentation and publication process Tamara Haller, David E. Hallam, Sharon M. Lawlor, Ella Zadorozny University of Pittsburgh Correspondence: Tamara Haller Trials 2017, 18(Suppl 1):P32 Tamara Haller, David E. Hallam, Sharon M. Lawlor, Ella Zadorozny University of Pittsburgh Correspondence: Tamara Haller Trials 2017, 18(Suppl 1):P32 Correspondence: Tamara Hall Trials 2017, 18(Suppl 1):P32 P31 Will you walk a little faster? - joining the database development dance Mary Rauchenberger1, Kenneth Babigumira2, Chiara Borg2, Emma Little2, Nancy Tappenden2, Matthew R. Sydes2, Nadine Van Looy2 1MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL, London, UK; 2MRC London Hub for Trials Methodology Research, London, UK Correspondence: Mary Rauchenberger Trials 2017, 18(Suppl 1):P31 Our approach The technical solution concentrates on auto-generation. In our model, Excel is used to document the user requirements as meta- data. This allows users to engage with a familiar tool to specify con- ditions in a structured method. Once reviewed and finalised, the spreadsheet is uploaded into the database and the metadata is used to generate the database tables, triggers and procedures that pro- vide the necessary functionality (such as audit trail, query generation, etc.). The metadata also provides the input to a customised code generator which produces the front-end code for data entry screens and validations. Common code modules and standard field names produce a consistent interface, with core functionality and generic el- ements that can be easily reused across projects. The tempo of the dance for the database developers moves from a waltz to a quick- step. We have also looked at development methodology, moving away from waterfall approaches and adopting elements of agile pro- ject management and development into our processes. Key to this is the phased approach, concentrating on what needs to be included in the initial release and keeping to firm release dates, prioritising the product backlog for each release. Self-organising teams, feasible in larger trials units, bring more resource to a project at critical timepoints. Workshops with the trial team help with metadata The PNP system was designed to allow the user to quickly configure the system based on the study’s requirements through a set-up wiz- ard. The wizard does not require that all elements be pre-defined, but allows the users to configure the system throughout the process. Reports created in Crystal Reports or SAS are supported by the PNP system and allow the users to create customized reports at the pro- ject and proposal level. Access to the PNP system is restricted ac- cording to a user’s project role and permissions assigned. In summary, the PNP system is a tool that can help to organize the process of writing a manuscript, abstract, or research grant while re- ducing the personnel time and effort needed for communication and coordination among the collaborators. In summary, the PNP system is a tool that can help to organize the process of writing a manuscript, abstract, or research grant while re- ducing the personnel time and effort needed for communication and coordination among the collaborators. Page 14 of 235 Trials 2017, 18(Suppl 1):200 Page 14 of 235 Background The NIHR (National Institute for Health Research) is the research arm of the NHS and is the most integrated clinical research system in the world. It invests about one percent of the NHSD budget in research to improve the health and wealth of the nation. 1University of Bristol; 2Ulster University; 3Institute of Clinical Trials and Methodology Correspondence: Alex Nicolson Trials 2017, 18(Suppl 1):P33 The NIHR funds the RDS (Research Design Service) to provide design and methodological support to health and social care researchers across England to develop grant applications to the NIHR (Programme Grants for Applied Research, Research for Patient Benefit, Health Tech- nology Assessment, Public Health Research, Invention for Innovations, Health Services and Delivery Research etc.) And other national peer- reviewed funding programmes. NIHR research design service Mark Mullee University of Southampton Trials 2017, 18(Suppl 1):P34 Alex Nicolson1, Anne Daykin1, Karen Coulman1, Clare Clement1, Helen Cramer1, Carrol Gamble1, Rhiannon Macefield1, Sharon McCann1, Gillian W. Shorter2, Mathew R. Sydes3 More than the emperor’s new clothes: enhancing meaningful patient and public involvement in trial oversight committees through qualitative research with eight clinical trials facing challenges More than the emperor’s new clothes: enhancing meaningful patient and public involvement in trial oversight committees through qualitative research with eight clinical trials facing challenges NIHR research design service Mark Mullee University of Southampton Trials 2017, 18(Suppl 1):P34 Public involvement in research The RDS has been at the forefront of the NIHR drive to ensure that members of the public play an important role in developing success- ful grant applications. The RDS has been particularly active and pio- neering in the area of Public Involvement, from design of the research study through to dissemination of research findings. The RDS recently worked in partnership with the Wessex Institute, University of Southampton on a successful bid to host INVOLVE (funded by NIHR to support active public involvement in research). The expertise and regional networks of the RDS were recognised as an important component of the partnership. NIHR RDS metrics h l Analysis revealed the importance interviewees placed on the role of PPI to provide a patient voice within trial oversight. PPI was gen- erally favoured within TOCs, but several tensions arose relating to meaningful PPI implementation at TSC and TMG levels. Lack of clar- ity about what PPI is and whether it was needed led to inclusion of those representatives who, perhaps, were not best equipped with the appropriate skills, experience and attributes. Representatives who lacked detailed knowledge or familiarity with trial method- ology and technical language found it difficult to understand and contribute to meetings. Interviewees felt it was important when selecting representatives to consider whether they truly had em- pathy for the trial population or had possible ‘hidden agendas’. Consideration of PPI representatives’ commitments and circum- stance outside of trial oversight was important for ongoing engage- ment and attendance. Participants saw a need for training and or mentoring of PPI representatives to foster appropriate involvement and contribution. However, there was no clear consensus of who was or should be responsible for enabling or providing such train- ing and support. The RDS remit includes increasing the quality and quantity of re- search applications. Since 2009, the RDS has supported: The RDS remit includes increasing the quality and quantity of re- search applications. Since 2009, the RDS has supported: 17,949 projects, 2,705 outline applications submitted with 1,111 shortlisted (43% success rate), 6,432 full applications submitted with 2,209 funded (36% success rate). search applications. Since 2009, the RDS has supported: 17,949 projects, 2,705 outline applications submitted with 1,111 shortlisted (43% success rate), 6,432 full applications submitted with 2,209 funded (36% success rate). The RDS also provides triage for under-prepared or misplaced fund- ing applications. Thus, reducing waste in terms of the time and re- source used by NIHR funding programmes to review poor quality applications. pp One NIHR The RDS is recognised as the ‘local face of the NIHR’. It has become an intermediary between national NIHR structures (Collaboration for Leadership in Applied Health Research and Care (CLAHRC), Clinical Research Networks, Clinical Trials Units, Biomedical Research Centres and NHS Trust R&D etc.) and local investigators and organisations. The RDS is recognised as the ‘local face of the NIHR’. It has become an intermediary between national NIHR structures (Collaboration for Leadership in Applied Health Research and Care (CLAHRC), Clinical Research Networks, Clinical Trials Units, Biomedical Research Centres and NHS Trust R&D etc.) and local investigators and organisations. The RDS has often facilitated local partnerships, to pursue ‘One NIHR’. It has brought together various components of the NIHR at local and regional levels, to share good practice, look for efficiencies of delivery and to enable investigators and organisations to have a more streamlined access to support and advice. The RDS has often facilitated local partnerships, to pursue ‘One NIHR’. It has brought together various components of the NIHR at local and regional levels, to share good practice, look for efficiencies of delivery and to enable investigators and organisations to have a more streamlined access to support and advice. g pp Conclusion To truly enable PPI representatives to speak on behalf of patient or public voice and ensure meaningful contributions of such represen- tatives within trial oversight, more thought needs to be given to de- signing the involvement of PPI in TOCs. This includes clarification around roles and what would constitute optimal involvement at dif- ferent oversight levels and stages of trials. To ensure ongoing worth- while PPI, training and support needs of contributors needs to be reflected upon and provided, and consideration needs to be given to PPI selection and TOC meeting conduct to ensure attendance and engagement is maintained. Background The value of Patient and Public Involvement (PPI) in trial oversight is increasingly recognised; at present it is a requirement for most UK re- search funding bodies to involve PPI members in Trial Oversight Committees (TOCs) including Trial Steering Committees (TSCs) and Trial Management Groups (TMGs). However, there is little evidence- based guidance to optimise their roles and inputs. The actions and experiences of TOC members including PPI representatives were cap- tured to inform recommendations about enhancing PPI contribution to trial oversight. This was carried out within the context of a larger multi-method study which aimed to explore the role and function of TOCs, and their contribution to trial conduct. h d The RDS is a national service delivered by ten regions covering England. NIHR RDS (research design service) expertise Methodological advice is provided to researchers by teams of Ad- visers whose expertise includes statistics, qualitative research methods, health economics, systematic reviews, health psychology and behavioural science. The RDS has an important role in referring individuals to appropriate sources of advice, outside of the RDS, where appropriate. For ex- ample, referrals to those with specialist expertise in intellectual prop- erty or to a local Clinical Research Network for practical help in identifying and recruiting patients to studies. TOC meetings of eight large phase III UK trials that were undergoing challenges (e.g. Recruitment issues, protocol deviation or amend- ments) were observed by a qualitative researcher and audio- recorded. Interviews explored PPI in interviewees’ trials and where they thought PPI contributors were best placed. PPI representatives also reflected on their personal experience of TOC meetings, their understanding of their roles and how they felt they had influenced trial conduct. Data (meeting transcripts, field notes and interview transcripts) was analysed thematically using techniques of constant comparison. Public involvement in research p Results Seven TSC and six TMGs (n = 13) were observed and six of the meet- ings had PPI present (3 TSC, 3 TMG). Sixty-six semi-structured inter- views were carried out with fifty-two members of these TOCs which included three PPI representatives. NIHR RDS metrics Methods Different level of efforts including government, society, university, and industry have been devoted to increasing the number of “qualified” biostatisticians in Japan, and in October 2016, Japan Agency for Med- ical Research and Development (AMED) have decided to fund the two universities, Kyoto University (KU) and University of Tokyo to develop a new program for training the next generation biostatisticians with em- phasis in clinical trials, under the public and private partnership with the Japan Pharmaceutical Manufacturers Association (JPMA). Each of two universities formed the alliance to develop the program: KU with KU Hospital and National Cerebral and Cardiovascular Center (NCVC), and UT with UT Hospital and National Cancer Center. A 16-item questionnaire assessing signs, symptoms and interventions potentially indicative of SSI was developed using standard methods. Patients undergoing general abdominal surgery and women under- going caesarean section were recruited from three UK hospital trusts. Participants were sent the new questionnaire to complete approxi- mately 30 days after surgery and return by post (self-assessment). Short “debriefing” questions to assess ease of completion were in- cluded. Healthcare professionals attempted to contact participants approximately 30–35 days after surgery and complete the new ques- tionnaire via telephone (observer assessment). A proportion of partic- ipants (limited by study resources) were seen face-to-face 4–8 weeks after surgery and classified as having an SSI or not using the Center for Disease Control (CDC) classifications for wound infection (refer- ence standard). These assessors were blinded to participants’ self- assessment and observer assessment. Analyses, which are ongoing, will: 1) compare participant (self-assessment) and healthcare profes- sional (observer assessment) responses, 2) examine the sensitivity of the questionnaire for identifying symptoms compared to similar cri- teria in the reference standard, 3) test a clinician-lead hypothesised scale structure and scoring system for determining SSI outcome, 4) examine the discriminative ability of the questionnaire to identify po- tential SSI “problems” Using a set of receiver operating characteristic (ROC) curves and 5) assess the reliability of the questionnaire. Results In this presentation, we briefly review the current issues in MPH-level biostatistical education and training in Japan, and outline our plan and activities for developing the educational and training program for the next generation biostatisticians. Assessing the impact of a funder’s recommendation on consideration and uptake of core outcome sets in funding applications 1 1 2 P37 Clinical and psychometric validation of a new outcome measure: methods to assess measurement properties in the absence of a 'gold' standard Rhiannon Macefield, on behalf of the Bluebelle Study Group University of Bristol Correspondence: Rhiannon Macefield Trials 2017, 18(Suppl 1):P37 P37 Clinical and psychometric validation of a new outcome measure: methods to assess measurement properties in the absence of a 'gold' standard Rhiannon Macefield, on behalf of the Bluebelle Study Group University of Bristol Correspondence: Rhiannon Macefield Trials 2017, 18(Suppl 1):P37 Karen Barnes1, Jamie J. Kirkham1, Mike Clarke2, Paula R. Williamson1 1University of Liverpool; 2Queen’s University Belfast Correspondence: Karen Barnes Trials 2017, 18(Suppl 1):P38 Karen Barnes1, Jamie J. Kirkham1, Mike Clarke2, Paula R. Williamson1 1University of Liverpool; 2Queen’s University Belfast Correspondence: Karen Barnes Trials 2017, 18(Suppl 1):P38 Conclusion Examination of the clinical validity and measurement properties of a new SSI outcome measure is ongoing. Different thresholds for SSI “problem” scores may be needed when assessments are made by participants or healthcare professionals. Qualitative work to further understand the difference in agreement between participant and healthcare professional reports of symptoms would be beneficial. P35 P35 A program for training the next generation of biostatisticians in japan: developing on-the-job training at NCVC Toshimitsu Hamasaki1, Haruko Yamamoto1, Shiro Tanaka2, T. Shun Sato2 1National Cerebral and Cardiovascular Center; 2Kyoto University School of Public Health Correspondence: Toshimitsu Hamasaki Trials 2017, 18(Suppl 1):P35 A program for training the next generation of biostat japan: developing on-the-job training at NCVC 1 1 2 j p p g j g Toshimitsu Hamasaki1, Haruko Yamamoto1, Shiro Tanaka2, T. Shun Sato2 1National Cerebral and Cardiovascular Center; 2Kyoto University School of Public Health Correspondence: Toshimitsu Hamasaki Trials 2017, 18(Suppl 1):P35 Correspondence: Toshimitsu Hamasaki Trials 2017, 18(Suppl 1):P35 Page 15 of 235 Page 15 of 235 Trials 2017, 18(Suppl 1):200 Statistical contributions to clinical trials and medical product devel- opment have been well-recognized in Japan since the ICH-E9 guide- line “Statistical Principles for Clinical Trials” Was implemented in 1998, where the guideline helped trigger the revelation that there is a shortage of qualified statisticians who can comprehend and imple- ment the principles outlined in the guideline and improve the quality and integrity of the trials being conducted. Although the number of educational programs for Master and phd level biostatisticians at uni- versities have been greatly increased during the last two decades, at this period, the supply of new graduates in biostatistics in Japan is relatively steady while the demand is increased dramatically. over a certain threshold can be interpreted as clinically meaningful or “problematic”. Standard methods to identify such thresholds re- quire an established reference standard and the use of receiver oper- ating characteristic (ROC) curves. We have developed a new questionnaire to assess wounds for surgical site infection (SSI), with a view to it being used as an outcome measure in a future trial. Valid- ation, however, is challenging because the diagnostic accuracy of the established reference standard is imperfect and estimates of sensitiv- ity and specificity may therefore be biased. The aim of this study is to explore the clinical validity and measurement properties of the new measure in the absence of a “gold” standard. Methods over a certain threshold can be interpreted as clinically meaningful or “problematic”. Standard methods to identify such thresholds re- quire an established reference standard and the use of receiver oper- ating characteristic (ROC) curves. Methods Our developed program is very unique to combine the two learning approach to gain skill and knowledge of clinical trials-related biostatistics: learnings (i) by being taught, by study- ing it, or by researching it through structured courses/modules at KU School of Public Health and (ii) by experiencing it in practical situations (i.e., On-the-Job (OJT) Training) at KU Hospital or NCVC. We describe our developed OJT training program at NCVC. P36 Efficient group-sequential designs for monitoring two time-to-event outcomes in clinical trials Toshimitsu Hamasaki1, Koko Asakura1, Tomoyuki Sugimoto2, Scott R. Evans3, Haruko Yamamoto1, Chin-Fu Hsiao4 1National Cerebral and Cardiovascular Center; 2Kagoshima University; 3Harvard T.H. Chan School of Public Health; 4National Health Research Institutes Correspondence: Toshimitsu Hamasaki Trials 2017, 18(Suppl 1):P36 36 Efficient group-sequential designs for monitoring two time-to-event outcomes in clinical trials 1 1 2 3 416 participants were recruited. Participants completed and returned 300/414 (72.5%) questionnaires (self-assessments). Healthcare profes- sionals successfully contacted 306/414 (73.9%) participants and com- pleted questionnaires via telephone (observer assessments). Face to face assessments were made for 115 (27.7%) participants (reference standard). Participants found the questionnaire quick and straightfor- ward to complete, with few missing data. Initial analyses of partici- pant and healthcare professional responses show that symptoms are reported a little more severe in self-assessments compared to obser- ver assessments; a consistent trend observed for all eight symptom- related items. Other planned analyses are ongoing, pending add- itional data from a pilot RCT where all participants (n = 330) were scheduled to receive a reference standard assessment. C l i Correspondence: Toshimitsu Hamasaki Trials 2017, 18(Suppl 1):P36 We discuss logrank test-based methods for early efficacy or futility evaluation in group-sequential clinical trials designed to compare two interventions using two time-to-event outcomes. We consider three typical situations (1) both events are non-composite and non- fatal, (2) both events are non-composite but one event is fatal, and (3) one event is composite but other is fatal and non-composite. We outline strategies for rejecting the null hypothesis associated with two inferential goals, evaluating if a test intervention is superior to a control intervention on: (1) both outcomes (multiple co-primary end- points: MCPE), and (2) at least one outcome (multiple primary end- points: MPE). We provide an example to illustrate the methods and discuss practical considerations when designing these trials. Background Background Patients’ health outcomes and experiences are often measured using validated questionnaires. Responses are usually scored and values P35 We have developed a new questionnaire to assess wounds for surgical site infection (SSI), with a view to it being used as an outcome measure in a future trial. Valid- ation, however, is challenging because the diagnostic accuracy of the established reference standard is imperfect and estimates of sensitiv- ity and specificity may therefore be biased. The aim of this study is to explore the clinical validity and measurement properties of the new measure in the absence of a “gold” standard. Methods Background g A systematic review published in 2014 [1] identified 198 published core outcome sets (COS) and a recent update found that this figure had increased to 227 by the end of that year [2]. The details of these Trials 2017, 18(Suppl 1):200 Page 16 of 235 Page 16 of 235 COS, along with others that are planned and in development, are re- corded in the COMET (Core Outcome Measures in Effectiveness Tri- als) database. As the number of COS grows, it is important to assess their uptake by clinical trialists because the continued development of COS, without their implementation, could add to waste in re- search, and would mean that those using the results of trials to make decisions about healthcare will not realise the benefits that using COS can provide. Methods and results All published articles reporting at least one short-term composite outcome assessed within three months of cardiac surgery were identified. One hundred and fifty four papers were identified, reporting 166 composite outcomes; 64 different adverse events were included across the composite outcomes. Death was a com- ponent in the majority of composites (135/166, 81%), as were cerebrovascular events (105/166, 63%), myocardial infarction (MI) (81/166, 49%), renal failure/acute kidney injury (AKI) (78/166, 47%) and reoperation/revascularisation (42/166, 25%). Two “established” composite outcomes were identified in the review, Major Adverse Cardiac Events (MACE) and Major Adverse Cardiac and Cerebro- vascular Events (MACCE), but the definitions for both differed across studies. Assuming MACCE includes death, cerebrovascular events, MI and reoperation/revascularisation, 16/166 composites included these four components; 12 of these 16 also included other adverse events, suggesting that the currently used compos- ite outcomes are based on, but not restricted to, existing MACCE definitions. Other adverse events that were commonly included together in composite outcomes were renal failure and death/ cerebrovascular event, and prolonged ventilation and death/cere- brovascular event. Analysis Following extraction of the above data, the following analysis will be performed: – Assessment of the number and proportion of NIHR HTA applications referencing the COMET database or a COS published in the COMET database pp g published in the COMET database – Assessment of the number and proportion of NIHR HTA applications using a COS, if one exists, in their research Results and Conclusions Results and Conclusions Results and conclusions will be presented following examination of all funded and non-funded applications to the NIHR HTA researcher-led, commissioned and themed call funding streams from January 2012 to December 2015 (n = 281). The sample con- sists of applications for both randomised trials (n = 189) and evi- dence syntheses (n = 92). Method The completed application form and detailed project description of each NIHR HTA application will be examined for: Evidence that the COMET database had been searched to establish whether or not a COS exists Reference to a COS study published in the COMET database Evidence that a COS was included in the application if one exists Evidence that a COS was not included in the application where one exists Evidence that the COMET database had been searched to establish whether or not a COS exists Reference to a COS study published in the COMET database E id h COS i l d d i h li i if i Reference to a COS study published in the COMET database Evidence that a COS was included in the application if one exists Reference to a COS study published in the COMET database Evidence that a COS was included in the application if one exists Evidence that a COS was not included in the application where one exists Reasons given for not including a COS where one exists Reasons given for not including a COS where one exists Reasons given for not including a COS where one exists Rationale for outcome choice in the absence of a COS Rationale for outcome choice in the absence of a COS The majority of composite outcomes were binary outcomes (any event vs. none) that gave equal importance to all components. Two studies investigated the relative weighting assigned to ad- verse events in MACCE, both among patients and one among trialists, and reported that respondents assigned different weight- ings to each of the adverse events within the composite. Differ- ences between the weightings assigned by patients and clinical trialists were also reported, with patients rating MI and stroke the same as or worse than death, but trialists rating death as the most severe. Background Composite outcomes are often reported in randomised controlled tri- als, particularly for safety endpoints. Use of a composite endpoint can allow a study to provide information about safety when the rates of component adverse events are low, but risks aggregating events that are not affected by the intervention. We undertook a literature review to explore the variability in composite outcomes used in car- diac surgery studies, to inform the development of an objective measure of recovery. p In January 2012 the guidance for NIHR HTA funding recommended ‘details should include justification of the use of outcome measures where a legitimate choice exists between alternatives. Where estab- lished Core Outcomes exist they should be included amongst the list of outcomes unless there is good reason to do otherwise. Please see The COMET Initiative website at www.comet-initiative.org to identify whether Core Outcomes have been established.’ This study will as- sess the extent to which this recommendation has been followed by NIHR HTA applicants from January 2012, when the recommendation was introduced, to December 2015. References [1] Gargon E, et al. Choosing important health outcomes for comparative effectiveness research: a systematic review. Plos ONE 2014; 9: e99111. [2] Gorst SL, et al. Choosing important health outcomes for comparative effectiveness research: an updated review and user survey. Plos ONE 2016; 11: e0146444. [2] Gorst SL, et al. Choosing important health outcomes for comparative effectiveness research: an updated review and user survey. Plos ONE 2016; 11: e0146444. P40 Evaluation of interventions for informed consent for randomised controlled trials (ELICIT): results from a systematic review and interviews towards developing a core outcome set Katie Gillies1, Heidi Gardner1, Alex Duthie1, Cynthia Fraser1, Vikki Entwistle1, Shaun Treweek1, Paula Williamson2, Marion Campbell1 1University of Aberdeen; 2Liverpool University Correspondence: Katie Gillies Trials 2017, 18(Suppl 1):P40 Discussion These assessments will be used to draw conclusions about the po- tential impact on the use of COS of a research funder’s recommenda- tion about their use. This review has highlighted the variability in the way composite outcomes for cardiac surgery studies have been defined. The range of events included supports the need for the development of a composite outcome including a range of adverse events to give a more complete picture of recovery. Furthermore, these findings support the need for composite outcomes to incorporate weightings, particularly when adverse events differ in their impact on patient recovery, and for the views of both patients and clini- cians to be considered when assessing the relative importance of different adverse events if the composite outcome is intended to give an overall assessment of recovery. Variation was seen in the definitions used for some events (e.g. renal failure) across studies; there is a need for consistent definitions to be agreed to aid syn- thesis of results from different cardiac surgery studies in meta- analyses. Results A total of 9375 participants were requested to complete diary cards over the three time periods. Generally, diaries were well completed with 69% of participants completing all four diaries, and 83% completing at least one diary card. Completion rates were consistent across each of the three time intervals. There was a small but statistically significant increase in the proportion of people not returning a diary over the three successive time periods (p < 0.001). Those allocated to complete diary cards were more likely to withdraw from follow-up questionnaires than those not allocated to complete diaries in the same 4 month period. This was a small but con- sistent effect over the entire study (difference in rates of ~2%). In those participants who returned all diary cards and a corresponding question- naire, falls were underreported in the questionnaire. People who returned no diaries were older, had poorer levels of physical and mental health, and had poorer cognitive function as well as a higher number of falls and fractures reported in their corresponding follow up questionnaires. Conclusions Jared Foster1, Ranran Dong2, Qian Shi1 1Mayo Clinic; 2The Ohio State University Correspondence: Jared Foster Trials 2017, 18(Suppl 1):P41 Most contemporary methods in the field of surrogate endpoint evaluation involve assessing the degree to which average treat- ment effects on the surrogate and true endpoints are correlated (i.e. The trial-level surrogacy), using data from a (generally) small number of randomized clinical trials RCT). Because the number of relevant clinical trials is generally small, these methods may pro- duce estimates of trial-level surrogacy that are highly variable. To this end, we consider the evaluation of potential surrogate end- points within a personalized medicine framework. In particular, we consider a two-step procedure. In step 1, the surrogate and true endpoints are modeled as a function of treatment received, and other patient characteristics. Using these models, we obtain esti- mated, conditional (on patient characteristics), subject-specific treatment effects on the true and potential surrogate endpoints for each patient. In step 2, the estimated, subject-specific treatment ef- fects on the true endpoint are modeled as a function of those on the surrogate endpoint using linear regression, and the trial-level surrogacy is estimated using the R-Squared from this model. P41 Surrogate endpoint evaluation in a personalized medicine framework 1 2 1 P41 Surrogate endpoint evaluation in a personalized medicine framework P39 Variability in composite outcomes reported in cardiac surgery studies: a literature review Rachel Maishman1, Barnaby C. Reeves1, Umberto Benedetto2, Chris A. Rogers1 1University of Bristol Clinical Trials and Evaluation Unit; 2Bristol Heart Institute, University of Bristol Correspondence: Rachel Maishman Trials 2017, 18(Suppl 1):P39 This abstract is not included here as it has already been published. Page 17 of 235 Trials 2017, 18(Suppl 1):200 Page 17 of 235 P41 Surrogate endpoint evaluation in a personalized medicine framework Jared Foster1, Ranran Dong2, Qian Shi1 1Mayo Clinic; 2The Ohio State University Correspondence: Jared Foster Trials 2017, 18(Suppl 1):P41 P43 Designing trial outcomes for rare diseases 1 1 P43 Designing trial outcomes for rare diseases Eftychia Psarelli1, Trevor F. Cox1, Lakshminarayan Ranganath2 1Liverpool Cancer Trials Unit, University of Liverpool; 2Royal Liverpool University Hospital Correspondence: Eftychia Psarelli Trials 2017, 18(Suppl 1):P43 Background ll Falls are a substantial health risk in older people. The collection of ac- curate falls data is problematic within clinical trials at several levels1. In particular there are issues with reporting falls when these events are associated with recall bias. Different data collection methods have been proposed to minimise bias. In the prefit trial2 we per- formed a study within a trial (SWAT3) to compare two common methods’ daily falls diaries and retrospective reporting within quar- terly questionnaires. Swats are an increasingly popular method to in- vestigate uncertainties faced by researchers when conducting and designing randomised controlled trials. P43 Designing trial outcomes for rare diseases 1 1 P42 Comparison and impact of prospective and retrospective falls data completion methods in the prefit trial: results of a randomised methodology study within a trial (SWAT) James Griffin1, Emma J. Withers2, Ranjit Lall2, Julie Bruce2, Susanne Finnegan2, Sallie E. Lamb3, prefit Study Group2 1Warwick Clinical Trials Unit; 2Warwick Clinical Trials Unit, University of Warwick; 3Kadoorie Centre for Critical Care Research and Education, John Radcliffe Hospital, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford Correspondence: James Griffin Trials 2017, 18(Suppl 1):P42 The selection of appropriate endpoints is of paramount importance for a clinical trial to meet its objectives. For some diseases it is difficult to choose a single endpoint or a few multiple endpoints that measure the disease from which a comparison of treatments can be made. This can be especially true for some rare diseases, where a major challenge in clin- ical trial design is the lack of a validated well-characterised efficacy end- point. In order to assess disease severity in people with a rare condition such as alkaptonuria (AKU) - an orphan inborn homogentisate dioxygen- ase enzyme deficiency resulting in accumulation of homogentisic acid - a new tool was developed. The AKU Severity Score Index (AKUSSI) incorpo- rates multiple, clinically meaningful outcomes that can be described in a single score. AKUSSI consists of both subjective and objective features that have been selected on current knowledge of the disease and it is sensitive to all morbid features of the condition. This score is a quantifi- able, multidisciplinary assessment system, with the potential of reflecting changes in disease severity over time. Clinical experts, patients and statis- ticians were part of the development team. Tools like AKUSSI that de- scribe disease manifestations can be used to compare disease across patients at different time points for other complex and multi-systemic diseases. Details and rationale of the AKUSSI tool that is now used as an outcome in a Phase III efficacy study (SONIA 2) will be described, with special attention to issues arising from the rarity of the disease. P44 A systematic search of clinicaltrials.gov to assess the uptake of core outcome sets Jamie J Kirkham1, Mike Clarke2, Paula R Williamson1 1MRC North West Hub for Trials Methodology Research, Department of Biostatistics, University of Liverpool, Liverpool, UK; 2Northern Ireland Hub for Trials Methodology Research, Centre for Public Health, Queen’s University Belfast, Belfast, UK Correspondence: Jamie J Kirkham Trials 2017, 18(Suppl 1):P44 Results Pre- liminary simulation studies suggest that, in many cases (when ap- propriate models are selected for the surrogate and true endpoint, and when certain other assumptions hold), this estimate of trial- level surrogacy has dramatically lower variance than some more traditional estimates of trial-level surrogacy. This SWAT provides evidence that allocation to complete prospective diary cards alongside four-monthly retrospective postal questionnaires has a small but significant effect on withdrawal from the main trial. Retrospective and prospective falls data are not consistently reported when collected simultaneously. People who did not return diaries were systematically in poorer health than those who completed all allocated diary cards. Swats are an efficient additional component of RCT design and should be considered to improve the design of future trials. Concealing the randomised allocation in trials: experience from the thermic trials Using a logic model and quantitative and qualitati feasibility study incorpora Daniel Hind University of Sheffield Trials 2017, 18(Suppl 1):P46 y y p Daniel Hind University of Sheffield Trials 2017, 18(Suppl 1):P46 Background Correspondence: Julia Edwards Trials 2017, 18(Suppl 1):P45 Correspondence: Julia Edwards Trials 2017, 18(Suppl 1):P45 Funders often encourage the use of both qualitative and quantitative data in evaluations. Such evaluations are sometimes seen as limited without formal approaches to the integration of qualitative and quantitative data [1], and dismissed as multi-method rather than truly mixed-method. Qualitative research is encouraged during feasi- bility/pilot work [2]. We used a version of a protocol suggested by Farmer and colleagues [3] to integrate and compare quantitative and qualitative findings (methodological triangulation of data sets) in a mixed-methods feasibility study of a hydrotherapy intervention for Duchenne Muscular Dystrophy (NIHR HTA 12/144/04). Methods Background I di i In paediatric open-heart surgery body cooling during cardiopulmonary bypass (CPB) is commonly used to help protect vital organs. However, hypothermia can have detrimental effects. Thermic-1 was a parallel- group open randomised controlled trial which recruited 59 children undergoing heart surgery between 2002 and 2004. Patients were randomised to receive either hypothermic (28 °C) or normothermic (35 °C −37 °C) CPB. Thermic-2 followed on from Thermic-1, rando- mising 141 patients between 2012 and 2014. The co-primary out- comes included intubation time and length of post-operative stay. Methods A logic model, a tool used to evaluate the implementation of a care programme [4], was developed with collaborating interventionists. We reviewed qualitative and quantitative datasets to identify compo- nents of the intervention logic model (“sorting”). A convergence cod- ing matrix summarized similarities and differences between data sets for each of 17 logic model components, selecting examples to dem- onstrate how each had contributed to the intervention’s success or failure (“convergence coding”). We applied a convergence coding scheme: “agreement”; “partial agreement”; “silence”; or, “dissonance”. We quantified the level of agreement between data sets (“conver- gence assessment”) And highlighted their different contributions to the research question (“completeness comparison”). We shared the triangulated results with team members and other selected stake- holders at a face-to-face meeting, for feedback, allowing points of disagreement to be discussed and changes in interpretation incorporated. Randomisation: The 10-year gap between phases saw changes in randomisation systems. In Thermic-1 allocations were placed in opaque sequentially numbered sealed envelopes, which were given to the clinical fellow managing the study. Thermic-2 was managed by the clinical trials unit with allocation determined by secure com- puterised system. Data capture: Data capture processes also changed between the two phases. A clinical fellow collected data on Excel spreadsheets in Thermic 1, whereas data were collected by research nurses in Thermic-2 and then entered into a purpose-designed data- base. Statistical analysis: Data from the two trials were pooled in one overall analysis adjusted for study phase. Interaction terms were added to the models to examine differences between trial phases. Res lts Discussion The imbalance in baseline characteristics suggests that Thermic-1 re- sults are at high risk of bias due to inadequate concealment of ran- domisation. Allocation compliance was only collected in Thermic-2, so the true extent of non-compliance could not be determined. Add- itionally, the differing results for post-operative stay suggest the study was also at risk of detection bias; the age and gender differ- ences did not account for the difference observed. While the deci- sion to extubate is protocol driven, the decision to discharge patients lies with the clinical team. The results illustrate the import- ance of methodological rigour in the design and conduct of clinical trials and provide a valuable example of the importance of working with methodologists. We selected a different methods appropriate to the commissioning brief, but did not implement methods independently. A formal mixed-methods approach allowed the robust use of qualitative data used to explain quantitative findings. References 1. O’Cathain A: BMJ 2010, 341(sep17 1):C4587-C4587. 2. O’Cathain A. Health Technol Assess 2014, 18:1–197, V-Vi. 3. Farmer T. Qual Health Res 2006, 16:377–94. 4. Mclaughlin JA. Eval Program Plann. 1999 Mar;22(1):65–72. References 1. O’Cathain A: BMJ 2010, 341(sep17 1):C4587-C4587. 2. O’Cathain A. Health Technol Assess 2014, 18:1–197, V-Vi. 3. Farmer T. Qual Health Res 2006, 16:377–94. 4. Mclaughlin JA. Eval Program Plann. 1999 Mar;22(1):65–72. p Results Baseline characteristics: Imbalances in patient demographics were observed in Thermic-1; participants allocated to the normothermic group were on average 3 years older (median 7.5 years [IQR 3.5-10.6] vs 4.3 [2.2-11.5]) and more likely to be male (68% vs. 48%). In con- trast, in Thermic-2 no imbalance was observed; the median age was 2.3 years (0.5-5.2) in the normothermic group vs 2.9 (0.5-6.0) and there were similar proportions of males in the two groups (43% vs. 44%). Primary outcomes: Pooling the data across both phases, intubation time was slightly shorter in the normothermic group (median 10.6 hours [IQR 5.9-25.3] vs 16.4 [6.1-26.6]), although this was not statistically significant (hazard ratio [HR] 1.14, 95% CI 0.86-1.51, p-value = 0.36). The median duration of post-operative stay was 6.0 days in both groups (IQR 5.0-7.0); HR 1.06 (95% CI 0.80-1.40), p-value = 0.70. Examining the results by phase found no difference in treat- ment estimates for intubation time. However a significant difference between the two phases was found for length of stay (p-value for inter- action = 0.079). The estimated HR was 1.57 (95% CI 0.93-2.64) in Thermic-1, i.e. Marginally favouring the normothermic group, compared to 0.90 (95% CI 0.65-1.26) in Thermic-2. There was agreement on six components, silence on eight (areas only amenable to qualitative assessment), and dissonance on two. The areas of dissonance concerned session attendance and interven- tion optimisation. In each case, a naïve reading of the quantitative data could lead to an overly simplistic attribution of cause. For ses- sion attendance, quantitative sub-studies pointed to illness or simple non-appearance of the family; the qualitative data revealed that the convenience of available timeslots played a strong role in non- attendance for some families. Similarly, quantitative data identified an apparent failure, on the part of several physiotherapists, to opti- mise the intervention; the qualitative data revealed this to be part of a misunderstanding, with therapists wrongly assuming that the study required them to apply the manual prescriptively or extensively, ra- ther in a focused and more achievable way proposed at training. Those same therapists were aware and concerned that therapy was not optimised. Qualitative research contributed data to 15/17 logic model components; quantitative components contributed to nine. Samples from the convergence coding matrix are presented in the presentation. Feedback from stakeholders confirmed the account of- fered and adequate explanation of events observed in the study. Discussion g g Methods The prefit trial recruited community dwelling older people from pri- mary care. We compared alternative falls reporting methods to assess the impact on the likelihood of response, prevalence and pattern of missing values, and agreement between data sources. We also com- pared baseline participant characteristics by completion status. Par- ticipants were asked to complete a four month period of prospective fall diary completion; participants were randomly allocated to one of the periods (baseline to 4 months, 5 months to 8 months or 9 months to 12 months). Falls diaries were produced in a calendar format, posted to participants in a pack of four, with a covering instruction letter. Participants also completed follow-up questionnaires, contain- ing a retrospective question on number of falls in the preceding months at 4, 8, 12 and 18 months post randomisation. core outcome sets Jamie J Kirkham1, Mike Clarke2, Paula R Williamson1 1MRC North West Hub for Trials Methodology Research, Department of Biostatistics, University of Liverpool, Liverpool, UK; 2Northern Ireland Hub for Trials Methodology Research, Centre for Public Health, Queen’s University Belfast, Belfast, UK Correspondence: Jamie J Kirkham Trials 2017, 18(Suppl 1):P44 Correspondence: Jamie J Kirkham Trials 2017, 18(Suppl 1):P44 This abstract is not included here as it has already been published. Trials 2017, 18(Suppl 1):200 Page 18 of 235 Concealing the randomised allocation in trials: experience from the thermic trials Julia Edwards1, Katie Pike1, Sarah Baos2, Massimo Caputo3, Chris A. Rogers1 1Clinical Trials and Evaluation Unit, School of Clinical Sciences, University of Bristol; 2School of Social and Community Medicine, University of Bristol; 3Bristol Royal Hospital for Children, Division of Women and Children University Hospitals Bristol NHS Foundation Trust Using a logic model and a triangulation protocol for integrating quantitative and qualitative research data in a mixed-methods feasibility study incorporating an external pilot RCT References 1. O’Cathain A: BMJ 2010, 341(sep17 1):C4587-C4587. 2. O’Cathain A. Health Technol Assess 2014, 18:1–197, V-Vi. 3. Farmer T. Qual Health Res 2006, 16:377–94. 4. Mclaughlin JA. Eval Program Plann. 1999 Mar;22(1):65–72. References Page 19 of 235 Page 19 of 235 Trials 2017, 18(Suppl 1):200 p Conclusions Data demonstrate that PNs were generally recording responses to case-finding questions using the ENHANCE EMIS template as intended, suggesting that this process within the ENHANCE study was feasible and accurate. PNs were asked to record patient re- sponses on a new computer template while maintaining a patient- centred dialogue and completing an integrated ENHANCE review within the available timeframe, so it is unsurprising that some typing errors or discrepancies may occur. Nonetheless, it is helpful to ac- knowledge that these may exist, as template data is often used for fi- delity checking of intervention delivery within trials. We have identified difficulties in the use of case-finding questions that could be addressed through PN training in a future main trial. P49 Exploring understanding of neural stem cell transplantation (NSCT) as an intervention for Huntington’s Disease (HD) Richard Hellyar Exploring understanding of neural stem cell transplantation (NSCT) as an intervention for Huntington’s Disease (HD) Richard Hellyar School of Healthcare Sciences, Cardiff University Trials 2017, 18(Suppl 1):P49 Objectives Objectives Background Neural Stem Cell Transplantation (NSCT) has been identified as a po- tential therapeutic intervention for the treatment of Huntington’s dis- ease (HD) (Dunnett and Rosser, 2007). This neurosurgical procedure utilises stem cells, which are injected into the mid-brain of affected individuals and are intended to improve symptoms (Lindvall and Bjorkland, 2000). Previous research, utilising NSCT, has briefly ac- knowledged ethical sensitivity including the source of cells utilised, alongside the hopes of HD patients surrounding the intervention (Bachoud-Levi et al., 2000). The understanding of NSCT amongst po- tential beneficiaries has however yet to be explored in depth. With future clinical trials being planned to explore the intervention, this proposed qualitative project seeks to redress this gap and it is envis- aged that the understanding gained will inform information giving, recruitment strategies and care pathway planning in such a way as to augment any future participant experience. p p Conclusions Methods Methods Patients and PNs in four general practices were asked to give consent for their ENHANCE consultations to be audio-recorded for fidelity checking (24 patients and seven PNs consented). 12 patients also gave consent for the research team to access their medical record data, which included the ENHANCE template data (entered by six PNs during ENHANCE reviews). Consultation recordings for these 12 patients were compared with corresponding ENHANCE EMIS template data entered by the PNs, to identify and explore any discrepancies. Background In depth semi structured interviews were conducted with a purpose- ful sample of healthcare and research professionals responsible for recruitment in three RCTs in different disease sites in surgical oncol- ogy (oesophgago-gastric, thoracic and colorectal). Interviews were audio recorded, transcribed verbatim and analysed thematically. Sampling, data collection and analysis were undertaken iteratively and concurrently. The ENHANCE pilot trial aimed to test the feasibility and acceptability of integrating case-finding for osteoarthritis, anxiety and depression within extended primary care nurse-led long-term condition (LTC) re- view consultations. Training was delivered to general practice nurses (PNs) to deliver the ENHANCE reviews, supported by an adapted EMIS LTC computer template. Results This analysis explored the extent to which data recorded by the PNs in the ENHANCE EMIS template reflected the content of discussions and case-finding in ENHANCE LTC review consultations. The findings form part of a process evaluation exploring the ways in which PNs delivered ENHANCE LTC reviews. Thirty six interviews were conducted with recruiters at seven differ- ent hospital sites. Sites in which a culture of clinical collaboration within and across disciplines existed recruited more participants than those in which individual clinicians tended to work in isolation. The multidisciplinary team meeting (tumour board meeting) appeared to facilitate cross disciplinary collaboration and was an important factor in determining the ability of individual sites to effectively recruit. The degree to which individual specialty teams within each centre were in equipoise influenced study engagement. Exploring discrepancies between long-term condition review consultation audio-recordings and computer template data (the ENHANCE pilot trial) Trials in surgical oncology frequently experience issues with recruit- ing adequate numbers of participants. This is particularly difficult within RCTS involving interventions which are routinely delivered by different clinical specialties (such as surgery and oncology based treatments). Teamwork between individual healthcare professionals and specialty and research teams has been highlighted as a signifi- cant factor in recruitment. This study evaluated aspects of teamwork which were important in recruitment to three RCTs in surgical oncology. Jennifer Liddle, Sarah A. Lawton, Carolyn A. Chew-Graham, Emma L. Healey, Christian D. Mallen, Clare Jinks Keele University Correspondence: Jennifer Liddle Trials 2017, 18(Suppl 1):P47 Jennifer Liddle, Sarah A. Lawton, Carolyn A. Chew-Graham, Emma L. Healey, Christian D. Mallen, Clare Jinks Jennifer Liddle, Sarah A. Lawton, Carolyn A. Chew-Graham, Emma L. Healey, Christian D. Mallen, Clare Jinks Correspondence: Jennifer Liddle Trials 2017, 18(Suppl 1):P47 q p Discussion This study has demonstrated several aspects of teamwork that ap- pear to be important for recruitment in trials in surgical oncology. Understanding these aspects of teamwork will aid the development of guidance on team relevant issues that should be considered in trial management and the development of interventions that will fa- cilitate teamwork and improve future recruitment to RCTs. Use of the ENHANCE case-finding questions in the audio-recorded ENHANCE LTC review consultations was high. The majority of patient responses to case-finding questions/tools in the audio-recordings matched those recorded by PNs through the new ENHANCE EMIS template, however, 12 discrepancies between the audio-recordings and EMIS computer template data were identified, arising from five of the consultations (with three PNs). Discrepancies included: re- sponses to case-finding questions not matching; responses recorded in the template data for questions not asked in the audio-recording; missing template data for questions that were in the audio- recording. Some discrepancies appeared to arise from PNs’ under- standings of what constituted a legitimate “Yes” or “No” response to the case-finding questions for depression and anxiety. There was also evidence that PNs sometimes attempted to question, dismiss or nor- malise patients’ initial responses. References h d To the best of our knowledge this is the first training toolkit that has been developed to assist trial managers in planning, designing, doc- umenting and evaluating staff training within clinical research. Fur- ther validation of the toolkit is required to assess its practical use in a variety of clinical trial settings. Bachoud-Levi, A,C. Et al. 2000. Safety and tolerability of interstriatal neural allografts in five patients with Huntington’s disease. Experimental Neurology 161, pp 194–202. Dunnett, S, B. Rosser, A, E. 2007. Stem cell transplantation for Huntington’s disease. Experimental Neurology 203, pp 279–292. Lindvall, O. Bjorklund, A. 2000. First steps towards cell therapy for Huntington’s disease. The Lancet. 356, pp 1945–1946. Lindvall, O. Bjorklund, A. 2000. First steps towards cell thera Huntington’s disease. The Lancet. 356, pp 1945–1946. P51 Use of simulations to explore recruitment and the impact of late availability of experimental arms in the pivotalboost trial Nuria Porta1, Clare Griffin1, Isabel Syndikus2, John Staffurth 3, Peter Hoskin4, David Dearnaley5, Ann Henry6, Brendan Carey6, Alison Tree5, Rebecca Lewis1 1The Institute of Cancer Research; 2The Clatterbridge Cancer Centre; 3Velindre NHS Trustl; 4Mount Vernon Cancer Centre; 5Royal Marsden NHS Foundation Trust; 6Leeds University Teaching Hospitals Correspondence: Nuria Porta Trials 2017, 18(Suppl 1):P51 y Results As future clinical trials of NSCT are due to be undertaken in the United Kingdom in the near future, it is important that this (anon- ymised) information from this thesis is shared with Professionals, working within Clinical Trials, in order to support recruitment strat- egies and information provision. Methods The content of the training toolkit was developed by combining i) qualitative data obtained from semi-structured interviews with trial managers (n = 6) and healthcare professionals (n = 13) working on six purposefully selected case studies of the ATLAS project; ii) responses to questionnaires (n = 120) used to evaluate site staff and facilitators’ experience of site initiation training sessions, iii) a review of existing regulations and guidance documents from various regulatory bodies (MRC, HRA, MHRA, FDA) on training requirements in clinical research; and iv) a review of existing literature on the processes of learning, training and development. The training toolkit was then evaluated in two-stages. Firstly, semi-structured follow-up interviews with the trial managers (n = 6) facilitating trial-specific training sessions in the six participating ATLAS studies were undertaken and the toolkit was amended in light of the feedback received. At the second stage, feedback was sought on the revised toolkit from trial managers at- tending scheduled meetings (n = 2) in two established Clinical Trial Units in Bristol. Aims To develop and validate the ATLAS training toolkit that can be uti- lised by trial management teams when planning training of staff within clinical trials. Part of the training toolkit also aims to evaluate the process of trial-specific training provided to site staff during the site initiation process. g Method The primary aim of this research is to gain insight into the perceptions and understanding about Neural Stem Cell Transplantation (NSCT) amongst potential recipients. The information gained is then intended to inform and underpin the development of information giving ap- proaches for potential NSCT recipients and ensure their issues are ad- dressed in the development of consent procedures and care pathways. Firstly three purposively targeted Specialist Professionals, from the field of NSCT, have been approached via email, consented and interviewed (semi-structured) in order to explore their past experience. The re- corded interviews addressed their recollections of the recipient Effective teamwork is crucial to maximising recruitment to randomised controlled trials in surgical oncology h l ll Sean Strong, Sangeetha Paramas Jane Blazeby University of Bristol Correspondence: Sean Strong Trials 2017, 18(Suppl 1):P48 Page 20 of 235 Trials 2017, 18(Suppl 1):200 Page 20 of 235 Page 20 of 235 selecting appropriate mode of delivery; ii) Designing the training plan; iii) Delivering and documenting training; iv) Evaluating training and v) Identifying additional training needs and re-training of staff. Each element is supplemented by support documents (including flowcharts and template documents) that can be utilised by trial managers as guides when planning trial-specific staff training. Over- all, the training toolkit was positively received and was considered a useful reference document encouraging active thinking of staff train- ing during the early stages of study design. Most trial managers felt that the decision-making flowchart provided useful prompts to assist trial managers in selecting the appropriate mode of training during the decision-making process. The training plan template was also viewed as a helpful document for recording decisions about the ap- propriate level of training required for each study. The training feed- back forms were regarded as invaluable documents in identifying key areas where additional training is required and improving future training sessions. experience, their understanding, questions, queries and concerns with regards to NSCT. A thematic analysis of these interviews has been undertaken and used to inform and guide the development of minimally-structured interviews with six, genetically positive, individuals who have yet to show symptoms of HD. Emergent themes thus far in- clude Making Sense via Contrast, Chronological Risk, Ethical Dissonance and Familial/Community Drivers and Brakes. This second phase, using minimally-structured qualitative interviews, is intended to elicit the per- ceptions and understanding surrounding NSCT as an intervention amongst potential recipients. Aims To investigate, via simulation of recruitment, how the three elements above impact recruitment and imbalance between treatment arms and how adaption of the allocation ratio could minimise imbalances. Methods We assumed recruitment would take 54 months and with staggered opening, all 40 sites would be open by 24 months. Expected site- specific monthly accrual rates and availability of boost technologies were obtained via site feasibility questionnaires. An expected month of opening and when the boost arms would be available at each site was inferred using survey results and clinician input. Recruitment was assumed to follow a Poisson distribution with site-specific monthly accrual rates. For each patient accrued at a specific month we simulated the boost volume, risk group and suitability for receiv- ing a boost, so the patient could be allocated to the 2-arm or 4-arm randomisation accordingly. We performed initial simulations with a Development and first evaluation of the atlas training toolkit for clinical trials 1 1 1 1 Athanasia Gravani1, Marcus Jepson1, Caroline Wilson1, Athene Lane1, Chris Rogers2 1MRC conduct-II Hub for Trials Methodology Research, School of Social and Community Medicine, University of Bristol, Bristol, UK; 2Clinical Trials and Evaluation Unit, School of Clinical Sciences, University of Bristol, Bristol, UK Correspondence: Nuria Porta Trials 2017, 18(Suppl 1):P51 Correspondence: Athanasia Gravani Trials 2017, 18(Suppl 1):P50 Background pivotalboost (CRUK/16/018) is a 4-arm phase III randomised trial in patients with node negative, high or intermediate risk localised pros- tate cancer currently in set-up. 1952 patients will be randomised to receive (A) standard prostate intensity modulated radiotherapy (IMRT); (B) A with pelvic node IMRT; (C) A with a prostate boost; or (D) A with pelvic node IMRT and a prostate boost. The prostate boost can be delivered by IMRT or by high dose rate brachytherapy (HDRB). Availability of the boost arms C and D depends on: (1) A suitable boost tumour volume identified by functional MRI. (2) Availability of boost technologies - participating sites can open initially to A vs B randomisation with the boost arms (i.e. 4-arm randomisation) open- ing later. (3) Patient suitability and fitness. The study is powered to compare each experimental arm with control. Due to the above re- cruitment restrictions for the boost arms, power was reduced for the boost comparisons (85% power for A vs B, 80% A vs C and A vs D), giving a design where the trial population will be split 9:9:8:8 across arms. Background Trial-specific training is highly varied across trials and there is great uncertainty on the best ways to provide training to facilitate trial conduct. g Method These participants will be recruited from an Asymptomatic Huntington’s Disease clinic. experience, their understanding, questions, queries and concerns with regards to NSCT. A thematic analysis of these interviews has been undertaken and used to inform and guide the development of minimally-structured interviews with six, genetically positive, individuals who have yet to show symptoms of HD. Emergent themes thus far in- clude Making Sense via Contrast, Chronological Risk, Ethical Dissonance and Familial/Community Drivers and Brakes. This second phase, using minimally-structured qualitative interviews, is intended to elicit the per- ceptions and understanding surrounding NSCT as an intervention amongst potential recipients. These participants will be recruited from an Asymptomatic Huntington’s Disease clinic. Conclusions Results indicate that drug reduction is possible and safe. However focused support and alternative interventions are required. The results of the qualitative study and reflections on the challenges faced provide important insights into the experiences of people taking part in drug reduction studies that should influence future trial development. Simulation of recruitment proved useful to understand the potential imbalances that may occur during the trial and led to a cost-effective strategy of different allocation ratios during the trial to correct for ini- tial imbalance. A pilot randomised controlled trial of community led anti- psychotic drug reduction for adults with learning disabilities: ANDREA-LD P53 Barriers to recruitment from primary care into a trial in secondary care settings: experience from the feasibility study of IBIS-3 trial Adedayo Oke1, Jill Knox1, Jermaine Tan1, Benoit Aigret1, Peter Schmid1, Robert E. Coleman2, Jack Cuzick1, Mangesh A. Thorat1 1Queen Mary University of London; 2University of Sheffield Correspondence: Adedayo Oke Trials 2017, 18(Suppl 1):P53 Elizabeth Randell, Rachel McNamara, Lianna Angel, David Gillespie, Andrea Meek, Mike Kerr Cardiff University Correspondence: Elizabeth Randell Trials 2017, 18(Suppl 1):P52 Elizabeth Randell, Rachel McNamara, Lianna Angel, David Gillespie, Andrea Meek, Mike Kerr Elizabeth Randell, Rachel McNamara, Lianna Angel, David Gillespie, Andrea Meek, Mike Kerr Elizabeth Randell, Rachel McNamara, L Andrea Meek, Mike Kerr Cardiff University Correspondence: Elizabeth Randell Trials 2017, 18(Suppl 1):P52 y Correspondence: Elizabeth Randell Trials 2017, 18(Suppl 1):P52 Results The toolkit has five components each focusing on a particular elem- ent of the training cycle: i) Specifying initial training needs and Page 21 of 235 Trials 2017, 18(Suppl 1):200 1:1:1:1 allocation ratio and reviewed monthly mean recruitment per treatment, allocation ratio to control (X:A) and probability of complet- ing recruitment before the planned time. We explored the impact of delays in opening the boost arms on prolonging the recruitment period. Results Simulations showed that using a 1:1:1:1 allocation ratio causes an ini- tial imbalance with more patients allocated to A and B as expected by late opening of boost arms, which could result in an imbalance at the end of recruitment in favour of the control arm; though infre- quent, some simulated trials had up to 25% more patients in one group than another. Initial use of 2:2:3:3 allocation ratio appeared to protect against such imbalances. Recruitment by arm will be moni- tored as the trial progresses with a planned adaption to a 1:1:1:1 allo- cation ratio part way through recruitment. Methodological challenges faced in setting up and delivering the trial included: recruitment of principal investigators and sites equipped to distribute medication; recruitment of participants and carers; obtain- ing consent according to regulations surrounding trials for this vul- nerable population; ensuring maintenance of the blind and dispensing medication to participants; carers subjective assumptions of trial arm allocation. g Method ANDREA-LD was a two arm individually randomised (1:1) double blind placebo controlled drug reduction trial. The majority of recruit- ment was through community learning disabilities teams in South East Wales and South West England. Participants were adults with LD prescribed risperidone for treatment of challenging behaviour with no known current psychosis or previous recurrence of psychosis fol- lowing prior drug reduction. Carers also consented to their involve- ment in the trial. ment rate Method Five Clinical Research Networks (CRNs) invited GP practices close to trial’s participating Secondary Care Sites (SCS) to join as Participant Identification Centres (PICs) on our behalf. GPs who agreed to partici- pate as PICs screened their databases to identify potentially eligible patients and wrote to these patients inviting them to participate in the trial by contacting the trial’s central coordinating office (CCO). The CCO further checked eligibility and referred patients to their local SCS. After 6 months of original request, a brief survey to identify main reasons for non-participation was sent to all GPs who declined participation. Results Of the 22 participants randomised, 13 (59.1%) achieved progression through all four stages of reduction. Follow-up data at six and nine- months post-randomisation was obtained for 17 participants (77.3% of those randomised) with 10 intervention and seven control partici- pants followed up. There were no significant changes in participants’ levels of aggression or challenging behaviour at the end of the study. participat Results The level of support provided to both the CCO and GPs varied across 5 CRNs potentially impacting GP participation rate. Overall, only 5% GPs agreed to participate and only 23 of 800 (3%) subsequently responded to the survey. The main reasons identified for non- participation were lack of time/resources to carry out database search (61%) and/or review medical records to confirm eligibility (48%), request coming at a busy time (9%) e.g. Calendar or financial year-end, and insufficient funding (26%). Encouragingly, 26% of GPs that completed the survey indicated willingness to participate at the time of the survey. The intervention was a double blinded drug reduction programme leading to full withdrawal within six months. The control group main- tained baseline treatment. Treatment achieved at six months was maintained for a further three months under blind conditions. The blind was broken at nine months following final data collection. Feasibility outcomes were number and proportion of: (i) sites progres- sing from initial approach to participant recruitment (ii) recruited partic- ipants who progressed through the trial. Trial arms were also compared regarding; Modified Overt Aggression Scale; Aberrant Behaviour Check- list; PAS-ADD checklist; Antipsychotic Side-effect Checklist; Dyskinesia Identification System Condensed User Scale; Client Service Receipt In- ventory; use of other interventions for challenging behaviour; use of as required medication; psychotropic medication use. Background Data suggests there are 50,000 adults with learning disabilities (LD) in England and Wales currently prescribed antipsychotic medication. Illness in this population is high, including significant rates of challen- ging behaviour and mental illness with particular concern over use of anti-psychotic drugs prescribed for reasons other than treatment of psychosis. Control of challenging behaviour is the primary reason why such medications are prescribed, despite the absence of good evidence of therapeutic effect for this. This innovative study was ini- tially conducted in primary care however due to complexities sur- rounding set up and recruitment, continued in community learning disabilities teams as a feasibility study. The primary objective was to assess feasibility of recruitment and retention, and explore non- efficacy based barriers to a blinded anti-psychotic medication with- drawal programme for adults with LD without psychosis compared to treatment as usual. A secondary objective was to compare trial arms regarding clinical outcomes. g Oestrogen Receptor (ER) positive breast cancer (ERBC) is now a chronic disease with high 5-year survival rates. However, a large pro- portion of cases continue to be at a substantial risk of recurrence up to 20 years from diagnosis. Trials of interventions designed to pre- vent late recurrences in ERBC face a unique challenge. These inter- ventions often need to be carried out in secondary care setting when patients have already been discharged back into primary care. Therefore recruitment from the primary care setting is important for such trials. The IBIS-3 feasibility study is a randomised controlled trial (RCT) of interventions in long-term survivors of ERBC with recruit- ment rates from primary case as one of its objectives. Conclusion This is the largest embedded trial to test the impact of a recruitment or PPIR intervention. Advertising PPIR using a leaflet had no benefits for improving recruitment or response rates. Our findings contrast with the literature suggesting advertising PPIR benefits trial recruit- ment. We will discuss implications of our findings for trial recruit- ment, research and policy. Background Correspondence: Adwoa Hughes-Morley Trials 2017, 18(Suppl 1):P54 g Recruitment to randomised controlled trials (RCT) remains one of the key challenges in trial management. Patient aversion to ran- domisation is often cited as a reason why patients choose not to enroll in RCTs. For many recruiters and patients alike, ‘randomisa- tion’ appears a challenging concept, yet one that requires commu- nicating and understanding given its centrality to informed consent and trial recruitment. The UK National Research Ethics Service (NRES) has produced guidance on how to describe randomisation simply and clearly in written patient information. We investigated how recruiters described randomisation in recruitment appoint- ments and compared this with a framework based on the NRES guidance. Methods d We undertook a cluster randomised controlled trial, embedded in an ongoing “Host” Mental health trial (the “EQUIP” Trial). EQUIP was a cluster randomised controlled trial recruiting service users with a diagnosis of severe mental illness. In EQUIP, mental health teams in England were randomised to an intervention group to receive train- ing to improve service user and carer involvement in care planning, or to a “no training” control group. The recruitment intervention ad- vertising PPIR was informed by a systematic review, a qualitative study of patients who declined a trial, social comparison theory and a workshop that included mental health service users and trialists. Using Participatory Design methods, we collaborated with PPIR part- ners (service users and carers) to design a recruitment intervention using a leaflet format to advertise the nature and function of the PPIR in EQUIP to potential participants. Professional graphic design aimed to optimise the intervention’s readability and impact. Service users being approached into EQUIP were randomised to the PPIR intervention or not, alongside the standard trial information. The pri- mary outcome was the proportion of participants enrolled in EQUIP. The secondary outcomes included the proportion expressing interest in enrolling. Analysis was by intention to treat and used generalised linear mixed models. Aims A maximum variation sample of 64 audio-recorded recruitment ap- pointments was purposefully sampled from five RCTS to encompass a range of recruiters, surgical and non-surgical trials and cancer and non-cancer conditions. Using the NRES guidance for written patient information as a hypothesised ideal explanation of randomisation, an analytical framework was developed identifying five interlinked concepts considered necessary for a clear exposition of randomisa- tion. This analytic framework was applied to extracts from consulta- tions during which randomisation was discussed using content analysis, assessing whether the concepts were absent or present and explicit or implicit, according to coding rules derived from the data. We aimed to develop an intervention directly advertising PPIR in a trial to potential participants and evaluate its impact on trial recruit- ment and response rates. Results We randomised 34 mental health teams and 8182 potential partici- pants were invited. For the primary outcome, 4% of patients receiv- ing the PPIR leaflet were enrolled vs. 5.3% in the control group. After adjusting for mental health team cluster size, levels of deprivation and care quality rating, the intervention was not effective for improv- ing recruitment rates (adjusted OR = 0.75, 95% CI = 0.53 to 1.07, p = 0.113). For the secondary outcome 7.3% of potential participants re- ceiving the PPIR leaflet responded positively to the invitation to par- ticipate, vs. 7.9% in the control group. The intervention was not effective for improving response rates (adjusted OR = 0.74, 95% CI = Does advertising patient and public involvement in a trial to potential participants improve recruitment and response rates? An embedded cluster randomised trial Adwoa Hughes-Morley1, Mark Hann2, Claire Fraser3, Oonagh Meade4, Karina Lovell3, Bridget Young5, Chris Roberts6, Lindsey Cree3, Donna More3, Neil O’Leary7 Adwoa Hughes-Morley1, Mark Hann2, Claire Fraser3, Oonagh Meade4, Karina Lovell3, Bridget Young5, Chris Roberts6, Lindsey Cree3, Donna More3, Neil O’Leary7 2 P55 How do recruiters present randomisation during trial recruitment? An evaluation of current practice Carmel Conefrey, Julia Wade, Marcus Jepson, Daisy Elliott, Jenny Donovan, The Quintet Team University of Bristol Correspondence: Carmel Conefrey Trials 2017, 18(Suppl 1):P55 1University of Manchester and University of York; 2NIHR School for Primary Care Research, University of Manchester; 3School of Nursing, Midwifery and Social Work, University of Manchester; 4School of Health Sciences, The University of Nottingham; 5MRC North West Hub for Trials Methodology Research, University of Liverpool; 6Department of Biostatistics, University of Manchester; 7Centre For Medical Gerontology, Trinity College Dublin Biostatistics, University of Manchester; 7Centre For Medical Gerontology, Trinity College Dublin Results Two key findings emerged. Firstly, recruiter explanations of and for randomisation tended to be incomplete when evaluated against the NRES informed framework: in nearly 45% (29) of cases, three or fewer components were present. Only five of the 64 encounters in- cluded mention of all five concepts and in only two of these were all five concepts made explicit. Secondly, recruiters referred to some concepts more frequently than others to articulate the ration- ale for randomisation. Whilst most recruiters referred to ‘clinical equipoise’ and ‘the need for a number of patient treatment groups’, few referred to ‘the need for patient groups to be similar except for the treatment allocated’. Where expressed, recruiters tended to convey ‘the need to compare treatment effects’ and ‘that chance determines assignment to a treatment allocation’, implicitly rather than explicitly. Background There is emerging evidence that patient and public involvement in research (PPIR) may increase participant recruitment into randomised controlled trials. However, it is not clear how to use PPIR to improve trial recruitment. Whilst publicly funded trials in the UK and else- where routinely use PPIR to improve design and conduct, such trials on the whole do not advertise their use of PPIR to potential partici- pants. Effective advertising of PPIR in trials to potential participants might increase enrolment rates, through trials being perceived to be more trustworthy, relevant and socially valid. Conclusions Wide variations exist in the level of support provided to GPs across CRNs. Ensuring uniform and higher levels of support including fund- ing to help overcome time/resources scarcity barriers is likely to Page 22 of 235 Page 22 of 235 Page 22 of 235 Trials 2017, 18(Suppl 1):200 0.53 to 1.04, p = 0.082). The intervention was not effective for any other outcomes measured. improve GP participation as PICs for trials in secondary care settings. A re-request for participation from CRNs, made at a time when prac- tices are less busy should also be considered as a measure to im- prove participation. p Conclusion An evaluation of recruiter practice during recruitment consultations across a range of trials showed that recruiters did not explicitly communicate key concepts identified by NRES as fundamental to a clear definition of randomisation. There is a need to understand whether all aspects of the NRES guidance are necessary for the communication of randomisation, and which are the key concepts that are essential to facilitate patient understanding and assure in- formed consent. Page 23 of 235 Trials 2017, 18(Suppl 1):200 Page 23 of 235 Obtaining consent in the pre-hospital setting: consent procedures from the rapid intervention with glyceryl trinitrate in hypertensive stroke trial-2 (right-2) Paramedics are equipped to assess and recognise patients with sus- pected stroke in the out-of-hospital setting. Treatment is highly time- dependent but definitive intervention for stroke is currently limited to in-hospital therapies. Commencing treatment in the pre-hospital set- ting could dramatically reduce time to treatment. The rapid interven- tion with glyceryl trinitrate in hypertensive stroke trial (RIGHT-2) is assessing the safety and efficacy of pre-hospital ambulance-based paramedic-delivered glyceryl trinitrate (GTN) when administered ultra- acutely after stroke. Whilst ambulance-based paramedic-delivered stroke trials have been done in the UK in single site pilot trials, they have not been done across multiple ambulance services and hospital sites in the UK. It is important for paramedics to have awareness of Good Clinical Practice (GCP) principles and to be trained in trial proce- dures. Methods used to train paramedics need to account for the fact that paramedics have little time at work not on shift to complete train- ing and may be reliant on them completing training in their own time. Methods ( g ) Polly Scutt, Mark Dixon, Jason P. Appleton, Philip M. Bath University of Nottingham Correspondence: Polly Scutt Trials 2017, 18(Suppl 1):P57 Sample size adaptations in a randomised controlled biomarker based strategy (hybrid) trial: experience from the OUTSMART trial Dominic Stringer Kimberley Goldsmith Janet L Peacock Leanne M Gardner Proxy consent can ensure patients are enrolled rapidly into emer- gency clinical trials. In the RIGHT-2 trial, patients with a severe stroke, who may benefit from the intervention, are able to take part in the study when they would otherwise be excluded, which boosts recruit- ment and ensures the trial population is representative of the popu- lation the intervention is intended for. y Caroline Murphy, Anthony Dorling Kings College London Correspondence: Dominic Stringer Trials 2017, 18(Suppl 1):P59 Methods h Ethics approval was obtained to allow proxy consent to enable ran- domisation into the RIGHT-2 trial within the 4 hour recruitment win- dow. Informed consent or proxy consent is taken at the stroke scene or in the ambulance. Brief assessment of capacity is performed by the paramedic explaining to the patient that they have had a sus- pected stroke, their BP may need lowering, and that a patch will be applied that might lower their BP. The paramedic then asks the pa- tient what the suspected diagnosis is (‘stroke’), what might need to be done to their BP (‘lower’), and how this will be done (‘patch’). Pa- tients with capacity give written or witnessed oral consent to the paramedic. If a patient lacks capacity, proxy consent is obtained from a relative, carer or friend acting as a personal consultee, if available, or by the paramedic witnessed by another member of the ambu- lance staff at the scene. For participants who did not have capacity at the time of randomisation, consent is verified in hospital with themselves or a relative (if the participant still lacks capacity). Results co te t Result Five ambulance services are currently recruiting into the RIGHT-2 trial. From these, 958 paramedics expressed interest in being involved with the RIGHT-2 trial, of which 628 (65.6%) have completed the online training as- sessment. Feedback from paramedics suggests that face-to-face is their preferred method of training, however, sessions need to be repeated sev- eral times to allow paramedics who are on rotating shift patterns to at- tend. This takes up a considerable amount of time for the research paramedic who has to travel large distances across the ambulance ser- vice. Remote webinars were well attended, with some paramedics attend- ing multiple sessions to recap on key points. As with face-to-face sessions, multiple sessions are required for a reliable uptake of paramedics. Conclusion As of 28th October 2016, 247 participants have been enrolled into the RIGHT-2 trial. 127 (51.4%) participants gave their own consent. Proxy consent was given by a relative/carer/friend for 97 (39.3%) par- ticipants, and by a paramedic for 23 (9.3%) participants. The median time to consent for all participants was 58 minutes. After the partici- pants reached hospital, 141 (61.8%) gave their own consent, 45 (19.7%) had continued consent by a relative or close friend and 42 (18.4%) had no further consent after proxy consent was taken in the ambulance. Patients who had proxy consent in the ambulance had a more severe stroke, median [IQR] National Institutes of Health Stroke Scale score 12.5 [5, 18] versus 4 [2, 7] for those who gave consent themselves. Training for paramedics who recruit patients into clinical trials needs to cover the necessary elements of GCP as well as trial procedures. It must be easy to access and succinct in order for them to complete training around their normal work schedule. A remote webinar pro- vides balance between accessibility for paramedics and ability to interact with those delivering the training. Background Obtaining consent in the pre-hospital setting: consent procedures from the rapid intervention with glyceryl trinitrate in hypertensive stroke trial-2 (right-2) ing and m Methods Paramedics working in ambulance services involved with RIGHT-2 who express interest in recruiting patients into the trial are invited to watch the training video. The training video lasts for 1 hour and contains de- tails on trial procedures and elements of GCP relevant to paramedics recruiting patients. The video is available over the internet so para- medics are able to watch and revisit it whenever they choose so they are able to complete the training in the small amount of time they have available. Training has been delivered in face-to-face small group sessions by Research Paramedics from participating ambulance trusts and members of the RIGHT-2 team. Training has also been delivered by a remote webinar, where the RIGHT-2 team deliver the training over the internet, which allows for interaction between paramedics and those delivering the training. Once paramedics have been trained they must complete an online assessment questionnaire based around the content of the video before they are able to recruit patients. R lt Conclusion P59 Sample size adaptations in a randomised controlled biomarker based strategy (hybrid) trial: experience from the OUTSMART trial Dominic Stringer, Kimberley Goldsmith, Janet L. Peacock, Leanne M. Gardner, Caroline Murphy, Anthony Dorling Kings College London Correspondence: Dominic Stringer Trials 2017, 18(Suppl 1):P59 Background Stroke is a severe and often fatal or disabling condition. Despite treat- ment effects in acute stroke being predominantly time dependent (e.g. Thrombolysis and thrombectomy), proven treatments are hospital based. Commencing treatment in the pre-hospital setting could dra- matically reduce time to treatment. The rapid intervention with glyceryl trinitrate in hypertensive stroke trial-2 (RIGHT-2) recruits patients in the pre-hospital setting within 4 hours of stroke onset. Obtaining consent in emergency situations can be difficult, especially when the time win- dow for recruitment is short. Proxy consent allows patients to be re- cruited when they lack capacity to give consent themselves, a common scenario in people suffering a stroke. Conversely, a waiver of consent offers the opportunity to include all eligible patients but may disregard the initial choice of patients who have capacity to make an informed decision regarding participation in research. h d Background The Pulmonary Metastasectomy in Colorectal Cancer (pulmicc) trial completed its feasibility phase in 2015. Surgically treated colorectal cancer patients, with newly diagnosed lung metastases, were rando- mised to continued active monitoring or pulmonary metastasectomy followed by active monitoring. Randomisation was a two stage process; Stage 1 investigations assessed fitness for surgery and eligi- bility for Stage 2 randomisation. A key trial criterion was clinician un- certainty regarding the benefit of surgery in the light of the patient’s test results. The trial was anticipated to be challenging for both clini- cians and patients. Both patient information, and healthcare profes- sional, training DVDs were produced to assist with trial discussions and decision making. Additionally a patient survey was conducted to examine patients’ views about the trial. The first strategy used to improve efficiency was the incorporation of rescreening HLA Ab- participants in the trial design, with participants converting to HLA Ab + in the unblinded group moving on to opti- mised immunosuppression. The known prevalence of HLA Ab + in renal transplant recipients is ap- proximately 25%, DSA account for 1/3rd of these, and the known inci- dence of de novo DSA development is 3%. Sample size calculations based on these known rates estimated requiring 2800 randomised partici- pants for 324 DSA+ participants to allow comparison of the effect of bio- marker led immunosuppression optimisation in these patients. Sixteen months into recruitment both the prevalence and incidence of DSA+ was lower than expected. This led to the application of a second strategy for efficiency improvement; a change in the primary outcome from binary to time to event (time to graft failure, approved by the DMC and TSC). The sample size calculation was amended both to reflect this change and to take into account the lower than expected DSA+ rates, retaining trial power. The amended sample size required 165 DSA+ participants from an estimated 2357 to be recruited. This change also meant there was already sufficient numbers of HLA Ab + DSA- and HLA Ab- participants recruited. As recruitment continued, the overall proportion of DSA+ participants in the trial increased as the DSA prevalence and incidence rates normal- ised, and also as a consequence of the increasing pool of participants to screen as more HLA Ab- participants were randomised. The latter sort of complexity would generally only be simplistically accounted for in sample size calculations. P60 Heidi Gardner, Cynthia Fraser, Graeme MacLennan, Shaun Treweek University of Aberdeen Methods for training paramedics to recruit patients into the rapid intervention with glyceryl trinitrate in hypertensive stroke trial-2 (right-2) OUTSMART is an ongoing multi-centre randomised controlled trial test- ing whether a combined structured biomarker screening programme and optimized immunosuppression treatment regimen can reduce risk of graft failure in kidney transplant patients. HLA antibodies (HLA Ab+), particularly if they contain donor specific antibodies against the graft (DSA+) are prognostic biomarkers for graft failure. However it is unclear how best to treat patients positive for the biomarkers. Trials 2017, 18(Suppl 1):200 Page 24 of 235 Page 24 of 235 The study design involves screening kidney transplant patients to de- termine if they are positive or negative for HLA antibodies and if HLA Ab+, whether they have donor specific antibodies (DSA+) against the graft. Antibody screening results are initially blinded, and then participants are randomised 1:1 to blinding or unblinding of their HLA Ab status. The blinded group remain treated as they were at baseline (standard care) and the unblinded HLA Ab + group are treated with an optimized immunosuppression intervention. Perceptions of the utility and barriers of using electronic health records for patient screening in clinical trials: an in-depth interviews study of site coordinators Perceptions of the utility and barriers of using electronic health records for patient screening in clinical trials: an in-depth interviews study of site coordinators 1 1 2 3 Correspondence: Heidi Gardner Trials 2017, 18(Suppl 1):P60 Emily O’Brien1, Diane Bloom1, Carol Koro2, Jamie Lorimer3, Sudha R. Raman1, Robert J. Mentz1, Bradley G. Hammill1, Lisa G. Berdan1, Ty Rorick1, Salim Janmohamed3 1 2 3 Emily O’Brien1, Diane Bloom1, Carol Koro2, Jamie Lorimer3, Sudha R. Raman1 Robert J. Mentz1, Bradley G. Hammill1, Lisa G. Berdan1, Ty Rorick1, Salim Janmohamed3 This abstract is not included here as it has already been published. 1Duke University; 2Glaxosmithkline & Merck & Co.; 3Glaxosmithkline Correspondence: Emily O’Brien Trials 2017, 18(Suppl 1):P62 P61 Surveying patients: views on trial information provision and decision making using the ‘accept/decline’ clinical trials questionnaire Kathryn Monson1, Tom Treasure2, Chris Brew-Graves3, Valerie Jenkins4, Lesley Fallowfield4 1Brighton & Sussex Medical School, University of Sussex; 2Clinical Operational Research Unit, University College London; 3Surgical & Interventional Trials Unit, University College London; 4Sussex Health Outcomes Research & Education in Cancer (SHORE-C), Brighton & Sussex Medical School, University of Sussex Correspondence: Kathryn Monson Trials 2017, 18(Suppl 1):P61 Method Following pulmicc stage 1 tests, patients eligible for randomisation (pulmicc stage 2) were offered an 'Accept/Decline’ Questionnaire to complete following their decision to either proceed to randomisation or decline pulmicc stage 2. This 16 item, Likert scale, self-report ques- tionnaire explores aspects of trial information provision, patients’ con- cerns about their illness, influence of friends, family and doctor, and concerns regarding randomisation (V Jenkins, L Fallowfield, 2000). It en- ables the collection of patients’views on key issues surrounding trial in- formation provision and decision-making in a structured, quantitative manner. Patients also identify their most important reason for accept- ing or declining study participation. The questions are worded generic- ally to enable widespread use in randomised trials. R lt Background Close monitoring of biomarker rates and on- going sample size updates can address such issues and improve trial ef- ficiency. This has led to outsmart meeting recruitment targets earlier than expected, avoiding unnecessary randomisation of participants. Conclusions The known prevalence of HLA Ab + in renal transplant recipients is ap- proximately 25%, DSA account for 1/3rd of these, and the known inci- dence of de novo DSA development is 3%. Sample size calculations based on these known rates estimated requiring 2800 randomised partici- pants for 324 DSA+ participants to allow comparison of the effect of bio- marker led immunosuppression optimisation in these patients. Sixteen months into recruitment both the prevalence and incidence of DSA+ was lower than expected. This led to the application of a second strategy for efficiency improvement; a change in the primary outcome from binary to time to event (time to graft failure, approved by the DMC and TSC). The sample size calculation was amended both to reflect this change and to take into account the lower than expected DSA+ rates, retaining trial power. The amended sample size required 165 DSA+ participants from an estimated 2357 to be recruited. This change also meant there was already sufficient numbers of HLA Ab + DSA- and HLA Ab- participants recruited. y Result p p As recruitment continued, the overall proportion of DSA+ participants in the trial increased as the DSA prevalence and incidence rates normal- ised, and also as a consequence of the increasing pool of participants to screen as more HLA Ab- participants were randomised. The latter sort of complexity would generally only be simplistically accounted for in sample size calculations. Close monitoring of biomarker rates and on- going sample size updates can address such issues and improve trial ef- ficiency. This has led to outsmart meeting recruitment targets earlier than expected, avoiding unnecessary randomisation of participants. Conclusions Questionnaires were returned by 54 randomised patients and 57 who declined randomisation. The majority 106/111 (95%) indicated that they had received sufficient written information about the study and 110/111 (99%) indicated that the doctor had told them what they needed to know about the trial. Of patients who agreed to randomisa- tion, 43/54 (80%) thought the trial offered the best treatment available and 48/54 (89%) were satisfied that either treatment in the trial would be suitable for them. Twenty five patients (44%) who declined random- isation were satisfied that either treatment in the trial would be suitable for them but 40/57 (70%) wanted the doctor to choose their treatment rather than be randomised by a computer. The results did not highlight significant problems such as patients feeling unable to say ‘no’ or con- cerns that their illness might get worse unless they joined the study. We have been able to use the information, together with clinicians’ views on their experiences of the feasibility phase of the trial, to iden- tify potential barriers to recruitment and enable strategies to be put in place to address these. Conclusion: We found the questionnaire easy to administer and acceptable to both patients who declined or agreed to join pulmicc stage 2. It is an efficient tool for collecting relevant views from patients regarding potential drivers and barriers to recruitment. Pre-planned ongoing rescreening of biomarker negative participants and flexible reconsideration of the primary outcome allowed both en- richment for individuals with the biomarker of interest and dynamic modification of the sample size, leading to improved trial efficiency. Where possible, time-to-event or continuous primary outcomes should be used in trials, especially where recruitment might be difficult and/or biomarkers of interest are rare. Background The Electronic Health Record (EHR) is a rich source of clinical data that holds promise for improving clinical trial conduct. However, little information is available on site-level barriers to optimal use of EHR systems in contemporary trials, particularly with respect to screening and enrollment. More data is needed on the current use and associ- ated challenges of using the EHR to identify trial participants. Objective j We described existing site-level processes for using the EHR for screening and recruitment of potential participants for an ongoing Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 25 of 235 Page 25 of 235 clinical trial. We also ascertained information on successful recruit- ment strategies and key barriers to using the EHR for trial recruit- ment from the perspective of site coordinators. Methods In this study, the statements included on the cards relates to the in- formation items recommended by HRA for inclusion in a PIL. Twenty trial stakeholders were recruited (10 potential trial participants [PTPs] and 10 research nurses [RNs]) completed the card sort within one-to- one think-aloud interviews. To contextualise the card sort, PTPs were asked to imagine they had been approached to participate in a phase III RCT comparing treatments A and B for a chronic condition. RNs were asked to think about potential participants making the de- cision to take part in the same phase III RCT. In this study, the statements included on the cards relates to the in- formation items recommended by HRA for inclusion in a PIL. Twenty trial stakeholders were recruited (10 potential trial participants [PTPs] and 10 research nurses [RNs]) completed the card sort within one-to- one think-aloud interviews. To contextualise the card sort, PTPs were asked to imagine they had been approached to participate in a phase III RCT comparing treatments A and B for a chronic condition. RNs were asked to think about potential participants making the de- cision to take part in the same phase III RCT. Qualitative focus groups were conducted with 18 study coordinators and site investigators at sites actively participating in the global multi- center HARMONY Outcomes trial, an ongoing randomized controlled study to evaluate the effect of albiglutide on cardiovascular events in patients with Type 2 diabetes (Clinicaltrials.gov ID: NCT02465515). Inter- views were conducted by a professional moderator using a semi- structured, open-ended topic guide and were analyzed to identify com- mon cross-site themes. Background Focus group participants represented research sites in the United States (n = 14), the United Kingdom (n = 2), Canada (n = 1), and Denmark (n = 1), with the majority based in multi-physician or hospital-based practices. Both stakeholder groups ranked the following three statements in their top four most important statements: “possible disadvan- tages and risks of taking part”, “possible advantages of taking part” and “possible side effects of trial treatment”. Both stake- holder groups ranked “who is funding the research” among their least important statements. There were differences between groups in the other statements ranked as least important. RNs in- cluded “who has approved the study”, “how have patients and the public been involved in the design of the study” and “has the scientific quality of the study been checked” among their least important statements. PTPs ranked “will expenses be reim- bursed”, “will there be any impact on any insurance policies” and “will I receive any payments for taking part” among their least important statements. Objectives To review the consent, recruitment and retention rates for single and multi-centre randomised control trials funded by the United King- dom’s National Institute for Health Research (NIHR) Health Technol- ogy Assessment (HTA) Programme. Background Substantial amounts of public funds are invested in health research worldwide. Publicly funded randomised controlled trials (RCTs) often recruit participants at a slower than anticipated rate. Many trials fail to reach their planned sample size within the envisaged trial time- scale and trial funding envelope. A recent survey amongst the Direc- tors of the Clinical Trials Units registered with the UK NIHR Clinical Research Network identified priorities for research into the method- ology of trials. The top three priorities were improving recruitment, choice of outcomes, and improving retention. Correspondence: Karen Inne Trials 2017, 18(Suppl 1):P63 p Trials 2017, 18(Suppl 1):P63 A Patient Information Leaflet (PIL) is a requirement for UK health- related research studies. Health Research Authority (HRA) guidance lists 36 topic areas for inclusion in a PIL. However, there is limited evidence about whether stakeholders believe these items to be of importance when considering participation in a Randomised Con- trolled Trial (RCT). This study identified and assessed which items of information trial stakeholders ranked as most important and the rea- sons for this. P64 Recruitment and retention of participants in randomised controlled trials: a review of trials funded by the United Kingdom health technology assessment programme Stephen Walters, Inês Bonacho dos Anjos Henriques-Cadby, Oscar Bortolami, Laura Flight, Daniel Hind, Richard M. Jacques, Christopher Knox, Nadin, Joanne Rothwell, Michael Surtees University of Sheffield Correspondence: Stephen Walters Trials 2017, 18(Suppl 1):P64 P64 Recruitment and retention of participants in randomised controlled trials: a review of trials funded by the United Kingdom health technology assessment programme Stephen Walters, Inês Bonacho dos Anjos Henriques-Cadby, Oscar Bortolami, Laura Flight, Daniel Hind, Richard M. Jacques, Christopher Knox, Nadin, Joanne Rothwell, Michael Surtees University of Sheffield Correspondence: Stephen Walters Trials 2017, 18(Suppl 1):P64 The majority of study coordinators in an ongoing diabetes trial re- ported that the EHR was the primary modality used to identify po- tential trial participants. Key barriers to full use of the EHR for recruitment included limitations on access to medical records and lack of research modules designed to support screening. Despite these barriers, the use of EHR systems for screening is viewed as an improvement over non-EHR-based methods. P63 The importance of informational items in a randomised controlled trial participant information leaflet: a mixed method study Karen Innes, Katie Gillies, Seonaidh Cotton Marion Campbell Health Services Research Unit, University of Aberdeen, Aberdeen, UK Correspondence: Karen Innes Trials 2017, 18(Suppl 1):P63 p Results f Most focus group participants reported that the EHR was the primary modality used for screening, with the application of study-specific EHR queries in conjunction with medical chart review to generate a list of potentially eligible patients. In addition to EHR-based screen- ing, most site coordinators reported using a multipronged approach of high- and low-yield trial recruitment strategies, including asking non-study investigators at the site to refer potentially eligible partici- pants, posting fliers in clinics, sending mass mailings, and consulting lists of names of past participants for future studies. Several key bar- riers to use of the EHR system for recruitment were reported, includ- ing limitations on accessing individual patient records without informed consent, access to billing-only modules rather than re- search modules, limitations on the number of search parameters, and site-level restrictions on cold-calling patients meeting study cri- teria. Coordinators reported that, despite these barriers, using an EHR system has dramatically improved the perceived yield and timeframe relative to traditional, paper-based screening methods. l This study is one of the first to explore how different stakeholder groups rank the information contained in an RCT PIL. Similarities exist between both stakeholder groups in statements ranked as most im- portant, but there are differences in the least important statements. These results have implications for researchers developing PILs for RCTs. Further research is required to identify any association between the information provided in PILs and the decision-making process around RCT participation. References Based on this review for most publicly funded trials the recruitment rate is likely to be between 1 and 2 participants per centre per week (4 to 10 a month). There is considerable variation in the consent, re- cruitment and retention rates in publicly funded RCTs. In practice, re- cruitment rates will vary, depending on whether the target population is acute, where opportunistic recruitment will target inci- dent cases, or chronic, where database recruitment can effectively target prevalent cases. It will also vary according to whether the intervention is therapeutic or preventive and the base incidence and prevalence rate of the condition. Investigators should bear this in mind at the planning stage of their study and not be overly optimis- tic about their recruitment projections. 1. Edwards P, Roberts I, Clarke M, diguiseppi C, Wentz R, Kwan I, et al. Methods to increase response to postal and electronic questionnaires. Cochrane Database Syst Rev. 2009;3, MR000008. 2. Brueton V, Tierney J, Stenning S, Nazareth I, Meredith S, Harding S, et al. Systematic review of strategies to reduce attrition in randomised trials. Cochrane Database Syst Rev. 2013;12, MR000032. gy Methods Our mixed method study used aspects of Q-methodology (a card sort technique) and simultaneous cognitive interviews (think aloud). This mixed methods approach captures data on subjective opinions held around a particular area of interest. The card sort technique pro- vides participants with a set of “cards” (statements describing specific information items) which they rank according to their opinion of rela- tive importance. A specially formatted grid is used to capture the relative rankings. While the participant completes the card sort, they are encouraged to use the think-aloud technique to verbalise their thoughts. HTA reports of individually randomised single or multi-centre rcts published from the start of 2004 to the end of April 2016 were reviewed. Data extraction Information was extracted, relating to the trial char- acteristics, sample size, recruitment and retention by two independ- ent reviewers. Main outcome measures Target sample size and whether it was achieved; recruitment rates (number of participants recruited per centre per month) and retention rates (randomised participants retained and assessed with valid primary outcome data). Page 26 of 235 Trials 2017, 18(Suppl 1):200 Page 26 of 235 Page 26 of 235 increase in response rates (before 71.4%, after 75.9%) but it was not statistically significant, 95% CI [0.87,1.80]. Background l Non-muscle invasive bladder cancer (NMIBC) is a locally recurring dis- ease for which patients undergo long term surveillance following ini- tial diagnosis. CALIBER is a multicentre phase II feasibility study comparing intravesical chemotherapy (chemoresection) with surgery (standard of care) in patients with recurrent low risk NMIBC (2:1 che- moresection:surgery randomisation). The primary aim is to assess complete response to chemoresection and the trial is randomised to test feasibility of recruitment to a larger randomised phase III trial. It was anticipated that patient recruitment would be challenging due to the need to identify potential participants at the time of recur- rence prior to treatment, complex risk stratification criteria and varied treatment pathways across participating sites. As such we developed recruitment aids with the aim of raising awareness amongst potential participants, ensuring site staff remain aware of the trial and promot- ing effective liaison between site staff when suitable patients are identified. Background During routine monitoring in the ethos study (a surgical trial compar- ing Stapled Haemorrhoidopexy with Traditional Haemorrhoidectomy for the treatment of grade II-IV haemorrhoids), it was found that the response rates to the 12 and 24 month follow-up postal question- naires were lower than expected. Literature reviews looking at methods to increase response rates identified monetary incentives as one potential way to boost response rates1-2. Two Studies With-in a Trial (swats) were conducted within ethos to assess the effectiveness of a small unconditional voucher and a higher value conditional vou- cher on response rates to the postal questionnaires. Following no ef- fect of a lower value voucher incentive (£5.00) being found in increasing response rates in the study (SWAT1), the team designed an additional study to evaluate if a higher value monetary incentive would be more effective in increasing questionnaire response rates. Methods Methods From the outset of the trial, ethics approved short patient informa- tion leaflets and posters have been available to highlight the trial to patients attending surveillance visits. A staff poster was also provided to raise awareness amongst staff conducting surveillance. A CALIBER specific risk calculation tool was introduced in March 2016 as an aid to assess eligibility. We surveyed 34 participating centres about their use of these aids and their use of the tools was compared to their average recruitment. Participants enrolled in ethos who had not yet received their 12 and/ or 24 months follow-up questionnaires were included in SWAT2. All participants were sent a covering letter with their postal question- naires which informed them that they would receive a £30 high street voucher as a token of appreciation upon receipt of a com- pleted questionnaire. The primary analysis was a before and after analysis of the effect of the voucher in increasing response rates at each time-point. A sensitivity analysis was also carried out due to the overlapping influence of SWAT 1. Results increase in response rates (before 71.4%, after 75.9%) but it was not statistically significant, 95% CI [0.87,1.80]. This review identified 151 individually randomised controlled trials from 778 NIHR HTA reports. The final recruitment target sample size was achieved in 56% (85/151) of the RCTs and more than 80% of the final target sample size was achieved for 79% of the RCTs (119/151). For 34% (52/151) of trials the original sample size target was revised (downward in 79% (41/52)). The median recruitment rate (partici- pants per centre per month) was found to be 0.92 (IQR 0.43 to 2.79) and the median retention rate (proportion of participants with valid primary outcome data at follow-up) was estimated at 89% (IQR 79% to 97%). y g Discussion Both studies highlight that, despite current literature to the contrary, the use of monetary incentives may not increase questionnaire re- sponse rates in all study populations and could even have a negative impact. There are a number of contextual aspects which may explain this unexpected finding. Care is needed when introducing a new intervention into an ongoing trial. Future evaluations of incentives are needed to explore the impact of contextual issues which may moderate their impact and influence in different study settings. P66 Recruitment aids for a phase II randomised trial in low risk bladder cancer 1 1 2 3 Steven Penegar1, Rebecca Lewis1, James Catto2, Joanne Cresswell3, Leyshon Griffiths4, Micki Hill1, John Kelly5, Allen Knight6, John McGrath7, Laura Wiley1 1 2 3 Do higher monetary incentives improve response rates part-way through a randomised control trial? 1The Institute of Cancer Research; 2University of Sheffield; 3South Tees Hospitals NHS Foundation Trust; 4University Hospitals of Leicester NHS Trust; 5University College London; 6Patient representative; 7Royal Devon & Exeter NHS Foundation Trust Do higher monetary incentives improve response rates part-way through a randomised control trial? Jessica Wood1, Jonathan A. Cook2, Jemma Hudson1, Alison McDonald1, Hanne Bruhn3, Angus J. M. Watson4 1Centre for Healthcare Randomised Trials (chart), Health Services Research Unit, University of Aberdeen; 2Nuffield Department of Orthopaedics, Rheumatology & Musculoskeletal Sciences, University of Oxford; 3Health Economics Research Unit, University of Aberdeen; 4Department of Surgery, NHS Highland Correspondence: Jessica Wood Trials 2017, 18(Suppl 1):P65 Do higher monetary incentives improve response rates part way through a randomised control trial? Jessica Wood1, Jonathan A. Cook2, Jemma Hudson1, Alison McDonald1, Hanne Bruhn3, Angus J. M. Watson4 1Centre for Healthcare Randomised Trials (chart), Health Services Research Unit, University of Aberdeen; 2Nuffield Department of Orthopaedics, Rheumatology & Musculoskeletal Sciences, University of Oxford; 3Health Economics Research Unit, University of Aberdeen; 4Department of Surgery, NHS Highland Correspondence: Jessica Wood Trials 2017, 18(Suppl 1):P65 Correspondence: Steven Penegar Trials 2017, 18(Suppl 1):P66 g Results Responses were received from 26/34 centres. 25/26 (96%) are using at least one of the short patient information leaflet, patient poster, clinician poster or eligibility. Average monthly recruitment does not appear to increase with increased use of the tools, with a median re- cruitment of 0.21 for the 8/26 (31%) sites using two tools and 0.03 for the 6/26 (23%) sites using all four. Since distributing the CALIBER risk calculator, the number of eligibil- ity queries received by the coordinating clinical trials unit has sub- stantially decreased. Initial feedback from centres suggests it is a useful tool for local pre-screening. Centres are advised to print the Responses were received from 26/34 centres. 25/26 (96%) are using at least one of the short patient information leaflet, patient poster, clinician poster or eligibility. Average monthly recruitment does not appear to increase with increased use of the tools, with a median re- cruitment of 0.21 for the 8/26 (31%) sites using two tools and 0.03 for the 6/26 (23%) sites using all four. A cross-cutting approach to clinical trial success: the US Department of Veterans Affairs’ network of dedicated enrollment sites (NODES) model A cross-cutting approach to clinical trial success: the US Department of Veterans Affairs’ network of dedicated enrollment sites (NODES) model Marcus Johnson1, Aliya Asghar2, Tawni Kenworthy-Heinige3, Debra Condon4, Karen Bratcher5, Meghan O'Leary6, Danielle Beck7, Cyenthia Willis8, Grant D. Huang9 1 2 Background Clinical trials are a critical component of biomedical research and provide valuable insights into effective means for enhancing patient care and establishing new therapies. Recruitment into clinical trials remains a key determinant to study completion and success. Barriers to achieving enrollment targets include distrust of the medical com- munity and clinical research, lack of awareness or understanding about clinical trials and eligibility criteria, and concerns about the lo- gistics of participation, such as required travel, the time involved with participating, and potential costs. While various strategies have been proposed, it is unclear how broadly they apply when different popu- lations, diseases, and/or study goals are involved. The ability to ef- fectively overcome challenges may require approaches that focus more on addressing shared interests among sites in overcoming clin- ical trial barriers. g CSP NODES piloted a "mentoring" (or hub-spoke) model in which a Node site would more directly work with a study site to identify and overcome barriers to recruitment, compliance, and data quality. Aims 1. Determine the impact of an external research site mentoring model on study recruitment. 2. Examine the study site-level charac- teristics that facilitate or impede study recruitment. M h d The Colonoscopy Versus Fecal Immunochemical Test in Reducing Mortality From Colorectal Cancer (CONFIRM) (CSP #577)3 study is a large, simple, multicenter, randomized, parallel group trial directly comparing screening colonoscopy with annual FIT screening in aver- age risk individuals. Each of the 9 Node sites was paired with a low performing (recruitment) CSP #577 study site. One Node site was assigned two low recruiting sites for a total of 10 pilot sites. The re- spective Node Manager then worked with the study site and the West Haven CSP Coordinating Center (WHCSPCC) 4 to perform the following: Research site mentoring: a novel approach to improving study recruitment 1US Department of Veterans Affairs; 2VA Long Beach Healthcare System, US Department of Veterans Affairs; 3VA Portland Health Care System; 4Minneapolis VA Health Care System; 5VA Palo Alto Health Care System; 6Durham VA Medical Center, US Department of Veterans Affairs; 7VA San Diego Healthcare System, US Department of Veterans Affairs; 8VA North Texas Healthcare System, US Department of Veterans Affairs; 9VA Cooperative Studies Program Central Office, US Department of Veterans Affairs Marcus Johnson, Margaret Antonelli, Lynn Tommessilli, Beata Planeta US Department of Veterans Affairs Correspondence: Marcus Johnson Trials 2017, 18(Suppl 1):P67 Marcus Johnson, Margaret Antonelli, Lynn Tommessilli, Beata Planeta US Department of Veterans Affairs Methods The Department of Veterans Affairs (VA) Cooperative Studies Pro- gram (CSP) is a clinical research infrastructure embedded within the nation’s largest integrated health care system. The VA Network of Dedicated Enrollment Sites (NODES) is a consortium of nine sites intended to provide systematic site-level solutions to issues that arise during the conduct of VA CSP clinical research [1]. Each NODES site is represented at each VA Medical Center (VAMC) by a Director (or team of co-directors/associate directors) and a Manager. Additionally, each site has clinical support staff, including nurses and research as- sistants, designated to assist multiple CSP clinical trials locally. Within the context of a large, integrated health care system, NODES goals are to: 1) enhance recruitment for clinical trials; 2) create study effi- ciencies; 3) improve communication and disseminate best practices; and 4) provide broader expertise in the design and conduct of VA clinical research. Initial pilot activities were conducted for establish- ing more cross-cutting approaches to improving recruitment. Results Created a site management checklist to determine the current state of local study operations; Used the site management checklist to conduct interviews with site study staff; Used the feedback that was gathered during the site interviews to create study improvement plans that contained performance metrics to measure criteria related to recruitment, compliance, and data quality; Held regular conference calls independently with study sites and WHCSPCC to monitor pro- gress. The pilot was conducted over a 6-month period from February 2016-June 2016. Limitations The impact of introduction of different tools on recruitment could not be confirmed as most have been available since the trial com- menced. The reduction in eligibility queries since introduction of the recurrence calculation tool may be a result of increased centre ex- perience. In addition, the use of tools may be confounded with fac- tors such as centre size and frequency of patient screening for the trial. Background The VA Cooperative Studies Program's (CSP)1 Network of Dedicated Enrollment Sites (NODES) is a consortium of 9 VA medical centers (VAMCs) that have teams (Nodes) in place dedicated to conducting CSP studies to enhance the overall performance, compliance, and management of CSP multi-site clinical trials. Each Node site has a Dir- ector (MD/PhD), Manager (Clinical Trial Nurse, Research Project Man- ager), and Research Assistant(s). Correspondence: Marcus Johnson Trials 2017, 18(Suppl 1):P68 pp Results In total 586 and 562 participants were included in the 12 and 24 month analyses respectively in SWAT2. Results showed no statis- tical evidence of an effect on the response rates at both 12 and 24 month time-points. Similarly, the sensitivity analyses results showed no evidence of a difference in the 12 month response rates after the incentive was given. At 24 months there was a slight Since distributing the CALIBER risk calculator, the number of eligibil- ity queries received by the coordinating clinical trials unit has sub- stantially decreased. Initial feedback from centres suggests it is a useful tool for local pre-screening. Centres are advised to print the Page 27 of 235 Trials 2017, 18(Suppl 1):200 Page 27 of 235 Trials 2017, 18(Suppl 1):200 completed score calculation and retain in the patient notes to docu- ment this eligibility assessment. of recruitment at community-based outpatient clinics, lack of utilization of the full spectrum of recruitment materials e.g. Letters, flyers, participant screening algorithms (electronic medical records), etc., unmotivated/disengaged study staff, lack of clinical referrals, and uneven distribution of duties across study teams. Having a sub- ject matter expert that was external to the CSP coordinating center and could serve as a mentor was beneficial for the pilot sites. The pilot provided a resource to the site that worked within a similar en- vironment and could provide specific, site-level guidance on how to resolve some of the recruitment issues/barriers that they faced. Conclusion of recruitment at community-based outpatient clinics, lack of utilization of the full spectrum of recruitment materials e.g. Letters, flyers, participant screening algorithms (electronic medical records), etc., unmotivated/disengaged study staff, lack of clinical referrals, and uneven distribution of duties across study teams. Having a sub- ject matter expert that was external to the CSP coordinating center and could serve as a mentor was beneficial for the pilot sites. The pilot provided a resource to the site that worked within a similar en- vironment and could provide specific, site-level guidance on how to resolve some of the recruitment issues/barriers that they faced. Conclusion Conclusions With provision of targeted recruitment aids, centre staff training and ongoing support from the coordinating clinical trials unit, potential barriers to recruitment in a trial with challenging patient identifica- tion pathways and complex eligibility criteria can be managed effect- ively. However there is no obvious increase in recruitment with increased use of recruitment aids. In order to robustly evaluate the impact of recruitment aid interventions they should be introduced in a controlled manner to facilitate assessment of within and between centre pre- and post- intervention accrual rates. The site mentoring model was successful in increasing participant re- cruitment at study sites in a large, simple, multicenter, randomized, parallel group trial in the VA healthcare system. Conclusion NODES addressed barriers in various aspects of clinical trial recruit- ment and management by working collaboratively to solve problems with multiple stakeholders. Varied practices and operational changes in CSP research related to recruitment, staff training and research methodology were implemented by taking an overall, system wide approach. Many challenges with patient recruitment experienced within CSP are similar to those encountered by other multi-site gov- ernment or private industry clinical trials. As a result, the solutions to these recruitment problems presented by NODES may be transfer- able to other healthcare settings. Results Results The ten Study sites that participated in the pilot mentorship had an average improvement of 4.9 participants enrolled per month vs. An average improvement of 1.3 participants enrolled per month at the 27 study sites that were not part of the pilot. Some common issues/ barriers to recruitment that the pilot sites faced are as follows: lack NODES addressed key barriers affecting clinical trial outcomes at study-specific and organizational levels. Results of these activities are presented in categories related to 1) implementing innovative partici- pant recruitment strategies, 2) creating site-level efficiencies for study Trials 2017, 18(Suppl 1):200 Page 28 of 235 Page 28 of 235 P70 Small sample corrections for cluster randomised trials: implications for power and type I error rate P70 Small sample corrections for cluster randomised trials: implications for power and type I error rate Clemence Leyrat1, Katy Morgan1, Baptiste Leurent1, Brennan Kahan2 1London School of Hygiene and Tropical Medicine; 2Queen Mary University of London Correspondence: Clemence Leyrat Trials 2017, 18(Suppl 1):P70 g Tackling health inequalities has been a Scottish Government priority since 2007. General Practitioners (GPs) at the Deep End is a collabor- ation of general medical practices serving the 100 most deprived populations in Scotland, based on the proportion of patients on the practice list with postcodes in the most deprived 15% of Scottish data zones. Eighty-six of these 100 practices are in the NHS Greater Glasgow and Clyde Health Board area. The Deep End Links Worker Programme was designed to prevent and reduce health inequalities in Scotland, and support people living in the most deprived areas of Scotland to ‘live well’ In their communities. It provides resources to General Practices serving these populations to develop a ‘links ap- proach’. Practices can refer patients to a Links Worker, who works with the patient to identify and enable access to appropriate community-based resources. The argument is that increasing and en- abling local activities can enhance community connection, trust and cohesion. Hence, the aim of Links Worker is to act as catalyst to hope and self-determination, by using the strong relationship with patients that exists in General Practices as a natural community hub. Part of the evaluation of the programme involved a comparison of patient and staff outcomes measured in 7 Intervention practices, with those from 8 Comparator practices. These 15 practices were allocated to the Intervention and Comparison arms at random, prior to the in- volvement of the evaluation team. Methods We performed a simulation study to assess both the type I error rate and the power of parallel two-arm CRTs with a continuous outcome analysed with cluster-level methods, mixed models or GEE. For cluster-level analysis, we studied the performance of a linear model of cluster means without correction, a linear model weighted by the cluster size or weighted by the variance components and a Wilcoxon test. For mixed models, we assessed the performance of a Z-test, as well as three degree of freedom corrections: Satterthwaite, Kenward- Roger and the between-within method. Finally, for GEE, we com- pared the performance of a Z test using model-based and robust standard errors and a small sample correction proposed by Fay and Graubard. We studied the impact of varying the intraclass correlation coefficient (ICC), the number of clusters randomised and the vari- ability in cluster size. Results operations and management, and 3) establishing metrics to more ef- fectively evaluate site and network performance. Initial network ef- forts produced several lessons and best practices for common clinical trial problems. Additionally, innovations for wider adoption across CSP studies were developed. Such strategies include mobile recruitment, algorithmic inclusion/exclusion data programs for re- cruitment activities, staff cross-training and mentorship, and stan- dardized performance reporting. Some metrics were also used for overall network performance. The results confirmed that when only few clusters are randomised, inflated type I errors are observed and this inflation increases with the ICC and with the variability in cluster size. Amongst the com- pared methods, only the cluster-level model weighted on the vari- ance components and mixed models with Satterthwaite or Kenward- Roger corrections maintained the type I error rate at or below 5% in all scenarios. Second, in terms of power, individual-level analyses out- performed cluster-level analyses, but the power remained low for fewer than 20 clusters randomised. Moreover, when the number of clusters was very small (<8), the degree of freedom corrections lead to very low type I errors (<2%), thus reducing the power to between 30 and 50%. Background Cluster randomised trials (CRTs) are increasingly implemented to as- sess the effectiveness of interventions in settings in which individual randomisation is impossible or challenging. Three main analysis strat- egies have been proposed to analyse CRTs: cluster-level analysis, mixed-models and generalised estimating equations (GEE). Whereas the former approach maintains the nominal type I error rate, that is, the chance to detect an effect when there is not, the last two lead to inflated type I error rates when the number of clusters is small or the cluster size varies. Small sample corrections have been proposed for mixed models and GEE to circumvent this problem, but the impact of these methods on power is still unclear. Objective The primary aim of the evaluation was to assess the effectiveness of the Links Worker Programme in achieving the intended outcomes at patient, practice and community levels. Methods Conclusions When the number of randomised clusters is small, appropriate cor- rections must be used to maintain an appropriate type I error rate. GEE approaches are not recommended, but a weighted cluster-level analysis or a mixed model with a Satterthwaite or Kenward-Roger de- gree of freedom correction can maintain an appropriate type I error. However, these approaches lead to an important decrease in power when fewer than 20 clusters are randomised, so adjustment of the sample size is required at the design stage when such corrections are used. No common denominator: a review of outcome measures in IVF RCTs Jack Wilkinson1, Stephen A Roberts1, Marian Showell2, Daniel R Brison3, Andy Vail1 The Glasgow deep end links worker programme evaluation: analysis implication of an unusual randomised study design Andisheh Bakhshi, Alex McConnachie, Bridie Fitzpatrick, Kate O’Donnell, Mhairi McKenzie, Kathyrn Skivington, Nai Chng, Mathew Smith, Sally Wyke, Stewart Mercer University of Glasgow Correspondence: Andisheh Bakhshi Trials 2017, 18(Suppl 1):P71 y 1University of Manchester; 2University of Auckland; 3Central Manchester University NHS Foundation Trust Correspondence: Jack Wilkinson Trials 2017, 18(Suppl 1):P69 1University of Manchester; 2University of Auckland; 3Central Manchester University NHS Foundation Trust This abstract is not included here as it has already been published. p , p Methods A quasi-experimental evaluation design was decided by the Scottish government as part of their funding conditions. Among 15 General Practices that submitted a formal expression of interest in having a Links Worker attached to their team, patients’ outcomes in 7 Gen- erals Practices with an attached Links Worker will be compared with those from 8 General Practices that do not have an attached Links Worker. The intervention group are patients who were referred to Trials 2017, 18(Suppl 1):200 Page 29 of 235 Page 29 of 235 the Links Worker, but the comparator group are a random sample of the practice population. By design, the intervention group is very dif- ferent from the comparator group. Therefore, simple between-group comparisons, which would be appropriate for most randomised stud- ies, will not be appropriate. The main statistical analyses have been defined in terms of mixed effects regression models, to account for clustering of outcomes at the practice level, with adjustment for dif- ferences in baseline covariates. In addition, other methods will be ex- plored, aiming to control for the selection bias inherent in the study design (e.g. With respect to age, gender, deprivation and comorbidi- ties), such as nearest neighbour matching and propensity score analysis. whether the treatment warrants further investigation. A limitation of single-arm designs is that the results of the trial may be unreliable predominantly due to selection bias. Therapeutic benefits may gener- ally be smaller than differences in outcome due to baseline charac- teristics. Detailed description of the baseline prognostic factors is of little help in determining for example whether a group of patients with poor prognosis has been recruited into the trial or whether the treatment is not truly worthy of further study. Buyse (2000) argues that randomisation should be considered more often for early trials of experimental treatments. The calibrated design (Herson & Carter 1986) is a randomised single- stage design utilising a binary endpoint. The design differs from the typical randomised trial, as the “control” Group is not employed to provide a comparison for the experimental treatment, but to evalu- ate whether the sample population who receive the experimental treatment has the capability of showing a response. The design es- sentially constitutes two separate designs; one for the experimental arm and one for the calibration arm. Results The final analysis is underway. Some results of the evaluation will be presented, with particular focus on the implications of this unusual de- sign, and the results obtained using alternative analysis approaches. P72 Group sequential methods for monitoring multi-arm clinical trials Danni Liu1, Michele Melia1, for the Diabetic Retinopathy Clinical Research Network2 1Jaeb Center for Health Research; 2DRCR.net Correspondence: Danni Liu Trials 2017, 18(Suppl 1):P72 Trials 2017, 18(Suppl 1):P72 The Myeloma UK Clinical Trials Network developed the muktwelve trial; a randomised phase II study to assess the progression-free sur- vival of a potential treatment regimen in relapsed and refractory multiple myeloma. A calibration group will receive a control treat- ment, and will be used to evaluate the validity of the historic control used in the experimental group. Sixty patients are randomised on a 3:1 basis to experimental and control arms respectively. The sample size was based primarily upon the experimental arm. Due to prag- matic constraints, there is insufficient power to conduct formal hy- pothesis testing in the calibration arm. However the inclusion of this arm is necessary to safeguard against selection bias and give context for the historic control rate. In comparison with conventional two-arm clinical trials, multi-arm clinical trials provide investigators with the ability to directly compare more than two competing treatments in a single trial setting. The multi-arm trial design optimizes the efficiency of a trial, and even re- duces the required total sample size if all experimental treatments share a single control arm. It has been shown that a multi-arm trial design can have economic and ethical benefits, such that it allows for more efficient resource allocation and cost savings compared with two separate trials, as well as the possibility of dropping an in- ferior experimental treatment in the interim or even stopping the study early if there is strong evidence for efficacy or futility. y y y y It is common in multi-arm trials that researchers are more interested in simultaneously monitoring all pairwise comparisons than just a global test. Given that the probability of falsely rejecting the null hy- pothesis is inflated when multiple pairwise hypothesis tests are per- formed repeatedly in a single study, several multiple comparison procedures have been generalized to the context of group sequential testing to preserve the experiment-wise type I error rate, even when an inferior treatment arm is dropped during interim monitoring. Results Some examples are the generalization of the simple Bonferroni pro- cedure, the Pocock’s procedure, the O’Brien-Fleming procedure, the Fisher’s Least Significant Difference method, and the Newman-Keuls procedure. We review the use of a calibration arm in phase II oncology trials and the reasons for implementing such an approach, with an application to the muktwelve study. We also assess the possibility of designing the trial in a Bayesian setting, with the intention of more formally using the calibration arm. It is postulated that the uncertainty around the historic control rate can be better modelled by using this ap- proach. One possibility would be to adapt the use of commensurate priors, which can be utilized in a two-arm setting with a formal com- parison to assess historic and concurrent heterogeneity (Hobbs, Car- lin & Sargent 2013). p In this presentation, we will discuss group sequential methods for monitoring clinical trials with multiple treatment groups. Two re- cently completed Phase III multi-center randomized clinical trials evaluating treatments for diabetic macular edema conducted by the Diabetic Retinopathy Clinical Research Network (DRCR.net) will be used as examples. We will apply several group sequential methods to these two fixed-sample studies and demonstrate the design and conduct of statistical interim monitoring for efficacy under two dis- tinct scenarios: comparisons with a control group and all pairwise comparisons. Sample size re-estimation in cross-over trials: a simulation study investigating the type I error, power and sample size in the AIM HY-INFORM study Sample size re-estimation in cross-over trials: a simulation study investigating the type I error, power and sample size in the AIM HY-INFORM study y Julie Wych, Adrian Mander University of Cambridge Correspondence: Julie Wych Trials 2017, 18(Suppl 1):P74 Hypertension is the single biggest contributor to the global burden of disease in the UK. It is widely accepted that ethnicity is one factor that is associated with hypertension and which influences the re- sponse to existing first-line antihypertensive treatments. In the UK hypertension treatment is stratified by age and self-defined ethnicity (SDE), problems associated with this include a lack of data from UK populations supporting the current SDE stratification and no refer- ence to South Asians – the largest ethnic minority group in the UK. The primary objective of the AIM HY-INFORM study is to determine if the response to existing first-line anti-hypertensive drugs differs by ethnic group for patients on mono- or dual therapies. p , p Methods The experimental arm is set up using a single-stage design using an exact binomial distribution (A’Hern 2001) with corresponding “recommend” and “abandon” cut points specified against a historic control. Outcomes in the calibra- tion arm are used to assess whether the assumptions for the historic control are acceptable and whether the response rate lies within the expected range. If the response rate is not comparable, the conclu- sions from the experimental arm are questioned. Results The work pre- sented here will consider patients on a monotherapy treatment re- gime. The study design is a 3-period 3-treatment cross-over trial in a P73 Calibrated phase II oncology design in a bayesian setting Jessica Kendall, Duncan Wilson, Sarah Brown, Andrew Hall University of Leeds Correspondence: Jessica Kendall Trials 2017, 18(Suppl 1):P73 p Trials 2017, 18(Suppl 1):P73 Phase II oncology designs commonly seek to assess the potential ef- ficacy of a treatment. Single-arm trials can be utilised comparing against a historic control, with decision-making cut points indicating Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 30 of 235 Page 30 of 235 Page 30 of 235 multi-ethnic cohort of hypertensives using a linear mixed effects model with subject as a random effect. With the absence of prior es- timates of the within-subject SD, one problem with this multilevel design is the calculation of the required sample size to ensure the desired power to detect a single ethnic by treatment interaction. Sample-size re-estimation designs can be used in this context to change the sample size according to accrued information in a statis- tically robust way. Ahead of trial recruitment, a simulation study was carried out with the main aims of (i) Assessing the properties of the hypothesis tests for the sample size defined in the AIM HY-INFORM study protocol; (ii) Estimating the fixed (single ethnic by treatment interaction) and random (within-subject SD) estimates of interest along with their summary and performance measures; (iii) Simulating an interim analysis after 50 subjects have completed their treatment sequence to re-assess the sample size calculation. Results and performance measures will show that the hypothesis-generating procedure attains a size of 0.05 for 1000 simulations with a protocol sample size of 660 subjects for different within and between-subject SDs. The estimated power is in line with that achieved in the study protocol and an underestimated assumed within-subject SD requires a larger than planned study sample size; providing a means of preserving the power of the study, a distinct advantage over a fixed design. We propose to compare the result of the primary analysis with alterna- tive methodologies, including time series analyses, which treat the data continuously and incorporate the time-dependent correlation between measurements. In addition, we are interested in determining if the CGM data and clinical data on subject treatment can be used to further optimize the current clinical algorithm and improve glucose control. Methodological challenges in the react study, a randomized controlled trial of real time continuous glucose monitoring in neonatal intensive care 1 2 3 3 Many strategies for treating cancers use combinations of drugs or mul- tiple forms of treatment (e.g. Concurrent chemoradiotherapy). In most early phase trials of two therapies (labelled as A and B, say) a fixed dose of agent A is administered and only the dose of agent B is escalated. The aim is to identify a recommended phase II dose combination (RP2DC), i.e. The largest dose of agent B that, when combined with the fixed dose of agent A, has a chance of causing a dose-limiting toxicity (DLT) close to some predefined limit. Currently, more combination ther- apy trials allow both treatments to be escalated; patients entering the trial receive the best dose from a grid of possible combinations, and one or more RP2DCs can be considered for use in phase II. However, modelling the relationship between dose combination and toxicity can be challenging since most parametric models require many parameters to be flexible enough, and model assessment is impossible given the limited data available in phase I trials. 1University of Cambridge and Cambridge Clinical Trials Unit, Addenbrooke’s Hospital; 2Cambridge Clinical Trials Unit; 3University of Cambridge and Cambridge University Hospitals NHS Foundation Trust Trials 2017, 18(Suppl 1):P75 P77 A model-free Bayesian adaptive design for combination therapy dose-finding trials with censored time-to-toxicity data 1 2 3 Graham Wheeler1, Michael J. Sweeting2, Adrian P. Mander3 1University College London; 2University of Cambridge; 3MRC Biostatistics Unit Hub for Trials Methodology Research Correspondence: Graham Wheeler Trials 2017, 18(Suppl 1):P77 P75 Methodological challenges in the react study, a randomized controlled trial of real time continuous glucose monitoring in neonatal intensive care Julia Forman1, Simon J. Bond2, Catherine Guy3, David Dunger3, Kathryn Beardsall3 1University of Cambridge and Cambridge Clinical Trials Unit, Addenbrooke’s Hospital; 2Cambridge Clinical Trials Unit; 3University of Cambridge and Cambridge University Hospitals NHS Foundation Trust Trials 2017, 18(Suppl 1):P75 Methodological challenges in the react study, a randomized controlled trial of real time continuous glucose monitoring in neonatal intensive care 2 3 3 Background In utero, glucose levels are normally maintained between 4–6 mmol/L. Infants born preterm are at risk of both hyperglycaemia (20-86%) and hypoglycaemia (17%), both of which have been associated with in- creased mortality and morbidity. In neonatal intensive care, clinical practice relies on intermittent blood glucose monitoring. The REACT Trial aims to evaluate the role of real time continuous glucose monitor- ing (CGM) in the detection and management of hyperglycaemia and hypoglycaemia in these babies. REACT will thus provide a wealth of in- formation, combining both continuous and clinical data, and present challenges in statistical analyses regarding how to optimally interpret and use these results. The product of independent beta probabilities escalation (PIPE) de- sign, a model-free approach for identifying multiple RP2DCs in dual- agent phase I trials, is being used in ORCA-2, a phase I trial seeking optimum dose and schedule combinations of olaparib in patients with advanced head and neck cancer. However, the DLT observation period for each patient is 13 weeks, with up to 40 patients planned for a dose-escalation phase and another 10–30 patients planned for a dose-expansion cohort. In trials using radiotherapy, some toxicities can occur months after treatment, so long DLT observation periods are required to ensure dlts are accurately captured and future pa- tients are not at risk of excessive toxicity. In both settings, it is im- practical to wait until all current patients have completed follow-up before treating the next patient. Using data from patients that have partially and fully completed DLT follow-up at the current time could potentially reduce study duration and costs, particularly for trials of combination therapies where sample sizes should be larger than single-agent trials and recruitment is faster than expected. Trial design REACT is an international multicentre randomised controlled trial and will recruit 200 subjects within 24 hours of birth, with a birth weight <1200 g. Subjects will be randomized equally to two parallel arms. All babies will have an Enlite sensor (Medtronic, Northridge, CA, USA) inserted and linked to a Medtronic Minimed 640G monitor for data collection. In the intervention arm the real time sensor data will be used for clinical management. In the control arm the data will be col- lected blind to the clinical team caring for the baby. In both study arms the data will be recorded every five minutes for six days. The primary endpoint for the trial is the difference between the arms in the percentage of time sensor glucose is in the target range of 2.6- 10 mmol/l within the first six days of life. y Methodological challenges We therefore propose an extension to the PIPE design that uses censored toxicity and complete follow-up outcomes to assist with dose-escalation decisions. We show how different recruitment rates affect both trial con- duct and results relative to a trial that requires complete patient follow- up. We consider scenarios where time-to-toxicity is distributed earlier, uni- formly, or later in the DLT observation window and use an adaptive weighting procedure that alters the amount of information each patient provides based on previous DLT times. In the context of the ORCA-2 trial, we obtain substantial reductions in trial duration, with comparable ex- perimentation and recommendation properties to the planned design. Methodological challenges Results Trial registration and funding This trial is registered with the ISRCTN (number 12793535) and funded by the NIHR Efficacy and Mechanism Evaluation Programme. The Efficacy and Mechanism Evaluation programme is funded by the MRC and NIHR, with contributions from the CSO in Scotland, NISCHR in Wales and the HSC R&D, Public Health Agency in Northern Ireland. This report is managed by the NIHR Evaluation, Trials and Studies Co- ordinating Centre (NETSCC) (Efficacy and Mechanism Evaluation, 11/ 133/07 Real Time Continuous Glucose Monitoring in Neonatal Inten- sive care). The views expressed in this publication are those of the author(s) and not necessarily those of the MRC, NHS the National In- stitute for Health Research or the Department of Health. P77 A model-free Bayesian adaptive design for combination therapy dose-finding trials with censored time-to-toxicity data Graham Wheeler1, Michael J. Sweeting2, Adrian P. Mander3 1University College London; 2University of Cambridge; 3MRC Biostatistics Unit Hub for Trials Methodology Research Correspondence: Graham Wheeler Trials 2017, 18(Suppl 1):P77 Methodological challenges Evaluating continuous data, upon which dynamic treatment deci- sions are made, presents challenges for analysis. The primary analysis has a clear interpretation, but it ignores the time dependence of the data, and dichotomises the results: within/outside the target range. Therefore it cannot detect any time-dependent treatment effects. Furthermore, as with all dichotomisations, it is unduly influenced by the choice of boundaries that define the target range. There is a pau- city of alternatives that correct both of these issues, and offer a clin- ically valuable interpretation. Page 31 of 235 Trials 2017, 18(Suppl 1):200 P78 A two-stage, phase II clinical trial design with nested criteria for early stopping and efficacy Michelle DeVeaux, Michael J. Kane, Daniel Zelterman Yale School of Epidemiology and Public Health Trials 2017, 18(Suppl 1):P78 To overcome these challenges multiple imputation using chained equations (“Ice” Command in Stata v14.0) was used to impute the missing data items. Subsequently multivariable fractional polynomial models were fitted on the imputed dataset (“Mfpmi” Command), within a Cox model (“Stcox” Command). This command performs backwards selection to select variables that are predictive of mortal- ity, whilst finding the most suitable functional form of such covari- ates, within a time to event model. Finally, the proportional hazards assumption was checked in the final model, using standard methods. Discussion We propose a two-stage design for a single arm clinical trial with an early stopping rule for safety. This design employs different criteria to assess early stopping and efficacy. The early stopping rule is based on a criteria that can be determined more quickly than that for effi- cacy. These separate criteria are also nested in the sense that efficacy is a special case of, but usually not identical to, the early stopping cri- teria. The addition of a curtailed sampling scheme allows for early decisions to be made before all of the data has been observed. The design readily allows for planning in terms of statistical significance, power, and expected sample size. This method is illustrated with a Phase II design comparing patients treated for lung cancer with a novel drug combination to a historical control. In this example, the early stopping rule is based on the number of patients who exhibit progression-free survival (PFS) at 2 months post treatment follow-up. Efficacy is judged by the number of patients who have PFS at 6 months. Background Infection is a major concern in UK hospitals and it is estimated that up to 8% of inpatients have an infection. The death rate from these infections can reach 10-30% depending on the patient and patho- gen. A number of non-modifiable patient factors (e.g. Comorbidities and infection severity) are known to impact adversely on outcome. However there are limited data available from large multicentre stud- ies to investigate the importance of modifiable risk factors. ‘Blood Stream Infection: Focus on outcomes’ is a multicentre, prospective observational study with the primary aim of identifying modifiable risk factors associated with all-cause mortality in patients with one of six key pathogens. Modifiable data collected includes staffing levels, antibiotic use (in particular the timing of appropriate therapy) and use of intravenous lines. Analysis approach Although the primary purpose of the study is to identify and esti- mate the effect of modifiable factors, the analyses need to take the non-modifiable risk factors into account. To maximise the degrees of freedom available, the analysis was split into two stages. For the first stage, a Cox model was fitted for the non-modifiable risk factors only with the aim of deriving a risk scorer which quantifies each patient’s risk of mortality. The second stage of the modelling is investigating the modifiable risk factors and the risk score derived in the first stage is included as a covariate (analysis underway). Methodological challenges The use of multivariable fractional polynomials within survival ana- lysis models using imputed data can be useful to find a best fitting model for a dataset with missing data. The code in order to achieve this is available in standard statistical packages, such as Stata. Frac- tional polynomials can be difficult for non-statisticians to grasp and may not be the best choice in settings where straightforward inter- pretability of coefficients is required. Methods We used patient-level data from the English NHS Patient Reported Outcome Measures between 2012/13 and 2014/15. We included data from 126,064 patients who responded to all seven pain component questions of the six-month post-operative OKS questionnaire. The pain component includes questions on level of pain, night pain, pain while walking, standing up, limping, interference with work, and con- fidence. All questions had five response levels from 0 (most pain) to 4 (no pain) so that the pain component subscale ranges from 0 (most pain) to 28 (no pain). Dataset Background Background About 20% of patients who undergo primary total knee replacement (TKR) surgery in the UK experience chronic pain after their operation. The provision of healthcare services for these patients has been found to be patchy and inconsistent in the NHS. Although chronic pain is understood to be pain persisting for several months, the level that pain must persist for a patient to be considered in chronic pain is not defined. The aim of this work was to identify a cut-off point in the pain component subscale of the Oxford Knee Score (OKS) that could be used to identify patients in chronic pain following a primary TKR. Dataset A total of 1,676 patients were included in the study across 5 centres. The dataset comprises 48 modifiable and 38 non-modifiable factors. The overall mortality rate is 20.8% (95% CI: 18.8% - 22.8%). We adopted a data-driven approach in order to derive groups with different levels of pain using cluster analysis. We applied a hierarch- ical method of clustering whereby a multi-level hierarchical tree was created by repetitively splitting data into clusters. “similar” observa- tions (based on inter-observation distance) were placed in the same cluster. Clusters were then split until no further dissimilarity could be found, or until the maximum number of clusters was reached. The cluster analysis was run for an increasing maximum number of clus- ters from two to 10. Clusters were then examined based on their dis- tribution over the range of the pain component subscale to identify if the cluster with the lowest scores (highest pain) was stable as the number of clusters increased. If a stable cluster was found, its highest values would be identified as the cut-off point. An oxford knee score cut-off point to identify patients with chronic pain after knee replacement for a complex intervention trial Sara Khalid1, Vicky Wylde2, Rachael Gooberman-Hill2, Andrew Judge1, Rafael Pinedo-Villanueva1 Sara Khalid1, Vicky Wylde2, Rachael Gooberman-Hill2, Andrew Judge1, Rafael Pinedo-Villanueva1 1University of Oxford; 2University of Bristol Correspondence: Sara Khalid Trials 2017, 18(Suppl 1):P80 1University of Oxford; 2University of Bristol Correspondence: Sara Khalid Trials 2017, 18(Suppl 1):P80 P79 Analysis challenges in blood stream infection: focus on outcomes study Rebecca Evans1, Katie Pike1, Alasdair MacGowan2, Chris A. Rogers1 1Clinical Trials and Evaluation Unit, University of Bristol; 2Southmead Hospital, North Bristol NHS Trust Correspondence: Rebecca Evans Trials 2017, 18(Suppl 1):P79 Results The first challenge encountered was missing data; data were missing for between 10% and 45% of patients for some of the key data items. Secondly, in order to ensure the calculated risk score provides the best summary of the relationship between the non-modifiable factors and survival, it was important to ensure the most suitable functional form of each continuous covariate was used. Finally, the modelling required a survival analysis framework, which included en- suring that the appropriate model assumptions were met. The distribution of the hierarchical clusters over the pain component subscale showed a changing shape for the highest pain group when the maximum number of clusters was set to two or three, but a largely consistent distribution was observed when the number of clusters was set to four or higher. The highest score for the pain component subscale for the high-pain cluster was 24 for two or three clusters, but it converged to 14 for four clusters and above. Trials 2017, 18(Suppl 1):200 Page 32 of 235 Trials 2017, 18(Suppl 1):200 Page 32 of 235 P82 The web tool provides an interactive graphical user interface that al- lows users to easily conduct simulations and assess the best design for meeting the primary objective of the proposed trial. Adaptive designs and further information are available at http://cqs.mc.vanderbilt.edu/ shiny/adaptivedesigns/and will be updated soon. Background In phase I oncology clinical trials, the operating characteristics of adaptive designs are used to evaluate the performance of adaptive designs via a simulation study. Research has shown that no single es- calation method has proven superior in all circumstances. Thus, an interactive web application with a comprehensive score has been de- veloped to find an appropriate adaptive design for conducting an oncology phase I trial. Correspondence: Susan Hala Trials 2017, 18(Suppl 1):P81 Residuals in the proportional hazards (PH) model are useful in detect- ing outliers or influential points in clinical trials by testing the propor- tional hazards assumption and exploring functional form. Assuming proportional hazards and non-informative censoring, the full likeli- hood approach is used to obtain score and deviance residuals. The first residual is based on the ideas used in obtaining the score-type residuals in partial likelihood approach. The second type of residual is based on the concept of the deviance residuals. We conduct simu- lations and compare the performance of the full likelihood residuals with other common residuals that are based on the partial likelihood approach. In addition, the graphical approaches are used to illustrate the applications of these residuals using some real life examples in clinical trials. gy Methods The web application evaluates twelve different designs: two versions of the 3 + 3 design, accelerated titration design (ATD), biased coin design (BCD), k-in-a-row (KIR) design, two versions of the continual reassessment method (CRM) design, escalation with overdose control (EWOC) design, escalation based on toxicity interval (EBTI) design, the modified toxicity probability interval (mtpi) design, Bayesian opti- mal interval design (BOIN) and T-statistics design. The dfcrm, bcrm and BOIN packages in R software are used for CRM, EWOC, EBTI and BOIN designs. Through simulation studies with a matched sample size, a comprehensive score is used to evaluate the performance of selected adaptive designs with desired parameters as well as differ- ent scenarios. Susan.halabi@duke.edu P82 Application of recent NEJM recommendations for supplementary analyses of positive or failed randomized clinical trials Mark Van Natta, Laura Wilson, Katherine Yates, James Tonascia Johns Hopkins Bloomberg School of Public Health Correspondence: Mark Van Natta Trials 2017, 18(Suppl 1):P82 The cohort multiple randomised controlled trial: methodological lessons from a practical application (the protects trial) 1 2 3 3 Recently, Pocock and Stone (NEJM, 2016) proposed recommenda- tions for reporting the results of randomized clinical trials depending on whether the primary outcome was "positive" and another set of recommendations when the outcome was "failed." We show that these recommendations are difficult to implement in the body of pa- pers for journals with limits on words, tables, and figures (e.g., 2700 words and 5 tables + figures for NEJM) in the main article; however, the recommendations can be effectively implemented in online sup- plementary tables and figures to add value to the reporting of the RCT. Conclusions acid for non-cirrhotic, non-alcoholic steatohepatitis trial (Lancet, 2014) and Nortriptyline for Idiopathic Gastroparesis trial (JAMA, 2013). Our study identified a stable high-pain group with consistent OKS pain component scores between 0 and 14 using the hierarchical clus- ter method. This cut-off point will provide a useful way to identify patients for our trial on post-operative management of chronic pain after TKR in the UK, and we expect that it will be equally useful for other trials focused on patients with chronic pain after knee replace- ment. Further work to better understand the uncertainty around this cut-off point is recommended before adoption. Conclusions Recommendations from Pocock and Stone can be incorporated into the original article’s RCT’s main or supplementary analyses. Background P84 The cohort multiple randomised controlled trial: methodological lessons from a practical application (the protects trial) David Reeves1, Kelly Howells2, Amy Blackmore3, Mark Hann3, Maria Panagioti3, Peter Bower3 1University of Manchester; 2Centre for Primary Care, University of Manchester; 3Manchester, Manchester UK, University of Manchester Correspondence: David Reeves Trials 2017, 18(Suppl 1):P84 Acknowledgement p gy Sheau-Chiann Chen, Yu Shyr Vanderbilt University Medical Center Correspondence: Sheau-Chiann Chen Trials 2017, 18(Suppl 1):P83 This abstract presents independent research funded by the UK NIHR. Sheau-Chiann Chen, Yu Shyr Vanderbilt University Medical Center Correspondence: Sheau-Chiann Chen Trials 2017, 18(Suppl 1):P83 P81 Two new residuals in survival analysis with full likelihood Susan Halabi, Sandipan Dutta 1Duke University Correspondence: Susan Halabi Trials 2017, 18(Suppl 1):P81 Methods h l Controlled Randomised Trials often struggle to recruit and there is interest in innovative trial designs that can more effectively recruit and retain patients and make the trials more efficient and patient- centred. One innovation is the ‘cohort multiple randomised con- trolled trial’ (CMRCT). Under a standard pragmatic RCT (pRCT) all pa- tients are told about the different treatments in the trial arms, including any new treatment, but only half are randomised to that new treatment. The CMRCT design aims to make the trial consent procedure more like standard health care, where people are only asked to consent to treatments they are being offered and are not told about treatments they cannot access. Under this design a sub- stantial cohort of participants is recruited, then followed up at regu- lar time intervals. To trial a new treatment, all eligible participants are identified and a random sample offered the treatment. The remaining eligible patients (those not offered the treatment) The analysis recommendations for ‘positive’ RCTs include: 1) display of both relative and absolute risk of primary outcome; 2) use of 95% confidence intervals; 3) analysis of parts if a composite primary out- come; 4) analysis of secondary outcomes; and 5) subgroup analysis. The recommendations for ‘failed’ RCTs include: 1) display of exact p-value to assess trend; 2) use of 95% confidence intervals; 3) power calculations; 4) subgroup analysis; 5) analysis of secondary outcomes; 6) alternative analyses including covariate adjustment and as-treated analysis; and 7) meta-analysis using external data. Results We show how these recommendations can be implicated in practice using data from two published trials sponsored by the National Insti- tute of Diabetes and Digestive and Kidney Disease (NIDDK), one posi- tive and one failed: Farnesoid X nuclear receptor ligand obeticholic Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 33 of 235 Page 33 of 235 constitute the control arm. These patients are not informed about the trial or about treatments they will not receive. Advocates of the CMRCT design claim significant advantages regarding recruitment, patient centredness, and efficiency. Since the design was first pro- posed a number of patient cohorts and related CMRCT s have been established but very few have yet reported and good evidence for these claims is lacking. We established the CLASSIC cohort of 4,377 patients with long-term conditions and are currently conducting a CMRCT (PROTECTS) of a telephone-based health coaching interven- tion. Split-plot designs: sample size considerations 1 2 Correspondence: Beatriz Goulao Trials 2017, 18(Suppl 1):P85 Correspondence: Beatriz Goulao Trials 2017, 18(Suppl 1):P85 Methods h l In the process of conducting PROTECTS, use of the CMRCT de- sign has raised many methodological and statistical issues so far not addressed in the literature. In this paper we consider these issues, re- port how we tackled them within CLASSIC and PROTECTS, and their implications for the design, conduct and analysis of CMRCTs. Results p Conclusion We have discovered many challenges to the use of the CMRCT de- sign in actual practice. Primary amongst these are issues around power and sample size calculation, and the nature of the treatment effect being estimated, which have not previously received adequate attention. The rate of patient non-consent to treatment is a critical factor in determining sample sizes for both the CMRCT and the host cohort, and also efficiency relative to a pRCT. We have also found that some sampling practices commonly applied in pragmatic trials, when applied to a CMRCT, can result in selection bias and the intro- duction of unintended differences between trial arms. The fixed data collection points that are a feature of CMRCTs can result in high vari- ation in the intervals between measurement and treatment that is less controllable than in more conventional designs and can cause problems in analysis. CMRCT -specific CONSORT guidance may be indicated. Current sample size calculations in S-P RCTs are either non-existent or incomplete. Researchers calculating a sample size for an S-P de- sign should: 1. Indicate explicitly the target differences expected for the cluster and individual-level interventions 2. Base sample size cal- culations on the cluster-level intervention if the target differences ex- pected at both levels are similar 3. Use simulation if a smaller target difference at the individual-level is expected to estimate the number of participants that need to be recruited The S-P design is an effi- cient way to assess two interventions, when one of the interventions needs to be randomised at the cluster-level. Observational versus randomised data: using a fractional factorial RCT to compare results Jennifer Bell1, Rhian M. Daniel2, Pollyanna Hardy1, Peter Brocklehurst3 1University of Oxford; 2London School of Hygiene and Tropical Medicine; 3University of Birmingham Observational versus randomised data: using a fractional factorial RCT to compare results Jennifer Bell1, Rhian M. Daniel2, Pollyanna Hardy1, Peter Brocklehurst3 1University of Oxford; 2London School of Hygiene and Tropical Medicine; 3University of Birmingham Correspondence: Jennifer Bell Trials 2017, 18(Suppl 1):P86 Background The split-plot (S-P) design is historically associated with agriculture studies, but more recently used in healthcare research. The S-P is a complex design that has both cluster randomised and factorial ele- ments, but is distinguished by two levels of randomisation: one at a cluster-level and one at a lower or individual level. In a previous re- view, we identified twelve S-P randomised controlled clinical trials (RCTs). Nine reported a sample size calculation and all 9 based the sample size on the cluster randomisation element ignoring the indi- vidual randomisation element of the S-P design. Background Background In clinical research randomised controlled trials (RCTs) are seen as the ‘gold standard’ for providing evidence to evaluate health care in- terventions. Randomisation can ensure balance of baseline character- istics between the intervention groups. In observational studies there are often systematic differences between groups, typically analysed using regression adjusting for measured confounders. However, pro- pensity scores (PS) are an increasingly used alternative. This analysis uses the unique opportunity provided by a large fractional factorial RCT to investigate whether similar conclusions can be reached from the results of the randomised interventions and their equivalent ob- servational data from the non-randomised interventions within the same RCT. The observational data are analysed using regression ad- justment and propensity scores methods. The RCT investigated was CORONIS, a 2x2x2x2x2 fractional factorial RCT comparing caesarean section techniques on 15,935 women. Methods Methods We used Monte-Carlo simulations to investigate the relationship be- tween the number of clusters (5–45 per arm) with: intra-cluster cor- relation (0.02, 0.06 and 0.1); intervention target differences at the cluster-level and individual-level (0, 7.5, 15 for each); and statistical power in a S-P design for both for the cluster and patient-level inter- ventions. Current simulations assumed no interaction between inter- ventions and a fixed cluster size of 25 but this will be extended to varying levels of interaction and cluster sizes. Simulated data sets were analysed using a mixed-effects model with a random-effect at the cluster level in Stata 14. section te Methods One intervention pair from CORONIS (repair of uterus: exteriorisation vs. Intra-abdominal) is assessed for its effect on the primary outcome, death or maternal infectious morbidity. The results of five analyses are presented: using the randomised intervention: the unadjusted, marginal risk ratio (RR), and using the non-randomised intervention: logistic regression to derive the marginal RR, and an inverse probabil- ity of treatment weighting propensity score (IPTW PS) model to de- rive the marginal RR. The results of these analyses are compared. Results To estimate the optimal sample size for S-P designs and how sample size calculations should be reported. Results We found that power for the cluster and individual-level depended on the intervention target differences expected. If both target differences were similar, a sample size based on the cluster-level intervention leads to an overpowered comparison at the individual –level. The cluster- level power is similar using simulation or applying a standard cluster RCT formula. For a fixed cluster-level target difference, as the ICC in- creased, there was an increase in power to detect target differences at the individual –level due to increased overall sample size. However, if the individual-level target difference was smaller than the cluster-level there was a point at which the sample size and power should be based upon the individual rather than the cluster-level target difference. For example, we observed that when the individual-level target difference is smaller than the cluster-level by 10% or more for an ICC of 0.01, the sample size should be based on the individual-level difference. Conclusion Conclusions The CMRCT research design is an intriguing development that may offer several potential advantages over conventional designs. How- ever, there are many challenges to the use of this design in actual practice. Further research and methodological developments are needed to determine whether, and in which contexts, the design can live up to its initial promise. Background Often randomised controlled trials measure an outcome repeatedly over the study period. An area under the curve (AUC) approach sum- marises serial measurements using a single measure. Missing data can occur at one or more time-points; it is unclear what the optimum method to use is when missing data are present. The aim of this work was to explore different analysis strategies for dealing with missing data for an AUC outcome in the ethos trial that compared two surgical interventions, stapled haemorrhoidopexy (SH) and traditional haemor- rhoidectomy (TH) for treating haemorrhoids. An AUC approach was used because it was hypothesised that the interventions would have different recovery trajectories with respect to quality of life. h d As anticipated, results varied depending on the true underlying tox- icity profile. Under the conventional assumption that toxicity grad- ually increases with increasing dose level, all non-standard designs had higher MTD and overall selection rates than the standard 3 + 3 design. However, the non-standard design with the highest MTD se- lection rates, the CRM, was too aggressive and over-estimated the true MTD most frequently, resulting in the lowest overall selection rates among the non-standard designs. Overall selection rates for the CCD, BOIN, mtpi, and mtpi2 were all within two percentage points of one another and consideration of logistical complexities as well as design familiarity in choosing among these is reasonable. For a fairly constant toxicity profile that is safe, all non-standard designs resulted in higher MTD and overall selection rates than the standard 3 + 3 de- sign. The CRM appeared most aggressive in escalating and recom- mending the highest dose level which resulted in higher overall selection rates, followed in order by BOIN, CCD/mtpi, and mtpi2. With a jump in toxicities between adjacent dose levels, the standard 3 + 3 design more often recognized when the MTD had been exceeded compared to the non-standard designs; non-standard designs too frequently over-estimated the MTD, resulting in overall success rates that were either similar to or lower than the overall success rate when using the standard 3 + 3 design. Thus, the conservative nature of the standard 3 + 3 design was preferable under these scenarios. rive the m Results The unadjusted analysis of the randomised interventions provides no evidence of a difference in effect of repair method on the primary outcome (RR 0.94, 95% CI (0.80 to 1.11), SE = 0.08, n = 5925). Regres- sion analysis on the non-randomised interventions adjusting for mea- sured confounding shows similar results to that of the randomised interventions, but is less precise (RR 1.01, 95% CI (0.72 to 1.44), SE = 0.18, n = 5894) and very similar to the IPTW PS analysis (RR 0.98, 95% CI (0.70 to 1.39), SE = 0.18, n = 5925). Trials 2017, 18(Suppl 1):200 Page 34 of 235 Trials 2017, 18(Suppl 1):200 Page 34 of 235 A comparison of methods to handle missing data in the analysis of an area under the curve outcome A comparison of methods to handle missing data in the analysis of an area under the curve outcome Jemma Hudson1, Jessica Wood1, Angus Watson2, Jonathan Cook3 1University of Aberdeen; 2NHS Highland; 3University of Oxford Trials 2017, 18(Suppl 1):P87 Six Phase I designs (3 + 3, CCD, BOIN, mtpi, mtpi2, and CRM), each followed by a standard Phase II (Simon’s optimal 2-stage) and Phase III (1:1 randomized group sequential with two interim analyses) study were implemented. Dose limiting toxicity and response data were as- sumed binomially distributed, and survival data exponentially distrib- uted. Eight toxicity profiles representing gradually increasing toxicity across dose levels, fairly constant toxicity that is considered safe, and a jump in toxicity between two adjacent dose levels were each eval- uated with a linear response/survival profile using 4000 simulations. Results Jemma Hudson1, Jessica Wood1, Angus Watson2, Jonathan Cook3 1University of Aberdeen; 2NHS Highland; 3University of Oxford Trials 2017, 18(Suppl 1):P87 Methods A comparison of methods to handle missing data in the analysis of an area under the curve outcome Methods The primary outcome in ethos was health related quality of life mea- sured using the EQ-5D over a 24-month follow-up period (baseline, 1, 3 and 6 weeks post-surgery and 12 and 24 months post-randomisation). The AUC was derived using the trapezoidal rule. There was a substantial amount of participants with missing data (30%). The analysis strategies were; 1) complete-case analysis (defined as data available at each time- point); 2) minimal data analysis (including all participants with at least one shorter-term and one longer-term follow-up measure); 3) last ob- servation carried forward (LOCF); 4) simple imputation (intervention group mean at that time-point); and 5) multiple imputation (MI). We used linear regression with adjustment for design variables in Stata 14. Simulations will be carried out to assess the statistical properties of each methods for a range of follow-up profiles and missing data patterns. Seven hundred seventy-seven participants were randomised to SH (389) or TH (388). Analysis of 570 participants with minimal data favoured TH: mean difference in AUC −0.073 95% CI (−0.140, −0.006); p = 0.034. The complete case (N = 400: −0.057 95% CI (−0.113, −0.001); p = 0.046), simple imputation (N = 774: 0.054 95% CI (−0.089, −0.019); p = 0.004) and MI (N = 774: −0.054 95% CI (−0.107, −0.000); p = 0.049) analyses were similar. The LOCF analysis was not consistent with other approaches (N = 774: 0.025 95% CI (−0.058, 0.109); p = 0.538). Conclusion Extending the two-stage patient preference design for binary outcomes with stratification Background The standard 3 + 3 Phase I design remains the most widely used Phase I design in practice, despite an increasing number of both rule-based and model-based designs that range in complexity, but generally outperform the standard (Yuan et al., 2016). Few have inte- grated common measures of Phase I design performance such as correct selection of the maximum tolerated dose (MTD), average number of dose-limiting toxicities and average number of patients treated above the MTD with a measure of overall success. We aimed to characterize the ability to recognize a safe and efficacious drug by the end of Phase III using six Phase I clinical trial designs, each followed by a standard Phase II and Phase III design. Methods The results from the analysis on the non-randomised interventions il- lustrate the importance of adjusting for confounders when analysing observational data. Results based on regression adjustment and pro- pensity score analysis are comparable, but the latter has the added benefit of greater transparency when assessing balance of the base- line characteristics between the groups. It is possible for confounding to be controlled for using standard regression adjustment or IPTW propensity scores, though this may depend on data quality, which this study benefited from. Extending the two-stage patient preference design for binary outcomes with stratification The complete-case, minimal data, simple imputation and MI analyses were in broad agreement, but LOCF was not. This was because SH had a shorter recovery compared to TH, using LOCF to impute the longer-term missing outcome biased estimates. LOCF should not be used to impute missing data for an AUC outcome when interven- tions have potentially different recovery trajectories. Denise Esserman, Briana Cameron, Peter Peduzzi Yale University Denise Esserman Trials 2017, 18(Suppl 1):P90 Denise Esserman, Briana Cameron, Peter Peduzzi Denise Esserman, Briana Cameron, Peter Peduzzi Yale University Denise Esserman Trials 2017, 18(Suppl 1):P90 Patient preference plays a role in clinical practice, and is at the heart of patient-centered outcomes research, thus ignoring the impact on outcomes would be unrealistic. Furthermore, a patient may have a different psychological response to a treatment he/she deems more favorable. Patient preference can have a substantial impact on a study’s outcome, particularly when it is not feasible to conduct a blinded study. While the completely randomized design, the trad- itional clinical trial setting where individuals or clusters are randomly allocated to one of multiple treatment groups, is the gold standard for assessing a treatment effect (the average effect a particular Background In short, selection of a Phase I design should be based on the under- lying toxicity profile that is anticipated, with appropriate safeguards, and should consider the MTD selection rate in conjunction with over-dosing and under-dosing errors that influence the overall selec- tion rate of a favorable drug. P88 P88 Overall success rate of a safe and efficacious drug: results using six phase I designs, each followed by phase II and III trials using standard designs Amy Ruppert, Abigail B. Shoben The Ohio State University Correspondence: Amy Ruppert Trials 2017, 18(Suppl 1):P88 Overall success rate of a safe and efficacious drug: results using six phase I designs, each followed by phase II and III trials using standard designs standard designs Amy Ruppert, Abigail B. Shoben The Ohio State University Correspondence: Amy Ruppert Trials 2017, 18(Suppl 1):P88 Page 35 of 235 Page 35 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 35 of 235 evidence of efficacy, while observational data provides evidence of effectiveness (Piantadosi 1997). It is not surprising that the overall ef- fectiveness of a treatment would be attenuated when used in the general population. The discrepancy between the unadjusted and adjusted findings highlight the importance of developing standards for evaluating quality of observational data analyses to ensure proper control of confounding. We are currently developing a software as- sistant that will assist in standardization of studies using linked SEER- Medicare data. Recently published trial findings have similar compari- sons with observational data. Hamdy et al. (10-Year Outcomes after Monitoring, Surgery, or Radiotherapy for Localized Prostate Cancer, NEJM, Advance release) found no statistically significant differences in mortality among men with localized prostate cancer randomized to monitoring, surgery, or radiotherapy. However, fewer than 1.2% of men in all groups died from prostate cancer at 10 years. The point estimates were similar to a SEER-Medicare analysis (Wong et al. Sur- vival Associated with Treatment vs Observation of Localized Prostate Cancer in Elderly Men JAMA 2006). Unlike the randomized trial, the observational data findings were statistically significant, perhaps be- cause the sample size was over 50 times larger. Conclusion treatment will have in a specified population), this design ignores the role patient preference may have on study outcomes; they are not estimable in this design. The two-stage trial design first proposed by Rucker, also known as the doubly randomized preference trial, enables researchers to disentangle the treatment effect from those effects resulting from choosing a treatment. , Methods We identified women diagnosed with node positive breast cancer treated by mastectomy from 1990 to 2008 in the SEER database. We examined the effectiveness of radiotherapy on survival outcomes using adjusted Poisson regressions, confirmed by Cox or Fine & Grey competing risk regressions. We compared our findings with those re- ported in a meta-analysis of clinical trials as reported in the Journal of Clinical Oncology. Decomposing the effects of the study population on null results: the look ahead trial Peter Kaufmann University of Colorado College of Nursing Trials 2017, 18(Suppl 1):P92 Decomposing the effects of the study population on null results: the look ahead trial Peter Kaufmann University of Colorado College of Nursing Trials 2017, 18(Suppl 1):P92 Peter Kaufmann Obesity continues to be a major risk factor for diabetes and is esti- mated to cause 365,000 deaths annually in the US, thus placing it as the third cause of death after Heart Disease and Malignant Neo- plasms. The Look AHEAD trial sought to determine whether the highly successful lifestyle intervention employed in the Diabetes Pre- vention Program to reduce incident diabetes among pre-diabetic in- dividuals is effective in reducing the incidence of major acute cardiovascular events. The Look AHEAD trial randomly assigned 5,145 overweight or obese individuals with type 2 diabetes to either a control intervention consisting of Diabetes Support and Education (DSE) or an intensive lifestyle intervention (ILI) consisting of a weight loss program and increased physical activity. The six-month intensive intervention was tapered to a maintenance phase for the remainder of the trial, which had a maximum follow-up of 13.5 years and 90% statistical power to detect an 18% reduction in cardiovascular dis- ease. After the trail was launched, the event rate in the control arm for the primary outcome measure (a composite of death from cardio- vascular causes, non-fatal MI and non-fatal stroke) was observed to be only 0.7%, per year, much lower than the design event rate of 3.125% per year. In spite of an adjustment to the primary outcome by adding hospitalization for angina as defined by American Heart Association criteria, and extending the design follow-up from 10.5 to 13.5 years, the trial was stopped for futility after 11 years maximum follow-up. Trials 2017, 18(Suppl 1):P91 Results The published meta-analysis found that radiotherapy had a protect- ive effect on breast cancer specific survival (Rate Ratio [RR] = 0.84, 95% Confidence Interval [CI] 0.76 to 0.94), and overall survival (RR = 0.89, 95% CI 0.81-0.97). In the SEER data, we found that the un- adjusted effect of radiotherapy on breast cancer specific death was harmful, RR = 1.29 (95% CI 1.25-1.33), but that the adjusted effect was beneficial, RR = 0.91 (95% CI 0.88-0.94). The unadjusted effect of radiotherapy on overall survival was RR = 1.08 (95% CI 1.05-1.11), while the adjusted effect was 0.87 (95% CI 0.84-0.89). Adjustment for the number of positive nodes had a primary confounder impact re- sponsible for the qualitative discrepancy between the unadjusted and adjusted findings. In an effort to understand the null results, we examined the baseline characteristics of the enrolled trial participants and compared them to baseline characteristics of participants in other contemporary major clinical trials in diabetes. Look AHEAD participants were found to be of younger age, with a higher proportion of females, a lower proportion of history of cardiovascular disease, and shorter duration of diabetes that those enrolled in comparable clinical trials. The pro- file suggests a much lower risk for cardiovascular events than had been anticipated, approaching a floor effect for benefit of an inter- vention. The Look AHEAD experience has considerable implications not only for estimating event rates for the purpose of sample size calculations but for methods of managing safety concerns, screening for eligibility as well as monitoring the characteristics of enrolled par- ticipants in real time. These factors will be discussed in the context of other clinical trials with null results. P88 While the use of the two-stage trial design is becoming more prevalent, especially as the emphasis for use of decision aids continues to grow and the number of trials testing behavioral interventions increases, there is still a large gap in the methods available to design and ultimately analyze these trials. Often the primary outcome of interest is not measured on a continuous scale; typically, binary outcomes (e.g. Are patients satis- fied with their treatment) are used. In addition, we are often inter- ested in accounting for important covariates (e.g. Age, gender, and/ or type of insurance coverage) that may have an impact on the out- come of interest, or may influence the preference rate (e.g. Men may have a stronger preference for a surgical intervention, while woman may have a stronger preference for a medical intervention). Cur- rently, no methods exist to accommodate these scenarios. We present our extensions of the two-stage clinical trial design for sam- ple size determination and analysis for binary outcomes with stratifi- cation and give closed form sample size formulas. evidence of efficacy, while observational data provides evidence of effectiveness (Piantadosi 1997). It is not surprising that the overall ef- fectiveness of a treatment would be attenuated when used in the general population. The discrepancy between the unadjusted and adjusted findings highlight the importance of developing standards for evaluating quality of observational data analyses to ensure proper control of confounding. We are currently developing a software as- sistant that will assist in standardization of studies using linked SEER- Medicare data. Recently published trial findings have similar compari- sons with observational data. Hamdy et al. (10-Year Outcomes after Monitoring, Surgery, or Radiotherapy for Localized Prostate Cancer, NEJM, Advance release) found no statistically significant differences in mortality among men with localized prostate cancer randomized to monitoring, surgery, or radiotherapy. However, fewer than 1.2% of men in all groups died from prostate cancer at 10 years. The point estimates were similar to a SEER-Medicare analysis (Wong et al. Sur- vival Associated with Treatment vs Observation of Localized Prostate Cancer in Elderly Men JAMA 2006). Unlike the randomized trial, the observational data findings were statistically significant, perhaps be- cause the sample size was over 50 times larger. Conclusion Using properly analyzed observational data can provide generalizable effectiveness information in a non-trial population. Background ll l Well-run clinical trials represent the gold standard for assessing effi- cacy of interventions. Using observational (i.e. Non-randomized) data to determine treatment efficacy requires statistical adjustment and often untestable assumptions. P88 P91 Pragmatic verification of cancer clinical trial efficacy findings using the surveillance epidemiology and end results (SEER) database Brian Egleston1, Elin Sigurdson2, Elizabeth A. Handorf2, Eric A. Ross2, Slobodan Vucetic3, Yu-Ning Wong2, Richard J. Bleicher2 1Temple University Health System; 2Fox Chase Cancer Center; 3Temple University Correspondence: Brian Egleston Trials 2017, 18(Suppl 1):P91 Objective We determined the degree to which SEER (a large generalized co- hort) could replicate findings from the Early Breast Cancer Trialists' Collaborative Group Meta-Analyses of Randomized Trials (a highly se- lective cohort). This was in part motivated by a research letter on the subject (McGale et al. Can Observational Data Replace Randomized Trials, Journal of Clinical Oncology 2016, 34(27):3355–3356). Background Permuted Block Randomisation (PBR) was first suggested in 1952 by Austin Bradford Hill [1] to restrict the possible degree of imbalance that might occur in a stratified randomised trial. In the following sixty years, several alternative methods have been proposed and well- studied as regards the trade-off between imbalance control and pre- dictability. Such methods include - but are not limited to - the class of biased coin designs, the big stick design, the class of urn designs, the maximal procedure and the block urn design, almost all of which have shown to have lower predictability than their permuted block counterparts. Despite the developments, recent review articles have found that individually-randomised trials performing stratified ran- domisation (using randomisation within mutually-exclusive strata, rather than covariate-adaptive randomisation methods) almost exclu- sively use PBR to generate the allocation sequence. In addition, a re- cent article on the subject [2] stated that ‘there is no argument in the literature to suggest that the permuted block design is better than or even as good as the [Maximum Tolerable Imbalance] procedures’. The question must be asked as to why statisticians continue to recommend the use of the inferior PBR method over any other that has been shown to be better at reducing the risk of selection bias. M th d y We will present best practices for implementation of sex as a bio- logical variable in the various phases of a clinical trial, and will in- clude examples from the National Institute on Drug Abuse (NIDA)- funded grants. The first step in implementation involves a rigorous literature review that would explain how sex may influence the study design based on previous preclinical or clinical research. If there is a reasonable foundation of existing research, sex-specific hypotheses could be generated, including primary, secondary or exploratory hy- potheses. The next step entails developing a study design, which should include a statistical analysis plan that provides for subgroup analyses identifying differences in the intervention effect by sex. Such subgroup analyses should be implemented using interaction models to test whether the treatment effect differs across sexes, or an analogous approach if there is no modeling. Recognizing that most clinical trials are not powered to detect differences based on gender, these analyses are exploratory in nature. In progress reports and publications, the study findings should include whether sex dif- ferences were, or were not, detected. j Discussion j g Discussion Although our adjusted SEER estimates were less beneficial in magni- tude than the clinical trial estimates, they were consistent in direc- tion and significance. Of note, clinical trial data often provides Page 36 of 235 Trials 2017, 18(Suppl 1):200 Page 36 of 235 Page 36 of 235 In January 2016, a new National Institutes of Health (NIH) policy went into effect requiring that researchers consider sex as a biological vari- able in animal and human studies (see NOT-OT-15-102 Guidance). This requires grant applicants of NIH-funded research studies to ex- plain how sex will be factored into research design, data analyses and reporting, requiring strong justification for studying only one sex. The policy is meant to increase the quality and generalizability of biomedical research, thus enhancing the reproducibility and trans- latability of research in the biomedical field. In January 2016, a new National Institutes of Health (NIH) policy went into effect requiring that researchers consider sex as a biological vari- able in animal and human studies (see NOT-OT-15-102 Guidance). This requires grant applicants of NIH-funded research studies to ex- plain how sex will be factored into research design, data analyses and reporting, requiring strong justification for studying only one sex. The policy is meant to increase the quality and generalizability of biomedical research, thus enhancing the reproducibility and trans- latability of research in the biomedical field. q Results Justifications given by statisticians for using PBR and varying-size PBR in- cluded questions around the motivation of clinicians to subvert the ran- domisation sequence, ability to correctly implement the alternative methods, a belief that weaknesses of fixed-size permuted blocks are over- come by use of randomly-varying block sizes and that alternative methods do not yield sufficient reductions in predictability to justify their use. Conclusions Phase I clinical trials are typically set up to establish the safety of a proposed drug, study the pharmacokinetics and pharmacodynamics of this drug and to identify a dose which is suitable for taking for- ward to a further trial. Good design of Phase I studies is often chal- lenging, due to limited evidence to inform study protocols. The traditional approach for Phase I trials uses prespecified rules to assign patients to dose levels and choose the recommended dose for the next study, typically the ‘3 + 3’ design or variations. However, while easy to implement, the operating characteristics of rule-based de- signs tend to be unattractive. Not only can such designs lead to poor decision making regarding the future investigation of the drug, but they may also expose unnecessary numbers of participants to in- appropriate doses. Adaptive designs, such as the Continual Reassess- ment Method (CRM), which seek to model a dose–response curve using all available information offer an alternative, and can be con- veniently carried out within a Bayesian framework. These model- based designs are now well-established in cancer, but much less so in other clinical areas. Since statisticians make recommendations about appropriate methods of allocation sequence generation, take up of better randomisation methods depends on educating statisticians as to existence of alterna- tive methods, that they can be readily implemented, and that the con- vergence strategy is as big a risk of selection bias as the ‘perfect prediction strategy’, if not moreso. P93 What barriers exist to statisticians recommending alternative methods to permuted block randomisation? Colin Everett University of Leeds Trials 2017, 18(Suppl 1):P93 References [1] Hill AB. The Clinical Trial. N Engl J Med 1952; 247:113–119. [2] Berger VW, Bejleric K, Agnor R. Comparing MTI randomization procedures to blocked randomization. Statistics in Medicine. Statist. Med. 2016, 35 685–694. doi: 10.1002/sim.6637 P94 Considering sex as a biological variable in research design, data analyses and reporting Eve Jelstrom1, Abigail G. Matthews1, Dikla Shmueli-Blumberg1, Robert Lindblad1, Paul Van Veldhuisen1, Carmen Rosa2 1The EMMES Corporation; 2The National Institute on Drug Abuse (NIDA) Correspondence: Eve Jelstrom Trials 2017, 18(Suppl 1):P94 Background Grantees are also required to report annually on the enrollment of males and females so that this can be monitored throughout the implementation of the trial. Presentations were given to statisticians at the Leeds Institute for Clin- ical Trials Research. In one, presented to an unscientifically-selected sample of statisticians, two alternatives were introduced in a scenario where members of independent oversight committees objected to use of Varying-size PBR in an open-label randomised trial, requesting justifi- cation for not using either of the suggested alternatives. (Soares and Wu’s Big Stick Method and Zhao and Weng’s Block Urn Design) In the second - open to all statisticians - any alternatives were presented in a meeting, and statisticians were invited to discuss reasons for current PBR use and invited to discuss justifications either to continue using PBR, or to use other methods in trials involving restricted randomisa- tion sequences within mutually-exclusive strata. This new policy guides researchers to take into account whether there are biological factors related to sex which should be explored in the study design or approached differently than originally planned. The NIH policy will have an impact on the planning and the conduct of clinical trials in humans and potentially new rela- tionships between disease entities and sex will be explored and given more weight. 96 Developing a Bayesian adaptive design for a phase I clinical trial: a case study for a novel HIV treatment Alexina Mason1, Alan Winston2, Deborah Ashby2 1London School of Hygiene and Tropical Medicine; 2Imperial College London Correspondence: Alexina Mason Trials 2017, 18(Suppl 1):P96 Tradeoffs of a randomize, then consent approach to improving cluster participation rates in cluster randomize trials Abigail Shoben , cluster participation rates Abigail Shoben The Ohio State University Trials 2017, 18(Suppl 1):P97 Cluster randomized trials (CRTs) are increasingly being used in public health research to test interventions that are delivered at a cluster level (e.g., in schools, hospitals, clinics, etc.). From a statistical per- spective, the ideal for these trials is first to consent (recruit) partici- pating clusters, then obtain consent from individuals participating from each cluster (if needed), and finally randomize each cluster to a treatment arm. This ideal minimizes the possibility of bias in the esti- mated treatment effect and preserves the internal validity of the trial. However, this ideal requires that clusters agree to study participation and randomization, which in practice may be difficult and instead re- sult in clusters simply choosing not to participate in the study. Such nonparticipation by clusters may limit the external validity of study findings. Motivated by a school-based trial of physical activity promo- tion programs in Ohio Appalachia, we discuss strengths and weak- ness of an alternative approach where clusters are randomized first and then approached for consent to study participation. This ap- proach has the potential to improve cluster consent rates, but risks bias due to differential participation rates by factors that may also in- fluence the outcome. By providing a framework for when this bias may occur and its potential magnitude, we provide guidance for fu- ture studies about the statistical tradeoffs between (1) the traditional consent then randomize approach and (2) the alternative randomize then consent approach under various assumptions about cluster par- ticipation rates and factors influencing a cluster’s decision to consent. P99 Biomarker-guided clinical trial designs: sample size calculations with survival endpoints Miranta Antoniou, Ruwanthi Kolamunnage-Dona, Andrea L Jorgensen Department of Biostatistics, Institute of Translational Medicine, University of Liverpool & MRC North West Hub For Trials Methodology Research Correspondence: Miranta Antoniou Trials 2017, 18(Suppl 1):P99 Biomarker-guided treatment is a rapidly developing area of medicine, where treatment choice is personalised according to one or more of an individual’s biomarker measurements. A number of biomarker- guided trial designs have been proposed in the past decade, includ- ing both adaptive and non-adaptive trial designs which test the ef- fectiveness of a biomarker-guided approach to treatment with the aim of improving patient health. Considering sex as a biological variable in research design, data analyses and reporting A phase I study to assess the safety, pharmacokinetic profile and antiretroviral efficacy of C34-PEG4-Chol, a novel once-weekly pep- tide fusion inhibitor for the treatment of HIV-infection, was set-up with MRC funding. During the study work up, Bayesian adaptive de- signs based on the CRM were compared with a more standard rule-based design using simulation studies based on seven test Page 37 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 37 of 235 While randomised controlled trials remain the gold standard for the evaluation of new therapies, the development of targeted therapies, disease monitoring, and the concomitant increase in stratification of patients presents new challenges in trial design. Increasingly, patient eligibility becomes dependent on on variables which are known after trial enrolment. Especially in conditions such as haematological ma- lignancies, where delays in therapy can prove costly, treatment mo- dalities may need to be amended in real time to take into account molecular or genetic factors, where the test result is only available some days after the start of treatment, or a patient’s minimal residual disease status, again only available at the start of the next course of treatment. It is therefore imperative to be able to direct therapy ap- propriately, and allow prompt randomisation to the correct targeted therapy. In the context of Acute Myeloid Leukaemia, there are a number of potential targeted therapies which would typically start after a 7–10 day course of chemotherapy. To avoid the dilution of re- sults from patients not suitable for the therapy, randomisation needs to take place once eligibility is known, but before targeted treat- ment is due to start. Similarly, in an evaluation of minimal residual disease monitoring, only those patients with a suitable target should be randomised between monitor and no monitor. There is therefore a need to ensure communication between clinicians, trials office and the different accredited laboratories so that prompt and appropriate randomisation takes place, allowing patients access to an appropriate targeted therapy, and giving maximal power to de- tect treatment differences. We present a generalised approach to managing such trials, based upon an integrated computer system, automated notification emails, and monitoring of take-up rates. The approach is flexible enough to allow for several different targeted therapies given in addition to chemotherapy in a factorial design, real-time risk adapted therapy options, and the evaluation of the benefits of sequential disease monitoring using digital PCR. Considering sex as a biological variable in research design, data analyses and reporting The approach builds upon the AML15 trial, which was an early rando- mised evaluation of targeted therapy, and has been successfully used with a network of over 100 sites in the UK, Europe, Australia and New Zealand. scenarios, with the aim of choosing a design that would maximise the scientific information gained from the study [1]. A dose- inefficacy curve rather than the more usual dose-toxicity curve was modelled. In determining the implementation details, five key ques- tions were addressed: 1) how is the endpoint defined? 2) how will the target dose be identified? 3) what are plausible scenarios? 4) which drug doses should be available? And 5) what cohort sizes are practical? While the results showed no optimal design for all cir- cumstances, the trial team concluded there were clinical advan- tages in choosing an adaptive design over the originally proposed rule-based design. The process of specifying and evaluating the design options was time-consuming, and required the active involvement of all members of the trial’s protocol development team. However, the effort was worthwhile as the originally proposed rule-based design was re- placed by a more efficient Bayesian adaptive design. While the out- come to be modelled, design details and evaluation criteria are trial specific, the principles behind their selection are general. This case study illustrates the steps required to establish a design in a novel context. Reference 1. Mason AJ, Gonzalez-Maffe J, Quinn K, Doyle N, Legg K, Norsworthy P, Trevelion R, Winston A and Ashby D. Developing a Bayesian adaptive design for a Phase I clinical trial: a case study for a novel HIV treatment. Stats Med in press P100 A systematic mapping review of methods used for the evaluation of implementation fidelity in primary care trials Rebecca Barnes, Catherine Jameson, Alyson Huntley, Cindy Mann, Alison Heawood, Athene Lane University of Bristol Correspondence: Rebecca Barnes Trials 2017, 18(Suppl 1):P100 Background The feasibility and effectiveness of novel procedures for improving viability of organs for transplantation is a growing area of research and assessing the various outcomes of these procedures is vital to understanding the overall benefit relative to current practices. Under- standing the implications of reducing discard rates, particularly in kidney and liver transplantation, would aid in the development of more practical analytical frameworks for assessment of these novel procedures. p Methods The following databases were searched: MEDLINE via PubMed, EMBASE, the Cochrane Central Register of Controlled Trials (CEN- TRAL), the Transplant Library and the National Institute of Health Clinical Trials database. Reference lists were also hand searched for other relevant trials/reviews. Research focusing on Kidney or Liver transplantation was reviewed. To be included in the review, the re- search results needed to specify the status of and/or analytical as- sessment of discard rates. Extracted information from studies meeting the inclusion criteria included: author, year, title, organisa- tion, country, study type, organ type, donor type, categorisation of reference to discarded organs within research and outcomes re- ported. The quality of the studies included was assessed using the Cochrane Risk of Bias Tool; for assessing the quality of reviews, the ROBIS bias assessment tool was applied. Tradeoffs of a randomize, then consent approach to improving cluster participation rates in cluster randomize trials Abigail Shoben A better understanding of them is needed as challenges occur in terms of trial design, analysis and practical application, including the control of the false-positive rate, power of the study, prevalence of the biomarker, treatment effect es- timation and the potential increases in cost and time. We have undertaken a comprehensive literature review based on an in-depth search strategy with a view to providing the research community with clarity in definition, methodology and terminology of the vari- ous reported biomarker-guided trial designs from a total of 211 in- cluded papers. Of these 211 included papers, 107 papers related to biomarker-guided adaptive trial designs were reviewed in our pub- lished paper Antoniou et al. (2016) [1]; biomarker-guided non- adaptive trial designs were referred to in 100 papers and are reviewed in our more recent paper to be published shortly. Navigating the literature to gain an understanding of which trial de- sign to implement in a given situation, and the practical implications P98 A system for real-time integration of laboratory data into trials of targeted therapy with applications to acute myeloid leukaemia Sophie Betteridge1, Robert Kitching1, Helen Clark1, Sarah Burns1, Robert K. Hills1, Nigel Russell2, Sylvie Freeman3, David Grimwade4 1Cardiff University; 2University of Nottingham; 3University of Birmingham; 4King’s College Correspondence: Sophie Betteridge Trials 2017, 18(Suppl 1):P98 Navigating the literature to gain an understanding of which trial de- sign to implement in a given situation, and the practical implications Page 38 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 38 of 235 Page 38 of 235 of doing so is difficult as our reviews revealed. Hence, in order to im- prove the understanding of the biomarker-guided trial designs and provide valuable and much-needed guidance on their implementa- tion we are developing a user-friendly online tool (www.bigted.org) informed by our literature review. Bigted will provide an easily ac- cessible resource to inform on the most optimal design when embarking on a biomarker-guided trial including easy to navigate graphical displays of the various trial designs. Knowledge on how to design, implement and analyse these trials is essential for testing the effectiveness of a biomarker-guided approach to treatment. Hence, in this study, we focus on key statistical aspects of several of the identified trial designs with particular focus on examining the sample size requirement under different settings where outcome is time-to- event. Background g Implementation fidelity has been described as the extent the intervention-as-delivered matched the intervention-as-planned. The primary goal is to increase scientific confidence that an intervention under evaluation has been adequately tested and that the measured outcomes are a reliable indication of its effectiveness. This decreases the likelihood of incorrectly accepting the null hypothesis as a result of inadequate implementation, and of potentially effective interven- tions being discarded. Under-evaluation or under-reporting of fidelity can also make it difficult to replicate an intervention, and to be sure the outcomes could be reproduced. Primary care may be particularly vulnerable to low implementation fidelity due to a tendency towards pragmatic trials of complex interventions being delivered by multiple implementers across multiple settings. Although important reasons exist for why we should invest in high quality evaluations of imple- mentation fidelity, there is little guidance available, and little evi- dence for which might be the optimal method in various contexts. The aim of this study was to conduct a systematic mapping review of methods currently in use for the assessment of implementation fi- delity across primary care trials. p Objective j The primary objective of this systematic literature review is to iden- tify research which assesses the clinical and economic impact of kid- ney and liver discard rates in transplantation research in order to understand how kidney or liver discard rates impacts on the risk of disease progression and mortality as well as cost of care. Methods Tradeoffs of a randomize, then consent approach to improving cluster participation rates in cluster randomize trials Abigail Shoben To achieve this, we applied statistical simulation methods and here we report on our findings. extraction will be performed by one reviewer and checked by a second. Data extraction will include information on study type, the nature of the planned intervention, the extent of implementation fidelity assess- ment including methods of data collection and analysis, and level of in- tegration into outcomes evaluation. Preliminary results suggest that implementation fidelity has been under-evaluated and under-reported however these results will be refined through the formal mapping. Discussion of doing so is difficult as our reviews revealed. Hence, in order to im- prove the understanding of the biomarker-guided trial designs and provide valuable and much-needed guidance on their implementa- tion we are developing a user-friendly online tool (www.bigted.org) informed by our literature review. Bigted will provide an easily ac- cessible resource to inform on the most optimal design when embarking on a biomarker-guided trial including easy to navigate graphical displays of the various trial designs. Knowledge on how to design, implement and analyse these trials is essential for testing the effectiveness of a biomarker-guided approach to treatment. Hence, in this study, we focus on key statistical aspects of several of the identified trial designs with particular focus on examining the sample size requirement under different settings where outcome is time-to- event. To achieve this, we applied statistical simulation methods and here we report on our findings. In light of the evidence produced by our review, we will share our recommendations for practical steps towards high quality evaluation of implementation fidelity in the design of future primary care trials. We will also discuss the strengths and limitations of methodological reviews of trial conduct and how the quality of implementation fidel- ity evaluation might be more formally appraised going forward. Reference P101 A systematic review of analytical methods applied for discarded organs in kidney and liver transplantation research Richéal Burns, Peter Skerritt, Simon Knight, David Nasralla, Ally Bradley, Virginia Chiocchia, Rutger Ploeg, Peter Friend University of Oxford Correspondence: Richéal Burns Trials 2017, 18(Suppl 1):P101 1. Antoniou M, Jorgensen AL, Kolamunnage-Dona R. Biomarker-Guided Adaptive Trial Designs in Phase II and Phase III: A Methodological Review. Plos ONE. 2016;11(2):e0149803. doi:10.1371/journal.pone.0149803. P102 Outcome measurement in paediatric proton beam radiation therapy studies: is greater standardisation needed? Caroline Main1, Simon P. Stevens1, Roger E. Taylor2, Barry Pizer3, Nick Thorp4, Keith Wheatley1, Pamela R. Kearns5, Robert Phillips6, Martin English5, Sophie Wilne7 1University of Birmingham; 2Swansea University; 3Alder Hey Children's NHS Foundation Trust; 4The Clatterbridge Cancer Centre; 5Birmingham Children's Hospital NHS Foundation Trust; 6Centre for Reviews and Dissemination; 7Queen's Medical Centre Correspondence: Caroline Main Trials 2017, 18(Suppl 1):P102 y Methods The purpose of this study is to verify the robustness of Bayesian NMA with respect to different imputation strategies through simulations. Fifty trials are simulated in full databases by including baseline, follow-up and Delta variation in- formation. Baseline data are obtained by sampling from bounded 0– 100 normal distributions (X ~ N(41.8,21.5)) (Cannon, 2000), to mimick the support of WOMAC score. Delta variation data are simulated from normal distributions with parameters provided by a review about 6 FANS treatments. Follow up variability data are provided from gener- ated Delta and baseline variability measures setting hypotheses on pre-post correlation and considering, in each scenario, a sequence from 0.3 to 0.95 by 0.05. Sample size are obtained by sampling from an uniform 50–100 distribution. Between trial heterogeneity has been included as a variability measure by following, for each simula- tion setting, a sequence from 0.1 to 5 by 0.1. Each scenario provides different combinations of heterogeneity between trials and pre-post correlation creating 700 scenarios. For each scenario 2 imputed data- bases are generated. In the first case, information about Delta vari- ation are randomly removed, from full database, leaving only baseline end follow up data, then variability of mean change is im- puted using the correlation method. In the other case, also informa- tion at baseline and follow up are removed, then Delta variability isimputed with maximum standard deviation method. On simulated dataset, NMA, with random effect and Uniform (0,5) prior on hetero- geneity parameter, has been performed (MCMC method, 200000 iter- ations, 4 chains). To investigate robustness of NMA, under several scenarios and different imputation methods, the bias of rank prob- abilities estimates has been computed in order to check models per- formance in ranking treatments. For each scenario, the mean, bias and the standard deviation of the first rank probability, for full and imputed databases, have been computed. The results show that the bias is very small for every scenario, then ranking provided by models is robust with respect to different imputation methods. The method is more robust to imputation in a low heterogeneity frame- work, especially if considered trials are conducted on similar popula- tion. Small bias is observed also for heterogeneity values similar to expectation of NMA heterogeneity prior, indicating more robustness if a priori knowledge is well specified. y Methods A search strategy was developed and agreed with input from all members of the research team, two information specialists, represen- tatives from a local group of trial managers and the extant literature. The databases tested for citations were Medline®, Excerpta Medica Database (Embase), and Cumulative Index to Nursing and Allied Health Literature (CINAHL®). For each database, search terms were adapted according to the search capabilities of that database. To be included in the review, studies or trial protocols had to have been published in the last 10 years and report on any primary care inter- vention undertaken in a general practice setting in the context of a complex effectiveness trial. This included full trials, feasibility studies and/or pilot RCTs. Studies had to state in title or abstract that they had included assessment or reported on implementation fidelity. The initial searches resulted in 6253 citations across the three databases. Electronic abstract screening by two reviewers is underway. Next steps TBC Outcome measurement in paediatric proton beam radiation therapy studies: is greater standardisation needed? Caroline Main1, Simon P. Stevens1, Roger E. Taylor2, Barry Pizer3, Nick Thorp4, Keith Wheatley1, Pamela R. Kearns5, Robert Phillips6, Martin English5, Sophie Wilne7 1 2 3 1University of Birmingham; 2Swansea University; 3Alder Hey Children's NHS Foundation Trust; 4The Clatterbridge Cancer Centre; 5Birmingham Children's Hospital NHS Foundation Trust; 6Centre for Reviews and Dissemination; 7Queen's Medical Centre Discordant decisions will be discussed and persistent discordant deci- sion will be referred to a third reviewer for decision. Following the re- trieval and screening of full papers and reference checking, data Correspondence: Caroline Main Trials 2017, 18(Suppl 1):P102 Correspondence: Caroline Main Trials 2017, 18(Suppl 1):P102 Page 39 of 235 Page 39 of 235 Page 39 of 235 Trials 2017, 18(Suppl 1):200 information due to incomplete study data retrieved through a sys- tematic review, which are therefore excluded from the analysis. Sev- eral methods are provided in literature to handle missing or incomplete data in a NMA. It is often the case that only baseline and follow-up measurement are available; to obtain data about mean change it is necessary to consider pre-post study correlation. In a Bayesian framework, some authors (Abrams, 2005), suggest imput- ation strategies of pre-post correlation. In other cases, a variability measure associated to mean change score might be unavailable. Dif- ferent imputation methods are suggested, as those based on max- imum standard deviation MSD imputation. Aims The Core Outcome Measures in Effectiveness Trials (COMET) initiative was instigated to develop the minimum set of standardised key out- comes that should be assessed in studies, making it easier for results of different studies to be compared, contrasted and combined. Within a systematic review of the effects of proton beam radiation therapy (PBT) in children with malignant Central Nervous System (CNS) tumours we assessed the standardisation of outcome measures utilised. P103 Background A cluster randomized clinical trial (CRCT) is a trial that randomizes clusters of people, but collects data on individuals. Concerns about the quality of reporting of results from CRCTs led to the publication of Consolidated Standards of Reporting Trials (CONSORT) statements for CRCTs in 2008 and 2010. Additional CONSORT criteria for CRCTs include: identification as a CRCT (title), the numbers of clusters and individuals randomized to each group (abstract), the intracluster cor- relation used for sample size calculation, and how clustering was taken account in the statistical methods. A review of CRCTs pub- lished in 2000–2008 concluded that reporting improved after CON- SORT publication but remained suboptimal. No reviews have looked P103 Handling missing or incomplete data in a Bayesian network meta-analysis framework Danila Azzolina1, Ileana Baldi1, Paola Berchialla2, Clara Minto1, Dario Gregori1 1University of Padua; 2University of Turin Correspondence: Danila Azzolina Trials 2017, 18(Suppl 1):P103 Cluster randomized clinical trials: can they be found in pubmed and clinical trial registries and do they adhere to consort guidelines? There is a need to standardise outcomes and their measurement within studies of children undergoing radiotherapy including PBT. In particular, survival outcomes need to be measured from the com- mencement of PBT in order to facilitate comparisons between differ- ent studies. Long-term adverse events need to be better defined and measured using standardised scales. Elizabeth Wright, Kenneth J. Wilkins National Institute of Diabetes Digestive and Kidney Diseases/NIH Correspondence: Elizabeth Wright Trials 2017, 18(Suppl 1):P104 Elizabeth Wright, Kenneth J. Wilkins National Institute of Diabetes Digestive and Kidney Diseases/NIH Correspondence: Elizabeth Wright Trials 2017, 18(Suppl 1):P104 Methods Twelve electronic databases were searched from 1985 onwards. Comparative and non-comparative studies were included. Outcomes included overall survival (OS), surrogate survival outcomes, local/dis- tant failure rates (LFR and DFR), response rates, toxicities, long-term adverse events, neurocognitive outcomes and quality of survival. Standard systematic review methods were used to minimise bias in study identification, selection and data extraction. R lt Seventeen studies (one single arm phase II trial and 16 case series) included a total of 492 patients. Mean sample size of 29 (range: 6– 109), with a mean length of follow-up of 3.1 years (range: 0.1- 27.2 years). All treatment regimens assessed were multi-modality. Of the 492 patients, 442 (90%) were newly diagnosed and 53 (10%) had relapsed disease. Tumour types included were low-grade gliomas, medulloblastoma or supratentorial primitive neuroectodermal tumours(SPNET’s), ependymoma,atypical teratoid rhaboid tumours (AT/RT), germ cell tumours, and pineoblastomas. g p Fifteen studies (460 patients; 93%) reported OS, eight progression- free survival (265 patients; 54%), three disease-free survival (144 pa- tients; 29%) one event-free survival (8 patients; 2%), one time-to- progression (59 patients; 12%), 12 LFR (342 patients; 70%), eight DFR (190 patients; 39%) and five response rates (39 patients; 8%). How- ever, all survival outcomes were measured from different time points across the studies. Two studies (5%) measured survival from diagno- sis, five (38%) from the start of PBT, six from completion of PBT (28%), whilst the remaining three studies (29%) did not report base- line timing of survival outcomes. This can potentially alter results. One study of 31 patients (6%) reported 2-year OS from both the time of diagnosis [68.3% (95% CI: 52.9%-88.1%)] and the end of PBT [52.9% (95% CI: 36.0% - 77.8%)] indicating the differences in results obtained from when baseline PBT is measured. Endocrinopathies and ototoxicities were the most commonly reported medium-term ad- verse events, both reported in seven studies with 229 (47%) and 126 (26%) patients respectively. There was little standardisation in the measures used to assess either outcome. In relation to endocrinopa- thies six different scales, including any type of endocrinopathy (un- specified), had been utilised, and the studies employed different criteria to define these outcomes. In terms of ototoxicities, a total of five different scales had been used across seven studies, with only two using standardised outcome measures. Results on neuro- cognitive outcomes (85 patients) were highly limited. Conclusions Handling missing or incomplete data in a Bayesian network meta-analysis framework 1 1 2 1 meta analysis framework Danila Azzolina1, Ileana Baldi1, Paola Berchialla2, Clara Minto1 Dario Gregori1 1University of Padua; 2University of Turin Correspondence: Danila Azzolina Trials 2017, 18(Suppl 1):P103 meta analysis framework Danila Azzolina1, Ileana Baldi1, Paola Berch Dario Gregori1 1University of Padua; 2University of Turin Correspondence: Danila Azzolina Trials 2017, 18(Suppl 1):P103 y Danila Azzolina1, Ileana Baldi1, Paola Berchialla2, Clara Minto1, Dario Gregori1 1University of Padua; 2University of Turin Correspondence: Danila Azzolina Trials 2017, 18(Suppl 1):P103 A Bayesian NMA model is often used to estimate the effect of each intervention compared to others synthesizing results using rank probabilities. In several cases, a NMA is associated to a loss of Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 40 of 235 Page 40 of 235 at the identification of CRCTs in clinicaltrials.gov and other registries. We sought to determine whether adherence to CONSORT guidelines has improved and whether CRCTs could be identified in trial registries. Methods at the identification of CRCTs in clinicaltrials.gov and other registries. We sought to determine whether adherence to CONSORT guidelines has improved and whether CRCTs could be identified in trial registries. Methods Conclusions RELEASE is the largest systematically developed, evidence synthesis study in the field of aphasia, and is more complex than most IPD trial meta-analyses. Our research will not only provide important evidence relating to the recovery of people with aphasia, but will also be an exemplar to researchers who plan to create databases to analyse complex individual patient data. The quality of CRCT publications has increased, but there con- tinue to be publications that are underpowered and do not ac- count for the effect of clustering in the analysis. Trial registries do not currently include a code for CRCT or a structured means of recording the number of clusters in results. The description of CRCTs in registries needs to be improved so these trials can be included in systematic reviews. Background The implementation of adaptive design methods in phase II, phase III or phase II/III has increased over the years [1]. There is a need for a set of guidelines to report adaptive design method- ology used in clinical trials in addition to the CONSORT guide- lines [2] to ensure full transparency of trials implementing predetermined or concurrent adaptations. The aim of this litera- ture review is to understand the current application of adaptive design methodology in oncology trials, and to ascertain how this methodology is reported. Trials 2017, 18(Suppl 1):P105 Background The number of stroke rehabilitation trials reported is rapidly increasing. Efficient trial design contributing to advances in rehabilitation should be informed by completed trials in the field. More than 50,000 people in the UK each year acquire aphasia: a stroke related language impairment affecting the ability to speak, understand speech, read and write with significant consequences for quality of life. Existing Cochrane systematic review evidence indicates that speech and language therapy (SLT) bene- fits language recovery in people with aphasia, however, the specific patient and intervention factors which predict optimal recovery and rehabilitation are unclear. By using a wider dataset with individual pa- tient data (IPD) analysis we are enhancing the evidence synthesis process with the aim of addressing these evidence gaps. RELEASE (rehabilitation and recovery of people with Aphasia after stroke) is an international collaboration of aphasia researchers which seeks to achieve this goal. Methods We invited contributions of primary datasets from members of the Collaboration of Aphasia Trialists (cats). We also conducted a system- atic search of existing published research to identify a comprehen- sive set of potentially existing aphasia research datasets which met our inclusion criteria. Research datasets were required to include a minimum of 10 people, a measure of aphasia severity as a conse- quence of stroke and information on time since stroke. We invited researchers from these studies to contribute data and to create a unique multilingual, international, interdisciplinary resource in this clinical field. p Results Building on the past: systematic identification, data extraction and synthesis of pre-existing individual stroke patient datasets to inform the development and design of future clinical trials Andrew Elders1 the RELEASE2 gy Pankaj Mistry, Janet Dunn, Andrea Marshall Warwick Clinical Trials Unit, University of Warwick Correspondence: Pankaj Mistry Trials 2017, 18(Suppl 1):P106 gy Pankaj Mistry, Janet Dunn, Andrea Marshall Warwick Clinical Trials Unit, University of Warwick Correspondence: Pankaj Mistry Trials 2017, 18(Suppl 1):P106 gy Pankaj Mistry, Janet Dunn, Andrea Marshall Warwick Clinical Trials Unit, University of Warwic p Andrew Elders1 the RELEASE2 1 NMAHP Research Unit, Glasgow Caledonian University Nursing Midwifery and Allied Health Professions Research Unit Collaboration, The RELEASE (www.aphasiatrials.org/index.php/research/ release) Results Our search identified 557 English language publications between 2000 and 2015, 349 of which included patients with diabetes. 262 (75%) were reports of CRCT. Excluded publications used terms like “parallel group randomized”, “cluster of risk factors”, “cluster sam- pling”, and “Cluster analysis” or were reviews. A few excluded publi- cations called themselves CRCT but were trials where individuals were randomized to receive treatment in groups. An additional 54 publications were found for a total of 316 publications from 186 tri- als: 143 primary results, 81 design, and 92 secondary. We grouped the 143 results publications by year published: 44 in 2000–2007, 39 in 2008–2011, and 60 in 2012–2015. The percent with CRCT in the title (18%, 51%, 63%) and the percent registered (11%, 79%, 83%) in- creased over time. 86% had the number of clusters in the abstract, 78% discussed clusters in the statistical analysis plan, and 49% in- cluded the sample size intracluster correlation. Only 39/86 registered trials included the word cluster in the registration (clinicaltrials.gov 17/47, ISRCT 14/26, ACTRN 4/6, others (4/7). Following a systematic search of the literature, we screened 5276 ti- tles (including 2346 abstracts and 1152 full texts), from which we identified 874 eligible studies. We have received 76 study datasets contributing IPD from 4597 people with aphasia (56 through the sys- tematic search and 20 via cats). These data have been contributed from 23 countries and we have identified a further 2400 IPD in the public domain. The substantive challenge is our planned IPD meta- analysis to examine recovery, predictors of recovery and effective- ness of intervention approaches. Our statistical analysis plan states that a one-stage approach will be conducted for the primary ana- lyses, although a two-stage approach will also be explored. Network meta-analyses and meta-regression (some of which includes sub- group analyses) are also planned. We will discuss the methodological challenges, particularly which arise when there are non-standardized data, some non-randomized data, a large number of outcome mea- surements and some degree of sparse data. C l i Objectives Funded by the National Institute for Health Research (Health Services and Delivery Research - 14/04/22) we have systematically gathered IPD from pre-existing aphasia research datasets to examine the nat- ural history of recovery from aphasia, the predictors of recovery and optimal interventions (by rehabilitation regimen, delivery model and the aims and content of treatment). We focused our review on CRCTs designed to improve the care of patients with diabetes through interventions aimed at either patients or health care providers. We searched pubmed in September 2016 using the terms Diabetes AND ((cluster randomized) OR (cluster ran- domised) OR (group-randomized) OR (group-randomised)). Reviews, bibliographies, and registries were searched for additional publica- tions. Publications were classified as: diabetes treatment, diabetes prevention, or not diabetes and as CRCT or not. We extracted data on the adherence to CONSORT guidelines and determined the trial registration status for each publication (included in the publication, registered but not included, or not registered). This information was used to group publications by trial and we selected the primary re- sults publication, if any, for each trial. A literature review of the use of adaptive design methods in oncology trials P105 Building on the past: systematic identification, data extraction and synthesis of pre-existing individual stroke patient datasets to inform the development and design of future clinical trials Andrew Elders1 the RELEASE2 1NMAHP Research Unit, Glasgow Caledonian University 2Nursing Midwifery and Allied Health Professions Research Unit Collaboration, The RELEASE (www.aphasiatrials.org/index.php/research/ release) et odo Methods A literature search of PubMed, Embase and Ovid databases for full text publications of phase II, phase III or phase II/III cancer trials using adaptive design methodology during 2015 was conducted. The key words used for the literature search are as follows: adaptive design, flexible design, group sequential, sample size re-estimation, MAMS, adaptive randomisation, interim analyses, adaptive seamless, bio- marker adaptive, two-stage adaptive, dose escalation, ‘Drop the loser’, ‘Pick the winner’, multiple adaptive, adaptive enrichment. Page 41 of 235 Trials 2017, 18(Suppl 1):200 Page 41 of 235 Relevant full text articles were reviewed to identify the type of adap- tive methodology applied, if the publication explicitly stated the use of adaptive designs and whether adaptive design methodology was applied prospectively or concurrently. R l Stage 1, the Idea phase where formal data collection should begin. This requires quality recording of data using standardised outcome measures. The emphasis is on explanation and description. Stage 2a, the Development phase, is a period of iterative improvement and ad- justment with thorough prospective data recording. It focuses on technical details and feasibility. Stage 2b, the onset of formal Explor- ation evaluation using systematically collected group or cohort data. This stage is a bridge or a pilot to a full RCT. It further refines out- come measures and takes account of learning curves. Stage 3, is a formal comparative Assessment phase of treatment usually involving randomised studies. It involves a full assessment of efficacy. Stage 4; Long term follow up involves monitoring outcome, particularly in long term conditions. Stage 1, the Idea phase where formal data collection should begin. This requires quality recording of data using standardised outcome measures. The emphasis is on explanation and description. Stage 2a, the Development phase, is a period of iterative improvement and ad- justment with thorough prospective data recording. It focuses on technical details and feasibility. Stage 2b, the onset of formal Explor- ation evaluation using systematically collected group or cohort data. This stage is a bridge or a pilot to a full RCT. It further refines out- come measures and takes account of learning curves. Stage 3, is a formal comparative Assessment phase of treatment usually involving randomised studies. It involves a full assessment of efficacy. Stage 4; Long term follow up involves monitoring outcome, particularly in long term conditions. P107 P107 Evidence based evaluation of practice using the ideal-physio framework. A strategic method of evaluating innovation and current practice David Beard1, Arsenio Paez2, Loretta Davies1, Jonathan Cook1, Allison Hirst1, Peter McCulloch1 1University of Oxford; 2Northeastern University Correspondence: David Beard Trials 2017, 18(Suppl 1):P107 Conclusion This review has highlighted that adaptive design methodology is rarely explicitly stated and hence supports the argument for needing a set of guidelines to report the adaptive design methodology used in clinical trials. Furthermore specific reporting guidelines will assist in the consistency of reporting and ensure the ease of future identifi- cation of trials implementing any prospective or concurrent adaptive design methodology. et odo Methods The databases produced a total of 14544 phase II, phase III, phase II/ III cancer trial related articles that were published in 2015. Of which 99/2127 (5%) for the PubMed database, 519/9573 (5%) for the Embase database and 116/2844 (4%) for the Ovid database articles included the search terms related to adaptive design methodology. After the removal of duplicates, 464 articles were remaining. Of the 464 articles, 92 (20%) were full text trial related publications, 261 (56%) were abstracts, 32 (7%) were methodology or review papers and 79 (17%) were not related to the search criteria. Background Whilst there have been recent improvements, the practice and pro- fession of physiotherapy (Physical Therapy) has suffered from the un- controlled introduction and proliferation of treatments which have an inadequate scientific basis, little or poor evaluation, and under- exposure to rigorous scientific method. New treatment modalities can be developed and introduced without evidence of efficacy, regu- lation or governance. There is no requirement to collect prospective data to support any claims or demonstrate efficacy. This approach has resulted in numerous disparate practices which may not stand up to rigorous evaluation or evidence based commissioning. p Results Results Within the software, we specified five scenarios for the event-rates across doses. For the ‘upper dose is just safe’ scenario, BCRM outper- formed 3 + 3 in discovering the highest dose (72% versus 62%). For the ‘upper dose is just unsafe’ scenario, the BCRM recommended the upper dose 54% versus 48% times, but 3 + 3 incorrectly recom- mended the lowest dose more often (22% versus 10%). For the ‘all event rates are ascending but lower than the target’ scenario, BCRM recommendations across doses were superior (5%,14%,81%) relative to 3 + 3 (34%,35%,31%). For the ‘highest dose is unexpectedly high’ scenario, both approaches recommended it in just under 10% of sim- ulations. For the ‘highest dose is plausibly high’ scenario, both ap- proaches recommended it approximately one-third of the time, and BCRM recommended the correct dose more frequently than 3 + 3 (41% versus 30%). The IDEAL framework is an established method of formalising the systematic evaluation of innovation (and existing practice) in com- plex clinical interventions. It has been useful for setting out an inter- nationally based evaluation framework for surgical procedures. This framework lends itself to other complex, non-pharmacological inter- ventions such as Physiotherapy (Physical Therapy). We outline the application of this framework to Physiotherapy (Physical Therapy) in a new IDEAL-Physio framework. A journey from statistical consultation to funded adaptive trial Toby Prevost y Conclusion g Methods There was a consensus to keep the sample size near 20. A 20% target toxicity event rate for the Maximum Tolerated Dose was set, below the 33% relevant in oncology trials. The performance of the 3 + 3 and BCRM methods was assessed through setting a range of likely and unlikely scenarios for the event rates at the 3 doses, followed by simulation (3000 trial repetitions). Primary assessment was the percentage of trials recommending each of the three doses. The 3 + 3 with a sample size of 18 was contrasted with a BCRM of sample size 16 specifying a one-parameter power model and a Gamma prior. p David Beard1, Arsenio Paez2, Loretta Davies1, Jonathan Cook1, Allison Hirst1, Peter McCulloch1 p David Beard1, Arsenio Paez2, Loretta Davies1, Jonathan Cook1, Allison Hirst1, Peter McCulloch1 1 2 1University of Oxford; 2Northeastern University Correspondence: David Beard Trials 2017, 18(Suppl 1):P107 1University of Oxford; 2Northeastern University Correspondence: David Beard Trials 2017, 18(Suppl 1):P107 Background A funding board assessed an application for a Phase 1 three-doses dose escalation trial of a treatment in Crohn’s Disease, but disliked the deterministic 3 + 3 design. The principle applicant sought statis- tical advice which led to an adaptive design with extra doses. An- other board disliked this adaptive design and the 3 + 3 was reinstated. The boards jointly requested a three-dose adaptive design which was funded. This incorporated a Phase II stage, and a cross- over design to allow placebo-controlled periods in blocked patient pairs. The aim is to describe the use of the adaptive Bayesian Contin- ual Reassessment method available in BCRM Software [1], for design- ing dose escalation trials. 1. Hatfield, Isabella. Adaptive designs undertaken in clinical research: a review of registered clinical trials. Trials (2016): 1273–9 2. www.consort-statement.org/consort-2010 Recommendations The adaptive design methodology used in over half of the trials (48/92) was applied by performing interim analyses due to safety, efficacy or futility, 21 out of 92 trials incorporated dose escalation methods and 14 out of 92 implemented a two-stage design. The remaining 9 trials applied the following methods: Bayesian adap- tive design (3/92), group sequential design (3/92) change in pri- mary endpoint (1/92), seamless phase II to phase III (1/92), multiple adaptive (1/92). Despite using adaptive design method- ology, only four trials explicitly stated that it had an adaptive de- sign. There were 89 out of 92 trials that had prospectively planned adaptations, of which two of these also incorporated an ad-hoc interim analysis. We recommend the use of IDEAL - Physio to help guide and evaluate innovation with the overall strategy of providing better evidence based care and foster innovation in Physiotherapy (Physical Therapy). This paper outlines the principles of IDEAL - Physio and describes its utility in changing practice on a global level. Keywords: Physical Therapy Innovation Evaluation Keywords: Physical Therapy, Innovation, Evaluation, Background g Serious Adverse Events (SAEs) are routinely collected in many trials and processed by Clinical Trials Units on a daily basis. In the past at Nottingham Clinical Trials Unit (NCTU), saes were processed by the Trial Manager (TM), however this could lead to problems if they were absent, with the potential that Ss were not handled in a timely man- ner. In 2013 a database was developed to track all incoming SAEs Unit-wide, so that members of the team could efficiently process SAEs on any trial within the Unit. This is done on a rolling rota basis and TMs are responsible for ensuring cover if they are unable to undertake their duties. This ensures all TMs have exposure to SAE handling, irrespective of whether their own trial collects SAEs or not and is therefore good for staff development. It also ensures full cover across all trials, irrespective of whether the specific trial team are available. The roll-out of this system was initially successful, however problems later developed with standard procedures being followed inconsistently and a lack of consistent oversight with no formal pro- cedures in place. Cellular therapy for hematologic malignancies is an emerging area of cancer therapeutics. One of the major challenges of cellular therapy re- search is the ability to make specialized processes widely available. BMT CTN protocol #1401 was designed to translate a single-center manufac- turing process of a vaccine with dendritic cell/myeloma fusions to a model where the investigational product is manufactured locally at each participating institution. The study provides a framework for implement- ing multi-center cancer vaccine studies requiring a unique approach for clinical protocol and manufacturing process development, Investiga- tional New Drug (IND) submission, site selection, training and qualifica- tion, and data collection. The primary challenge was adapting a single- center process to a multi-center clinical protocol. Study specific standard operating procedures (sops) for cell collection and manufacturing were developed utilizing the institutional sops from the single center study and managed centrally via an SOP management process. Laboratory staff at participating sites performed an in-depth review of the manufac- turing sops and identified processes requiring generalization based on available equipment and institutional-specific processes. Participating sites were invited based on past participation and experience with den- dritic cell vaccine clinical research. Following site selection, each institu- tion attended training in-person and via teleconference to discuss SOP development and familiarize key staff with the production process. Background Two successful mock runs of the vaccine product were required prior to site activation. Initiating these training procedures concurrent with protocol development and FDA submission allowed for faster site activation upon protocol release. The IND application included an in-depth overview of the site training, qualification, and selection procedures to assure that all sites were adequately trained in vaccine manufacturing. There are sev- eral unique components of data collection and monitoring in the con- text of cellular therapy. It is integral to the integrity of the study and safety of the participants to monitor compliance with the manufacturing sops to ensure that each product meets criteria for release. An electronic system was developed to monitor vaccine production and release cen- trally in real time. Sites are required to submit electronic reports of any deviation from the study sops via the data system within 24 hours of knowledge of the event. These reports are reviewed by medical monitors, and recommendations for corrective actions and future prevention of deviations are provided to the site within five business days. All sites will enter electronic data on the vaccine re- lease criteria and upload the completed study specific worksheets. These data will be reviewed and approved centrally prior to local release of the vaccine for administration to patients. Any deviations Results Streamlining the existing process took approximately 6 months and was fully introduced in August 2016. Feedback from users has been positive. The trial-specific handling instructions have been helpful to all safety handlers and design and functionality changes to the data- base have been received positively. Oversight is now done consist- ently across all trials with appropriate accompanying documentation filed, essential for audit and inspection purposes. P109 Translation of a single-center cellular therapy manufacturing approach to a multi-center, center-specific manufacturing platform: the experience of the blood and marrow transplant clinical trials network (BMT CTN) protocol #1401 Courtney Nelson1, Kelly O'Brien1, Adam Medizabal1, Iris Gersten1, Lynne Uhl2, David Chung3, Nina Shah4, David Avigan2, Marcelo Pasquini5 1The Emmes Corporation; 2Beth Israel Deaconess Medical Center; 3Memorial Sloan-Kettering Cancer Center; 4University of Texas, MD Anderson Cancer Center; 5Medical College of Wisconsin Correspondence: Courtney Nelson Trials 2017, 18(Suppl 1):P109 Translation of a single-center cellular therapy manufacturing approach to a multi-center, center-specific manufacturing platform: the experience of the blood and marrow transplant clinical trials network (BMT CTN) protocol #1401 Courtney Nelson1, Kelly O'Brien1, Adam Medizabal1, Iris Gersten1, Lynne Uhl2, David Chung3, Nina Shah4, David Avigan2, Marcelo Pasquini5 1The Emmes Corporation; 2Beth Israel Deaconess Medical Center; 3Memorial Sloan-Kettering Cancer Center; 4University of Texas, MD Anderson Cancer Center; 5Medical College of Wisconsin Correspondence: Courtney Nelson Trials 2017, 18(Suppl 1):P109 Methods Updates were made to the database design to enhance the user ex- perience and additional reports were added. Emails were then chan- ged from manual to automated to speed up the process of receipt acknowledgement and sending SAEs for medical review. A thorough training session was provided to TMs on the rota, delivered by the Senior Trial Managers and the database developer. All trials now have trial-specific SAE handling instructions stored centrally for easy access; these instructions give step-by-step guidance on how to han- dle an SAE for a particular trial. An oversight process was then put in place which involves, at a trial level, the specific-Trial Manager and, at a Unit-level, the team of Se- nior Trial Managers and QA Manager. A range of easy-to-use check- lists were produced to ensure consistent regular oversight, undertaken on a rota basis. Oversight also includes running a series of regular reports through the tracking database and ensuring, via the TMs, that the database and other documentation is kept fully up- to-date. TMs are also encouraged to use in-built reports to allow ac- curate and timely reconciliation of SAEs. Conclusions Some funding boards are encouraging of researchers to adopt and exploit the advantages of novel designs. The availability of the BCRM software enabled a range of event-rate scenarios to be examined for a three-dose example with moderate target rate. Across scenarios, y Similarly to IDEAL for surgery, five stages exist; each representing a letter of the acronym. Page 42 of 235 Trials 2017, 18(Suppl 1):200 Page 42 of 235 Page 42 of 235 noted in the worksheets will be reported and reviewed by the medical monitors to ensure the final product is not compromised. While there continue to be ongoing challenges, BMT CTN 1401 pro- vides a framework for the successful implementation of a multi- center cellular therapy clinical trial by utilizing a single-center ex- perience in conjunction with an established clinical trials network. Further efforts are needed to explore the application of this frame- work to additional therapeutic areas. this adaptive approach was seen generally to outperform the 3 + 3 design, with a smaller sample size. The software was flexible in allow- ing patients to be recruited and dose-allocated in pairs, accommo- dating a crossover element. The scenario results indicate that with samples of a typically small size in Phase 1, there is surprisingly quite a lot of sampling variability in the dose recommended. Simulation is therefore an important part of adaptive and deterministic design planning. Consideration could be given to exploring modestly raised sample sizes, and/or a further stage of dosing around the initially rec- ommended dose, and/or carrying forward an extra dose to Phase 2. this adaptive approach was seen generally to outperform the 3 + 3 design, with a smaller sample size. The software was flexible in allow- ing patients to be recruited and dose-allocated in pairs, accommo- dating a crossover element. The scenario results indicate that with samples of a typically small size in Phase 1, there is surprisingly quite a lot of sampling variability in the dose recommended. Simulation is therefore an important part of adaptive and deterministic design planning. Consideration could be given to exploring modestly raised sample sizes, and/or a further stage of dosing around the initially rec- ommended dose, and/or carrying forward an extra dose to Phase 2. P110 Streamlining SAE handling in a busy CTU Eleanor Mitchell, Kirsty Sprange, Clare Brittain, Isobel Hawley University of Nottingham Correspondence: Eleanor Mitchell Trials 2017, 18(Suppl 1):P110 Reference [1] Sweeting M, Mander A, Sabin T. Journal of Statistical Software, 2013;54(13) [1] Sweeting M, Mander A, Sabin T. Journal of Statistical Software, 2013;54(13) and stream Methods A ‘Lessons Learnt’ database specification was written following the generation of ideas by a group of experienced Trial Managers and Data Managers. The database was built using Access, and iterative piloting was undertaken until the group were satisfied with the data- base functionality. TAPUR’s pragmatic approach incorporates the clinician and patient perspective into the design. For example, enrollment is expanded through use of broader eligibility criteria, including participants with performance status (PS) 0–2, prior malignancies or HIV, and previ- ously treated, but stable brain metastases. To date, 19% of partici- pants have PS = 2 and 22% reported prior malignancies. This approach not only provides greater trial access to patients and maxi- mizes accrual, but also increases generalizability. Drug dosing is ac- cording to the drug label and modification is allowed through clinician judgment. Data collection centered around routine clinical care reduces site burden and costs, while also allowing for rapid en- rollment since the trial can leverage existing data, critical for patients and clinicians looking for timely treatment options. Within the database, lessons are grouped by study and are recorded by: study registration details, stage of study (e.g. Planning and de- sign), process area (e.g. Consent), summary of the event, impact of the event, statement of the lesson learnt and action taken. Lessons are searchable by others using these key items. Each lesson has a blog, which all staff are encouraged to use to make comments, for example, to share similar experiences or suggest solutions. Lessons are reviewed by a core review team on a monthly basis. The review team record their recommendations, request further actions and/or disseminate findings to all research staff. A lesson is closed once all actions and dissemination have been completed. Reports can be printed from the database by the review team which, amongst other functions, provide metrics such as stage of study where lessons are reported, making it easier to identify recurring issues. Results However, there are operational challenges associated with this level of flexibility. For example, due to the heterogeneity of tumor types and genomic alterations, dozens of cohorts are generated that enroll slowly (to date 82 cohorts exist for 102 participants). Therefore, total sample size for any cohort, site or drug are difficult to estimate. Conclusions Lessons learnt from past studies can prevent problems being re- peated and promote adoption of positive experiences. Keele CTU is in the process of engaging staff fully with the ‘Lessons Learnt’ process by embedding a culture of sharing and learning using a sys- tematic approach. Future work will aim to measure the impact of the database on the quality of research processes. Conclusion Implementing the database and rota system in 2013 was a step in the right direction. However, streamlining the process and imple- menting more robust oversight ensures that SAEs are handled appro- priately and efficiently to ensure regulatory timelines are met. Including TMs in reviewing and updating the tracker database and relevant documentation has been essential to ensure buy-in from users. Clear communication and training whilst updating the process has also been crucial. Trials 2017, 18(Suppl 1):200 Page 43 of 235 Page 43 of 235 P111 Operational advantages and challenges with pragmatic precision medicine trials: the TAPUR study Operational advantages and challenges with pragmatic precision medicine trials: the TAPUR study Staff within large research organisations who are responsible for managing a number of complex studies gain a vast amount of know- ledge and experience that is not always systematically and effectively shared with others. Embedding a ‘Lessons Learnt’ culture within an organisation can provide staff with the opportunity for reflection and promote a culture of problem-solving, ensuring that best practice is applied and mistakes are not repeated. A systematic approach to capturing and communicating learning can help to streamline and improve the quality of research procedures. y Pam Mangat, Kaitlyn R. Antonelli, Suanna S. Bruinooge, Richard L. Schilsky American Society of Clinical Oncology Correspondence: Pam Mangat Trials 2017, 18(Suppl 1):P111 The American Society of Clinical Oncology’s Targeted Agent and Pro- filing Utilization Registry Study (TAPUR) is a Phase 2, non- randomized, precision-medicine, basket trial founded in real-world clinical practice. The TAPUR study includes investigational therapies that are approved by the U.S. Food and Drug Administration to tar- get genomic alterations in patients with advanced cancer known to be a drug target or predict drug sensitivity. TAPUR is a large multi- site study with an innovative design that allows for assignment of 1 of 15 possible study regimens by the treating physician according to protocol defined drug-genomic alteration matching rules or guidance from the study Molecular Tumor Board (MTB). TAPUR has broad eligi- bility criteria, flexibility in treatment administration, and collection of data that mirrors routine clinical care. Analysis of study endpoints re- quires completion of cohorts defined by study drug, genomic variant, and tumor type according to a Simon 2-stage design where cohorts are closed or expanded based on response rate. p Results The ‘Lessons Learnt’ database has been implemented, with staff starting to record their positive and negative lessons. The review team assesses the reported lessons and facilitates actions required to implement changes needed to improve procedures. Best practice is disseminated to all staff by e-mail, preventing reinvention of the wheel and raising awareness of any changes to processes as a result of learning from lessons. P112 Is there anyone so wise as to learn by the experience of others? Developing and implementing a ‘Lessons Learnt’ database in a clinical trials unit Helen Myers1, Ian Thomas2, Stephanie Tooth1, Tom Shepherd2, Jo Smith1, Rachael Heath1, Sarah Lawton1, Kris Clarkson1 1Keele Clinical Trials Unit; 2Institute for Primary Care and Health Sciences Correspondence: Helen Myers Trials 2017, 18(Suppl 1):P112 and stream Methods In turn, an over- all study budget is also difficult to assess since many costs, such as drug distribution and per-case reimbursements, are related to the num- ber of enrollments. It is important to allow for ongoing modification, in- form collaborators about the need for fluidity of the project and consider novel operational approaches. For example, due to challenges in estimating allocation of drug quantities to clinical sites, the study uti- lizes a specialty pharmacy as the drug distributor, with drug provided on a per-participant basis at the time of order to reduce waste and negate the need for pre-defined estimates. Lastly, while the broad eligi- bility criteria is a clear advantage, it does require regular retraining of clinicians to utilize their best clinical judgment when assessing eligibil- ity. Leniency in measures such as performance status may result in an enrolled participant unable to meet all study requirements or achieve key study endpoints, like response evaluation. Introduction P111 Operational advantages and challenges with pragmatic precision medicine trials: the TAPUR study Pam Mangat, Kaitlyn R. Antonelli, Suanna S. Bruinooge, Richard L. Schilsky American Society of Clinical Oncology Correspondence: Pam Mangat Trials 2017, 18(Suppl 1):P111 Background Keele Clinical Trials Unit (CTU) aims to promote a culture where staff learn from previous experiences and apply best practice to become more efficient. The aim of the ‘Lessons Learnt’ database developed at Keele CTU is to capture and learn from positive and negative events or pro- cesses which occur during the life of a research study, and to communi- cate these to others. The overall objective is to make improvements to, and streamline the processes of setting up and running a research study. Methods P113 Tailoring a comprehensive website to support a clinical research network This presentation will highlight the innovative features of the study design, as well as design-related operational advantages and chal- lenges for consideration. network Lauren Yesko, Dikla Blumberg, Paul vanVeldhuisen, Jennifer McCormack, Michael Frasketi, Jordan McNeil, Heather Sabina The Emmes Corporation Correspondence: Lauren Yesko Trials 2017, 18(Suppl 1):P113 Lauren Yesko, Dikla Blumberg, Paul vanVeldhuisen, Jennifer McCormack, Michael Frasketi, Jordan McNeil, Heather Sabina Lauren Yesko, Dikla Blumberg, Paul vanVeldhuisen, Jennifer McCormack, Michael Frasketi, Jordan McNeil, Heather Sabina The Emmes Corporation Correspondence: Lauren Yesko Trials 2017, 18(Suppl 1):P113 NeuroNEXT study design working group experiences Eric Foster1, Katy Mahoney2 1 2 NeuroNEXT study design working group experiences Eric Foster1, Katy Mahoney2 1The University of Iowa; 2Massachusetts General Hospital Correspondence: Eric Foster Trials 2017, 18(Suppl 1):P114 1The University of Iowa; 2Massachusetts General Hospital Correspondence: Eric Foster Trials 2017, 18(Suppl 1):P114 Adverse outcomes in non-drug intervention trials: what and how these are recorded Adverse outcomes in non-drug intervention trials: what and ho these are recorded Vichithranie Madurasinghe, Sandra Eldridge, Ayesha De Costa, Anitha Manivannan, Ann Thomson Queen Mary University London, UK Correspondence: Vichithranie Madurasinghe Trials 2017, 18(Suppl 1):P115 Vichithranie Madurasinghe, Sandra Eldridge, Ayesha De Costa, Anitha Manivannan, Ann Thomson Queen Mary University London, UK Correspondence: Vichithranie Madurasinghe Trials 2017, 18(Suppl 1):P115 Results All were multi-centre trials involving at least two sites, and conducted in the United Kingdom. Six were individually randomised and two cluster ran- domised, and all but two were conducted in hospitals or specialist centres. Assessing safety outcomes was a stated objective in two studies and one study included outcome measures on safety, though there were no specific study objectives relating to these. All trials included a section on safety reporting in their protocols, and the procedures for complying with report- ing requirements were described in adverse events sops. However, what adverse events were recorded and how these data were captured varied across the studies. Six studies reported capturing data on all “adverse events” (i.e. any untoward medical occurrence as per standard definition), one captured data on related adverse events only and one study was cap- turing serious adverse events (i.e. Adverse event which is fatal: Results in death, is life-threatening, requires inpatient hospitalisation or prolongation of existing hospitalisation, results in persistent or significant disability/incap- acity or is a congenital anomaly/birth defect) only. All used adverse events forms or logs for doing so. Two studies described implementing additional processes for collecting adverse outcomes as supplementary to the main procedure. These included regular data downloads from routine data col- lection systems, questionnaires and contacts (by telephone, text or email) from research team. Only two studies described how the standard criteria for assessing seriousness were applied in that particular trial. All but one trial described expected serious events in adverse events SOP, but none pro- vided insights into how these were researched or decided upon. Six studies had data monitoring committees in place for assessing safety parameters. Conclusions The design and implementation of this updated website for the NIDA CTN has been effective in meeting the needs of both protocol- specific as well as network-wide activities. NeuroNEXT study design working group experiences Eric Foster1, Katy Mahoney2 1 2 P112 The Emmes Corporation, which serves as the Data and Statistics Center and the Clinical Coordinating Center for the National Institute on Drug Abuse (NIDA)-sponsored National Drug Abuse Treatment Clinical Trials Network (CTN), has recently updated its website with the goal to im- prove overall study conduct, accessibility to critical information, and overall network cohesiveness and efficiency within the multiple-protocol NIDA CTN environment. Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 44 of 235 Each active CTN protocol has a designated dashboard which houses critical documents for study implementation and conduct, including training information, official communications, the protocol, the man- ual of operations, and materials for study medications and/or other interventions. Within each protocol-specific area of the website are comprehensive sets of web reports, updated nightly, that describe important study progress metrics of a protocol implementation sta- tus timeline, recruitment, retention, data quality measures, treatment exposure, availability of primary outcome, and regulatory compliance measures, presented in tabular and graphical formats. Summary reports that compare data from each protocol to the a-priori- defined expectations of the sponsor with color codes highlight study performance for the protocol investigative teams and spon- sor. Similarly, regulatory reports are developed to display the sta- tus of important regulatory documents, identifying expired or soon-to-expire documents such as IRB approvals. These reports allow for continuous monitoring, prompt identification and cor- rection of problems by investigators, site staff, the sponsor, and coordinating centers. and how t Methods We identified eight non-drug trials from the Pragmatic Clinical Trials Unit (PCTU) administrative database for inclusion. These were se- lected because their trial documents were available to researchers electronically. Trial protocols, standard operating procedures (SOP) for adverse event reporting and the trial reports (where available) were reviewed. Data were extracted on 1) study design, population and intervention characteristics, 2) stated study objectives and out- comes, 3) data recording, managing and reporting process regards to adverse outcomes and 4) the use of data monitoring committees. Results The website also serves as a resource for network-wide activities, in- cluding a monthly Web Seminar as a forum for network members to share and exchange clinical research knowledge. This webinar is highlighted on the website and materials are stored for future refer- ence. Committees within the network manage their own dashboard where agendas, minutes, and other communications are posted. There is also a website page known as the investigator toolbox where templates, tools, sample forms, and policies and procedures are stored and serve to guide investigators in the preparation and execution of their clinical trials. Background RCTs are an essential part of providing safe healthcare. They should be designed, conducted and analysed according to sound scientific princi- ples to achieve their objectives; should be reported appropriately [1], and should not cause unnecessary harms to trial subjects. As such trial proto- cols should include a clear safety section, with definitions, procedures and responsibilities for recording and reporting adverse outcomes. The aim of this work is to understand the current practice regards to what and how these outcomes are captured in non-drug intervention trials. Methods Background diagnosis and monitoring, including: BUS - Accuracy of Bladder Ultrasound (BUS) in the diagnosis of Detrusor Overactivity (DO): a study to evaluate if ultrasound can reduce the need for urodynamics. Egfrc - Accuracy of glomerular filtration rate (GFR) estimation using creatinine and cystatin C and albuminuria for monitoring disease progression in patients with stage 3 chronic kidney disease: Prospect- ive longitudinal study in a multi-ethnic population. ELATION - A ran- domised trial of the efficacy and cost effectiveness of Real Time Ultrasound Elastography in The Investigation Of Thyroid Nodules and the diagnosis of thyroid cancer. ENDCAP development and evalu- tions of a biomarker panel to detect enhanced neoplasia in chronic colitis, GBS-2- Accuracy of a rapid intrapartum test to screen for ma- ternal group B streptococcal colonisation and its potential to reduce antibiotic usage in mothers with risk factors MEDAL - MRI to establish diagnosis in women with pelvic pain rockets - Evaluation of diagnos- tic tools to diagnose ovarian cancer in women referred with symp- toms from primary care METRIC - Diagnostic accuracy for the extent and activity of newly diagnosed and relapsed Crohn s disease STREAMLINE-COLON, STREAMLINE-LUNG. Comprehensive staging of newly diagnosed lung and colorectal cancer MROC - Impact of multi- parametric MRI on staging and management decisions in women with ovarian cancer. At our institution we are establishing a research nurse-led programme to collect in-hospital complication data on all cardiac surgery patients who provide consent. This initiative is part of a wider programme of research to investigate associations between phenotypic and genotypic characteristics with complications and speed of recovery after cardiac surgery. Data will be extracted from participant’s paper and electronic med- ical notes and collated using a purpose-designed dedicated data col- lection tool. Information can be obtained by either i) visiting the ward daily, with the opportunity to talk to nurses involved in partici- pants’ care, and recording events almost as they happen or ii) extracting data after the patient has been discharged home. g p g Nurses who collect data for randomised trials believe that method i) takes longer but method ii) results in events being missed. To investi- gate these beliefs we are planning to randomise nurses (and pa- tients) to collect the data either by method i) or method ii). Methods Research nurses will be assigned a random selection of patients and told the method of data collection to use for each. Background The Network for Excellence in Neuroscience Clinical Trials, or NEURO- NEXT, is a National Institute of Neurological Disorders and Stroke (NINDS, part of the U.S. National Institutes of Health, or NIH) initiative created to conduct studies of treatments for neurological diseases through partnerships with academia, private foundations, and indus- try. NEURONEXT is just one of several clinical trials networks sup- ported by NINDS (i.e., strokenet and NETT). In discussions among the members of the networks, it has been useful to share solutions and processes addressing problems that arise in the setting of a clinical trials network. A dialog with researchers external to NINDS-supported clinical trials networks may yield further benefits. Obj ti j At any given time, there are likely to be multiple proposals for stud- ies to be conducted within a clinical trials network. How these pro- posals move from an initial concept to a full grant submission varies by network as different solutions are crafted to the common prob- lems of how and when to spend a network’s proposal development resources. This work addresses the NEURONEXT approach to pro- posal development. An overview of the pathway for a proposal is given alongside a discussion of how the process has changed as NEURONEXT has matured. Full protocol development requires spend- ing a great deal of the network’s proposal development resources. The decision of when to spend resources on multiple proposals can be difficult with complexity increasing as the number of proposals in the pipeline increases. This work discusses the timing of NEURONEXT protocol development assistance and the corresponding ramifica- tions. Also discussed are several common misperceptions that inves- tigators have experienced during the NEURONEXT design working group process. All trials attempted to assess adverse outcomes, but there was little evi- dence of a clear or a consistent approach to doing so; what adverse outcomes were captured, how these were captured varied across trials, and how the expected adverse outcomes were researched and decided upon was unclear. Including these aspects in trial documentation can improve clarity. Collecting trial quality data about complications routinely: a study within a study to maximize efficiency g 1University of Bristol; 2University Hospitals Bristol NHS Trust Correspondence: Lucy Culliford Trials 2017, 18(Suppl 1):P116 Page 45 of 235 Trials 2017, 18(Suppl 1):200 Page 45 of 235 Background All nurses will use both methods (for different patients) and each patient will have their data collected by different nurses using the different approaches. Pa- tients and nurses will be assigned using a balanced incomplete block design to ensure balance across the nurses and by collection method. Each nurse will be blinded to the data collected by another member of the team on the same patient. We propose to compare data collected for completeness as well as the time taken to collect the data. From initial discussions, common themes of the challenges of test evaluation trials are appearing, including: Challenges of site set up, site finances, patient pathway, recruiting consecutive participants, eli- gibility biases, obtaining reference standard diagnoses, the role of adverse event monitoring, trial modifications, sample handling and future-proofing sample collections. While some of these themes also occur in RCTs, the relative importance or risks vary. These themes will be explored in more depth and strategies used to resolve or minim- ise the impact on the project will be reviewed. The study is planned to run over 2 months from December 2016 and will capture data on 96 patients. 72 patients will be reviewed twice, once by each method and 24 patients will be assessed 4 times, twice by each method. Six nurses in the cardiac surgery team have agreed to take part. Each nurse will undertake 40 reviews on 16 patients. Results and Conclusion P118 Informed consent process enhancement approaches for research participants who may be considered vulnerable populations Matthew Wright, Julia Lynne, Eric Hardter, Radhika Kondapaka, Kayla Williams, Dagmar Salazar, Dikla Shmueli-Blumberg, Eve Jelstrom, Robert Lindblad The Emmes Corporation Correspondence: Matthew Wright Trials 2017, 18(Suppl 1):P118 The study is ongoing. Full results will be presented at the meeting. This research was funded by the National Institute for Health Research Biomedical Research Unit in Cardiovascular Disease at the University Hospitals Bristol NHS Foundation Trust and the University of Bristol. Disclaimer This abstract presents independent research funded by the National Institute for Health Research (NIHR). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. The informed consent process is paramount to the legal and ethical conduct of clinical trials. Adequately explaining study details to po- tential participants in a manner that permits comprehension and promotes study retention is challenging; this is compounded when recruiting populations that Institutional Review Boards (IRBs) may view as potentially vulnerable populations, such as persons facing concurrent mental or physical illness resulting in diminished capacity for comprehension, or with socioeconomic issues such as unstable housing, limited educational status, or low functional literacy. Not- withstanding any applicable legal thresholds for a legally authorized representative’s involvement, these factors may not negate the par- ticipant’s autonomy and capacity to make an informed decision on trial participation. In planning studies that involve such potentially vulnerable populations, investigators should consider and implement additional safeguards to ensure that informed consent materials are readily understandable, and that the consent process balances ad- equate coverage of required elements while avoiding overwhelm for the participant and burden on the research team. Comparing the challenges of the management of trials of medical tests versus randomised controlled trials of health care interventions Caroline Rick, James Brown, William McKinnon, Ryan Ottridge, Andrew Palmer, Victoria Parker, Lee Priest, Susan Mallett, Jon Deeks University of Birmingham Correspondence: Caroline Rick Trials 2017, 18(Suppl 1):P117 Caroline Rick, James Brown, William McKinnon, Ryan Ottridge, Andrew Palmer, Victoria Parker, Lee Priest, Susan Mallett, Jon Deeks University of Birmingham Correspondence: Caroline Rick Trials 2017, 18(Suppl 1):P117 Trials of medical tests present a series of challenges that differ, or have differing significance, from randomised controlled trials (RCTs) of interventions. A lack of understanding of the test-specific chal- lenges in the design, set up and management of trials can lead to er- roneous results: tests can appear more or less accurate due to inappropriate application of the eligibility criteria and differences in care pathways; study conclusions may be compromised if disease prevalence differs in the study from the actual target population. Bir- mingham Clinical Trials Unit (BCTU) manages and/or provides statis- tical support for a wide range of test evaluation trials as well as RCTs of interventions. We have set up a working group to compare and contrast the different challenges of the management of these trial types to improve future trial design and management. The ten test evaluation trials under consideration cover a wide range of medical conditions with different tests being evaluated for screening, p p The National Institute on Drug Abuse National Drug Abuse Treatment Clinical Trials Network Clinical Coordinating Center (CCC) and the Na- tional Cancer Institute’s AIDS Malignancy Consortium (AMC) develop clinical trials for populations that IRBs may consider vulnerable: people living with HIV/AIDS (PLWHA) and/or persons who inject drugs (PWID). Common challenges that CCC and AMC investigators face during the consenting process include designing informed con- sent materials that communicate required regulatory elements sim- ply yet effectively, engaging the individual in the consent discussion, and assessing comprehension. Methods to be discussed include: Page 46 of 235 Trials 2017, 18(Suppl 1):200 Page 46 of 235 Page 46 of 235 Consent design factors for readability; Ancillary visual and written aids; Consent process standard operating procedures; Patient advocate feedback and involvement; Participant comprehension assessment. external validity of the protect trial. The data suggest that those randomised are broadly representative of the source population, with limitations in relation to the inclusion of more deprived and ethnic group participants. The extended comprehensive-cohort pro- vides assurance that the outcomes of the randomised group will be clinically relevant and widely generalizable. Demonstrating the external validity of a randomised controlled trial: the design and generalizability of the protect prostate cancer trial P120 Informed consent in acute trauma resuscitation: experiences of a major trauma centre P119 P119 Demonstrating the external validity of a randomised controlled trial: the design and generalizability of the protect prostate cancer trial Jenny Donovan1, Grace Young2, Eleanor Walsh3, Chris Metcalfe4, Athene Lane5, Richard Martin6, David Neal7, Freddie Hamdy8, on behalf of CAP and protect study groups 1Professor of Social Medicine; 2RA Statistics; 3SRA Data management; 4Reader in Statistics; 5Reader in Trials; 6Professor of Epidemiology; 7Professor of Surgical Oncology; 8Professor of Surgery Correspondence: Jenny Donovan Trials 2017, 18(Suppl 1):P119 Background Research investigating the resuscitation and management of un- stable trauma patients is essential to improve care and save lives. The decision to take part in a research study is voluntary and the ethical and legal codes that govern medical practice also apply to clinical trials. Informed consent is a vital part of the research process, however, in emergencies this is challenging. In this review the issue of consent in emergency research is presented with emphasis on ex- periences from a major trauma centre. Methods Methods A population-based cluster-trial of PSA screening using a Zelen de- sign created an intervention arm comprising a prospective cohort study of 80,000 men undergoing PSA testing leading to an embed- ded trial of treatments for localised prostate cancer (protect), and a control arm comprising usual care without formal PSA testing. All men agreeing to participate in the intervention arm entered a pro- spective cohort study of PSA testing and prostate cancer diagnosis. Socio-demographic, clinical and patient-reported symptom and qual- ity of life data were collected at recruitment and when eligible for prostate biopsies. Men diagnosed with localised prostate cancer who agreed to be randomised, and those who declined and chose a treat- ment (‘preference’ group) were followed up in a comprehensive- cohort design. This was extended to include those diagnosed with prostate cancer but excluded from the treatment trial with advanced cancer or excluded other group. We investigated differences in response and/or clinical eligibility at each stage of testing and diagnosis in the prospective cohort study. The characteristics of the randomised men were compared with the preference, advanced and excluded other groups in the extended comprehensive cohort study. Informed consent in acute trauma resuscitation: experiences of a major trauma centre Jenny Donovan1, Grace Young2, Eleanor Walsh3, Chris Metcalfe4, Athene Lane5, Richard Martin6, David Neal7, Freddie Hamdy8, on behalf of CAP and protect study groups j Claire Rourke, Ross Davenport, Karim Brohi Queen Mary University of London Correspondence: Claire Rourke Trials 2017, 18(Suppl 1):P120 Claire Rourke, Ross Davenport, Karim Brohi Queen Mary University of London Correspondence: Claire Rourke Trials 2017, 18(Suppl 1):P120 Ethics Co Results More than 5000 trauma admissions have been screened for eligi- bility into the following trials; Activation of Coagulation & Inflam- mation in Trauma (ACIT –a prospective, observational study), CRYOSTAT (feasibility study, non-CTIMP), itactic (Phase II non- CTIMP), EFIT1 (feasibility study, non-CTIMP), MP4OX (Phase iia & iib CTIMP) and synapse (Phase III CTIMP). Over 1500 patients have been enrolled into ACIT and a further 125 patients recruited into one of the randomised controlled trials (RCT). Following our con- sent procedures only 70 (5%) of participants in the observational study and 2 (2%) subjects in the combined RCTs were withdrawn due to patient or relative refusal of consent. There was no differ- ence in withdrawal rates between observational studies versus rcts and no discrimination between ctimps and non-CTIMPs. Using PLAR/NC agreement rather than seeking consent from the patient or legally appointed representative, the average time from admission to randomisation in the CRYOSTAT and EFIT RCTs was 13 minutes (0–58 mins, n = 52). p Methods Since 2008, all adult trauma patients (16 years) who met local criteria for trauma team activation at The Royal London Hospital have been screened by the Centre for Trauma Sciences Clinical Trial Unit for eli- gibility into a portfolio of clinical trials. In all trials, agreement to enrol a patient into a trial was obtained using a professional legally appointed representative (PLAR) or nominated consultee (NC), in all cases this was the trauma team leader (a physician independent of the research study). Written consent from the patient or next of kin was sought as soon after enrolment as appropriate by a GCP trained researcher. All studies were reviewed and approved by a Regional Ethics Committee (REC). compreh Results The Zelen design produced balanced intervention and control groups. Prospective cohort study participants were more likely to be healthy and from urban and less-deprived areas than non- responders or non-attenders. At subsequent stages in the PSA test- ing and diagnostic pathway, there were few differences between those eligible or ineligible to proceed, or who continued or de- clined participation according to socio-demographic and clinical history characteristics. Men who declined randomisation ‘prefer- ence group’ were more likely to have managerial/professional occu- pations and less deprivation than those agreeing to randomisation. Expected clinical differences were found between the randomised men and the ‘excluded other’ and ‘advanced’ groups; ‘preference’ men were clinically very similar. Conclusion The data from the pro- spective cohort study and extended comprehensive-cohort study provide detailed information to enable consideration of the Background Randomised controlled trials deliver robust and internally valid evi- dence, but external validity is often neglected. This can limit the rele- vance of trial findings for routine practice and hinder the implementation of robust evidence. External validity is a complex concept involving reflection on trial evidence in relation to prior knowledge, statistical understanding, biological plausibility and the interpretation of the impact of the trial’s eligibility criteria. Design features built into the protect trial of prostate cancer treatments facil- itated the assessment of its external validity, and provided an oppor- tunity to consider how trial designs should be used to improve the external validity of trials. Comparing the challenges of the management of trials of medical tests versus randomised controlled trials of health care interventions Design features to en- hance external validity could be adopted more widely in pragmatic trials to investigate selection issues during eligibility assessment and the clinical relevance of the randomised group. This presentation will highlight these challenges and offer tools and best practices for research teams to develop informed consent mate- rials and augment consenting processes. Background An overview of the EME Programme, in terms of its development, purpose, objectives and vision will be presented. The Programme is seeking to encourage studies which have novel methodological de- signs that deliver results more efficiently, reduce the study timeline and maximise the knowledge gained. Collaboration from academic, clinical groups, industry and charities is encouraged. Plasmamatch is a multi-centre phase IIa umbrella trial platform con- sisting of a ctDNA screening component and a therapeutic compo- nent. The primary objective is to assess the safety and activity profile of targeted therapies in patients with targetable mutations identified by ctDNA screening. Patients with metastatic or recurrent locally ad- vanced BC who have received prior systemic treatment in the ad- vanced setting will be invited to participate. Consenting patients will be registered for ctDNA screening and a sample of their blood will be sent to the central laboratory for analysis. Patients with a target- able mutation identified will be invited to enter a treatment cohort and consenting patients will receive treatment targeted to the spe- cific mutation identified. The programme supports translational research evaluating a wide range of novel or re-purposed interventions. The interventions may include diagnostic or prognostic tests and decision-making tools, drugs or biological compounds, psychological treatments, medical devices, and public health initiatives delivered within the NHS. The EME Programme primarily supports clinical trials, and other robustly designed studies that test the efficacy of interventions. The interven- tions should have the potential to improve patient care or benefit the public. Plasmamatch will be opened across a network of ~50 UK Screening Sites, of which ~25 sites will also be designated as Treatment Sites. Screening Only Sites will refer patients to Treatment Sites for treat- ment cohort entry and trial treatment administration. After comple- tion of trial treatment patients will be transferred back to the Screening Only Site for follow-up. Innovative study designs involving stratification, the use of routinely collected digital data or novel methodologies are strongly encour- aged. Hypothesis-driven mechanistic studies, integrated within the efficacy study that explore the mechanisms of action of the interven- tion or the disease, the cause of differing responses, or improve the understanding of adverse effects are also encouraged. Patients with specific targetable mutations identified in tumour se- quencing performed outside of plasmamatch will be eligible to enter one of the treatment cohorts, thereby providing a therapeutic option for patients participating in alternative tumour sequencing initiatives. Funding innovative study designs - The efficacy and mechanism evaluation programme Lisa Douet University of Southampton Trials 2017, 18(Suppl 1):P121 Introduction To discuss the role of the Efficacy and Mechanisms Evaluation Programme in the funding of clinical research. The research funded by the EME Programme has the potential to make a step-change in the treatment of disease. EME research supports research primarily aimed at establishing clinical efficacy but also embedded within this, the Programme encourages the further understanding of treatment and disease mechanisms. Background Plasmamatch ctdna screening will also report on mutations to facili- tate entry into trials outside of plasmamatch, should the patient not be eligible to enter a treatment cohort in plasmamatch. A wide range of studies have been funded to date and examples of studies with novel study designs will be discussed, along with exam- ples of mechanistic studies and what the Programme means by the term ‘proof of concept’. p Conclusions S di f d The plasmamatch umbrella trial platform is dynamic in design, such that further genetic aberrations or molecular subtypes paired with targeted therapies may be added in the future, providing the addition would not compromise the completion of recruitment to existing cohorts. Studies funded through the EME Programme have the potential to have a considerable impact on the treatment of patients in the NHS. The Programme is keen to drive forward novel or infre- quently used study designs that increase the value of the study, by maximising the chances of demonstrating the benefit of an intervention, increasing the knowledge gained or by making the study more efficient. Plasmamatch adds to a growing portfolio of trials assessing the utility of ctDNA and will seek to demonstrate the feasibility of ctDNA as a screening tool for patients with advanced BC with the potential for future integration into routine clinical practice. The UK plasma based molecular profiling of advanced breast cancer to inform therapeutic choices (plasmamatch) trial: a multiple parallel cohort, open-label, multi-centre phase IIa clinical trial aiming to provide proof of principle efficacy for designated targeted therapies in patients with advanced breast cancer where the targetable mutation is identified through ctDNA screening (CRUK/15/010) Discussion Less than 5% of total enrolled patients or their next of kin de- clined consent to continue participation in a research trial. In re- cent years we have experienced a switch in REC opinion with discussions increasingly involving the use of waiver of consent in such trials to avoid potential delays in delivering an intervention or adversely affecting recruitment rates. Furthermore, feedback from the ethics committees has questioned the procedure used for obtaining informed consent as being potentially distressing and insensitive to relatives at a difficult time. In response to this, our protocols no longer pursue consent from bereaved relatives and the participants are allowed to remain in the study using the PLAR/NC agreement. Page 47 of 235 Page 47 of 235 Trials 2017, 18(Suppl 1):200 Circulating tumour DNA (ctDNA) is found in the plasma of over 90% of patients with advanced breast cancer (BC). Screening for the pres- ence of mutations in ctDNA provides a current assessment of the genetic profile of the patient’s recurrent cancer. This is important be- cause the mutations present in cancer cells often change over time when the disease spreads to other sites in the body or after treat- ment. Treatment of recurrent disease is often based on results from primary tumour biopsy as recurrent BC may not be re-biopsied in routine practice. Where a repeat tumour biopsy is performed due to the heterogeneity of cancer the genetic aberrations driving the tumour may not be present in the single biopsied sample to inform treatment decisions. ctDNA screening is more practical and may be more accurate than analysing tumour samples obtained through bi- opsy, is suitable for all patients including those with inaccessible dis- ease, more economical and more acceptable to patients. Ctdna screening can be carried out in a greater number of patients and could lead to a substantial reduction in the number of patients undergoing invasive biopsies. Funding innovative study designs - The efficacy and mechanism evaluation programme Background The Medical Research Council (MRC) recommends that once the ef- fectiveness of a fully-defined intervention has been established in a definitive Randomised Controlled Trial (RCT), the next phase is to de- termine whether others can replicate the intervention and results in clinical settings over the long-term. The Back Skills Training (best) programme is an intervention based on a cognitive-behavioural ap- proach for treating low back pain in primary care settings, and was found to be both clinically and cost-effective in a large RCT (Lamb et al., 2010). In order to deliver the intervention, clinicians in the trial underwent a 2-day training workshop delivered face-to-face. Thus, in order to replicate the intervention in clinical settings, the training needed to be available to clinicians. Given the large number of clini- cians and patients worldwide that could benefit from using this inter- vention, face-to-face training was deemed unfeasible. g Mail-based recruitment and follow-up is a cost-effective method of conducting randomized trials, but requires telephone support for queries from participants and their doctors. It is important to under- stand the resource implications of such a telephone service. Methods ASCEND is a randomized trial of aspirin, and of omega-3 fatty acids, for the primary prevention of vascular disease in people with dia- betes. Between 2005 and 2011, 423,403 potential participants were invited and 121,254 returned a screening form. Of these 26,462 par- ticipants entered a 2 month placebo run-in and 15,480 were subsequently randomized. Follow-up is by 6-monthly mailed ques- tionnaires (since November 2014 participants can complete their questionnaires online). A Freefone number is available for trial re- lated queries including a 24-hour service for urgent medical issues. A record is made of calls using a standard telephone summary within the trial participant management system. For information govern- ance reasons calls relating to people invited who did not consent to join the study are not included in this analysis. Furthermore, until March 2014, unsuccessful attempts to contact participants or their doctors were not recorded. Therefore, this analysis underestimates the total activity during the trial. For each call, the role of the staff member (medical [including doctor or nurse] or administrative), the individual calling or called (the participant, their GP or other person), the time and date, whether the call reached the intended recipient (for outgoing calls after March 2014) and whether the call occurred after randomization was extracted from the study database. Objective g Fang Chen, Marion Mafham, Lucy Fletcher, Allen Young, Jill Barton, Louise Bowman, Jane Armitage University of Oxford Correspondence: Fang Chen Trials 2017, 18(Suppl 1):P124 g Fang Chen, Marion Mafham, Lucy Fletcher, Allen Young, Jill Barton, Louise Bowman, Jane Armitage University of Oxford Correspondence: Fang Chen Trials 2017, 18(Suppl 1):P124 To describe the development and delivery of an online cognitive- behavioural education and training package for health professionals who treat patients with low back pain. The programme is intended support implementation of the evidence-based Back Skills Training programme in clinical practice. Practicalities of obtaining long term follow up data from international sites Sharon Ruddock, Julie Croft, Helen Howard Leeds Clinical Trials Research Unit Correspondence: Sharon Ruddock Trials 2017, 18(Suppl 1):P123 Correspondence: Sharon Ru Trials 2017, 18(Suppl 1):P123 (CRUK/15/010) Judith Bliss1, Sarah Kernaghan1, Charlotte Fribbens2, Laura Stevenson1, James Morden1, Claire Snowdon1, Iain Macpherson3, Andrew Wardley4, Rebecca Roylance5, Richard Baird6 1The Institute of Cancer Research Clinical Trials & Statistics Unit (ICR-CTSU); 2The Institute of Cancer Research; 3The Beatson West of Scotland Cancer Centre; 4The Christie NHS Foundation Trust; 5University College London Hospitals NHS Foundation Trust; 6Cambridge University Hospitals NHS Foundation Trust Correspondence: Judith Bliss Trials 2017, 18(Suppl 1):P122 Judith Bliss1, Sarah Kernaghan1, Charlotte Fribbens2, Laura Stevenson1, James Morden1, Claire Snowdon1, Iain Macpherson3, Andrew Wardley4, Rebecca Roylance5, Richard Baird6 1 Separate long term follow-up studies may be considered where there is interest in longer term outcomes that extend beyond the follow- up period of a trial. There is a worldwide interest in collecting long term cardiovascular and recurrence/survival data in breast cancer patients with ‘triple negative’ disease. BEATRICE was an international phase III trial asses- sing adjuvant treatment in ‘triple negative’ early breast cancer, in which patients were followed up to 5 years post randomisation. LOTUS is a separate long term follow up study which is prospectively Trials 2017, 18(Suppl 1):P122 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 48 of 235 Page 48 of 235 co-ordinating centre, 51% were recorded by medical staff and 49% by study administrators. Of the 36,584 outgoing calls, 24% were made by medical and 76% by administrative staff. Among 20,946 outgoing calls with information about whether the intended recipi- ent was reached, 41% were ‘successful’. The success rate was higher for calls after 5 pm compared to those at other times (56% after 5 pm vs 40% before 5 pm; 2 138, p < 0.001) irrespective of the partic- ipant’s age. evaluating longer term outcomes (up to 15 years post BEATRICE ran- domisation) in this specific population of patients. BEATRICE recruited over 2000 patients across 400 sites worldwide, providing a large, already identified population of potential patients with for LOTUS. The LOTUS trial management group agreed at the outset that recruit- ment into LOTUS would be limited to patients already recruited to the BEATRICE trial due to the availability of baseline data available from BEATRICE and to utilise the established research network of sites. Implementation into practice: the development, enhancement and delivery of an online training programme to support clinicians in the replication of the back skills training (best) programme in practice 1 2 3 3 Implementation into practice: the development, enhancement and delivery of an online training programme to support clinicians in the replication of the back skills training (best) programme in practice 1 2 3 3 Helen Richmond1, Amanda M Hall2, Bethan Copsey3, Gill Jones3, Esther Williamson3, Zara Hansen3, Sarah E Lamb4 1University of Warwick; 2The George Institute for Global Health, University of Oxford We will present further details on our experience of implementing the LOTUS trial and considerations for design of future longer term follow-up studies. 3Centre for Rehabilitation Research, University of Oxford; 4Centre for Rehabilitation Research, University of Oxford/University of Warwick) Correspondence: Helen Richmond Trials 2017, 18(Suppl 1):P125 3Centre for Rehabilitation Research, University of Oxford; 4Centre for Rehabilitation Research, University of Oxford/University of Warwick) Correspondence: Helen Richmond Trials 2017, 18(Suppl 1):P125 Telephone support for mail based recruitment and follow-up in a large randomized trial Fang Chen, Marion Mafham, Lucy Fletcher, Allen Young, Jill Barton, Louise Bowman, Jane Armitage University of Oxford Correspondence: Fang Chen Trials 2017, 18(Suppl 1):P124 p g Conclusion The telephone support for this mail-based trial requires substantial input from both medical and administrative staff, but allows a large study to be run cost-effectively. Contacting trial participants is more effective outside working hours. These findings have resource impli- cations for those planning similar studies. LOTUS is designed to present minimal burden to aid recruitment of both participating sites and patients for example, patient data is col- lected annually to coincide with routine clinic visits, or can be col- lected by phone. BEATRICE patient trial numbers are used in LOTUS for easier data collection at sites, and to enable data linkage between the BEATRICE and LOTUS datasets at analysis. Screening logs for sites were pre-populated for sites to facilitate screening and recruitment of patients. Out of the 53 BEATRICE sites that were approached to take part in LOTUS, 32 sites agreed to take part. Practicalities of obtaining long term follow up data from international sites In order to focus resources and efforts, only BEATRICE sites with 10 or more patients still in follow-up were invited to participate in LOTUS (except in the UK, where all sites were approached). This amounted to 53 sites across 19 countries and still yielded a potential pool of up to 901 patients to reach our planned LOTUS sample size of 250–500 patients. P125 P125 Implementation into practice: the development, enhancement and delivery of an online training programme to support clinicians in the replication of the back skills training (best) programme in practice Helen Richmond1, Amanda M Hall2, Bethan Copsey3, Gill Jones3, Esther Williamson3, Zara Hansen3, Sarah E Lamb4 1University of Warwick; 2The George Institute for Global Health, University of Oxford Background The pro- portion of successful calls before and after 5 pm was compared using a chi squared test. To ensure scalability and accessibility we translated the face-to-face training into an online format, entitled ibest. We pilot-tested the on- line training prototype (ibest) for feasibility, acceptability and credibil- ity with health professionals. During the pilot testing, we identified that despite being acceptable, feasible and credible compared to the face-to-face training, clinicians continued to struggle to implement the intervention in routine practice. Building on this evidence-base, we describe how we enhanced the ibest prototype to support clini- cians to use the evidence-based best programme in their practice. Methods We tailored ibest to address implementation barriers using the The- oretical Domains Framework and the Behaviour Change Technique (BCT) taxonomy. The selection of implementation strategies was in- formed by evidence, clinician and expert opinion and suitability for a sustainable online learning training package. R lt Reference Lamb et al., Group cognitive behavioural treatment for low-back pain in primary care: a randomised controlled trial and cost-effectiveness analysis. The Lancet, 2010;375:916–923. Results We identified 144 relevant primary study papers from a total of 317 records in the first search iteration. Intervention development papers were international: USA (56%), UK (25%), Europe (5%) and rest of the world (14%). We identified 7 approaches to intervention develop- ment (‘type’ in the typology) including theory-based, participatory, person-based and pragmatic. Each type included sub-types of ap- proaches. For example the theory-based approach included ‘Inter- vention Mapping’ (n = 5) and ‘The Behaviour Change Wheel’ (n = 7). Conclusions We have developed a typology to facilitate intervention developers in selecting the most appropriate approach to developing interven- tions for their context. This will feed into further parts of the INDEX study - Delphi, consensus workshops and qualitative interviews - to generate guidance for researchers on how to develop interventions. A systematic review of the different approaches to intervention development Katie Sworn1, Alicia O’Cathain1, Liz Croot1, Edward Duncan2, Pat Hoddinott2, Katrina Turner3, Lucy Yardley4 Katie Sworn1, Alicia O’Cathain1, Liz Croot1, Edward Duncan2, Pat Hoddinott2, Katrina Turner3, Lucy Yardley4 1 2 3 Aims As part the INDEX study (identification and assessment of different approaches to developing complex interventions), we aimed to re- view the range of approaches to intervention development and con- struct a typology to help researchers select the appropriate approach for their context. We also aimed to identify an initial set of core prin- ciples and processes of intervention development for use in a future Delphi Study. Conclusions Following guidance from the World Health Organisation, we have de- scribed the selection of strategies and how these were used to en- hance ibest for implementation. This process will inform the design and evaluation of further implementation interventions and replica- tion of complex interventions found to be effective from Phase III trials. Conclusions Results We included 15 studies that recruited 9035 participants, 7834 of whom provided data primary outcomes data, of which five were symptom-based or clinical outcomes, seven were functional mea- sures, two were combined measures and one assessed quality of life. Primary time points varied from immediately post-intervention to one year. Thirteen of the studies utilised a two-arm, parallel RCT de- sign, one used a four-arm factorial design of which only 2 arms re- lated to physical rehabilitation and one was a cluster RCT. The target populations, interventions and settings were diverse. Applying Djul- begovic’s classification, four studies were significantly in favour of the new treatment, one was significantly in favour of the control treat- ment, eight studies had a true negative outcome, one was inconclu- sive in favour of the new treatment was and one inconclusive in favour of the control treatment. Background Background Randomised controlled trials (RCTs) of rehabilitation interventions are generally complex, involve a broad population, multi-faceted inter- ventions delivered in a complex healthcare system, and measure multiple outcomes. The National Institute for Health Research Health Technology Programme (NIHR-HTA) has funded a number of UK trials of physical rehabilitation interventions. To date, no research has reviewed and synthesised the outcomes of this body of work. In this review we aimed to (a) establish the treatment outcomes of RCTs of physiotherapy (PT), occupational therapy (OT), or speech and lan- guage therapy (SLT) funded by the HTA, and (b) assess how often those interventions that undergo testing in RCTs result in establish- ing the effectiveness of new treatments. Methods g Methods Increasingly researchers are developing complex interventions which are evaluated later in randomised controlled trials. Researchers adopt a range of approaches when developing interventions. However, in this rapidly developing field there is little understanding of the ratio- nales, and strengths and weaknesses, of different approaches to intervention development for different contexts. We included all phase III superiority RCTs funded by NIHR-HTA from 1997 until July 2016, that evaluated a physical rehabilitation programme of PT, OT or SLT and that had reported their main find- ings either in a peer-reviewed journal or as an NIHR-HTA monograph. We extracted data on trial design, target population, intervention de- scriptions, primary outcome(s) and time point(s), any minimally clin- ical important difference (MCID) identified in support of the sample size, proposed and achieved sample size and between-group primary outcome results with 95% confidence intervals. We categorised pri- mary outcome data into one of six options as described by Djulbego- vic et al. (2008): (i) statistically significant in favour of the new treatment; (ii) statistically significant in favour of the control treat- ment; (iii) true negative; (iv) truly inconclusive; (v) inconclusive in favour of new treatment; or (vi) inconclusive in favour of the control treatment. We summarised the extracted data descriptively. Results Treatment success in randomised controlled trials of rehabilitation: a review Victoria Goodwin, Claire Pentecost, Katie Finning, Angelique Hilli, Jacqueline Hill, David Richards 1University of Exeter Correspondence: Victoria Goodwin Trials 2017, 18(Suppl 1):P127 Victoria Goodwin, Claire Pentecost, Katie Finning, Angelique Hilli, Jacqueline Hill, David Richards Victoria Goodwin, Claire Pentecost, Katie Finning, Angelique Hilli, Jacqueline Hill, David Richards This research was carried out at the Centre for Rehabilitation Research in Oxford and was supported by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care Oxford at Oxford Health NHS Foundation Trust. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. Correspondence: Victoria Goodwin Trials 2017, 18(Suppl 1):P127 q Results Up to November 2016, 52,696 calls were recorded. Of these, 8828 were recorded during the run-in period (0.33 calls per run-in partici- pant) and 43,868 after the participant was randomized (0.42 calls per randomized participant per year). Of 16,112 incoming calls to the trial We chose 11 BCTs to target domains of knowledge, skills, beliefs about capabilities, beliefs about consequences, professional role, mo- tivation and goals, and environmental context and resources. The enhanced version of ibest has been accredited with the British Trials 2017, 18(Suppl 1):200 Page 49 of 235 Page 49 of 235 Psychological Society and is currently being evaluated as part of a national level implementation study in the UK. Following an analysis of core methodology papers and books and primary research using each type, an initial core set of principles and processes for developing interventions was identified; these are be- ing refined using further search iterations. p Methods We undertook two interlinked systematic reviews. First, we con- ducted a Best Fit Framework Synthesis by developing an a-priori typ- ology of approaches to intervention development based on the team’s knowledge base. For our first iteration, we searched Medline, psycinfo, CINAHL, ERIC and ASSIA from July 2015 to June 2016 for methodological literature and primary studies on intervention devel- opment. We then reviewed our typology and for each ‘type’ identi- fied the rationale for its use, and its strengths and weaknesses. Further search iterations will be used to refine the typology. Sec- ondly, we the undertook Realist Synthesis on each ‘type’ to hypothe- sise and test key methodological principles and processes by considering the mechanisms at work to successfully inform interven- tion development in different contexts. Conclusions Although most trials reported conclusive findings (13/15) very few in- terventions tested in these trials achieved superior outcomes com- pared with controls (4/15). Despite, therefore, a considerable research effort in rehabilitation interventions, there are just a handful of new interventions that outperform existing approaches available Page 50 of 235 Page 50 of 235 Trials 2017, 18(Suppl 1):200 Page 50 of 235 for clinicians to use in their routine practice. Even in a situation of genuine uncertainty or clinical equipoise, the reasons why so few ex- perimental interventions in rehabilitation that are brought to trial achieve the results expected of them by trialists and intervention de- velopers requires further investigation so that future trials can be tar- geted at potentially more fruitful interventions. controlled, open label trial to compare the efficacy of ex-vivo normo- thermic machine perfusion (NMP) with static cold storage (SCS) for organ preservation prior to liver transplantation (ISRCTN 39731134). Fol- lowing confirmation of donor and recipient eligibility, livers were rando- mised (1:1) to either NMP or SCS, stratified by donor type (donation after brain death (DBD) or circulatory death (DCD) and centre. At the end of preservation, if not discarded, the liver was transplanted in the consented recipient who was managed according to standard local practice and protocols. P129 Design and analysis of the learning curve and clustering effects in randomised surgical trials - A review of current practice Conroy1, J. Blazeby2, G. Burnside1, J. Cook3, C. Gamble1 1Department of Biostatistics, MRC North West Hub for Trials Methodology Research, University of Liverpool; 2Centre for Surgical Research, School of Social & Community Medicine, University of Bristol; 3Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford Correspondence: Conroy Trials 2017, 18(Suppl 1):P129 p p Primary outcome of the study is the difference in peak serum aspar- tate transaminase level (AST) within 7 days post-transplant between the two treatment arms. Secondary outcomes include Primary Non Function, Early Allograft Dysfunction and Patient and Graft Survival. Results 334 livers were randomised with 222 transplanted into recipients from June 2014 to March 2016 in seven European liver transplant centres (UK (4), Spain, Germany and Belgium). Recipients have been followed up for 6 months and the main analysis is currently ongoing. Discussion Introducing a complex intervention using a non-CE marked medical device into what is already a complex organ retrieval, preservation and transplant process provides many methodological hurdles to overcome. Conclusions Furthermore, as the trial adopts an international multicen- tre setting with different regulatory practices in place, several chal- lenges were highlighted during the conduct of the trial which impact directly on the planned analyses, results and interpretation. We will describe the different challenges encountered and how these can be tackled so that it will be of help in the setting of other complex inter- vention trials in transplantation. The topics in this discussion include: trial design and regulatory affairs, statistical and health economic analysis. Randomised controlled trials (RCTs) are considered the highest level of evidence. Consequently, it is important to ensure that they are de- signed, conducted and analysed to the highest possible rigour so that the clinical decisions that they are used to inform are valid. There are many practical and methodological difficulties that a med- ical researcher must overcome to conduct a successful RCT. In trials that involve a surgical intervention, these difficulties are often magni- fied. However, there are also additional challenges specific to surgical trials. This work focuses on two highly prevalent challenges: g y p g 1. Surgical learning curve 2. Clustering within site and surgeon Statistical methods have been developed with low uptake to account for the surgical learning curve and clustering effects separately in surgical trials. This research aims to evaluate these and determine when and how to apply them, by considering these methodologies individually and then jointly. P130 P130 Machine perfusion in liver transplantation: practical issues to consider in the design and conduct of a complex multinational interventional trial and the potential impact on the analysis Virginia Chiocchia1, David Nasralla2, Ally Bradley2, Richeal M. Burns3, Susan Dutton4, Rutger Ploeg2, Peter Friend2 1Centre for Statistics in Medicine (CSM) and Surgical Intervention Trials Unit (SITU), University of Oxford; 2Nuffield Department of Surgical Sciences, University of Oxford; 3Health Economics Research Centre (HERC), University of Oxford; 4Oxford Clinical Trials Research Unit (OCTRU) and Centre for Statistics in Medicine (CSM), University of Oxford P130 Machine perfusion in liver transplantation: practical issues to consider in the design and conduct of a complex multinational interventional trial and the potential impact on the analysis Virginia Chiocchia1, David Nasralla2, Ally Bradley2, Richeal M. Burns3, Susan Dutton4, Rutger Ploeg2, Peter Friend2 1Centre for Statistics in Medicine (CSM) and Surgical Intervention Trials Unit (SITU), University of Oxford; 2Nuffield Department of Surgical Sciences, University of Oxford; 3Health Economics Research Centre (HERC), University of Oxford; 4Oxford Clinical Trials Research Unit (OCTRU) and Centre for Statistics in Medicine (CSM), University of Oxford g This presentation will describe how CTRU adapted its data manage- ment processes from data submitted on paper CRFs to data submit- ted via RDE. Several key topics of this adaptation to RDE will be covered comprising discussion of the central questions behind the change, challenges faced and solutions found. Specifically, these topics will include addressing the background questions, why we de- layed the decision to move to RDE, what factors eventually prompted the change and how we might interact differently with sites in terms of training. They will also cover the challenge of replacing Microsoft Excel based data management logs on which data management pro- cesses, such as data verification, had often become entirely dependent especially on larger studies. As a solution to these chal- lenges, the design and creation of a data management dashboard y Correspondence: Virginia Chiocchia Trials 2017, 18(Suppl 1):P130 Correspondence: Virginia Chiocchia Trials 2017, 18(Suppl 1):P130 Breaking up is hard to do: letting go of paper case report forms (CRFs) and the challenges of adapting data management processes to remote data entry (RDE) The ultimate aim of this project is to improve the design, analysis, and generalizability of surgical trials. This will be achieved by devel- oping understanding about when learning curves and clustering ef- fects impact on the conclusions of an RCT by determining; the available methodologies and the impact of incorporating these methodologies. This will inform the impact of adjusting for these ef- fects and their impact on interpretation. This will be done consider- ing each effect in isolation and then in combination. y ( ) Paul McGarry Leeds Institute of Clinical Trials Research (LICTR) Trials 2017, 18(Suppl 1):P131 The Clinical Trials Research Unit (CTRU) at the Leeds Institute of Clin- ical Trials Research (LICTR) is a large academic trials unit organised into three divisions; Cancer, Complex Interventions and Comprehen- sive Health Research. The unit was established in 1991 and now has almost 200 staff running over 30 studies as well as a strong meth- odological portfolio and other applied health research in each of the three divisions. All of our current studies collect their data on paper CRFs and we receive approximately 240,000 pages of data and man- age approximately 12,100,000 data items per year. In 2006 initial at- tempts at remote data entry proved unsuccessful largely due to poor internet connectivity at many recruiting hospitals and systems that were not intended to be used remotely. With recent improvements in connectivity, systems and more widespread use of RDE, particu- larly in the commercial setting, a decision was taken within the LICTR to reinvest resource in conducting RDE trials. For the data manage- ment team tasked with taking this change forward at LICTR the deci- sion came with a series of demands and challenges to overcome. To inform this development, current practice has been established as to how surgical learning and clustering are accounted for in trial de- sign and analysis. A survey to establish current practice and require- ments within UK registered Clinical Trials Units has been undertaken. A cohort of published RCTs within medical journals and HTA mono- graphs has been obtained establishing how statistical methodologies are currently being applied and reported. Results of this work will be presented with how these findings im- pact development of methods and guidelines. Central chart review of complex outcomes Central chart review of complex outcomes Trisha Boekhoudt1, Rebecca Clifton2 1George Washington University; 2George Washington University Biostatistics Center Correspondence: Trisha Boekhoudt Trials 2017, 18(Suppl 1):P132 p Trisha Boekhoudt1, Rebecca Clifton2 1 2 MTB members for each session are identified from a large pool of ex- perts who have been recruited to participate. Clinical site requests for MTB review are facilitated through the study’s electronic data capture (EDC) system. The TAPUR study team is notified of a pending case and materials, including a clinical case summary, genomic test report, and the pathology report for the specimen tested, are pre- pared and distributed in advance. Following the session, the treat- ment options with rationale are captured and reported back to the clinical site through the EDC. Prior experience has shown the importance of performing central chart reviews of complex outcomes to ensure correct classification. We conducted a randomized controlled trial of 10,000 pregnant women across 16 clinical centers. The primary outcome was any pregnancy associated hypertension associated with serious maternal and infant complications, and the foremost secondary outcome was preeclampsia. Both outcomes had many different criteria (e.g., hyper- tension severity, proteinuria, thrombocytopenia) that would lead to a diagnosis. Each clinical center completed an Outcome Diagnosis Form that captured each of the elements included in the outcome definitions. One of the more complex criteria was the abstraction of qualifying blood pressures to determine the severity of hypertension as each patient had multiple intrapartum measurements and some of these measurements were deemed too labile to count towards the definition (e.g. During labor or cesarean). This meant the start and end of labor and cesarean were critical to the diagnosis and any inaccuracy in the qualifying time period could result in errors. Due to the complexity of the primary outcome and to confirm the diagnosis of preeclampsia, the protocol subcommittee planned a central chart review for all women whose Outcome Diagnosis Form reported preg- nancy associated hypertension. De-identified participant medical re- cords were reviewed by a team which consisted of two medical doctors, a nurse coordinator, and one coordinating center represen- tative. A total of 3,161 charts were reviewed by 21 chart review team members who were blinded to treatment assignment. During the chart review, data elements were discussed and concurrent re- sponses were recorded on a new study form by the coordinating center representative. Navigating the clinicaltrials.gov database: a case study Gillian Gresham, Jill Meinert, Stephan Ehrhardt, Lawrence J. Appel, Curtis L. Meinert P133 Initial experience with a virtual molecular tumor board in a pragmatic precision medicine study Kaitlyn Antonelli, Pam K. Mangat, Suanna S. Bruinooge, Richard L. Schilsky American Society of Clinical Oncology Correspondence: Kaitlyn Antonelli Trials 2017, 18(Suppl 1):P133 P133 Initial experience with a virtual molecular tumor board in a pragmatic precision medicine study The establishment of databases to store information and results about a clinical trial, registered under an index number, has allowed for the identification and tracking of clinical trials. Consequently, the download and analysis of clinical trial information, whether published or unpublished, is possible and has allowed methodologists to take a closer look at characteristics and trends of clinical trials. While many trial databases exist, this report will focus on the clinicaltrials.gov registry. Clinicaltrials.gov became operational in 2000 and is man- dated by the United States Government. Kaitlyn Antonelli, Pam K. Mangat, Suanna S. Bruinooge, Richard L. Schilsky American Society of Clinical Oncology Correspondence: Kaitlyn Antonelli Trials 2017, 18(Suppl 1):P133 The American Society of Clinical Oncology’s (ASCO) Targeted Agent and Profiling Utilization Registry Study (TAPUR), a non-randomized, pragmatic precision medicine basket trial, provides a virtual multidis- ciplinary Molecular Tumor Board (MTB) to support participating clin- ical sites in identifying the appropriate study drug to target a genomic alteration in a patient’s tumor. The TAPUR study offers pa- tients with advanced cancer access to FDA-approved, targeted drugs in non-indicated cancers while capturing safety and efficacy out- comes. Drugs are matched to genomic alterations based on a set of genomic matching rules to help guide the physician’s treatment deci- sion. If a proposed drug-variant match is not accepted by the rules Background & Methods The Consortium for Organ Preservation in Europe (COPE) is conducting several clinical studies, one of which is a multicentre, randomised, Page 51 of 235 Page 51 of 235 Trials 2017, 18(Suppl 1):200 will also be discussed. Finally, the presentation will look forward and outline our ideas for further adaptations and innovations from paper crfs to RDE including plans for a site facing dashboard to be used by site research staff and data managers. engine or the treating physician wishes guidance in interpreting the tumor genomic profile, TAPUR offers a virtual MTB to review cases and identify treatment options. The MTB is available to all participating sites and meets weekly by webinar. MTB members at each session include at least two clinical oncologists, one molecular pathologist, and one patient advocate. The treating physician or other representatives from the clinical care team also participate in the session. The MTB offers interpretation of genomic test results and identifies potential treatment options either within TAPUR, on other clinical trials, or with other drugs outside of TAPUR. Central chart review of complex outcomes The adjudicated outcomes were then com- pared with the original outcomes reported by the site. Discrepancy listings were sent to each of the clinical centers to affirm the review committee decision. There were a total of 553 (17.5%) participants which required changes to the diagnosis of pregnancy associated hypertension and/or preeclampsia. Chart reviews were an important part of the outcome adjudication and resulted in the correct classifi- cation of the primary and major secondary outcomes. To date, 172 patients have been registered to the TAPUR study, and 102 have found a match to a TAPUR study drug and received treat- ment. Approximately 75% of enrolled participants matched to a study drug through the automated genomic matching rules and 25% of participants matched through the MTB review process. In 8 of the 34 cases reviewed, although no TAPUR drug match could be identi- fied, the MTB provided treatment options for drugs outside of TAPUR and on other clinical trials, if available. One of TAPUR’s goals is to de- scribe the concordance of the treatment proposed by the clinical site with the options identified by the MTB. Thus far, proposed treatment options are concordant in 38% of cases reviewed by the MTB. p y Some challenges with convening a weekly MTB include staff time in- volved in the coordination of sessions and variability in reviews due to rotating volunteer participants. To minimize variability in MTB case review outcomes, the study provides training to MTB members, and begins each session clarifying the MTB’s role, authority, and responsibilities. The presentation will discuss the rationale through which ASCO formed the TAPUR MTB, its positive impact on the study, and chal- lenges to date. The governing and guiding processes and workflows will also be described. P134 Navigating the clinicaltrials.gov database: a case study Gillian Gresham, Jill Meinert, Stephan Ehrhardt, Lawrence J. Appel, Curtis L. Meinert Johns Hopkins School of Public Health Correspondence: Gillian Gresham Trials 2017, 18(Suppl 1):P134 Results Nine observational cohort studies in gastrointestinal surgery were run through the Birmingham Surgical Trials Consortium between 2013–2016. Six (66.6%) were conducted internationally, across a total of 92 countries, with the others being UK-only. Study questions spanned topics in surgery and perioperative medicine; surgical site infection, post-operative mortality, analgesia, acute kidney injury, obesity, stoma closure, cholecystectomy and colectomy. A total of 58,500 patient-level records were included, containing 1,308,000 individual data points. The 6 studies that underwent data point valid- ation confirmed a >95% data accuracy, and >90% case ascertainment rate. Hypotheses generated from these studies have directly in- formed the preparation of two major grant applications; globalsurg Surgical Site Infection Trial (pilot work funded by a Wellcome Trust/ Medical Research Council grant), Modifying Inflammation using Drugs Around Surgery (MIDAS, unfunded), and 3 further grant appli- cations are in progress. All grant applications have included principle investigators from prominent sites contributing to observational data collection. i i In attempts to characterize NIH-funded clinical trials, we encoun- tered: (1) Difficulty importing files into the statistical programs due to formatting issues (e.g., presence of special characters, hard returns, commas, xml text); (2) Misclassification of trials due to misinterpret- ation of registration fields (e.g., “funder” vs. “collaborator”; “interven- tional” vs. “observational”); (3) Duplicate counts of same trial as a result “obsolete” NCT identification numbers; (4) Misclassification of trials as to funding source (e.g., trials that were funded by NIH were not listed as being NIH-funded in registry); (5) Missing registration fields (e.g. Number of sites, number of publications). While some of the limitations listed are dependent on how registrants interpret and enter information, there are steps that can be taken to improve regis- tration. One such step would involve improving the editing process to keep files “clean.” Providing a test environment for developers to explore data entry options and allowing for the creation of scripts to extract specific data from the website would also be useful. Second, clarifying definitions of registration fields and including examples would help avoid misinterpretation. Furthermore, refining the “other” Categories and using drop-down menus and checkboxes to avoid free-text would reduce incorrect entries and inconsistencies. Third, allowing “obsolete” NCT ids to be filtered out and providing reasons for why the ids were listed as obsolete would avoid double-counting trials. Discussion We have demonstrated the ability to generate high-quality, patient- level data across diverse settings, generating hypotheses for rando- mised clinical trials. Validated, observational data serves as high- quality internal pilot, and provides accurate baseline rates to power trial interventions, across specific collaborating centres. By successful submission of data to observational studies, site investigators dem- onstrate their capacity for research leadership and “Self-select” for in- volvement in future clinical trials. Corporate authorship flattens the traditional hierarchical model research delivery and publication, and fosters an environment of collaboration for high quality prospective studies and their consequent clinical trials. Trial registries, such as clinicaltrials.gov, have allowed people to ac- cess, download and analyze trials data that otherwise would not be possible. However, improvements can be made to increase its useful- ness as a tool to describe characteristics of trials and trends over time. y Objective y Objective Using a case example, we will describe some of the challenges encoun- tered when identifying and analyzing trials registered in clinicaltrials.gov. Methods We downloaded the clinicaltrials.gov dataset and imported it into two statistical programs (SAS and Stata). We limited the dataset to interventional studies funded by the National Institutes of Health (NIH). All methods and tabulations were performed and compared by two independent authors. Trials 2017, 18(Suppl 1):200 Page 52 of 235 Page 52 of 235 Reference [1] Bhangu A, Kolias AG, Pinkney T, Hall NJ, Fitzgerald JE. Surgical research collaboratives in the UK. Lancet. 2013 Sep 28;382(9898):1091–2. doi:10.1016/S0140-6736(13)62013-9. Pubmed PMID: 24075040. 35 Using trainee-led, collaborative research to generate hypotheses for multi-centre clinical trials James Glasbey1, Dmitri Nepogodiev1, Richard Wilkin2, Dion G. Morton1, Laura Magill3, Aneel A. Bhangu1, Thomas Pinkney1 1University of Birmingham; 2West Midlands Research Collaborative; 3Birmingham Clinical Trials Unit Correspondence: James Glasbey Trials 2017, 18(Suppl 1):P135 Using trainee-led, collaborative research to generate hypotheses for multi-centre clinical trials 1 1 2 1 P136 Footprints in primary care: using mixed methods to assess fidelity to a complex intervention for frequently attending patients Rebecca Barnes1, Helen Cramer1, Clare Thomas1, Marcus Jepson1, Sandra Hollinghurst1, Sue Jackson2, Charlie Record3, Chris Metcalfe1, David Kessler1 1University of Bristol; 2University of the West of England; 3Frome Valley Medical Centre Correspondence: Rebecca Barnes Trials 2017, 18(Suppl 1):P136 P136 Footprints in primary care: using mixed methods to assess fidelity to a complex intervention for frequently attending patients Rebecca Barnes1, Helen Cramer1, Clare Thomas1, Marcus Jepson1, Sandra Hollinghurst1, Sue Jackson2, Charlie Record3, Chris Metcalfe1, David Kessler1 1University of Bristol; 2University of the West of England; 3Frome Valley Results Finally, continuing to expand and update the registry will be important as more trials are registered in coming years, with an ul- timate goal of having a worldwide registry for all trials. C l i Aim The first regional research collaborative within general surgery was the West Midlands Research Collaborative. The methodological ex- pertise acquired within this region has been used to design and con- duct a number of observational cohort studies, through the Royal College of Surgeons-funded Birmingham Surgical Trials Consortium. In this project, we collate our progress to date. The aim of our feasibility study was to explore the main uncertainties to designing a full trial to evaluate effectiveness and cost- effectiveness of this intervention in a pilot cluster RCT involving six practices (4 intervention, 2 usual care). Two of our key objectives were to optimise the content and delivery of staff training to support the intervention; and to assess the extent of implementation fidelity. Methods Background 1University of Bristol; 2University of the West of England; 3Frome Valley Medical Centre 1University of Bristol; 2University of the West of England; 3Frome Valley Medical Centre Over the past 8 years, trainee-led surgical research collaboratives have evolved across the UK, giving national coverage across both general surgery and all surgical subspecialties [1]. Several of these groups have undertaken protocol-driven, “Snap-shot” audit and re- search projects across multiple centres. Patient identification and follow-up periods are deliberately short, facilitating trainee involve- ment alongside clinical schedules. This approach allows for a large number of patients to be included in less time, prevents repetition, and permits greater generalisability than single-centre studies. The collaborative model of authorship dictates publication of research output under a single, corporate authorship. In the UK, the NHS Re- search Ethics Committee do not require formal ethical approval for this study type, as they are fully anonymised and do not alter patient care. Background Background At present the NHS is struggling to meet the demands on the ser- vice. The idea for this study originated in a local primary care prac- tice who felt that improvements could be made regarding how it was managing and caring for its most frequent attenders. The idea was developed into a RCGP award-winning intervention consisting of several components including matching eligible patients with a named GP, and training GPs to unpack background psychosocial is- sues in a contained way during the consultation using the ‘BATHE’ technique. Methods The number of participants recruited to the BEADS trial was recorded and presented by centre and by month. Overall recruitment rate was calculated by centre-month along with the 95% confidence interval. Simulations were carried out to simulate extending the recruitment period up to 80 centre-months. Data were simulated from a Poisson distribution and further simulations were carried out using a negative binomial distribution due to overdispersion in the pilot data. Recruit- ment rates and their 95% confidence intervals were calculated for each month using the simulated data to examine the precision of es- timates from an extended pilot recruitment period. Three values of the mean (1, 2 and 4), the recruitment rate, were used. The moving recruitment rate was plotted against centre-month for each simula- tion. Percentage bias and percentage coverage were calculated for each centre-month. Bias and coverage were plotted against centre- month to examine how these changed as recruitment period increased. A common objective of an Internal Pilot is to assess the trial’s recruit- ment rate at a pre-specified time. This is with the aim of reassuring the Trial Steering Committee, Funders and Sponsors that the trial is well received by patients and health practitioners, and is likely to be completed on time and within budget. However, it is possible that a trial’s recruitment rate cannot always be assumed to be constant over time. Another issue is that on-going individual recruitment after cluster randomisation will likely introduce selection bias. For some medical conditions, such as rare, chronic conditions, the majority of patients may already be registered at the site, and there are very few patients presenting or leaving the care of the service. It is possible that assessing the recruitment from the number of pa- tients already registered at each site may tell us almost as much about final recruitment to the trial as if we had waited until the end of the planned recruitment period. P138 Analysis of electronic medical records data throughout the 12 month intervention period enabled us to monitor dose, reach, provide mo- tivational feedback and document the effects of subsequent imple- menter trainings. Observations of appointment-making in all four intervention practices and video-recordings of all 12 implementer trainings elicited practical barriers and facilitators that could be ad- dressed, as well as success stories. Conversation analyses of 20 con- sultation recordings enabled a dynamic assessment of the delivery and receipt of BATHE in situ, that revealed common pitfalls in deliv- ery; specified and added new dimensions to the underpinning theor- etical assumptions of the intervention; and provided valuable real- world examples for future training. P138 Estimating the underlying overall and centre recruitment rates in external pilot trials- how many centre-months of recruitment data do we need? A case study of the BEADS pilot trial Laura Mandefield1, Stephen Walters1, The BEADS Trial Team2 1University of Sheffield; 2University of Sheffield & University of Nottingham Correspondence: Laura Mandefield Trials 2017, 18(Suppl 1):P138 Background and Aims Pilot trials are recommended to estimate certain key parameters for a definitive randomised controlled trial (RCT). The recruitment rate (number of participants recruited in a unit time period) is usually of particular importance as it will inform the number of centres and re- cruitment period required for a full trial. The BEADS external pilot trial intended to recruit over 12 months at 3 centres (36 centre- months of recruitment) to assess the feasibility of a definitive trial comparing Behavioural Activation Therapy (BAT) and usual care for treatment of post-stroke depression. Due to delays during the trial set up, recruitment was delayed in all centres. A total of 28 centre- months of recruitment were completed. It was suggested that re- cruitment period should be extended to meet the original target of 12 months per centre. It was decided that the recruitment period would not be extended and we should use the centre-months com- pleted to estimate recruitment rate. We aimed to investigate how many centre-months would be required to estimate the underlying recruitment rate using simulations. Methods Protocols for collaborative studies conducted with the Birmingham Surgical Trials Consortium were collated from group websites, and publication records. Recruitment figures, data points, and validation statistics were extracted from secure electronic records, and pub- lished data sets. Data was analysed using descriptive statistics. As part of the feasibility work, qualitative interviews were held with the stakeholders to build a clear description of the intervention, how it was implemented and expected to work. To ensure a high quality evaluation of implementation fidelity our study design included the Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 53 of 235 then randomise clusters and start following up individual participants. collection a diverse range of implementation data (quantitative and qualitative) at multiple time points. Observations of implementer training sessions and of appointment-making between patients and reception staff, were conducted. A varied sample of face-to-face and telephone consultations of GPs using the BATHE technique with pa- tients were video and audio recorded and transcribed in all interven- tion practices. Conversation analytic methods were applied to assess fidelity to BATHE and the nature and extent of patient response in the consultation recordings Routine monitoring data was collected from practice records to determine how often study patients were being matched with their named gps and patient consultation re- cords were audited for presence of a BATHE code. All quantitative data was analysed descriptively to determine intervention dose and reach. Through the use of Gantt charts, and 3 contrasting research ques- tions recently commissioned by the National Institute of Health Re- search, we ask which internal study would be the most appropriate for a Cluster RCT. We evaluate this in terms of minimising bias, the confidence of the recruitment estimates, de-risking the investment made into a potentially unfeasible trial, capitalising on preliminary feasibility research, additional information gained (if a sample size re- estimation could also be done), the validity of site registry lists to in- form recruitment estimates, and other consequences of the timing in relation to the trial’s population needs, intervention delivery and measured outcomes. p Discussion The findings were used to provide tailored top-up trainings, to clarify and help address misunderstandings and problems in implementa- tion and to encourage implementer engagement via whole practice and individual level feedback. Mixed methods were valuable at dif- ferent timepoints in enabling a full exploration of what might deter- mine the success or failure of a future trial; to optimise training and implementation fidelity; and to understand how and why future par- ticipants might resist or engage with the intervention. Conclusion This review, which is pending final results, will determine how often feasibility studies are being used to inform SW- CRTs and identify which feasibility issues are being investigated. Any information that is gained on how these feasibility studies have informed the main tri- als, will allow us to gain an insight into how feasibility studies can benefit SW- CRTs. Future qualitative work will determine which as- pects of feasibility studies are considered most useful and what bar- riers are commonly encountered when conducting a SW-CRT. The use of feasibility studies for stepped-wedge cluster randomised trials: a review of impact and scope 1 2 1 p p We are interested in obtaining responses from stakeholders with a wide range of involvements in SW- CRTs. These may be individuals that have been involved in the design, conduct or analysis of SW- CRTs, individuals that are conducting methodological research into SW- CRTs, individuals that have sat on funding panels where deci- sions have been made on grant applications for SW- CRTs or individ- uals with any other involvement in SW- CRTs. In addition to conference attendees, invitations to complete the online question- naire will be sent to the authors of SW- CRTs and feasibility studies for SW- CRTs identified by our review, authors of methodological pa- pers on SW- CRTs, funding panel members, advisors (such as the Re- search Design Service) and the wider community (from lists of SW- CRT conference attendees, the Allstat mailing list, clinical trials units etc.). The snowball technique will be implemented by encouraging recipients to forward the questionnaire to their contacts who are also involved in SW- CRTs. Background h d The stepped-wedge cluster randomised trial (SW-CRT) is a complex design for which many decisions must be made during the design stage, such as the required number and length of steps. Feasibility studies might help to inform these decisions and increase the likeli- hood of the main trial’s success. However, there is currently no guid- ance on how feasibility studies for SW-CRTs should be conducted. This review, the first in a series of related projects, aims to establish how often feasibility studies are being conducted for SW- CRTs and determine which feasibility issues are currently being investigated. Ultimately this work will lead to guidance on how feasibility studies in SW- CRTs should be conducted. Identification of participants for a questionnaire and interview study investigating the feasibility issues encountered during stepped-wedge cluster randomised trials Caroline Kristunas1, Karla Hemming2, Helen C. Eborall1, Laura J. Gray1 1University of Leicester; 2University of Birmingham Correspondence: Caroline Kristunas Trials 2017, 18(Suppl 1):P140 g Caroline Kristunas1, Karla Hemming2, Helen C. Eborall1, Laura J. Gray1 1University of Leicester; 2University of Birmingham Correspondence: Caroline Kristunas Trials 2017, 18(Suppl 1):P140 Conclusion The questionnaire and interview studies will gain both a breadth and depth of information on the issues affecting the feasibility of SW- CRTs. These studies form the second part of a series of related pro- jects which will ultimately lead to guidance on how feasibility studies in SW- CRTs should be conducted. Background As part of a randomised controlled trial comparing the use of mul- tiple daily injections of insulin with pumped infused insulin in newly- diagnosed paediatric Type 1 Diabetes Mellitus patients, we investi- gated the availability and feasibility of PLICS data for estimating diabetes-related hospital inpatient stays. The stepped-wedge cluster randomised trial (SW-CRT) is a complex design for which many decisions must be made during the design stage. If mistakes are made when making these decisions then the trial might prove to be unsuccessful. If the barriers to success are known prior to the trial then a feasibility study can be conducted. Unsuccessful trials are unlikely to be published and so the issues that they faced, which might be common, may go unreported and there- fore steps cannot be taken to prevent them from occurring again. We have conducted a review of feasibility studies for SW- CRTs which Methods and analysis Searches for feasibility studies for SW- CRTs were conducted in Ovid MEDLINE, Scopus, and psycinfo. Relevant studies were identified via titles, abstracts and full-text retrievals according to pre-defined study inclusion criteria. Data were abstracted on the aims of these studies and how these studies were able to inform the main trial. In order to also identify unpublished feasibility studies for SW- CRTs, fully pub- lished SW- CRTs were identified from the most recent systematic re- views. The authors of these studies were contacted with the aim of determining whether any unpublished feasibility work was con- ducted prior to the main trial. In addition, the lead statisticians for registered UK clinical trials units were contacted to acquire informa- tion on feasibility work that is being undertaken by these units to in- form SW- CRTs. Participants with complementary experiences of SW- CRTs may be in- vited to take part in an interview study that will aim to build upon the findings of the questionnaire study by gaining a greater depth of information on the issues faced by SW- CRTs. C l i Introduction Trial-based economic evaluations rely on a number of methods for estimating resource use and costs. The use of routine data has hith- erto been limited, with accuracy of coding, confidentiality, ownership and access having been previously identified as significant barriers to access. The Department of Health in England has recommended the use of Patient Level Information and Costing Systems (PLICS) to understand financial drivers at patient, specialty and hospital levels. These provide an opportunity for estimating secondary care costs within economic evaluations. Patient-level information and costing systems (PLICS) as a source of routinely collected cost data for trial-based economic evaluations Colin Ridyard, Dyfrig Hughes Bangor University Correspondence: Colin Ridyard Trials 2017, 18(Suppl 1):P141 y Methods The questionnaire will consist of both closed questions and free-text responses and will be informed by the findings of our review. Partici- pants will be asked about the type of involvement that they have had in SW- CRTs, as well as the issues that they have known SW- CRTs to encounter and those that they are concerned that SW- CRTs may face. These questions will ask the participant either about a par- ticular issue that was identified by our review or will be open for the participant to discuss additional issues that we have not identified. We are interested in obtaining responses from stakeholders with a wide range of involvements in SW- CRTs. These may be individuals that have been involved in the design, conduct or analysis of SW- CRTs, individuals that are conducting methodological research into SW- CRTs, individuals that have sat on funding panels where deci- sions have been made on grant applications for SW- CRTs or individ- uals with any other involvement in SW- CRTs. In addition to conference attendees, invitations to complete the online question- naire will be sent to the authors of SW- CRTs and feasibility studies for SW- CRTs identified by our review, authors of methodological pa- pers on SW- CRTs, funding panel members, advisors (such as the Re- search Design Service) and the wider community (from lists of SW- CRT conference attendees, the Allstat mailing list, clinical trials units etc.). The snowball technique will be implemented by encouraging recipients to forward the questionnaire to their contacts who are also involved in SW- CRTs. The questionnaire will consist of both closed questions and free-text responses and will be informed by the findings of our review. Partici- pants will be asked about the type of involvement that they have had in SW- CRTs, as well as the issues that they have known SW- CRTs to encounter and those that they are concerned that SW- CRTs may face. These questions will ask the participant either about a par- ticular issue that was identified by our review or will be open for the participant to discuss additional issues that we have not identified. P139 The use of feasibility studies for stepped-wedge cluster randomised trials: a review of impact and scope Caroline Kristunas1, Karla Hemming2, Helen C. Eborall1, Laura J. Gray1 1University of Leicester; 2University of Birmingham Correspondence: Caroline Kristunas Trials 2017, 18(Suppl 1):P139 Results This raises the question: ‘When is the best time to internally assess recruitment in a Cluster RCT?’ We consider two different stages in a trial’s life to execute an assessment of recruitment, through an In- ternal Pilot Study and an Internal Feasibility Study. The number of participants recruited to the BEADS trial per month at each centre ranged from 0 to 6; with a mean of 1.75 (1.12, 2.38) per month. Results from the simulations showed that although the level of precision increased as centre-months increased, the level of preci- sion gained by extending pilot recruitment period from 28 to 36 centre-months was not substantial. This was the case for all values of underlying recruitment rate and both the Poisson and negative bino- mial simulations. Furthermore, the decrease in level of bias and in- crease in coverage were both relatively small. Conclusions: Although a reduction in the pilot recruitment period was not planned, an extra Internal Pilot Study: Randomise clusters, then start recruiting and fol- lowing up individual participants, and have a go/no-go decision based on the recruitment rate, at a point early in the planned recruit- ment period. Internal Feasibility Study: Start recruiting individual participants, assess a pre-specified go/no-go decision point at the end of the planned recruitment period based on recruitment numbers, Page 54 of 235 Trials 2017, 18(Suppl 1):200 Page 54 of 235 Trials 2017, 18(Suppl 1):200 identified the feasibility issues that are currently being investigated for SW- CRTs. However, further issues are likely to exist given the possible publication bias. We aim to identify conference participants to take part in an online questionnaire which aims to identify further feasibility issues that are encountered by SW- CRTs. Methods eight centre months would not have greatly increased the precision or accuracy of our estimate of underlying recruitment rate and its 95% confidence intervals. More general recommendations for pilot recruitment can be made based on our different expected recruit- ment rates and proposed underlying distributions. Background g The Incremental Cost Effectiveness Ratio (ICER) is widely used to inform resource allocation decisions concerning health technolo- gies. However, the ICER is a point estimate and subject to consid- erable variability. This variability is often presented in Cost- Effectiveness Acceptability Curves (CEAC), which have underlying assumptions relating to both the correlation between the incre- mental costs and the incremental effects and their distributions. Statistical methods have been proposed to account for this in trial-based economic evaluations. These include non-parametric bootstrapping, and frequentist approaches such as Ordinary Least Square regression (OLS). Bayesian methods such as Generalised Linear Models (GLM) with Normal, Beta and Gamma distribution in cost and effects are scarcely used in trial-based economic evaluation, but may have utility in certain contexts, such as when the patient-level data is non normally distributed, the analysis of the trial is based on a small sample size, and when dealing with imbalanced covariates. (infliximab). The EMA takes a ‘Totality of evidence’ approach to assessing the benefit-risk balance by requiring that a comparability exercise dem- onstrates similarity in terms of quality characteristics, biological activ- ity, safety and efficacy. This involves a stepwise approach to conducting non-clinical and clinical studies. Clinical studies are con- ducted in one population with extrapolation allowed to other indica- tions given adequate scientific justification. Sponsor data for infliximab biosimilars has highlighted differences to RMPs vis-à-vis the ability to induce antibody-dependent cellular cyto- toxicity (ADCC), which is a potential mechanism of action for anti- TNF-alpha drugs in CD, and the number of adverse events. Whilst regulators in Europe, Japan and Australia approved the biosimilar Inflectra, the USA and Canada were more risk averse, first rejecting the biosimilar before approving following sponsor submission of The EMA takes a ‘Totality of evidence’ approach to assessing the benefit-risk balance by requiring that a comparability exercise dem- onstrates similarity in terms of quality characteristics, biological activ- ity, safety and efficacy. This involves a stepwise approach to conducting non-clinical and clinical studies. Clinical studies are con- ducted in one population with extrapolation allowed to other indica- tions given adequate scientific justification. g j Sponsor data for infliximab biosimilars has highlighted differences to RMPs vis-à-vis the ability to induce antibody-dependent cellular cyto- toxicity (ADCC), which is a potential mechanism of action for anti- TNF-alpha drugs in CD, and the number of adverse events. Acknowledgements Funded by the MRC North West Hub in Trials Methodological Research (MR/K025635/1) and the NIHR Health Technology Assessment programme (08/14/39). Heather Catt1, Dyfrig Hughes2, Keith Bodger1, Jamie Kirkham1 1University of Liverpool; 2University of Bangor y p Correspondence: Heather Catt Trials 2017, 18(Suppl 1):P143 Correspondence: Heather Catt Trials 2017, 18(Suppl 1):P143 Background Background Crohn’s disease (CD) is a chronic condition causing inflammation of the digestive tract, requiring ongoing treatment. Biological drugs have improved the quality of life for many patients. However, they are expensive and are linked to serious adverse events. Biosimilar is the term given by the European Medicines Agency (EMA) to a bio- logical drug that contains a version of the active substance of an authorised biological reference medicinal product (RMP). As the pat- ents for RMPs expire, applications are being made for biosimilars. The first CD biological treatment patent to expire was for Remicade (infliximab). Giovanna Culeddu1, Nicky J. Welton2, Dyfrig Hughes1 1Bangor University; 2University of Bristol Correspondence: Giovanna Culeddu Trials 2017, 18(Suppl 1):P142 Method We obtained consent to access patients’ electronic records from 15 participating sites. Diabetes-related, patient-level data were recorded, on: HRG codes PA67Z and PA68Z, lengths of hospital stay, total cost Trials 2017, 18(Suppl 1):200 Page 55 of 235 and, where available, full disaggregation of PLICS data on items such as critical care and drug costs. Inpatient stays were costed using 3 methods based on: (i) bed days alone; (ii) Payment by results (PBR) National Tariff reimbursement; and (iii) PLICS-reported total cost. Confidence intervals were calculated using non-parametric bootstrap analysis with 10,000 replications. Results and, where available, full disaggregation of PLICS data on items such as critical care and drug costs. Inpatient stays were costed using 3 methods based on: (i) bed days alone; (ii) Payment by results (PBR) National Tariff reimbursement; and (iii) PLICS-reported total cost. Confidence intervals were calculated using non-parametric bootstrap analysis with 10,000 replications. Results Method Data from two randomised controlled trials are modeled. SYCAMORE is a double-blind, multicentre, randomised controlled trial in which 114 children with severe uveitis associated with juvenile idiopathic arthritis are randomised in a ratio 2:1 to receive adalimumab or pla- cebo in conjunction with methotrexate. Health utilities are obtained from the HUI2 questionnaires completed by the participants or their parents (or guardians). Healthcare resource use and costs are ob- tained from the patients’ diaries and hospital data (PLICS and HES). Folated was a three-centre, double-blind, randomised controlled trial in which 358 patients with moderate to severe depression were ran- domised on a ratio 1:1 to receive folic acid or placebo in addition to their routine antidepressants. Health utilities were measured with the euroqol EQ-5D-3 L, EQ-VAS and SF-6D questionnaires. Healthcare re- source use was collected from patients’ self-completed question- naires, GP records of prescribed medications and hospital data. Frequentist and Bayesian regression methods were employed for the health economic analysis of the trials. Results and robustness of the models were assessed and compared. R l Data relating to 82 hospital admissions were obtained for 74 patients at 5/15 sites. The remaining hospitals (10/15) were still in the process of setting up their PLICS systems and could only provide routine pa- tient admissions or legacy finance database outputs in time for the study. The diabetes-related inpatient stays (N = 67/82 episodes) were comprised of the codes PA67Z (admission related to diabetic ketoaci- dosis) (12/67) and PA68Z (admission related to diabetes mellitus, without ketoacidosis or coma) (55/67). Mean costs (95% confidence intervals) for the diabetes-related codes were: (i) bed days: £662 (£587, £741); (ii) PBR: £1252 (£1230, £1278); and (iii) PLICS: £1839 (£1339, £2425). Disaggregated PLICS costs comprised medical/ specialist nursing staff (47%), wards/overheads (30%), critical care (8%), other clinical supply and services (6%), pharmacy/drug costs (5%), therapies (1%) and pathology (1%) with the remainder comprising blood supplies, imaging, operating theatre and other diagnostic tests. Results There is no agreed gold standard for estimating inpatient costs for economic evaluations. Reliance on bed day costing alone risks underestimating the total cost of an inpatient stay, especially if the daily rate does not account for staff, critical care, wards and overhead costs. PBR, whilst giving a more accurate cost based on hospital reimbursement, lacks granularity and does not include unbundled costs such as critical care, expensive drug costs and overheads which are reimbursed at a local level. PLICS outputs have sufficient detail to account for these shortfalls and could provide a more robust method of inpatient cost estimation in trial-based economic evaluations, especially where the stay in- volves additional expensive bundles of care such as long oper- ation times and intensive care admission. This research is currently ongoing and findings will be presented at the conference. Discussion Frequentist regression methods are widely used in trial-based economic evaluations of health technolo- gies; however they have several limitations that may be overcome using Bayesian methods. These include factors intrinsic to the clinical trial data, such as population characteristics, study design, sampling methodologies, skewness of cost and utility data, etc. Bayesian methods, however, require their prior belief is generated. This re- search will contribute to the understanding of approaches for more efficient and robust health economic analysis of clinical trials. P143 Quantitative benefit-risk modelling of infliximab biosimilar inflectra versus reference product remicade in the treatment of Crohn’s disease Heather Catt1, Dyfrig Hughes2, Keith Bodger1, Jamie Kirkham1 1University of Liverpool; 2University of Bangor Correspondence: Heather Catt Trials 2017, 18(Suppl 1):P143 Objective The aim of this research is to explore the applicability of alternative regression methods, determine their precision in calculating the cost- effectiveness analysis of clinical trials, and to assess the merits and disadvantages of each method to reflect uncertainty. M h d , Conclusion HEAPs are currently developed inconsistently and there is an appe- tite for formal guidance. As it seems likely that the use of heaps will continue to increase in the future (and potentially, be required by funding bodies or regulators), clarity on the appropriate usage and content would be advantageous. We therefore plan to conduct a Delphi survey of practising health economists and other trialists to determine suitable content for a HEAP. Potential impact The ‘Totality of evidence’ approach to benefit-risk balance assess- ment is subjective and leads to uncertainties. Sponsor-provided data shows differences in the ability of infliximab biosimilars to induce ADCC and there is evidence to support ADCC as a mechanism of ac- tion in CD; certolizumab pegol, for instance, is unable to induce ADCC and has minimal efficacy in CD. However, etanercept does in- duce ADCC but is not effective, adding to the uncertainty. P145 LENS - a clinical trial embedded in routine clinical practice to reduce the burden of diabetic eye disease David Preiss1, Jane Armitage1, John Olson2, Graham Scotland3, Graham Leese4, Helen Colhoun5, Naveed Sattar6, Kevin Murphy1, Jennifer Logue6 1University of Oxford; 2NHS Grampian; 3University of Aberdeen; 4NHS Tayside; 5University of Edinburgh; 6University of Glasgow Correspondence: David Preiss Trials 2017, 18(Suppl 1):P145 The main concern with biosimilars is the potential for developing anti-drug antibodies (ADAs), something which clinical trials for other indications are unable to rule out for CD. The consequences of devel- oping adas include reduced efficacy and adverse events, particular serious events that are peculiar to CD including some cancers. The main benefit is the great potential for biosimilars to offer significant cost savings. This work will identify the conditions that impact the benefit-risk bal- ance for the biosimilar and will provide a methodology that could be developed to assess the benefit-risk balance of future biosimilars. Embedding clinical trials in routine clinical care provides the oppor- tunity to enhance efficiency and reduce costs. Diabetic retinopathy (DR) remains a common cause of blindness and impaired vision and treatment of advanced DR is costly. However, few trials have been designed to investigate treatments which may retard progression from observable DR to advanced DR because the condition usually progresses slowly, it is challenging to identify patients at risk of pro- gression to clinically significant DR without retinal imaging and stud- ies require medical professionals and specialist equipment to capture and grade retinal images. This highlights the need for streamlined tri- als which can identify large numbers of eligible patients and follow them cost-effectively for extended periods. P144 Health economics analysis plans: the current state of play Joanna Thorn1, Colin Ridyard2, Dyfrig Hughes2, Sarah Wordsworth3, Borislava Mihaylova3, Sian M. Noble1, William Hollingworth1 1University of Bristol; 2Bangor University; 3University of Oxford Correspondence: Joanna Thorn Trials 2017, 18(Suppl 1):P144 y Aims (a) To map current practice and beliefs about the appropriate imple- mentation (or otherwise) of heaps, with a view to drawing up good practice guidelines in future work and (b) to provide a forum in which health economists and other interested parties engaged in economic evaluations could open a dialogue on the need for heaps and methods of standardisation. The LENS (Lowering Events in Non-proliferative retinopathy in Scotland) trial is a streamlined randomized double blind placebo- controlled study of fenofibrate. The aim of LENS is to investigate the effect of fenofibrate on progression of observable DR to either clinically significant DR or to DR which requires specialist treatment. Fenofibrate is a generically available cholesterol-lowering medication and pooled findings from previous cardiovascular outcome trials have suggested that it may reduce DR progression by 30-40%. We aim to randomise 1,060 participants in LENS and follow them for an average of 4 years. Background Whilst regulators in Europe, Japan and Australia approved the biosimilar Inflectra, the USA and Canada were more risk averse, first rejecting the biosimilar before approving following sponsor submission of Page 56 of 235 Trials 2017, 18(Suppl 1):200 Page 56 of 235 Page 56 of 235 deviations are permissible, and the appropriate oversight and gov- ernance of heaps. further explanatory evidence. More recently, an application for an- other biosimilar, Flixabi, has been approved by the EMA but with a significant divergent position statement. Background The use of statistical analysis plans (SAPs), produced prior to un- blinded analysis, is an accepted means of reducing bias in rando- mised controlled trials (RCTs) by minimising selective analysis. However, while health economics analysis plans (HEAPs) to guide economic evaluation analysis alongside RCTs are becoming more common, they lag far behind saps in terms of their acceptance and standardisation, and there is a fundamental question over the value they add to trials. y p The infrastructure of NHS Scotland provides a unique setting in which to conduct a trial with (i) low cost recruitment and (ii) record linkage for assessments of treatment efficacy and safety. Key ele- ments include NHS Scotland’s Diabetic Retinal Screening service (DRS) and SCI-Diabetes. DRS provides regular retinal screening for all 250,000 patients with diabetes in Scotland. Retinal images are graded centrally in health boards, results are posted to patients and auto- matic referral to specialist eye clinics occurs if a patient develops clin- ically significant DR. SCI-Diabetes is NHS Scotland’s diabetes information system which collects and records diabetes-specific data from primary and secondary care. – Develop a model to estimate the benefit-risk balance of Inflectra versus Remicade. – Use statistical methods to evaluate the impact of the surrogate outcome ADCC on efficacy outcomes and serious adverse events. – Carry out, where necessary, elicitation of probability distributions for uncertain model parameters from experts and/ or patients. – Carry out, where necessary, elicitation of probability – Carry out, where necessary, elicitation of probability distributions for uncertain model parameters from experts and/ or patients. Results and discussion Aims and objectives Few guidelines are available to aid health economists in compiling heaps. Currently, substantial variation exists in the structure, format and content of heaps, and there are questions over their purpose and appropriate methods of oversight. Heaps may be published as part of a SAP, or as a standalone appendix, but are commonly unpublished. Although concerns remain over the impact of the research and bureaucratic burden involved in producing a plan in advance (particularly given the relatively small health economic workforce), the potential loss of useful post hoc analyses if a plan is too rigid, and the timing of completion, there was a general feeling that heaps would be useful. The majority (approximately 65%) of health economists at the workshop were in favour of a combined SAP and HEAP, rather than a standalone HEAP. To date there has been no approach to measure quantitatively the benefit-risk balance of biosimilars. This project aims to quantify the benefit-risk balance for Inflectra versus Remicade in CD. The objectives of the study are to: – Develop a model to estimate the benefit-risk balance of Inflectra versus Remicade. Methods of the PV team are highly skilled medical and safety professionals with regulatory and safety experience. However, due to the sheer magnitude of Adverse Events (AEs) collected in many clinical trials, it is vital to utilize innovative EDC technologies in order to improve quality and efficiency of PV activities. Developing the safety elec- tronic case report forms (ECRFs) in a thoughtful and systematic way before the start of a trial can enhance data quality, facilitate coding procedures, and enable more rapid drug safety decisions. Certain adverse event terms can be predetermined on the ECRFs to reduce the number of varied but similar terms, and to categorize into protocol- specific events of interest (e.g., seizures) to allow for desired organization of large amounts of data. This can greatly reduce the preparation time for Data and Safety Monitoring Board (DSMB) review and other reporting needs. Technical capabilities also allow for auto- mated notifications to be triggered based on the use of a specific term, severity grade, or causality designation. For example, the PV team can be notified whenever a “related” AE is entered in the EDC system, or when lab values exceed a certain threshold. Leveraging the EDC cap- abilities optimizes the function of the clinical trial pharmacovigilance practices, provides real time monitoring of safety events, and can be made accessible for sponsors or DSMB members as needed. This pres- entation will discuss best practices for utilizing EDC systems to facilitate clean well-organized safety data and increasing the efficiency of phar- macovigilance activities throughout the life of a trial. diabetic patients). After two face-to-face visits for screening and ran- domisation, either side of an eight-week active run-in, follow-up will be conducted remotely. This will involve regular linkage by pseudo- nymisation to multiple national registries and completion of ques- tionnaires by computer or telephone. Registries will include DRS (for pre-specified eye outcomes), Scottish Morbidity Records (for hospita- lisations and outpatient visits), National Records of Scotland (for flag- ging deaths), the Prescribing Information System and SCI-Diabetes (for various diabetes-related outcomes). Furthermore, SCI-Diabetes will be used after randomisation for central monitoring of biochem- ical safety in almost real-time, based on the availability of results from routinely collected blood samples. Study medication will be mailed to participants by registered post. Background g HIV-NAT 015 study is a pediatric HIV cohort, aimed to collect long term efficacy and safety data of treatment in HIV-infected children, established since 2004, at HIV-NAT, the Thai Red Cross AIDS Research Centre, Bangkok, Thailand (clinicaltrials.gov number NCT00476606). In the first few years of this study, we collected clinical and labora- tory data through the data entry person after each clinic visits. How- ever, this process took time for data entry, monitoring, and had some transcription errors. A review of commercial systems, open source solutions versus the merits of developing a bespoke system was undertaken by Glasgow Clinical Trials Unit. A list of requirements was compiled in order to compare each system reviewed and inform the decision. It was also considered whether a combination of systems would fulfil the entire list even if this was not the most elegant solution. The process was concluded by a decision to develop a bespoke system as this was the only solution that could match all of the requirements in a single system. Background Torsak Bunupuradah1, Chavalun Ruengpanyathip1, Chowalit Phadungphon1, Yupawadee Jummanee1, Thanyawee Puthanakit2, Kiat Ruxrungtham3 1HIV-NAT, Thai Red Cross AIDS Research Centre; 2HIV-NAT, Thai Red Cross AIDS Research Centre and Research Unit in Pediatric Infectious Diseases and Vaccines, Faculty of Medicine, Chulalongkorn University; 3HIV-NAT, Thai Red Cross AIDS Research Centre and Department of Medicine, Faculty of Medicine, Chulalongkorn University Making a decision regarding which system to host your clinical trial data presents a number of challenges. The scope of the system: is it just data entry, is it web enabled, does it include randomisation and is there a requirement for other trial management functionality, has to be considered. The cost of the system is very important to aca- demic clinical trials units (CTUs) not just at the stage of initial pur- chase but the resource to maintain and setup each trial. As technology develops multiple platform capability is becoming in- creasingly key to the service a CTU can provide in a competitive landscape. Methods A workshop was held in Bristol, UK in October 2015, to discuss issues associated with heaps. 50 predominantly university-based partici- pants heard presentations from speakers before breaking into smaller groups for discussion sessions. Presented sessions included accounts of practical experiences of using heaps in RCTs, alongside perspectives from SAP guidelines, NICE and wider non-trial based economics. In the discussion sessions, participants debated topics in- cluding the appropriate content of heaps, the circumstances in which g y For recruitment, DRS will include a trial information leaflet with a FREEPOST reply slip along with retinal screening results mailed to pa- tients whose DR grading indicates they are eligible (about 10% of all Page 57 of 235 Page 57 of 235 Page 57 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 of the PV team are highly skilled medical and safety professionals with regulatory and safety experience. However, due to the sheer magnitude of Adverse Events (AEs) collected in many clinical trials, it is vital to utilize innovative EDC technologies in order to improve quality and efficiency of PV activities. Developing the safety elec- tronic case report forms (ECRFs) in a thoughtful and systematic way before the start of a trial can enhance data quality, facilitate coding procedures, and enable more rapid drug safety decisions. Certain adverse event terms can be predetermined on the ECRFs to reduce the number of varied but similar terms, and to categorize into protocol- specific events of interest (e.g., seizures) to allow for desired organization of large amounts of data. This can greatly reduce the preparation time for Data and Safety Monitoring Board (DSMB) review and other reporting needs. Technical capabilities also allow for auto- mated notifications to be triggered based on the use of a specific term, severity grade, or causality designation. For example, the PV team can be notified whenever a “related” AE is entered in the EDC system, or when lab values exceed a certain threshold. Leveraging the EDC cap- abilities optimizes the function of the clinical trial pharmacovigilance practices, provides real time monitoring of safety events, and can be made accessible for sponsors or DSMB members as needed. This pres- entation will discuss best practices for utilizing EDC systems to facilitate clean well-organized safety data and increasing the efficiency of phar- macovigilance activities throughout the life of a trial. Methods LENS is co-ordinated by the Clinical Trial Service Unit and Epidemio- logical Studies Unit, University of Oxford, and run in collaboration with the Universities of Glasgow, Aberdeen, Edinburgh and Dundee, DRS and mainland NHS Scotland health boards. It represents the first trial to be embedded within an existing national DR screening program. P146 Decisions, decisions: bespoke EDC? Sharon Kean, Jonathan Gibb, Jane Aziz, Ian Ford Glasgow Clinical Trials Unit Correspondence: Sharon Kean Trials 2017, 18(Suppl 1):P146 y Conclusion Since 2010, we developed an in-house HIV-NAT electronic health record (EHR) to use in this study. Physician can review all medical history and see overall growth parameters, and real time laboratory results includ- ing CD4, plasma HIV-RNA through tables and graphs format. This EHR can capture all adherence data by self-report and percent adherence by pill count of antiviral therapy, HIV disclosure status, parental vital sta- tus which is used for adherence counselling and care. The EHR can auto-calculated body surface area after entering body weight and height which minimized error when calculating dosage of antiviral ther- apy in young children. Physician can directly print the auto-filled pre- scription after seeing each patient. The EHR is directly integrated to HIV-NAT laboratory reporting systems which reduced workload for data entry and ensuring data safety and completeness. Nurse assistance can process the lab request through the EHR, and make the next clinic ap- pointment in this paperless EHR. Due to limited man-power in number of pediatric study nurse and monitor team, this EHR is helpful in task shifting i.e. A trained nurse assistance can replace some roles of study nurse for administrative work, a trained data entry person can do basic data monitoring by identify missing or out range data. Moreover, we can perform a data search for a new research question and feasibility survey for the new trials within a few minutes. The authors will discuss the implications of this decision on their Unit resource, future business and the justification for their decision. Have they bitten of more than they can chew? Have they developed their perfect system? What decisions have other CTUs made? P149 Jane Aziz, Sharon Kean Robertson Centre for Biostatistics Correspondence: Jane Aziz Trials 2017, 18(Suppl 1):P150 Leveraging electronic data capture (EDC) systems to optimize pharmacovigilance in clinical trials p g Radhika Kondapaka, Anne Hoehn, Paul Van Veldhuisen, Dikla Shmueli-Blumberg, Robert Lindblad The Emmes Corporation Correspondence: Radhika Kondapaka Trials 2017, 18(Suppl 1):P147 Radhika Kondapaka, Anne Hoehn, Paul Van Veldhuisen, Dikla Shmueli-Blumberg, Robert Lindblad The Emmes Corporation Correspondence: Radhika Kondapaka Trials 2017, 18(Suppl 1):P147 Pharmacovigilance (PV), also referred to as drug safety, is defined as the pharmacological science and activities related to the detection, assessment, understanding and prevention of adverse effects or other drug-related problems (WHO, 2002). Monitoring patient safety is an essential component of conducting clinical trials and members Page 58 of 235 Trials 2017, 18(Suppl 1):200 Using OWL to classify adverse events Using OWL to classify adverse events William Stevens, Karl Wallendszus, Salvador Girbau, William Herrington, Michael Lay, Martin Landray University of Oxford Correspondence: William Stevens Trials 2017, 18(Suppl 1):P151 Using OWL to classify adverse events William Stevens, Karl Wallendszus, Salvador Girbau, William Herrington, Michael Lay, Martin Landray University of Oxford Correspondence: William Stevens Trials 2017, 18(Suppl 1):P151 p p The TEMPER management system To meet the specified requirements, we developed a data manage- ment system to allow: 1) the extraction of tailored trigger data from the participating trials databases; these had been developed in a third-party Clinical Data Management System; 2) the ranking and se- lection of triggered sites based on the extracted trigger data; 3) the pairing of the chosen triggered sites with control sites based on simi- larity criteria; 4) the collection and management of findings data gathered in the monitoring visits to the triggered and control sites. The system was developed in Visual Basic.NET with a SQL Server database, which served as the main study database for TEMPER and was used for analysis. Background For defining clinical trial endpoints and for producing reports, it is necessary to group adverse events. For example a line reporting the number of participants who had a stroke includes participants who had “Haemorrhagic Stroke”, “Ischaemic Stroke”, “Cerebral Infarction”, and several other types of stroke. Classification of events into groups can be done using the event code, but often other properties also need to be used. These properties include: "hospitalization required", "cause of death", "urgent or non-urgent". Large trials can have 10,000 s of events, and hundreds of event classes. Defining event classes and classifying events are important problems. Methods Background Background If patients can be followed up electronically rather than in person, then the cost of running a clinical trial can be significantly reduced. At study design it was estimated that 75% of patients would complete their follow-up online, with the other 25% opting for either postal or telephone. y pp , y , y g 1MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL, London, UK; 2MRC London Hub for Trials Methodology Research, London, UK Correspondence: Carlos Diaz-Montana Trials 2017, 18(Suppl 1):P149 Trigger data h For each participating trial, a set of specific triggers was defined by the trial team; each consisted of a narrative explaining the conditions under which it fires. An example of a trigger narrative is: ‘More than 0.5% of the values in the open forms are missing or queried based on total number of fields to be entered’. The majority of trigger narratives were implemented as automatic triggers in the system. When data was not available in the trial database to implement an automatic trigger, for example data was from external sources or based on subjective inter- pretation, manual triggers were instead created in the system allowing users to set their status to fire as necessary. The TEMPER system allowed the trigger data to be summarised following extraction, ranked by a trigger score based on the triggers fired, and presented to the trial teams to inform the selection of triggered sites to visit. Matching Algorithm A matching algorithm was also implemented in the system which ranked best matches to the selected triggered sites based on site similarity while prioritising sites with lower trigger scores. Site similar- ity was defined by participation: number of patients randomised and number of days since first randomisation. Background As the study design allowed for three different methods of collecting patient data, a system was required that would allow very different types of user to access and enter data. These users would require dif- ferent access levels to the system and to the data being entered. En- suring patients could only access their own data was imperative, while study staff would need access to the data of all patients that they were in contact with. Additional complexity was introduced, as the staff creating, maintaining and supporting the system never deal with patients, so needed to remain blind to the patient’s personal contact details. The TEMPER study (results submitted separately) evaluates the use of trial-specific triggered site monitoring, where centrally collected data inform the choice of which sites should undergo monitoring visits. It compared findings at triggered monitoring visits to findings at visits to matched, untriggered control sites that would not otherwise have been visited. Three multi-centre cancer trials at the MRC Clinical Trials Unit at UCL participated in the TEMPER Study. Conclusion unit. The TEMPER system design will also inform future implementa- tions and improvements on monitoring systems at the unit, including further research. unit. The TEMPER system design will also inform future implementa- tions and improvements on monitoring systems at the unit, including further research. This in-house EHR is efficient, reduces transcription error and illegible data issues, saves time and cost, requires less storage space, and pro- vides a broader set of research questions and future data analysis. We recommend using this in-house EHR in both clinical trial center and in general hospital. Managing our TEMPER: monitoring triggers and site matching algorithms for defining triggered and control sites in the temper study Carlos Diaz-Montana1, Rahela Choudhury1, Will Cragg1, Nicola Joffe1, Nancy Tappenden1, Matthew R. Sydes1, Sally Stenning1 1MRC Clinical Trials Unit at UCL Institute of Clinical Trials and Carlos Diaz-Montana1, Rahela Choudhury1, Will Cragg1, Nicola Joffe1, Nancy Tappenden1, Matthew R. Sydes1, Sally Stenning1 1MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL, London, UK; 2MRC London Hub for Trials Methodology Research, London, UK Results A total of 38 triggers, 31 automatic and 7 manual, were specified for the three participating trials. The matching algorithm paired 42 trig- gered sites with corresponding control sites. Conclusion We will discuss the different levels of access required for the differ- ent roles, how access was provided to patients and the challenges in creating a system to be used by patients, while remaining blind to their identity. P150 P150 Access and security considerations of using EPROMS to collect follow-up data for clinical trials Jane Aziz, Sharon Kean Robertson Centre for Biostatistics Correspondence: Jane Aziz Trials 2017, 18(Suppl 1):P150 Access and security considerations of using EPROMS to collect follow-up data for clinical trials Jane Aziz, Sharon Kean Robertson Centre for Biostatistics Correspondence: Jane Aziz Trials 2017, 18(Suppl 1):P150 classes an Methods We describe how to use the Web Ontology Language (OWL) to ad- dress these problems. OWL software tools exist that can represent OWL in both human-readable and machine-readable forms; an OWL document defining event classes can serve as both specification and implementation. OWL permits a class to be defined as a Boolean combination of other classes. For example STROKE can be defined as HAEMORRHAGIC STROKE or ISCHAEMIC STROKE. Medical coding dic- tionaries such as meddra can serve as predefined event classes. We have converted meddra into an OWL representation so that it can be used as the basis for defining other event classes. We deliberately use only those features of OWL that correspond to basic set theory because this is already understood by clinicians who define event classes, and programmers who use them. During the process of de- fining event classes, OWL tools can be used to check for errors. For Developing novel endpoints, generated using mobile technology, for use in clinical trials: a clinical trials transformation initiative (CTTI) j t ( ) p j Martin Landray University of Oxford Trials 2017, 18(Suppl 1):P153 We found this to be a practical way of classifying events. We expect that OWL can be used for other classification tasks, such as the classi- fication of drugs into related groups. The appropriate inclusion of mobile technology into clinical trials of- fers significant opportunity to improve clinical endpoint ascertain- ment. Mobile technology can provide unprecedented access to real- world situations where multiple precise measurements on trial partic- ipants could be made without interference to their daily life. Such endpoints can reduce patient participation burden, increase trial feasibility and address unmet need for endpoints in certain thera- peutic areas and patient populations. We propose recommendations to clarify the pathway for developing novel endpoints, generated using mobile technology, for use in clinical trials. We describe steps for appropriate novel endpoint selection and development, along with an analysis of how this approach differs to traditional endpoint development and recommendations for reducing friction in this process. Our approach is designed to meet the needs of clinical trial- ists, regulators and trial participants. Our multi-stakeholder team of experts pursued a two-pronged approach to evidence gathering. Our recommendations have been informed by both new, empirical evi- dence generated by writing four use cases and a synthesis of exist- ing, published approaches across therapeutic areas. Four discrete use case teams developed proposed novel endpoints for Parkinson’s dis- ease, heart failure and Duchenne’s muscular dystrophy using data generated using accelerometers and a novel endpoint for diabetes derived from data from a continuous glucose monitor. Each team in- cluded investigators and patient reps with expertise and experience in the disease state, engineers and mathematicians with expertise in the specified device, regulators, nonprofit consortia and statisticians. The systematic literature review identified 101 manuscripts where novel endpoints were included in clinical studies. Embedding randomized clinical trials within registries: how feasible? Pamela Tenaerts1, Steven J. Mikita2, Sara B. Calvert1, Christopher Dowd3 1Clinical Trials Transformation Initiative; 2Patient Advocate; 3Cystic Fibrosis Foundation Pamela Tenaerts1, Steven J. Mikita2, Sara B. Calvert1, Christopher Dowd3 1Clinical Trials Transformation Initiative; 2Patient Advocate; 3Cystic Fibrosis Foundation Correspondence: Pamela Tenaerts Trials 2017, 18(Suppl 1):P152 Correspondence: Pamela Tenaerts Trials 2017, 18(Suppl 1):P152 Results James Barrett, Catey Bunce King’s College London Correspondence: James Barrett Trials 2017, 18(Suppl 1):P154 Depending on its characteristics and capabilities (e.g. Interoperability, connectivity, flexibility, sustainability), a registry can be used either as an observational data source for generation of clinically actionable evidence and hypothesis generation, or as a critical reusable compo- nent of the clinical trial infrastructure within which prospective ran- domized studies can be performed. Questions exist about identifying appropriate registries, ensuring data quality/comparability, meeting regulatory requirements, and processes for implementing a random- ized registry trial. Methods The CTTI Registry Trials project team conducted a literature review, interviewed 25 experts, and then convened a multi-stakeholder ex- pert meeting. At the expert meeting attendees discussed recommen- dations for best practices to increase the value, acceptance, and success of registry based clinical trials. Background A Registry is an organized system that uses observational methods to collect uniform data on specified outcomes in a population de- fined by a particular disease, condition or exposure. At their core, registries are data collection tools created for the purpose of gener- ating clinically usable information and evidence. The data captured in a registry typically includes information such as medical history, demographics, disease diagnosis and outcome data. Patient data collected in registries often overlaps with data gathered for clinical trials. Integrating clinical trials within observational data registries may offer opportunities to avoid duplicative data collection, increase operational efficiencies, reduce time to database lock and accelerate time to critical decision making, while decreasing clinical trial costs. The objective of the Clinical Trials Transformation Initiative (CTTI)’s Registry Trials project was create recommendations to sup- port the practice of leveraging registries to facilitate high quality clin- ical trials. g Conclusions Conclusions The TEMPER system implemented a novel approach on how to manage monitoring trigger data and how to match triggered sites with control sites; this allowed the realisation of the TEMPER study protocol. Implications If the TEMPER study results show the triggered monitoring strategy to be effective, the system could be introduced to other trials in the Page 59 of 235 Page 59 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 59 of 235 example, it is possible to specify that two event classes should have no events in common, or that a group of event classes should be equivalent to some other event class. In order to use OWL event class definitions during the production of analyses and reports, some short ‘glue’ programs are required. Events (and their properties) that occur during the trial are converted into an OWL document and fed into an OWL reasoning tool. The output from this is a list of which event classes contain which other event classes. This is converted into a flat tabular form that can be stored in a database and easily queried to find out which events are in which classes. example, it is possible to specify that two event classes should have no events in common, or that a group of event classes should be equivalent to some other event class. In order to use OWL event class definitions during the production of analyses and reports, some short ‘glue’ programs are required. Events (and their properties) that occur during the trial are converted into an OWL document and fed into an OWL reasoning tool. The output from this is a list of which event classes contain which other event classes. This is converted into a flat tabular form that can be stored in a database and easily queried to find out which events are in which classes. If the historical data created by a registry are well established to be relevant/fit to purpose, robust and reliable, then the registry can have a clear role in creating a sustainable infrastructure within which regulatory trials can be conducted. Identifiable requirements and practical considerations have not been defined in the use or modifi- cation of existing registries and/or in design of new registries in order to make them fit for the purpose for conducting a Randomized Registry Clinical Trial (RRCT). g Conclusions Such normative standards are essential for consistent evaluation of a registry’s suitability for generating the clinical evidence needed for regulatory decision making in the vari- ous phases of drug and device development. CTTI has created rec- ommendations and tools to assist in 1) evaluation of an existing registry’s suitability for conducting clinical trials and 2) designing a new registry in which to conduct a clinical trial. If the historical data created by a registry are well established to be relevant/fit to purpose, robust and reliable, then the registry can have a clear role in creating a sustainable infrastructure within which regulatory trials can be conducted. Identifiable requirements and practical considerations have not been defined in the use or modifi- cation of existing registries and/or in design of new registries in order to make them fit for the purpose for conducting a Randomized Registry Clinical Trial (RRCT). Such normative standards are essential for consistent evaluation of a registry’s suitability for generating the clinical evidence needed for regulatory decision making in the vari- ous phases of drug and device development. CTTI has created rec- ommendations and tools to assist in 1) evaluation of an existing registry’s suitability for conducting clinical trials and 2) designing a new registry in which to conduct a clinical trial. Pros This approach uses well-supported public domain software, so little in-house coding is required. Since OWL has a well-defined meaning, event class definitions can be communicated unambiguously to others. Performance is reasonable: in a large trial (85,000 events, 200 event classes) it takes a few minutes to check class definitions for in- consistencies, and one minute to classify all events. C CTTI Registry Trials recommendations and tools can assist researchers in evaluating new and existing registries to determine if embedding randomized clinical trials is appropriate. It is sometimes necessary to take account of the open-world seman- tics of OWL in order to define some event classes correctly; it is rea- sonable to expect programmers to take the time to understand this, but not so reasonable to expect this of clinicians. P153 Developing novel endpoints, generated using mobile technology, for use in clinical trials: a clinical trials transformation initiative (CTTI) j t Results 21/26 TMs completed the survey. 15 trials (71%) included a PPI repre- sentative on the trial team, 5 used another method of PPI (e.g. Via in- put to advisory group (n = 3)/Trial Steering Committee (n = 2), consultation of patient group (n = 1)), and one TM reported s/he did not know of any PPI in the trial. The 15 trials that included PPI repre- sentatives recruited 1–20 PPI representatives (mean 6.4, 1 missing), but in practice 0–10 were regularly involved (mean 4.8, 2 missing). None used a formal process to recruit PPI representatives. Preferential recruitment of informative individuals can potentially lead to successful observational studies with smaller cohort sizes. Re- sults from numerical simulations have shown that a desired level of statistical power can be achieved with an informative cohort of 200 individuals compared to a randomly selected cohort of 300 individ- uals. In order to illustrate how our methodology can be used in prac- tice we simulated studies of patients with cardiovascular disease using electronic health records from the Clinical Practice Research Datalink. The most common tasks undertaken by PPI representatives were re- view of participant-facing materials and other study documents and advising on recruitment/retention strategies. Changes made as a re- sult of PPI related to trial documentation and design; 2 TMs reported no changes had been made. Twelve TMs reported that PPI represen- tatives were paid for their time, 4 said payment was not offered and 3 did not know. Payments ranged from £10-£50/hour, with 3 trials paying in vouchers. Only 5 TMs reported that training was provided for PPI representatives (12 reported no training, 3 did not know, 1 missing). Background Public engagement is the interface between research and wider soci- ety. By helping to bring these two sometimes disparate worlds together, engagement can increase public trust and enhance rele- vance, accountability and transparency of, and in, research pro- cess(es) and researchers. Engagement is also important because it can empower people to become involved through offering their in- sights into and feedback on our work to help ensure that the re- search we conduct is relevant to the societies in which we live and are striving to improve. Bringing together colleagues’ experiences of, and interest in, public engagement with research, our team’s aim was to employ a strategic approach to deliver public engagement ac- tivities around clinical trials and health services research to demystify our work and facilitate public involvement. Methods f Informativeness depends on an individual’s covariates (or "risk factors"). The statistical measure of entropy is used to quantify how much statistical information an individual is expected to provide on the study. More informative individuals have a higher probability of being selectively recruited onto the study. R lt p g Lucy Selman, Clare Clement, J. Athene Lane, Chris Metcalfe, Joanne Simon, Jeremy Horwood University of Bristol Correspondence: Lucy Selman Trials 2017, 18(Suppl 1):P157 Correspondence: Lucy Selman Trials 2017, 18(Suppl 1):P157 Background Big databases of patient health information offer a unique opportun- ity to be selective about which individuals are invited to participate in clinical studies. In particular, large databases can be searched for Page 60 of 235 Page 60 of 235 Trials 2017, 18(Suppl 1):200 Page 60 of 235 the "most informative" individuals. Conventional study designs typic- ally recruit individuals at random, usually by waiting for eligible pa- tients to present at one of the study centres. Electronic health records allow researchers to immediately target the most relevant and informative patients for further investigation. Interviews with Trial Management Group members and PPI represen- tatives from case study trials (estimated n = 10). Case studies were se- lected purposively to represent a range of trial designs, funding streams and trial initiation dates. Interviews explored the perceived value of PPI involvement and barriers to/facilitators of PPI. Quantita- tive survey data were summarised using descriptive statistics and interview transcripts analysed thematically. A project PPI group ad- vised on interview topic guides, provided feedback on findings and assisted with dissemination. Conclusions Survey findings show that PPI involvement in trials is currently highly variable. PPI representatives are recruited informally, are rarely provided with any training, and are paid inconsistently across trials. This abstract is not included here as it has already been published. Background There are ethical, sociopolitical and scientific reasons for patient and public involvement (PPI) in research, and many funders now require applicants to include PPI to improve the relevance, accountability and quality of research. However, there is evidence of challenges in implementing PPI, and formal guidance on how to involve service users in the conduct of trials is lacking. ‘Is that it?’ Using ‘explorachoc’ to engage the public with clinical trials and health services research ‘Is that it?’ Using ‘explorachoc’ to engage the public with clinical trials and health services research Heather Morgan, Becky Bruce, Gordon Fernie, Heidi Gardner, Beatriz Goulao, Joanna Kaniewska, Clare Robertson, Sharon Wren, Katie Gillies University of Aberdeen Correspondence: Heather Morgan Trials 2017, 18(Suppl 1):P158 Conclusion The advent of large databases of patient records represents an un- precedented opportunity for more efficient and cost effective stud- ies. Our approach can also be applied to follow-up studies after a clinical trial has ended in which an informative subset of the trial par- ticipants are followed for a longer period of time. In situations when it is prohibitively expensive to follow the full trial cohort over an ex- tended time period our methodology may offer a more feasible alternative. TMs reported that CTUs could assist with recruitment of PPI repre- sentatives and provide guidance on integrating PPI in grant applica- tions. Challenges reported included ‘professional PPI members’ having a different agenda to the study population, and lack of con- tinuity in trial staff. The interviews are currently underway; findings will be presented at the conference. P156 Vibrating vaginal balls to improve pelvic floor muscle performance in women after childbirth: a randomised controlled feasibility trial Claudia Oblasser1, Christine McCourt1, Engelbert Hanzal2, Shashivadan P. Hirani1 1City, University of London; 2Medical University of Vienna Correspondence: Claudia Oblasser Trials 2017, 18(Suppl 1):P156 Conclusions Aim To systematically investigate how PPI is approached within the Bristol Randomised Trials Collaboration’s (BRTC’s) clinical trials unit (CTU) portfolio of trials, and identify barriers to and facilitators of its successful implementation, to contribute to our understanding of PPI in trials and guidance and training in this area. P157 Patient and public involvement: an investigation Patient and public involvement: an investigation Lucy Selman, Clare Clement, J. Athene Lane, Chris Metcalfe, Joanne Simon, Jeremy Horwood University of Bristol Correspondence: Lucy Selman Trials 2017, 18(Suppl 1):P157 Background a g ou d Adverse events (AEs) reporting is essential in clinical trials. The current system for reporting (Common Toxicity Criteria and Adverse Events, CTCAE) relies on clinicians’ interpretation of symptoms. The value of patient self-reports of AEs and Patient Reported Outcome Measures (proms) is recognised but robust data collection methods are needed. Here we report the REPORT-UK study which developed and evaluated an electronic (internet/telephone) system for self- reporting AEs and proms during trials. Results Overall, the consent rate was 48%. System preference was 82% inter- net/17% IVR (telephone). Only 13 participants withdrew and 6 died whilst on study. 192 returned end of study questionnaires. Overall patient compliance was good for weekly AE and monthly proms reporting, but differed between treatment groups, and dropped over time. Both systems were perceived as easy-to-use. Time to complete was perceived by patients to be acceptable, although actual times show the internet is quicker (median time 9 minutes vs. 21.5 mi- nutes). Baseline comparisons between patient vs. Clinician-reporting of some AEs differed substantially. j We will determine what proportion of clinical trials address one or more of the prioritised research uncertainties, and whether there are differences between different sponsor/funding types. We will also in- vestigate what proportion of the National Institute for Health Re- search (NIHR) commissioned calls and themes reflect the priorities identified by PSPs. Furthermore, we will look at the wider picture of impact of PSPs, including how findings can be used to support non- research activities (e.g. Services), development of diagnostic criteria and outcome measures, and whether PSPs can act as a gateway for individual patients/cares to progress to further involvement in research. p Results We randomised 365 people (48.5% blue) across the two events (83% Explorathon). The median in the blue group was 4.0 Interquartile range- IQR (3.0-4.0) and 3.5 IQR (3.0-4.0) in the yellow group (Mann Whitney U p-value = 0.633). The resounding response was ‘Is that it?’ Regarding randomisation, which suggests that we were able to somewhat demystify the process of randomisation. Most participants understood the connections between clinical trials, health services re- search and the health and social care they receive. Many recounted anecdotes of surgical, drugs and other therapies they have experi- enced themselves or within their families and acknowledged the im- portance of engaging and/or being involved in the types of research we do. 71 people volunteered their details for future contact regard- ing public involvement. The results of PSPs are increasingly being used by funders to priori- tise research of importance to both patients and clinicians. However, it is acknowledged that conducting a PSP requires considerable time and resources, so we will evaluate the value of PSPs by assessing the evidence of impact of those conducted in dermatology. The results of this study should therefore be helpful for researchers considering undertaking a PSP, and for potential funders of PSPs. Background g The purpose of conducting a Priority Setting Partnership (PSP) is to identify and prioritise uncertainties for a specific condition. PSPs usu- ally follow a standardised transparent methodology, often supported by the James Lind Alliance (JLA). They are a true collaboration be- tween clinicians and patients/carers. It is expected that the results of a PSP will influence the subsequent research agenda for that condi- tion, ensuring that research meets the needs of both patients and cli- nicians and therefore contributes to reducing research waste. The overall aim of this study is to assess the impact on the research agenda of all PSPs that have been conducted for skin conditions. Objectives 249 varied diagnosis cancer patients undergoing treatment (chemo- therapy/targeted agents/hormone therapy/radiotherapy/surgery, and an ECOG group with performance status 2) were recruited. For 12 weeks patients were reminded (text/email) to complete weekly AEs (NCI PRO-CTCAE) and monthly proms questionnaires (EORTC QLQ-C30) on their preferred system. Acceptability and feasibility was measured by recruitment rates, attrition, compliance, and patient and staff feedback at end-of-study. P160 REPORT-UK (real-time electronic patient outcome reporting of adverse events in UK cancer trials): methods to optimise data collection Jane Blazeby1, Fiona Kennedy2, B. Clayton2, Kate Absolom2, Elaine O'Connell Francischetto1, Louise Flintoff3, K. Gordon2, W. Crocombe2, F. Samy2, V. Hiley2 Jane Blazeby1, Fiona Kennedy2, B. Clayton2, Kate Absolom2, Elaine O'Connell Francischetto1, Louise Flintoff3, K. Gordon2, W. Crocombe2, F. Samy2, V. Hiley2 Methods We designed a two-arm trial, which we called ‘explorachoc’ to: dem- onstrate the randomisation process used in clinical trials; engage members of the public in conversations about clinical trials and health services research to solicit their perceptions and views; and re- cruit to a public involvement panel. We piloted this activity at the University of Aberdeen’s May Festival (28–29 May 2016) and ran a We included all currently active BRTC portfolio trials, plus those which ended in the previous 2 years. A mixed-methods study design was developed, involving: (1) An online survey of Trial Managers (TMs), conducted August-September 2016, to determine how trials included PPI and the support required from CTUs for PPI; (2) Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 61 of 235 Page 61 of 235 or published clinical trials in these disease areas. Sources will include clinical trial registries, funder databases, PubMed, and the JLA web- site. Where necessary, we will contact the clinical trial teams to clarify where information is unclear, particularly around timing, to deter- mine whether or not the trial was funded prior to the PSP results being available. We will also survey the authors of PSPs and subse- quent research teams to assess any wider impact of the PSPs such as successful fellowship applications and involvement of patients as partners in further research. modified version as part of the University’s European Researchers’ Night/Explorathon event (30 September 2016). The trial involved: double-blinded selection of either a blue or yellow ball (with equal chance of selecting either); depositing the ball in a large transparent jar to demonstrate the distribution of selection; ringing a randomisa- tion bell; being given a chocolate with a blue or yellow (white or milk chocolate) wrapper, corresponding to the selected ball’s colour; and ranking the chocolate on a scale of 1 (low) - 4 (high). We then en- gaged participants in a range of conversations about the history of clinical trials (using costumes and props to re-enact James Lind’s scurvy trial) and our research portfolio (supported by flyers and printed information). Finally, we asked participants whether they would be willing to be contacted to contribute to our public involve- ment panel. Conclusion The study demonstrates a user-friendly electronic data collection sys- tem, which provides information on patient compliance in a general oncology setting but we recognise this is different to a real trial set- ting. The system could be implemented in practice in clinical trials alongside traditional approaches to improve data quality and safety. Conclusions h We have established an approach to delivering public engagement activities around clinical trials and health services research designed to demystify our work and facilitate public involvement. We have a proof of concept for an effective engagement model, enhanced by the use of chocolate, and are building on this to develop public engagement and involvement strategies. y y 1University of Bristol; 2University of Leeds; 3University Hospitals Bristol NHS Foundation Trust 1University of Bristol; 2University of Leeds; 3University Hospitals Bristol NHS Foundation Trust Correspondence: Jane Blazeby Trials 2017, 18(Suppl 1):P160 p Results Preliminary searches have identified seven PSPs in the field of skin disease; acne, cellulitis, eczema, hair loss, hidradenitis suppurativa, pressure ulcers and vitiligo. Work on this study is ongoing and the full results will be available by the time of the conference. Implications Exploring the impact of priority setting partnerships in skin disease Joanne Chalmers, Natasha K. Rogers, Kim S. Thomas University of Nottingham Correspondence: Joanne Chalmers Trials 2017, 18(Suppl 1):P159 Joanne Chalmers, Natasha K. Rogers, Kim S. Thomas University of Nottingham Correspondence: Joanne Chalmers Trials 2017, 18(Suppl 1):P159 Methods Under the assumption of non-differential assessment (i.e. That misclassi- fication rates are the same in each treatment arm, as would typically be the case when outcome assessors are blinded), we addressed three questions about adjudication: (a) How many assessors should we use? (b) When is it better to use on-site or central assessment? And (c) Should central assessors adjudicate all outcomes, or only suspected events? Results Methods Study objectives were addressed through a repeat assessment of bleed- ing exercise. Once the participants’ platelet count was 30x109/L they had their bleeding status assessed using the BAT by trained assessors. The aim was for the participant to have an assessment of bleeding repeated by two assessors on a maximum of three consecutive days, which would generate six individual assessments. Repeat assessments were then com- pared for concordance i.e. Agreement in site and severity of any bleeding observed. The two assessors were blinded to the findings of one another's assessments and a repeat assessment had to take place as soon as possible after the first to try to ensure that both assessors were observ- ing the same ‘bleeding window’. A qualitative survey was used to collect feedback from bleeding assessors as to how easy they found using the tool. Feedback was used to refine the design of the BAT. Correspondence: Brennan Kahan Trials 2017, 18(Suppl 1):P162 Correspondence: Brennan Kahan Trials 2017, 18(Suppl 1):P162 Methods We will search relevant databases and websites to identify all skin- related PSPs that have been conducted, and then identify ongoing Trials 2017, 18(Suppl 1):200 Page 62 of 235 Page 62 of 235 P161 Tocilizumab in pulmonary hypertension Jules Hernandez-Sanchez, Mark Toshner Papworth Hospital Correspondence: Jules Hernandez-Sanchez Trials 2017, 18(Suppl 1):P161 Background g Many platelet transfusion trials now use bleeding as a primary outcome. There are two important considerations when bleeding is used as an outcome measure: how signs and symptoms of bleeding are docu- mented and the translation of this information into a clinically significant grade. This is fundamental to the robustness of results reported and the ability to draw comparisons between different studies with confidence (Estcourt et al., 2013). Currently because of the heterogeneity in the methods used to assess, document and grade bleeding it is not always possible to compare studies with any great confidence. If bleeding is to be used as a main outcome measure for platelet transfusion trials, it is important that it is defined and documented in a consistent and stan- dardized way. This validation exercise was a prospective multi-centred, observational study for which the main objective was to validate a Bleeding Assessment Tool (BAT) that had been developed by the inter- national Biomedical Excellence for Safer Transfusion (BEST) collaborative. The BEST BAT is designed to describe the site, severity, duration and clinical consequences of bleeding events in a standardized manner and is intended for use in the malignant haematology patient population. Methods Prior elicitation is particularly useful in small trials because the amount of information contain in the data is limited. However, the prior can have strong effects on the posterior so removing biases in the elicitation process is paramount. A multi-centred study validating a bleeding assessment tool (BAT) developed by the biomedical excellence for safer transfusion (BEST) collaborative for use in adult patients with haematological malignancy Claire Dyer NHS Blood and Transplant Trials 2017, 18(Suppl 1):P163 This is an ongoing project. Potential posterior distributions given dif- ferent priors (dashed lines) and any possible result. Conclusions Methods pp g Methods The study will be a 6-month open label phase II trial of IV tocilizumab (8 mg/kg) in 21 patients with group I PAH. Subjects will be assessed for safety and efficacy at screening, baseline, week 4, week 8, week 12, week 16, week 20 and study end. The main outcomes are: safety (incidence and severity of adverse events) and pulmonary vascular resistance (dynes’s Cm-5) measured using invasive haemodynamic assessment by right heart catheter. Prior elicitation techniques will be used to transform experts’ knowledge about the effect of the drug onto a distribution. Bayesian analysis will take into account the experts’ prior to predict the mode and 95% credible interval of the effect of tocilizumab in pulmonary vascular resistance [2]. No one adjudication approach works best across all situations. Trialists should choose the most appropriate adjudication approach based on the specific characteristics of their trial. P163 A multi-centred study validating a bleeding assessment tool (BAT) developed by the biomedical excellence for safer transfusion (BEST) collaborative for use in adult patients with haematological malignancy l A comparison of adjudication approaches for clinical trials Brennan Kahan1, Vipul Jairath2 p 1Queen Mary University of London; 2Department of Medicine, Epidemiology and Biostatistics, Western University p 1Queen Mary University of London; 2Department of Medicine, Epidemiology and Biostatistics, Western University Objective We found that no one adjudication approach worked best across all situations. The best approach will depend on specific trial characteris- tics, mainly the misclassification rates of the site and central asses- sors, and the correlation between assessors. In general, there will rarely be much benefit to using more than three assessors, unless the correlation between assessors is extremely low; for outcomes with very high correlation between assessors, using one assessor should be sufficient. Both site and central assessors can be appropri- ate, and the best choice depends on which type of assessor has lower misclassification rates. Using a combination approach in which both the site and central assessors are involved in adjudication may be useful when misclassification rates are unknown. Having central assessors adjudicate only suspected events will typically increase bias, unless the threshold for sending suspected events to the central assessor for adjudication is extremely low. j Bayesian estimation the proportion of pulmonary arterial hypertension patients with a 30% reduction in pulmonary vascular resistance from baseline after Background Pulmonary arterial hypertension comprises a grouping of diseases associated with a poor prognosis. Four classes of drug therapy targeting vasoactive pathways have been studied in ran- domised controlled trials (RCTs) and licensed for the treatment of pre- dominantly group 1 pulmonary arterial hypertension (PAH). These therapies have demonstrated moderate success, with meta-analyses of all RCT data suggesting a short-term improvement in mortality at 14 weeks [1]. Despite this, PAH in the UK still carries a 5 year survival in idiopathic PAH of 61% and as low as 49% for PAH associated with con- nective tissue diseases. Therefore, there remains an urgent need for the development of new treatments, particularly as the results from com- bination studies of these different classes of vasoactive therapies has been to date mixed and disappointing. References 1. Galie N, Manes A, Negro L, Palazzini M, Bacchi-Reggiani ML, Branzi A. A meta-analysis of randomized controlled trials in pulmonary arterial hypertension. Eur Heart J. 2009 Feb;30(4):394–403. Pubmed PMID: 19155250. Pubmed Central PMCID: 2642921. 2. Anthony O’Hagan, Caitlin E. Buck, Alireza Daneshkhah, J. Richard Eiser, Paul H. Garthwaite, David J. Jenkinson, Jeremy E. Oakley, Tim Rakow (2006) Uncertain Judgements: Eliciting Experts’ Probabilities. Wiley. Background Many clinical trials involve binary outcomes which require adjudication to determine whether an event occurred or not (e.g. Myocardial infarc- tion, disease progression, patient response, etc.). Incorrectly classifying the outcome (e.g. By incorrectly adjudicating an event when the true outcome was no event, and vice versa) can lead to biased estimates of treatment effect and reduced power. Using adjudication approaches which minimise the misclassification rate of outcomes is therefore im- portant, however there is little evidence on which to base this decision. Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 63 of 235 Page 63 of 235 Concurrent versus sequential addition of diethylstilbestrol with dexamethasone in the treatment of castration-resistant prostate cancer: an exploratory analysis p y y Shah-Jalal Sarker1, Jo Whelan1, Stuart Mealing2, Jonathan Shamash3 1Queen Mary University of London; 2Oxford Outcomes; 3St Bartholomew's Hospital Correspondence: Shah-Jalal Sarker Trials 2017, 18(Suppl 1):P164 p y y Shah-Jalal Sarker1, Jo Whelan1, Stuart Mealing2, Jonathan Shamash3 1Queen Mary University of London; 2Oxford Outcomes; 3St Bartholomew's Hospital Correspondence: Shah-Jalal Sarker Trials 2017, 18(Suppl 1):P164 36 To assess if the MCID for the POEM is convergent for different anchor-based methods and distribution-based methods 2) To assess whether using a patient or investigator assessment as an anchor measure produces a different MCID for the POEM. 3) To compare the MCID for the POEM calculated using different time points. Methods Background Secondary analysis utilising an existing trial data set (CLOTHES Trial ISRCTN77261365). A range of methods to determine the MCID will be compared. Anchor-based methods (including: within-patient score change, between-patient score change, the sensitivity and specificity method and the predictive modelling approach) will be assessed. These anchor-based methods will be used with both a 6 point Likert scale patient/parent global assessment (How is your/your child’s ec- zema today?) And a 6 point Likert scale investigator global assess- ment (How is the child’s eczema today?) As the anchor measure. Distribution-based methods (including: effect size estimate and half standard deviation of the baseline distribution of POEM scores) will also be used. Where appropriate, there will be separate calculations for the MCID in improvement and worsening of the eczema. All ana- lyses will be repeated looking at the MCID for POEM scores at 2 months and 6 months of follow-up. Results will be available to present at the conference. l Diethylstilbestrol (DES) and dexamethasone (Dex) can be used alone or in combination to treat castration-resistant prostate cancer. This paper applies up-to-date reporting criteria and alternative statistical analyses to the data from a randomised trial carried out to determine optimal treatment sequencing (Dex plus deferred or immediate DES). Methods PSA data were reanalysed and reported according to the Prostate Cancer Working Group 2 criteria, using waterfall plots. The ability of PSA changes from baseline at 4, 8 and 12 weeks to predict survival outcome was assessed. Individual patient level survival and health re- lated quality of life (HRQOL) data were analysed using a flexible para- metric model and a mixed effects model for repeated measures, respectively. Conclusion MCID calculation methods are often described as two broad categor- ies: anchor-based methods and distribution-based methods. Anchor- based methods ascertain how change in the measurement instru- ment corresponds to another measure of change using an external criterion. Distribution-based approaches are based on the distribu- tional characteristics of the sample. Different methods measure a dif- ferent type of change, therefore it is recommended a variety of methods are used. The ultimate aim of this study is for the BEST collaborative to use their international influence to promote use of the validated and re- fined BAT by researchers and clinicians working in the field of trans- fusion medicine and clinical haematology. The standardized use of a BAT in studies using bleeding data as a main outcome measure will make it possible to draw reliable comparisons and to pool data from different studies. Ultimately this has the potential to answer research questions and to improve care for patients at a faster rate. Little research to date has explored the impact of applying different methodologies and anchors when calculating the MCID of the POEM, and there is currently no consensus on the best anchor measure to use to calculate the MCID for patient-reported outcomes. There has also been no exploration into whether the use of different data time points to calculate the MCID for the POEM may affect the results. Objectives P164 Concurrent versus sequential addition of diethylstilbestrol with dexamethasone in the treatment of castration-resistant prostate cancer: an exploratory analysis Shah-Jalal Sarker1, Jo Whelan1, Stuart Mealing2, Jonathan Shamash3 1Queen Mary University of London; 2Oxford Outcomes; 3St Bartholomew's Hospital Correspondence: Shah-Jalal Sarker Trials 2017, 18(Suppl 1):P164 Assessing the impact of independent adjudication of serious adverse events on the safety results of the efficacy of nitric oxide in stroke (ENOS) trial Peter Godolphin1, Lisa Woodhouse1, Daniel Bereczki2, Alan A. Montgomery1 Philip M. Bath1, Nikola Sprigg1 1University of Nottingham; 2Semmelweis University Correspondence: Peter Godolphin Trials 2017, 18(Suppl 1):P166 Peter Godolphin1, Lisa Woodhouse1, Daniel Bereczki2, Ala Philip M. Bath1, Nikola Sprigg1 1University of Nottingham; 2Semmelweis University Correspondence: Peter Godolphin Trials 2017, 18(Suppl 1):P166 p Results PSA changes from baseline at 4, 8 or 12 weeks did not predict overall survival (P = 0.966, 0.589, 0.415, respectively). Maximum PSA decline was associated with prolonged survival (P < 0.001), but the effect was clinically insignificant. The flexible parametric model showed that the hazard function was not completely proportional throughout the trial. Fitting a parametric function that better reflected the underlying hazard function resulted in a wider difference in median survival (3.9 months) between the two arms than seen in the original ana- lysis, but this was not statistically significant (P = 0.14). Immediate DES was associated with a greater improvement in ‘global health sta- tus’ HRQOL score (difference vs. Deferred DES = 7.85, P = 0.009). P165 The minimum clinically important difference (MCID) of the patient oriented eczema measure (POEM): do different methods of calculation give different MCIDs? Laura Howells, Sonia Ratib, Joanne Chalmers, Lucy E. Bradshaw, Kim S. Thomas University of Nottingham Correspondence: Laura Howells Trials 2017, 18(Suppl 1):P165 The minimum clinically important difference (MCID) of the patient oriented eczema measure (POEM): do different methods of calculation give different MCIDs? Laura Howells, Sonia Ratib, Joanne Chalmers, Lucy E. Bradshaw, Kim S. Thomas University of Nottingham Correspondence: Laura Howells Trials 2017, 18(Suppl 1):P165 Background g Knowing the Minimum Clinically Important Difference (MCID) en- hances the interpretability of a patient-reported outcome measure and is necessary for sample size calculations in clinical trials. The Pa- tient Oriented Eczema Measure (POEM) is a measure of eczema symptoms completed by patients or carers with a score of between 0 (no eczema) and 28 (very severe eczema). Previous studies have shown that the MCID for POEM is around 3 points. Forty patients consented to participate. Thirteen trained bleeding asses- sors collected the data. Bleeding assessments were carried out on 113 separate days, 225 bleeding assessments were compared for concord- ance. The study found good concordance (79%) overall in observations of bleeding. The study also highlighted key areas of focus such as train- ing and the importance of well laid out documentation to facilitate a standardized approach to the assessment of bleeding. Conclusion Implications This work will add to the knowledge around the MCID of the POEM, which can help inform sample size calculations in future clinical trials as well as enhance the interpretability of trial data and clinical prac- tice records. The findings of this study will also be informative for cal- culating the MCID of other patient-reported outcomes as it will further understanding of how the methodology used can affect MCID calculations. The results will determine to what degree the methods used, the anchor measure used and the time point selected affect the calculated MCID. The data were sensitive to the statistical approaches used. Interesting additional information was obtained. In particular, deferring DES may slightly reduce rather than improve patients’ HRQOL overall. All re- sults are exploratory only. Background Background Independent adjudication of serious adverse events is common in clinical trials, especially in open label studies where outcome assess- ment can be prone to bias. Few studies have investigated the impact Trials 2017, 18(Suppl 1):200 Page 64 of 235 Page 64 of 235 that adjudication of serious adverse events has on the results of a trial. The objective of this study was to explore the effect of inde- pendent adjudication of serious adverse events on the safety results of the Efficacy of Nitric Oxide in Stroke (ENOS) Trial. M h d interviews, trialists seemed unaware of the implications for the evi- dence base of not reporting all outcomes and protocol changes. Systematic reviewers facing these challenges should contact trialists to try to obtain the missing data. They may subsequently apply a statistical approach as part of a sensitivity analysis. Bias bound esti- mation, multivariate meta-analysis, and modelling the selection process have been proposed. ENOS was an international multicentre trial which randomly assigned patients with acute stroke and raised blood pressure to receive either transdermal glyceryl trinitrate (GTN) or no GTN. Non-serious adverse events were not recorded due to their high incidence in stroke pa- tients and the established nature of the trial interventions. Serious adverse events (SAEs) were reported by local investigators who were not blinded to treatment allocation using a web-based SAE form. The local investigators report included event classification, event diagno- sis and evidence used to determine diagnosis, expectedness of event, and likely causality. Independent adjudicators, blinded to treatment allocation, reviewed the investigators reports and used evi- dence available to confirm or alter the classification of event, with the adjudicator’s decision being treated as the gold standard and used in the trial analysis. As well as event classification, adjudicators independently assessed causality, diagnosis and expectedness of event. A list of known adverse reactions was defined in the protocol and provided to investigators and adjudicators. Trial registration and improved reporting should help to reduce ORB, but for findings to influence policy and practice, outcomes chosen for meas- urement need to be relevant to patients, public, healthcare professionals and others making decisions about health care. So much could be gained if an agreed core outcome set (COS) of appropriate and important outcomes was measured and reported in all clinical trials of effectiveness in a specific condition. Outcome selection in clinical trials – Looking back at the problems and moving forward with solutions 1 1 1 2 g Paula Williamson1, Jamie K. Kirkham1, Carrol Gamble1, Kerry Dwan2 1University of Liverpool; 2Cochrane Collaboration Correspondence: Paula Williamson Trials 2017, 18(Suppl 1):P167 Results Preliminary results show that of 4011 patients enrolled in ENOS, there were 1473 SAEs reported by local investigators, reduced to 1444 after review by adjudicators (unweighted kappa, k = 0.85). There was fair agreement between investigators and adjudicators on relatedness of event to treatment with 808 agreements and 644 dis- agreements (weighted kappa, k = 0.30). However, when the related- ness to treatment categories were dichotomised into Definitely not or Unlikely versus Possibly, Probably or Definitely, then there were 1305 agreements and 147 disagreements (90% crude agreement, kappa = 0.32). Repeating the trial safety analysis with investigator re- ported events indicated that adjudication made little impact to the majority of the results, with a similar number classified by both inves- tigators and adjudicators. Correspondence: Rachel McNamara Trials 2017, 18(Suppl 1):P168 Correspondence: Rachel McNamara Trials 2017, 18(Suppl 1):P168 g Conclusions Serious adverse events were largely classified correctly by local inves- tigators with the largest disagreements arising between relatedness of event to treatment. In a large trial, with many serious adverse events reported, independent adjudication of these events had little impact on trial conclusions. Background g Prevalence of epilepsy in adults with an intellectual disability (ID) is up to 20 times greater than in the general population. There is how- ever little research assessing side effects of anti-epileptic drugs (AEDs) in adults with ID and epilepsy. Screening tools are available to assess AED side effects in the general adult population, and research suggests that active monitoring is sufficient to change management and improve quality of life (QOL). It is not known however whether such tools can be used to identify side effects in adults with ID, or whether included items are important and relevant to patients and carers. Furthermore, available instruments tend to focus on the more theoretical concept of QOL rather than on side effects of medication per se, and their validity or suitability for use in ID populations has not been established. A Cochrane review concluded that measure- ment of side effects in this population was hampered by reliability of available measures. The aim of this systematic review is therefore to identify research on measurement and impact of AED side effects in the adult epilepsy population. The review seeks to identify measures developed for adults with ID where available, and also in the general epilepsy population (i.e. Without ID), in order to identify measures that could be adapted for an ID population. The review is the first stage of a larger study to develop Patient Reported Outcome Mea- sures (proms) to assess AED side effects in adults with epilepsy and ID. The aim of the larger study is to develop appropriate versions for patient and carers. patient an Methods Selection of outcomes to measure in trials designed to compare dif- ferent interventions is crucial. It has been estimated that less than half of all outcome data collected in trials is fully reported, with miss- ing data due to unpublished trials, poor reporting, and choosing not to include particular results within trial reports. A systematic literature search was conducted in MEDLINE In-Process, MEDLINE, EMBASE, SCOPUS and Web of Knowledge. The review iden- tified studies in adults with epilepsy (and a subset of adults with ID) taking an AED, which included a scale/outcome measure of a poten- tial AED side effect. Studies focused on seizures as a side effect of medical treatment (brain surgery or medication) and seizure disor- ders not specified as epilepsy were excluded. Difficulties caused by heterogeneity in outcome measurement across studies are well known. Empirical research provides strong evidence that outcome reporting bias (ORB), defined as the results-based se- lection for publication of a subset of the recorded outcome variables, is a significant problem in a quarter to a third of randomised trials and can have major impact in a fifth of systematic reviews. In Patient reported outcome measure development in epilepsy and intellectual disability: a systematic review of measures of anti-epileptic drug side effects We used unweighted and weighted kappa respectively to estimate agreement between local investigators and independent adjudicators on diagnosis and relatedness to treatment of SAEs. The safety ana- lysis of ENOS (chi-squared tests between treatment arms for SAE diagnosis) was replicated using investigator reported events, and these were compared to adjudicator reported events with a test of homogeneity. Preliminary results are provided in this abstract, with full results available for presentation in May. Background The COMET (Core Outcome Measures in Effective- ness Trials) Initiative, http://www.comet-initiative.org/, an innovative global project, brings together people interested in COS development and application. This talk will review progress made with both statistical and non- statistical solutions to this problem. P168 P168 Patient reported outcome measure development in epilepsy and intellectual disability: a systematic review of measures of anti-epileptic drug side effects Rachel McNamara1, Lauren Copeland2, Andrea Meek1, Mike Kerr3, Michael Robling1, Kerenza Hood4 1South East Wales Trials Unit (SEWTU), Centre for Trials Research; 2Division of Population Medicine, Cardiff University; 3Institute of Psychological Medicine and Clinical Neuroscience; 4Centre for Trials Research Results We conducted 12 interviews across the target groups. We identified four themes in the data, how evidence is used, aspects of trials which are considered important when assessing evidence, views on trial design across the nine domains of PRECIS-2, and attitudes to- wards pragmatic trials. Most interviewees were aware of pragmatic trials however different views existed as to what the term meant. For some aspects of a trial, such as the flexibility given to those de- livering the intervention, or the level of resources and expertise made available to deliver the intervention, emulating routine prac- tice may not be the best way to make results relevant to primary care. Across other aspects of a trials design, for example the popu- lation and trial setting, our work indicates a pragmatic approach is more appropriate. Mixed methods feasibility work to inform data collection in a main study: designing case report forms to capture variation in surgical techniques , g 1Queen Mary University of London; 2University of Stirling; 3University of Aberdeen Charlotte Murkin1, Jane M. Blazbey2, Leila Rooshenas2, Conor Jones3, Ian R. Daniels3, Neil J. Smart4, Natalie S. Blencowe2, on behalf of the CIPHER study group Correspondence: Gordon Forbes Trials 2017, 18(Suppl 1):P170 1University of Bristol; 2University of Bristol, Centre for Surgical Research, School of Social and Community Medicine; 3Exeter Surgical Health Sciences Research Unit (HESRU), Royal Devon & Exeter Hospital; 4Peninsula College of Medicine and Dentistry, University of Exeter Correspondence: Charlotte Murkin Trials 2017 18(Suppl 1):P169 Background Randomised trials are difficult and costly. Like most things that are hard, the effort expended is only worth it because we hope to make a difference. Sadly, the benefit to potential users such as patients, healthcare professionals and policy makers is often smaller than it should be because trial design decisions reduce the relevance of the trial results to their intended users and the contexts in which they work. Background Surgical procedures are considered to be complex healthcare inter- ventions. As such, multiple variations in the delivery of a procedure with the same label are possible. Variations can be small or large, and known or unknown, because there is often a difference between what surgeons say they do and what is done. This project developed and tested methods for identifying all possible variations in a particu- lar surgical procedure to inform the design of case report forms for use in a main study. To ensure trial results match the needs of potential users it is recommended that researchers design pragmatic trials, testing their intervention under conditions similar to those found in the real world. PRECIS-2 is a trial design tool which encourages trial- ists to consider how pragmatic their trial is across nine different domains covering the population in the trial, how the interven- tion is delivered and the outcomes measured. Whilst in principle, a pragmatic trial should aim to emulate routine practice across the PRECIS-2 domains, in practice compromises will have to be made when designing trials. Little research exists to guide these decisions. Methods To identify variations in the surgical procedure, two phases of work were undertaken: i) systematic literature review (to identify ‘known’ variations in technique) and ii) qualitative work (to identify ‘unknown’ variations and explore clinical professionals’ views on how these vari- ations may influence outcomes of interest). The qualitative work comprised of case studies using digital video data capture and non- participant observation, and interviews with healthcare professionals. To date, 6 case studies have been conducted, as well as 13 inter- views. Observation field notes and textual descriptions of the video- captured operations were coded and categorised into themes and subthemes. Verbatim transcripts of the interviews and observations were analysed through constant comparison approaches. Interview and observation themes were compared and contrasted to inform new lines of enquiry for exploration in further case studies and inter- views. A long-list of themes and subthemes was synthesised from the data collected in both phases of work. The final phase of the pro- ject (yet to be undertaken) will refine and rationalise these themes through consensus methods, to finalise the data items to be included in the case report forms. This research explores the views of people who influence practice in primary care towards assessing and using evidence from clinical tri- als. The aims of the study are to assess what is important in design- ing randomised trials, and to ascertain how the PRECIS-2 tool can be used to make research more relevant to primary care. Methods We carried out semi-structured interviews with individuals or small groups of people involved in implementing research in primary care in the UK. We interviewed people involved with journals, guideline development, research charities, research funders, primary care edu- cators, clinical commissioning groups, GPs and clinical effectiveness research. A thematic analysis of the data from the interviews was car- ried out using the framework approach. Results Conclusion This novel methodology incorporates multi-modal data collection to provide insights into the ‘black box’ of complex interventions such as surgery. It can be successfully used to identify and summarise tech- nical variations in the delivery of complex interventions, and non- technical factors that may influence this delivery. Both phases of this study identified themes that would have otherwise remained ‘un- known’ if performed in isolation. Resulting data can subsequently be used to comprehensively and systematically design case report forms. This study design therefore adds value by identifying and doc- umenting all key data and variations of an intervention. Screening tools are available to assess AED side effects in the general adult population, however only two outcome measures are designed for use in ID populations. The focus of these measures is broader than side effects alone and therefore may not pick up the full range of side effects of importance in this group. There is a clear lack of established and validated assessment scales for patients with ID and epilepsy, and a need to consistently measure and report patient- reported side effects of medication, both in clinical practice and in trials of new medication regimes. P170 Maximising the relevance of randomised trials to primary care Gordon Forbes1, Kirsty Loudon2, Megan Clinch1, Stephanie J. C. Taylor1, Shaun Treweek3, Sandra Eldridge1 1Queen Mary University of London; 2University of Stirling; 3University of Aberdeen Correspondence: Gordon Forbes Trials 2017, 18(Suppl 1):P170 0 Maximising the relevance of randomised trials to primary care Gordon Forbes1, Kirsty Loudon2, Megan Clinch1, Stephanie J. C. Taylor1 Shaun Treweek3, Sandra Eldridge1 Results 460 papers were identified and 93 met inclusion criteria. Of 107 mea- sures identified, six were appropriate for use with adult ID popula- tions. Seven studies investigated adults with epilepsy and ID and Trials 2017, 18(Suppl 1):200 Page 65 of 235 discussed at a consensus meeting, during which senior clinicians will agree on the data to collect during the main study. Conclusion examined side effect domains of behaviour, functionality and quality of life. Side effects of AEDs are inconsistently and inadequately mea- sured in ID populations and are overly reliant on carer report. The overall burden of side effects is therefore likely to be under-reported. Conclusions P172 P172 Motives, meanings and misconceptions: exploring the understandings and experiences of advanced lung cancer patients participating in a non-placebo clinical IMP trial Emily Harrop1, Simon Noble2, Michelle Edwards1, Stephanie Sivell2, Barbara Moore4, Annmarie Nelson2 1Cardiff University School of Medicine; 2Marie Curie Research Centre, Cardiff University; 3DECIPHER, Cardiff University; 4Health and Care Research Wales Support Centre Trials 2017, 18(Suppl 1):P172 Funders and those designing trials in should carefully consider de- sign decisions across the PRECIS-2 domains to maximize the rele- vance of research to primary care. Across most aspects of their designs trials should aim to be pragmatic however there are some important exceptions where design decisions are more complex. Dif- fering perceptions about what it means for a trial to be pragmatic could be helped by the use of the PRECIS-2 tool by those using evi- dence from clinical trials to influence practice in primary care. This abstract is not included here as it has already been published. This abstract is not included here as it has already been published. P171 Systematic review of the use of mediation analysis in randomised controlled trials in the primary healthcare setting Gordon Forbes1, Ceire Costelloe2, Patty Chondros3 1Queen Mary University of London; 2Imperial College London; 3University of Melbourne Correspondence: Gordon Forbes Trials 2017, 18(Suppl 1):P171 Conclusions The pilot was useful in establishing the potential impact of the intervention on smoking cessation outcomes, and testing the processes and procedures in place for definitive trial. We will discuss the challenges we encountered and their methodological implications. Results The literature review identified 138 themes relating to technical vari- ations in the surgical procedure of interest, and 50 non-technical fac- tors including patient characteristics (e.g. Obesity), contextual operation factors (e.g. Emergency surgery), and the grade of the op- erating surgeon. The case studies and interviews identified 150 themes relating to technical variations and 64 non-technical factors. The themes identified in both phases were combined, duplicates and overlaps excluded, leaving 180 technical and 85 non-technical fac- tors. Of these, 137 were common to both phases of work and 77 were identified during the case studies. These factors will be Page 66 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Results A total of 138 references were identified using the electronic search strategy and 23 studies were found to be eligible for the review. Most studies were published post 2010 (70%), were set in the USA (65%) and involved an intervention targeting a mental health condi- tion. 48% of analyses involved a single mediator measured at one time point and the others included multiple mediators or measure- ments over time. Ten (44%) studies reported adjusting for any covari- ates and only 2 studies discussed unmeasured confounding as a potential limitation of their results. Background Mediation analysis is a way to investigate the mechanisms by which an intervention affects an outcome. It has been proposed as a way to ‘open the black box’ of traditional epidemiology, shedding light on the causal pathways between interventions and outcomes. Methodological work, based on the causal inference framework, has helped formalise the assumptions required for mediation analysis to give valid causal conclusions. For most common approaches to mediation analysis of randomised trials, a key assumption is that there is no confounding of the mediator outcome association that is not controlled for in the ana- lysis. The UK Medical Research Council recommend mediation analysis be used as part of a process evaluation of complex interventions. Trials which are conducted in the primary healthcare setting are often of complex interventions, so mediation analysis could be an appropriate method to include in any analysis plan of clinical trials in this setting. The aim of this systematic review is to examine applications of medi- ation analysis in clinical trials taking place in primary health care set- tings and use the findings to provide guidance for future analysis. Methods P173 Challenges of conducting clinical trials in community pharmacies and their methodological implications: pilot trial of a training intervention for smoking treatment optimisation in pharmacies (STOP) , , y 1Queen Mary University of London; 2Imperial College London; 3University of Melbourne Correspondence: Gordon Forbes Trials 2017, 18(Suppl 1):P171 Vichithranie Madurasinghe, Sandra Eldridge, Ratna Sohanpal, Wai James, Liz Steed, Stephanie Taylor, Chris Griffiths, Robert Walton Queen Mary University of London, UK Correspondence: Vichithranie Madurasinghe Trials 2017, 18(Suppl 1):P173 Vichithranie Madurasinghe, Sandra Eldridge, Ratna Sohanpal, Wai James, Liz Steed, Stephanie Taylor, Chris Griffiths, Robert Walton Background Expanding the role of community pharmacists is a cornerstone of UK government policy and health promotion activities such as smoking cessation are fundamental to this expanded role. We developed a service optimisation and training intervention (STOP intervention), and a cluster randomised trial was planned to evaluate the effective- ness of this intervention. In 2015, a pilot trial was carried out to as- sess its feasibility. P172 Motives, meanings and misconceptions: exploring the understandings and experiences of advanced lung cancer patients participating in a non-placebo clinical IMP trial Emily Harrop1, Simon Noble2, Michelle Edwards1, Stephanie Sivell2, Barbara Moore4, Annmarie Nelson2 1Cardiff University School of Medicine; 2Marie Curie Research Centre, Cardiff University; 3DECIPHER, Cardiff University; 4Health and Care Research Wales Support Centre Trials 2017, 18(Suppl 1):P172 Methods Twelve community pharmacies were assigned at random to STOP inter- vention or usual practice using simple randomisation (allocation ratio 2:1). Results STOP training intervention has potential to increase the number of smokers retained in smoking cessation services (89.5% in interven- tion arm vs 75.0% in usual practice arm) and subsequently quit smoking (52.6% in intervention vs 21.9% in usual practice arm). It had limited impact on pre-specified primary outcome, throughput (on average 43 vs 80 service users per pharmacy joined the smoking cessation service in intervention and usual practice pharmacies, respectively). We searched the Medline, Embase and Cochrane Library databases using keywords that identify randomised trials, mediation analysis and primary healthcare settings. Our eligibility criteria were papers published between 2004 and 2014 which described a mediation ana- lysis of data from a randomised trial conducted in a primary health- care setting. Abstracts were screened for eligibility, where eligibility was unclear from the abstract we performed full text screening. Data extraction related to the study design features and type of mediation analysis and assumptions was carried out independently by two au- thors. Any disagreement was resolved with a third reviewer. More importantly, the pilot was useful in highlighting the challenges of conducting clinical trials in this important but under-researched healthcare setting and how best to overcome them. The key issues we identified include: complex organisational structures between and within community pharmacies; pharmacies as businesses vs healthcare providers; achieving balance for individual characteristics in cluster randomisation; selecting trial outcomes; identifying factors influencing outcomes; data collection -using case report forms vs routine data. p Conclusions Mediation analysis is used in research conducted in primary health- care settings to understand the mechanisms of how an intervention works. Most mediation analyses identified in this review failed to ad- equately control for confounding of the mediator outcome associ- ation. Even when potential confounding factors are included in the analysis the consequences of unmeasured confounding are rarely acknowledged as limitations. This could lead to invalid conclusions being drawn from many mediation analysis of randomised trials in primary health care settings. When planning a mediation analysis investigators should collect data on potential mediator outcome confounders and adjust for these variables in the analysis. I struggle with equipoise: a qualitative study exploring clinicians - Views of a randomised controlled trial (ACL SNNAP) prior to trial conduct I struggle with equipoise: a qualitative study exploring clinicians - Views of a randomised controlled trial (ACL SNNAP) prior to trial conduct Loretta Davies1, Francine Toye2, Jonathan Cook1, Andrew Price1, David Beard1 1 2 1University of Oxford; 2Oxford University Hospitals NHS Foundation Trust Correspondence: Loretta Davies Trials 2017, 18(Suppl 1):P174 Trials 2017, 18(Suppl 1):200 Page 67 of 235 Page 67 of 235 Methods The NIHR HTA Tranexamic acid for intracerebral Haemorrhage (TICH- 2) trial, is a large multi-centre international randomised controlled trial, aiming to recruit 2,000 patients over two phases. The start-up phase was planned to take 18 months, recruiting 17 patients a month to reach a target of 300; actual recruitment was higher than this and the start-up phase stopped six months early, when it reached target. The main phase originally aimed to recruit 68 pa- tients a month, reducing down to 55 patients a month after the main phase was brought forward. Recruitment was monitored continu- ously throughout the study in order to predict if and when the de- sired sample size would be reached. Methods to predict future y Methods Recruitment in large scale clinical trials can be hard to predict during planning and difficult to maintain throughout the study. Many trials fail to finish to time and target; hence, better methods for recruit- ment predictions are needed. Two phase studies with internal pilots and a stop-go decision before the main phase allow time for re- estimation of recruitment. It was proposed to recruit 12 to 14 clusters (PT clinics) to test feasibil- ity across a range of settings; six clusters in each arm participating in two waves of recruitment and enrolling six participants in each clus- ter per wave [i.e. 144 participants, 72 per arm]. A minimum of 96 par- ticipants [48 per arm] was required for sample size calculations. The recruitment procedure agreed with PTs involved their screening wait- ing lists to identify potential participants for an invitation letter, and researcher led telephone and face-to-face screening. The recruitment rate [total, study arm and wave] and ratio of number screened: num- ber enrolled and reasons for exclusion at each step was calculated after each wave. g , Results The average recruitment over the 12 month start-up phase was 28 patients a month from 53 centres; the mean (SD) per centre was 0.17 (0.19) patients a month, with a range of 0.02 to 0.7. The monthly av- erages from the main phase ranged from 29 to 61 patients a month, from 108 centres; centre averages ranged from 0.03 to 3.04 patients a month with a mean (SD) of 0.42 (0.45). The largest 15 centres re- cruited at least one patient a month per site, whereas, some of the smaller centres did not even recruit one patient in five months. The average for the first 12 months of the main phase lies near the mid- dle of the main phase averages at 42 patients a month, suggesting this could be an ideal time to re-evaluate recruitment predictions; a yearly average will also ensure any seasonal fluctuations are cap- tured. After analysing the available data the trial team found that they were unlikely to recruit to target and would need at least an- other nine months of recruitment to reach the target sample size. Conclusion Background The SOLAS cluster randomised controlled feasibility trial aims to as- sess the (1) acceptability of the novel 6 week group-based education and exercise SOLAS complex intervention to patients and physiother- apists (PTs) compared to usual individual physiotherapy, (2) feasibility of trial procedures and sample size for a definitive trial and (3) effect on secondary outcomes. The aim of this study was to evaluate the feasibility of trial recruitment procedures. Methods Katie Flaherty, Lee Haywood, Lelia Nikola Sprigg University of Nottingham Correspondence: Katie Flaherty Trials 2017, 18(Suppl 1):P175 P175 P175 Recruitment monitoring and predictions: data from the tranexamic acid for intracerebral haemorrhage (tich-2) trial Katie Flaherty, Lee Haywood, Lelia Duley, Zhe Law, Philip M. Bath, Nikola Sprigg University of Nottingham Correspondence: Katie Flaherty Trials 2017, 18(Suppl 1):P175 Results Several issues were identified which may impact on the feasibility of this trial. Despite indicating a willingness to randomise, clinicians expressed varying levels of uncertainty and preferences which may impact on their ability to deliver balanced descriptions of the treat- ment options. This was especially evident in relation to certain pa- tient subgroups: young and highly active patients with the potential to affect which patients clinicians approach to participate. Clinicians indicated that patients often have strong preferences for treatment of this injury, particularly towards surgery, and considered this as a potential barrier to trial recruitment. Various sources were thought to influence patients’ views towards treatment, such as how information on the injury and its management are currently portrayed through the internet and media. Predicting recruitment when planning a clinical trial is difficult, methods for monitoring and predicting future recruitment should be performed throughout the study. When designing trials, flexible re- cruitment phase and end dates should be considered to allow for dif- fering recruitment rates than originally planned. Monthly centre averages showed larger variation in the main phase compared to the start-up, reflecting differences between recruiting sites. Twelve months into the main phase of a study appears a reasonable time point to re-evaluate recruitment predictions. Similar systems are used in our other large trials: HTA TARDIS and BHF RIGHT-2. Conclusion Exploring clinicians’ views at the pre-trial stage enabled potential trial specific issues to be identified. As a result of these findings, appropri- ate training and support for recruiting teams prior to the start of re- cruitment is being developed. Trialists may wish to consider the use of pre-trial qualitative studies, particularly in trials where the inter- ventions being evaluated are markedly different, to enable issues specific to a particular trial to be identified and addressed. Methods S Semi-structured qualitative interviews were undertaken with a pur- posive sample of surgeons (n = 6) and physiotherapists (n = 6) from 6 NHS hospitals. All clinicians were experienced in the management of ACL injuries and had expressed an interest in participating in the ACL SNNAP trial. Interviews were analysed using an Interpretative Phenomenological Analysis (IPA) approach. 6 Evaluation of recruitment procedures to the self-management of osteoarthritis and low back pain through activity and skills (SOLAS) cluster randomised controlled feasibility trial [ISRCTN 49875385] Evaluation of recruitment procedures to the self-management of osteoarthritis and low back pain through activity and skills (SOLAS) cluster randomised controlled feasibility trial [ISRCTN 49875385] Deirdre Hurley, Isabelle Jeffares, James Matthews, Ricardo Seguardo University College Dublin Correspondence: Deirdre Hurley Trials 2017, 18(Suppl 1):P176 Background recruitment, using existing trial data and recruitment trends, were developed. A system was developed for the trial website which shows live recruitment predictions; changing with the numbers of new participants and active sites. Averages over different periods throughout the trial were also looked at; including cumulative aver- ages, monthly averages and phase averages. Results Strong treatment preferences of both clinicians and patients have been shown to impact on recruitment, adherence to treatment allo- cation and ultimately the success of a randomised controlled trial (RCT). This is particularly evident when the interventions being evalu- ated are markedly different, such as the comparison of surgery with a non-operative approach such as physiotherapy. The aim of this study was to explore clinicians’ views at the pre-trial stage of NIHR HTA funded RCT of anterior cruciate ligament (ACL) deficiency man- agement comparing surgery and rehabilitation (ACL SNNAP Surgical Necessity in Non-Acute Patients) and to identify issues that may influence trial feasibility. Results 14 clusters were recruited (7 per trial arm), each site participated in two waves of recruitment, resulting in three study waves (W1-W3). The average cluster size in each arm was below six (Intervention: mean (SD) = 4.92 (1.31), range 2–7; Control: mean (SD) = 5.08 (2.43), range 1–9) with no significant difference between arms (df = 16.909, Page 68 of 235 Page 68 of 235 Trials 2017, 18(Suppl 1):200 t = −0.209, p = 0.837). The cluster size increased from W1 (Interven- tion: 4.25 (1.71), 2.00-6.00; Control: 4.40 (1.95), 3.00-7.00) to W2 in both arms (Intervention: 5.17 (1.17), 4.00-7.00; Control: 5.80 (1.79), 4.00-8.00), with a further increase in W3 in the intervention arm only (Intervention: 5.50 (0.71), 5.00-6.00; Control: 5.00 (5.66), 1.00-9.00). 120 participants (83.3%; of n = 144 expected) were recruited (Inter- vention n = 59; Control n = 61). The recruitment rate according to tar- get increased in subsequent waves (W1 Target: 54, Actual: 39; W2 Target: 66, Actual: 60; W3 Target: 24, Actual: 21). Overall, 1708 poten- tial participants were identified from the waiting list: 1136 (66.5%) were excluded predominantly due to diagnosis (n = 879), age (n = 158), exclusion criteria (n = 133) and symptom duration (n = 53). Of 572 invitation letters, 375 (65.6%) participants responded [W1: 42.6%, W2: 76.3%, W3: 78.3], with 224 (59.7%) excluded by telephone screen [W1: 35%, W2: 81.3%, W3: 85.1%] mainly due to exclusion criteria (n = 69), preference for individual PT (n = 62), inability to attend SOLAS group (n = 30) or poor English (n = 30). A further 31 (20.5%) were ex- cluded at face-to-face screening with 120 participants recruited, repre- senting 21% of invitation letters [W2: 20.2%,W3: 21.6%, W4: 19.8%]. The invitation letter was simplified after W1 following communication with PTs, and telephone screening refined after W2. will embed the use of the multimedia information resources into six host trials in the United Kingdom. Patients and their parents approached to participate in the host trials will be randomly allo- cated to one of three arms: to use the standard printed participant information sheet; or the multimedia information resource; or both the standard printed participant information sheets plus the multi- media information resource. The primary outcome will be the effect of the multimedia information resources on rate of recruitment into the host trials. Methods The TRECA study has two phases. The first TRECA phase involves a qualitative study with children and adolescents and their parents to inform the development of multimedia information resources, followed by rounds of iterative user testing to refine the resources. The multimedia information resources will contain elements of audio, video, text and animations, including some aspects that are trial- specific and others that pertain to any trial. The second TRECA phase Objectives The first objective of the TRECA (trials Engagement in Children and Adolescents) study is to use participatory design to develop multi- media information resources for use in healthcare trials. The study will also evaluate the potential for multimedia information resources to improve the quality of decision-making about participation in healthcare trials involving children and adolescents with long-term health conditions, and then assess the impact of these multimedia information resources on rates of trial recruitment and retention. Do multimedia resources increase the quality of information provision and rate of recruitment in trials involving children and adolescents?: protocol for the TRECA study 1 2 3 4 1University of Liverpool; 2Department of Psychological Sciences, University of Liverpool; 3Department of Child Health, University of Liverpool; 4Neurology Department, Alder Hey Children's Hospital; 5School of Social and Community Medicine, University of Bristol; 6School of Public Health, University of Sydney; 7Department of Health Research, University of Liverpool; 8Department of Biostatics, University of Liverpool Correspondence: Louise Roper Trials 2017, 18(Suppl 1):P178 Correspondence: Louise Roper Trials 2017, 18(Suppl 1):P178 p p Trials 2017, 18(Suppl 1):P178 This abstract is not included here as it has already been published. Background Randomised controlled trials are the best method for testing health interventions whilst minimising bias. However, recruitment and sub- sequent retention of children and adolescents in healthcare trials can be challenging. Printed participant information sheets for potential trial participants are often lengthy and difficult to read and under- stand. Presenting key information about trials using multimedia may help to overcome these limitations and better support children, ado- lescents and their parents in deciding whether to participate in a trial. Results Other outcomes measured include the effect of multi- media information resources on retention of participants in the host trials and the impact on the quality of decision making about partici- pation of the patient (child or adolescent) and the parents, when compared to standard printed participant information sheets alone. A prospective meta-analysis of the outcomes from the six host clin- ical trials will be undertaken. C l i P177 P177 Do multimedia resources increase the quality of information provision and rate of recruitment in trials involving children and adolescents?: protocol for the TRECA study Jackie Martin-Kerry1, Peter Bower2, Bridget Young3, Jonathan Graffy4, Ian Watt1, Rebecca Sheridan1, Paul Baines5, Catherine Stones6, Jenny Preston5, Steven Higgins7 1University of York; 2University of Manchester; 3University of Liverpool; 4University of Cambridge; 5Alder Hey Children's Hospital; 6University of Leeds; 7University of Durham Correspondence: Jackie Martin-Kerry Trials 2017, 18(Suppl 1):P177 Background The importance of evidence based medicine is now widely recog- nised, and randomised controlled trials (RCTs) are the gold standard method for evaluating health technologies. Comprehensive and sys- tematic evidence reviews are essential before embarking on RCTs, and this information should be clearly presented to potential partici- pants to ensure that they are fully informed about the rationale for the trial and treatment options. However, little is known about how health professionals present current evidence to eligible patients in RCT consultations. Conclusion This study will inform whether multimedia information resources, when developed using participatory design principles, are able to in- crease rates of recruitment and retention of children and adolescents in healthcare trials. There is also the potential for patients to make better informed decisions about participation in trials (whether or not they decide to take part) through the use of multimedia informa- tion resources. The multimedia information resources also have the potential to assist with providing information on other healthcare de- cisions outside of clinical trials. P179 How evidence is presented to potential participants in RCT recruitment consultations Daisy Elliott, Leila Rooshenas, Samantha Husbands, Sangeetha Paramasivan, Marcus Jepson, Caroline Wilson, Jenny Donovan University of Bristol Correspondence: Daisy Elliott Trials 2017, 18(Suppl 1):P179 Correspondence: Daisy Elliott Trials 2017, 18(Suppl 1):P179 p Conclusions The sample size was below target but sufficient for sample size calcu- lations. Recruitment rate, cluster size and response to invitation let- ters increased across waves as procedures were improved, but the enrollment rate remained unchanged. Recruitment to trials of com- plex interventions outside routine practice is challenging and war- rants further research with patients to address their barriers and enablers to trial participation. Children and young people’s views on research without prior consent in life threatening situations: a qualitative study Louise Roper1, Frances Sherratt2, Paul McNamara3, Bridget Young2, Richard Appleton4, Esther Crawley5, Angus Dawson6, Lucy Frith7, Carrol Gamble8, Kerry Woolfall2 Children and young people’s views on research without prior consent in life threatening situations: a qualitative study Louise Roper1, Frances Sherratt2, Paul McNamara3, Bridget Young2, Richard Appleton4, Esther Crawley5, Angus Dawson6, Lucy Frith7, Carrol Gamble8, Kerry Woolfall2 Background g The study within a trial (SWAT) concept enables trialists to assess dif- ferent ways of designing, conducting, analysing and evaluating stud- ies through the conduct of research embedded within a larger trial. PRIMETIME is a prospective biomarker directed cohort study aiming to identify a subgroup of breast cancer patients who can safely avoid adjuvant breast radiotherapy following breast conserving surgery. This subgroup is deemed to be at such a low risk of local relapse that the potential benefits of radiotherapy are unlikely to outweigh the known risks. The current uncertainty regarding the absolute benefit of adjuvant radiotherapy (in this subgroup), the concept of avoiding treatment, and the offer of entry into a clinical trial can be over- whelming and challenging for patients to cope with. The uncertainty patients face regarding healthcare decisions is known as ‘decisional conflict’. We would like to optimise the decision making process for patients facing this uncertainty. Decision aids are ‘interventions de- signed to help people make specific and deliberative choices among options by providing information about the options and outcomes relevant to a person’s health status’. Evidence suggests decision aids reduce decisional conflict. This SWAT is designed to investigate the effect of a decision aid on patients’ decisional conflict over their un- certainty regarding the absolute benefit of radiotherapy and there- fore their decisional conflict over whether or not to enter PRIMETIME. Proposed Method Throughout the recruitment process, women were significantly less likely than men to continue to the next stage: of those invited, 45% of women vs. 51% of men attended the screening visit; of those attending screening, 70% of women were eligible and consented to enter run-in compared to 78% of men; and of those entering the run-in period, 82% of women vs. 87% of men were randomised (all p < 0.001). This led to only 18% of all women invited agreeing to enter run-in compared to 28% of men, and 11% of the overall total of women being randomised vs. 19% of men (both p < 0.001). These gender differences were still sig- nificant after adjusting for age, ethnicity, deprivation index as a meas- ure of socioeconomic status, and distance from screening site. After adjustment, women were still significantly less likely than men to at- tend screening (OR: 0.79, 95% CI: 0.77-0.81), to enter run-in (OR: 0.57, 0.55-0.59), and to be randomised (OR: 0.69, 0.66-0.73). Methods Using data from the MRC/BHF Heart Protection Study (HPS), the influ- ence of demographic factors was explored at each stage of the trial re- cruitment process (invitation, screening, pre-randomisation run-in, and randomisation). The HPS dataset was chosen as patients had been identified as potentially eligible from their hospital records and then were sent a postal invitation to attend a screening visit. This method of invitation allows large numbers of patients to be invited, and provides a true, unselected denominator for the recruitment effort. R l y g Correspondence: Indrani Bhattacharya Trials 2017, 18(Suppl 1):P180 Background The REVEAL study began recruitment 17 years after HPS. There has been a decline in the proportion of patients agreeing to take part in CTSU trials when identified from electronic records. Whilst the overall proportion of pa- tients invited that were randomised was much smaller (3% in REVEAL vs. 16% in HPS), the observed differences between men and women, after adjustment, at screening (OR: 0.43, 0.41-0.44), at run-in (OR: 0.55, 0.51-0.59) and at randomisation (OR: 0.59, 0.53-0.66), were at least as great as in HPS highlighting that this is an ongoing issue. Conclusion p Results Preliminary results suggest that there was considerable variation within and across RCTs in how recruiters discussed evidence. Some recruiters did not introduce the concept of uncertainty about optimal treatment or discuss any empirical evidence about treatment options at all. These recruiters had a tendency to instead draw on anecdotes, most commonly in the form of their experiences of patients' treat- ment outcomes. The majority of recruiters alluded to an absence of evidence to introduce the RCT but did not elaborate further. Where recruiters provided information about previous studies, they tended to summarise the findings without referring to the quality of the re- search. Final results will be presented at the conference. C l i Investigating the impact of a decision aid on decisional conflict in a SWAT allows us to answer important questions in an economic and efficient manner where we are able to conduct research within the context of a larger study. Given our hypothesis that the decision aid will reduce decisional conflict, the choice of a stepped wedge trial design ensures that by the end of the study all centres will have use of the decision aid as opposed to a parallel design which may be considered less favourably as some centres would never introduce the decision aid. If we are able to determine that the introduction of a decision aid reduces decisional conflict this would provide the evi- dence required to support increasing resources into the develop- ment and, ultimately, routine use of decision aids for patients facing complex treatment decisions. Clinicians and nurses play a vital role in providing detailed and accur- ate evidence-based information to patients to facilitate informed consent for participation in RCTs. However, this study highlights con- siderable variability in how recruiters present evidence. It therefore demonstrates the need for support and training to enable recruiters to present information clearly, both in regards to summarising find- ings and appraising the quality of this research. P181 Factors influencing recruitment in large randomised trials Danielle Edwards, Michael Lay, Martin Landray, Louise Bowman, Jane Armitage University of Oxford Correspondence: Danielle Edwards Trials 2017, 18(Suppl 1):P181 Background Recruitment into large clinical trials is difficult, with many trials not recruiting to target. This can cause financial, ethical and practical problems and undermine the quality of the research as large num- bers of participants are needed to accurately assess moderate effect sizes. Previous research has identified age, ethnicity, socioeconomic status, and education as important influences on the success of trial recruitment but little has been done to quantify these effects. Methods Indrani Bhattacharya1, Charlotte E. Coles2, Lisa Fox3, Anna Kirby4, Lesley Turner5, Hilary Stobart5, Lesley Fallowfield6, Judith Bliss3 1Institute of Cancer Research; 2Oncology Centre, Cambridge University Hospitals NHS Foundation Trust; 3Institute of Cancer Research - Clinical Trials and Statistics Unit; 4Royal Marsden Hospital; Independent Cancer Patients' Voice; Sussex Health Outcomes Research & Education in Cancer University of Brighton Methods Five UK-based RCTs were purposefully selected to include a range of trials from different clinical contexts and with different types of re- cruiters. Consultations in which recruiters presented information about the RCT to eligible patients were audio-recorded (n = 117). Data relating to any presentation of uncertainty or evidence Trials 2017, 18(Suppl 1):200 Page 69 of 235 Page 69 of 235 regarding optimal treatment were transcribed verbatim and analysed thematically by DE using constant comparative techniques derived from grounded theory methodology. A subset of consultations were independently coded by LR and SH to confirm reliability of coding. Results regarding optimal treatment were transcribed verbatim and analysed thematically by DE using constant comparative techniques derived from grounded theory methodology. A subset of consultations were independently coded by LR and SH to confirm reliability of coding. Results began recruiting to PRIMETIME using minimisation. The primary out- come is to assess whether the addition of a decision aid video to stand- ard patient information giving reduces patients’ decisional conflict. The secondary outcome is to assess acceptance of entry into PRIMETIME. Discussion Interim results Of 3114 articles within the ORRCA database, 39 were eligible. Dupli- cate screening did not produce any unresolvable discrepancies. One paper used a randomised recruitment study design to evaluate an intervention, 11 evaluated an intervention through a non- randomised study and 16 recruitment studies did not evaluate an intervention. A further 11 studies report results from community sur- veys of proposed hypothetical RCTs. 3832 records were screened and data extraction was carried out on 48 pa- pers. The key barriers: gatekeeping by professionals and family care givers, high refusal rates, the need for intensive resources and participants not meeting eligibility criteria. Key facilitators included lead clinician support and key messaging. Research staff on site, regular contact with clinicians and the use of scripts/role play were seen as important recruitment strat- egies. Most evidence is based on researchers own reports of experiences of recruiting to palliative care RCTs rather than independent evaluation. Conclusion Perceived barriers to recruitment included the clinical condition of the patient, patients impaired ability to provide valid informed con- sent and a narrow therapeutic time window. Further barriers to re- cruitment identified as the result of the recruitment study were clinician’s refusal for patients to be approached, workload of the clin- ical team, insufficient approach of eligible participants and the use of surrogate decision makers (SDMs). Types of recruitment interventions included obtaining consent in the pre-hospital setting (n = 3), the use of alternative methods of consent (n = 3), on-site training/support/ education for clinical teams (n = 3), modifying the treatment window (n = 1), the use of mobile alert technology (n = 1) and the use of a screening log/site monitoring (n = 2). Further analysis is ongoing. Conclusion More methodological research is needed to try and reduce the waste of resources associated with RCTs that fail to reach their desired re- cruitment targets. Embedded clinical trials of recruitment strategies are a possible way forward to help to quantify whether potential strategies suggested in the literature truly have an impact. Rigorous comparative methodological studies to evaluate recruit- ment interventions are lacking in this setting. Informed consent for trial participation was the most commonly identified recruitment bar- rier but specific methods to optimise this require further research. What are the barriers and facilitators to patient and carer recruitment to randomised controlled trials in palliative care? A systematic review with narrative synthesis Included in this review were any studies within ORRCA that reported on recruitment to RCTs in an UHC setting in patients >18 years. Abstracts, editorials, reports of mental health RCTs and studies of re- cruitment to non-RCTs were excluded. UHC was defined as the care received during an unpredictable admission to hospital at short no- tice because of clinical need. This includes pre-hospital care, inten- sive care (ICU) admissions and A&E attendances. Screening was performed by one author (CR) with duplicate screening of 10% of the database performed by a second author (KF). y Correspondence: Lesley Dunleavy Trials 2017, 18(Suppl 1):P182 Correspondence: Ceri Rowlands Correspondence: Ceri Rowlands Conducting trials with hard to recruit disabled populations: a systematic review of the methodological challenges reported in the literature Ceri Rowlands1, L. Rooshenhas1, Jonathan Rees2, Katherine Fairhurst1, Carrol Gamble3, Jane M Blazeby4 Ceri Rowlands1, L. Rooshenhas1, Jonathan Rees2, Katherine Fairhurst1, Carrol Gamble3, Jane M Blazeby4 y 1MRC conduct-II Hub for Trials Methodology Research, School of Social & Community Medicine, University of Bristol; 2Division of Surgery, Head & Neck, University Hospitals Bristol NHS Foundation Trust; 3MRC North West Hub for Trials Methodology Research, Institute of Translational Medicine, University of Liverpool; 4MRC conduct-II Hub for Trials Methodology Research, School of Social & Community Medicine, University of Bristol and Division of Surgery, Head & Neck, University Hospitals Bristol NHS Foundation Trust Peter Mulhall, Vivien Coates, Laurence Taggart, Toni McAloon Ulster University Peter Mulhall, Vivien Coates, Laurence Taggart, Toni McAloon Ulster University y Correspondence: Peter Mulhall Trials 2017, 18(Suppl 1):P184 Background Why so many RCTs fail to achieve their recruitment targets is an important area of clinical practice that is poorly understood. This is especially so in the field of palliative care as patients are often ‘hard to reach’. Palliative care patients have a diverse range of conditions, they are cared for in a wide variety of clinical settings and have unpredictable and complex needs. Ai All papers were categorised according to the recruitment study de- sign (randomised or non-randomised) and whether an intervention to optimise recruitment was evaluated. Additional categorisation ad- dressed whether the paper evaluated recruitment to a real clinical RCT (host RCT) or potential recruitment to a RCT that did not yet exist (a ‘hypothetical RCT’). To identify and synthesise knowledge about barriers and facilitators to recruitment to palliative care RCTs to develop recommendations to increase recruitment. Data extracted included i) perceived barriers to recruitment which formed the rationale for the study, ii) barriers to recruitment identi- fied as the result of the recruitment stud and iii) types of intervention evaluated. Methods A systematic review with narrative synthesis. Social marketing theory provided a theoretical framework for the review. Medline, Cinahl, pscyinfo and Embase databases (from Jan 1990 to early October 2016) were searched. Papers included: interventional and qualitative studies addressing recruitment, primary palliative care RCTs or re- ports containing narrative observations about the barriers, facilitators or strategies to increase recruitment to palliative care RCTs. Themes within the literature were developed using thematic analysis. Results Proposed Method The PRIMETIME SWAT will utilise a stepped wedge trial design. The de- cision aid will be in video format. Decisional conflict will be assessed using a validated decisional conflict scale in centres prior to and follow- ing implementation of the decision aid. All centres will receive the standard patient information sheets and be randomised to receiving the decision aid video at increasing intervals from when their centre Further research into demographic differences in adherence to randomised treatment and completeness of follow-up will provide a comprehensive Page 70 of 235 Trials 2017, 18(Suppl 1):200 Methods to optimise this have not been previously explored. The aim of this study is to summarise methods to optimise recruitment into trials in UHC settings to inform future research. Methods view of the influence of gender on participation throughout these cardio- vascular trials. In addition, qualitative research might provide insight as to why women are less likely to participate, and how this can be addressed to maximise the relevance of the results to both men and women. view of the influence of gender on participation throughout these cardio- vascular trials. In addition, qualitative research might provide insight as to why women are less likely to participate, and how this can be addressed to maximise the relevance of the results to both men and women. The ORRCA (Online Resource for Recruitment Research in Clinical Trials; www.orrca.org.uk) database includes studies of all designs, sys- tematically extracted from the literature, reporting on recruitment into RCTs and non-randomised clinical studies. P182 What are the barriers and facilitators to patient and carer recruitment to randomised controlled trials in palliative care? A systematic review with narrative synthesis Lesley Dunleavy, Catherine Walshe, Nancy Preston Lancaster University Correspondence: Lesley Dunleavy Trials 2017, 18(Suppl 1):P182 Background Approximately 15% of the world’s population have a disability. Many of these disabilities will have a profound effect of the person’s social, cognitive or mental functioning, often requiring high levels of costly health and social care support throughout the person’s life. As such, it is imperative that they receive treatments and services that are based upon a sound evidence base. As a case example, the evidence base for medical, health and social care interventions for those with Correspondence: Ceri Rowlands Conclusion A multifaceted retention strategy led to very low rates of missing clinical outcome data and participant attrition in a long-term cancer trial. Successful retention requires multiple strategies, including on- going staff training, regular newsletters and questionnaire reminders. These strategies are optimally included at the design stage and maintained throughout follow-up to reduce the potential for bias due to participant attrition and missing data. g y Conclusions g y Conclusions Conducting RCTs with people with disabilities, particularly intellectual disabilities, can present unique challenges that require creative solu- tions. To date researchers have not maximised the sharing of their ‘experience base’ regarding these challenges and solutions. As a re- sult, the conducting of RCTs and the development of robust evi- dence bases remains slow and the health inequalities of people with disabilities continues to grow. Implications for the dissemination of the ‘evidence base’ and ‘experience base’ are discussed. Online resource for recruitment research in clinical trials research (ORRCA) 1 1 2 3 Anna Kearney1, Nicola L Harman1, Naomi Bacon2, Anne Daykin3, Alison J. Heawood3, Athene Lane3, Jane Blazeby3, Mike Clarke4, Shaun Treweek5, Paula R. Williamson1, Carrol Gamble1, Peter Bower1, On behalf of the ORRCA group 1North West Hub for Trials Methodology Research/University of Liverpool; 2Clinical Trials Research Centre/University of Liverpool; 3Conduct-II Hub for Trials Methodology Research/University of Bristol; 4Centre for Public Health, Queen’s University of Belfast; 5Health Services Research Unit, University of Aberdeen) Results The primary outcome was ascertained for all participants and clinical outcome data for 99% (1639) men at a median of 10 years follow-up. Site monitoring and nurse training improved data collection. SDV identified training issues to improve data collection and CRFs, al- though staff time required was high. Background Recruitment to randomised controlled trials (RCTs) in unplanned hos- pital care (UHC) settings is more challenging than in the elective set- ting because there is less time and patients are often unwell. Trials 2017, 18(Suppl 1):200 Page 71 of 235 Page 71 of 235 a cognitive or developmental disability is very sparse. One of the rea- sons for this lack of robust evidence may be because the process of conducting RCTs with disabled or impaired populations is fraught with many methodological challenges. We need a better understand- ing of these methodological challenges if the evidence bases are to be developed. by data managers with feedback to centres. Proms were collected annually by postal questionnaires with a reminder letter to non- responders. Three interventions to reduce attrition were assessed: firstly, nurses commenced telephoning non-responders. A study pen was later included with reminders and a shortened questionnaire was sent to non-responders by recorded delivery. Questionnaire re- sponse rates were compared for a six month period before and after these interventions. There was a study website and annual partici- pant newsletters. 18 participants were also interviewed, including about follow-up, the transcriptions were analysed thematically. Results by data managers with feedback to centres. Proms were collected annually by postal questionnaires with a reminder letter to non- responders. Three interventions to reduce attrition were assessed: firstly, nurses commenced telephoning non-responders. A study pen was later included with reminders and a shortened questionnaire was sent to non-responders by recorded delivery. Questionnaire re- sponse rates were compared for a six month period before and after these interventions. There was a study website and annual partici- pant newsletters. 18 participants were also interviewed, including about follow-up, the transcriptions were analysed thematically. Results Background Participant attrition and missing data can introduce biases, yet there is limited evidence for successful retention strategies to maximise collection and analysis of clinical and patient-reported outcomes (PROMs). Correspondence: Anna Kearney Methods A systematic literature review was conducted of internationally pub- lished randomised controlled trials with people with intellectual dis- abilities from 2000 to 2015. From a total of 7795 search results, 34 RCTs with adults with ID were reviewed to ascertain which barriers, challenges and solutions the authors faced and reported. Quantita- tive data were extracted in the form of frequency of reporting and qualitative data were extracted in relation to the specific barriers faced by the authors. Questionnaire response rates over six years follow-up were over 85% for all proms and did not diminish over time. The reminder letter in- creased the response rate from 76.4% (1045/1367) to 86.8% (1187) and telephoning non-responders increased rates to 90.5% (1105/ 1221). The shorter version of the questionnaire had some impact (9/84 posted, 10.7%, overall 1033/1142, 90.5%). The study pen was ineffective (1026/1142, 89.8%). y Results In interviews, most men found the questionnaires acceptable and understood their purpose although they were less liked than the an- nual nurse appointment. Some men saw questionnaires becoming less relevant over time either because they felt they were cured or they reported the same information annually, however, they contin- ued to complete them. Participant newsletters were interesting and gave a sense of belonging to a group. The study website was infre- quently accessed, partly because it was assumed to contain no add- itional information. A number of themes arouse including: 1) that there was a lack of de- tail regarding how trialists made reasonable adjustments to enable consent to be obtained, 2) that there is a lack of validated outcome measures for people with communication or intellectual difficulties, 3) the importance of engaging with family members, carers and sup- port staff when recruiting, and retaining and 4) that sample sizes are regularly small and studies are often underpowered. p Objective To explore the methodological barriers which are hindering the de- velopment of the evidence base for treatments and interventions for people with cognitive or developmental disabilities, and to find pos- sible solutions to overcoming the barriers. As a case example, the lit- erature regarding RCTs for people with Intellectual Disabilities (ID) was used to highlight pertinent issues. Background The impact of a multifaceted retention strategy developed in a long- term cancer trial was investigated using mixed methods research. Methods With less than a third of UK trials and 40% of US cancer trials failing to achieve their recruitment targets, addressing recruitment chal- lenges has become an important methodological priority. However, while this focus has led to an increase in the quantity of published research, navigating this literature to identify recruitment strategies relevant to different types of trials has remained difficult. Aim 1643 men aged 50–69 years were randomised between 1999–2009 to three localised prostate cancer treatments with a median of 10 years follow-up (protect: ISCRTN 20141297). Prostate cancer mor- tality (primary outcome) was ascertained by an independent commit- tee following death certificate notification. Clinical secondary outcomes were collected annually in case report forms (CRFs) by re- search nurses in meetings with participants (or by telephone) and from medical records. Follow-up procedures included nurse training including study meetings every six months, standard operating pro- cedures, annual site monitoring visits, source data verification (SDV, total 161) on a representative sample of participants from each site The ORRCA project aims to provide an online searchable database, categorising recruitment research according to key themes. Data Sources An unrestricted search of Medline (Ovid), Scopus, Cochrane Database of Systematic Reviews (CDSR) and Cochrane Methodology Register, Science Citation Index Expanded (SCI-EXPANDED) and Social Sciences Trials 2017, 18(Suppl 1):200 Page 72 of 235 Page 72 of 235 Citation Index (SSCI) within the ISI Web of Science and ERIC in Janu- ary 2015. Database specific search strategies were developed based on previous work by Treweek et al. 2010. Inclusion Criteria addition to existing strategies to boost recruitment, the study man- agement team decided to embark upon the establishment of a trainee engagement exercise in EDNA similar to that seen in other clinical specialties. During summer 2016 the EDNA Study manage- ment team asked Principle Investigators at all EDNA sites to nomin- ate a site trainee for the opportunity to co-own EDNA locally. This trainee would typically be in the early stages of their career. In return for local co-ownership of the study, opportunities for authorship, and valuable insight into modern clinical research issues; the Co-PI’s are expected to assist practically and clinically at local level to identify ways in which they can positively enhance all study activities. Methods We will present an overview of the methods for developing the ORRCA database, a full analysis of recruitment themes following completion of the literature review and suggestions for how trial teams might use ORRCA to improve their recruitment strategies. Surgeons and research nurses with a range of recruiting experience were offered one of four workshops appropriate to their profession. The 1-day training focused on sharing skills and evidence-based knowledge to promote awareness and tackling of key recruitment challenges, and to enhance self-confidence in recruiting patients to RCTs. The workshops were broadly similar, comprising interactive presentations, group exercises and discussion based around recruit- ment difficulties and targeting the different needs of the different health professionals. Recruiters-levels of self-confidence in discussing trial recruitment with patients was assessed through 10 self- completed questions on a 0–10 rating scale before and up to three months after the workshop. Awareness of key recruitment challenges and perceived impact of training on practice were assessed through rating and Likert scales after training. Data were analysed using two- sample t-tests, and supplemented with findings from the content analysis of free text comments. P187 Networked for success: the establishment and maturation of a trainee research network within a UK based opthalmology study Claire Cochran1, Katie Banister1, Usha Chakravarthy2, Craig Ramsay1, Yan Ning Neo3, Emma Linton4, Rachel Healy5 1University of Abedeen; 2Queens University Belfast; 3Hillingdon Hospital; 4Manchester Royal Eye Hospital; 5Gloucestershire Royal Hospital Correspondence: Claire Cochran Trials 2017, 18(Suppl 1):P187 Correspondence: Claire Cochran Trials 2017, 18(Suppl 1):P187 Background With 71% of full text articles reviewed we have identified 87 rando- mised studies or systematic reviews evaluating recruitment strat- egies, 458 articles documenting the application of strategies and 1073 articles describing observations to inform future strategies. Randomised controlled trials (RCTs) are regarded as the most rigor- ous study design to evaluate healthcare interventions but recruit- ment to them can be challenging, particularly to trials involving surgery. Recruiter-related factors are often cited as key reasons for this yet few interventions have been developed to support them. The quintet Recruitment Intervention (QRI) has been embedded in RCTs to understand and address recruitment difficulties. A cross-trial synthesis of findings led to the identification of hidden emotional and intellectual challenges for recruiters. These findings have been translated into training material to improve the practice of front-line health professionals who recruit to surgical RCTs. The aim of this paper is to describe the training and evaluate its impact on recruiters. Maximising patient consent was the predominant theme amongst the 87 articles evaluating recruitment strategies with 30 (34%) asses- sing the delivery mode of recruitment information, 15 (17%) review- ing the format or content of patient information sheets and 14 (16%) evaluating other aspects of the consent process. Analysis of all recruitment themes shows that published literature fo- cuses on describing recruitment barriers and facilitators, exploring trial acceptability to patients and addressing cultural considerations. Few articles explore recruiter training (n = 31) the impact of trial reporting (n = 5) or blinding (n = 6). Results l Electronic searches identified over 40,000 articles of which 3979 re- quired full text review. The online database (www.orrca.org.uk) launched in August 2016 and is being updated periodically. We an- ticipate it will contain over 2000 articles once the review process is completed towards the end of 2016. Inbuilt search functionality al- lows results to be filtered using categories such as recruitment theme, level of evidence, health area, research methods, age and gender. Background While taking joint responsibility for proactive recruitment to EDNA we ex- pected all Co-PIs to promote and maintain high data completeness and quality as well as attend all key EDNA meetings. In autumn 2016 these Co-PI trainees were inducted to EDNA. This presentation will describe the process and experiences of establishing a Co-PI trainee network within a UK wide diagnostic accuracy study. Studies reporting or evaluating strategies, interventions or methods used to recruit patients to randomised control trials, early phase tri- als, qualitative interviews, focus groups, surveys, biobanks and cohort studies. Case reports of recruitment challenges or successes and studies exploring reasons for patient participation or refusal are also included. Articles were screened by title and abstract before a full text review by researchers from the Hub for Trials Methodology Research Recruit- ment Working Group (HTMR RWG) in the UK and the Health Re- search Board for Trials Methodology Research Network (HRB-TRMN) in Ireland. Eligible articles were categorised according to pre-defined recruitment themes and the following types of evidence: randomised evaluations of recruitment strategies; application of recruitment strat- egies with or without evaluation; observations to inform future re- cruitment strategies. Additional data were abstracted to enable search functionality. P188 An evaluation of the impact of quintet RCT recruitment training on the self-confidence and self-assessed recruitment practice of recruiters to surgical trials Nicola Mills1, Jane M Blazeby1, Daisy Gaunt1, Daisy Elliott1, Sam Husbands1, Peter Holding2, Bridget Young3, Catrin Tudor Smith3, Carrol Gamble3, Jenny L Donovan1 1University of Bristol; 2University of Oxford; 3University of Liverpool Correspondence: Nicola Mills Trials 2017, 18(Suppl 1):P188 Investigating recruitment imbalance in EXPONATE, a cluster randomised trial Wei Tan1, Oye Gureje2, Alan Montgomery1, Trish Hepburn1, Bibilola Oladeji2, Richardo Araya3, Lola Kola2 Background Cluster randomised trials that recruit individual participants after allo- cation of clusters may have increased risk of between-arm differ- ences in numbers and/or characteristics of recruited participants. This is particularly the case when recruiters are aware of cluster allocation and may introduce selection bias. The aim of this study was to inves- tigate recruitment imbalance in a cluster randomised trial. Methods Evolution of a recruitment strategy between secondary and primary care during the recruitment phase of the fourfold asthma (FAST) trial y , 1University of Nottingham; 2University of Ibadan; 3London School of Hygiene and Tropical Medicine Correspondence: Wei Tan Trials 2017, 18(Suppl 1):P190 y Results Establishment & maturation of Ophthalmology Trainee Research Networks within the UK Clinical Research Network (CRN) is currently being encouraged. Such trainee networks already exist in surgery, neurology & anaesthetics. Research studies supported by the trainee networks have consistently exceeded targets for recruitment in rec- ord time. EDNA (Early Detection of Neovascular Age Related Macular Degeneration) is a publicly funded UK wide prospective cohort diag- nostic study for the early detection of neovascular age-related macu- lar degeneration (AMD). Active within 24 sites UK wide, EDNA has struggled to recruit to target within the original timeframe. In 99 participants (67 surgeons, 32 nurses) attended a workshop. There was evidence of an increase in self-confidence scores following train- ing (range of mean scores before training 5.1 to 6.9 and after 6.9 to 8.2, with 10 being most confident; p-values all <0.05). The greatest increases in scores were in the areas in which they felt least confident in prior to training, obtaining authentic informed consent (nurses) and discussing trials with suspicious patients (surgeons). Im- mediately after training, participants felt well aware of the challenges of trial recruitment : Surgeons’ mean awareness score 8.8 (SD 1.2), Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 73 of 235 nurses’ 8.4 (SD 1.3) (with 10 being very aware). At follow-up, half of the surgeons reported that the training had made a lot of difference to their trial discussions with patients (19/38, 50%), with slightly fewer nurses reporting this (10/25, 40%). Only 2/36 surgeons and 2/ 25 nurses reported the training as making no difference. Several nurses who had received quintet RCT feedback previously felt the training was preaching to the converted, but valued the opportunity to reaffirm their practice. Attendees felt training had made them aware of their training needs and improved their recruiting skills, of- fering ideas for different approaches and explanations. strategy to meet targets. Throughout this process we worked closely with the NIHR HTA; this communication meant the funder was sup- portive of the 11 month extension in recruitment. Although the strategy was successful it was not without its draw- backs. Many of the primary care sites had little or no previous re- search experience, which meant they required significant support in comparison to the secondary care sites. Lessons learnt also will be presented. Method Recruitment trends showed that GP practices were only active for approx. 6 months after which the pool of potential participants was exhausted. It was agreed by the Trial Management Group (TMG) in February 2014 to open more RIS’ to replace inactive sites. In order to do this close communication with the CRNs was essential. Mixed- methods were used to train sites, including the Trial Manager train- ing sites face-to-face or via Skype, and, for larger CRN regions, a train-the-trainer approach was adopted in order to keep costs and time resources down. In addition, participant information was re- vised, the trial was promoted via various channels and the CI visited many CRN areas to publicise the trial in primary care. In January 2015 (three months before the projected end of recruitment) an 11 month extension of recruitment was agreed with the funder (NIHR HTA). In addition, following a sample size review at the request of the DMC, the target sample size was revised to between 1774 and 1850 due to a higher than expected primary outcome rate. Background The Fourfold Asthma (FAST) Trial planned to recruit 2300 participants over 22 months via 5 Research Networks (CRNs) across a mix of 8 secondary care hospitals, GP Participant Information Centres (PICs) and GP Research Initiative Sites (RIS); a strategy used in a previous asthma trial which involved doubling the dose of steroids [1]. EXPONATE was a two-arm parallel cluster randomised trial of a pri- mary care-based intervention for perinatal depression delivered by community midwives in Nigeria. The unit of allocation was maternal care clinic, stratified by local government area and with a recruitment ratio of 1:1. Fifteen clinics were allocated to the intervention arm, and 14 to usual care. All consecutive attendees were approached about the study, and those scoring over 12 on the Edinburgh Postna- tal Depression Scale (EPDS) were invited to participate. When recruit- ment ceased after 16 months, a total of 686 women had been recruited, 452 and 234 in intervention and control arms respectively. We calculated recruitment fraction for each clinic and compared these between arms, before and after exclusion of outlier clinics. We compared characteristics of clinics and women by treatment arm. We then considered whether the imbalance in recruitment could have been anticipated and avoided. g Recruitment to the FAST Trial opened in May 2013. After 6 months, recruitment was only 25% of the target due to a combination of de- lays with contracting and recruitment in secondary care being far lower than expected; it then became imperative to refocus our re- cruitment strategy on primary care. Reference 1 Harrison. T. W., J. Obourne et al. (2004). “Doubling the dose of inhaled corticosteroid to prevent asthma exacerbations: randomised controlled trial” Lancet 363 (9405): 271–275 P190 P190 Investigating recruitment imbalance in EXPONATE, a cluster randomised trial Wei Tan1, Oye Gureje2, Alan Montgomery1, Trish Hepburn1, Bibilola Oladeji2, Richardo Araya3, Lola Kola2 1University of Nottingham; 2University of Ibadan; 3London School of Hygiene and Tropical Medicine Correspondence: Wei Tan Trials 2017, 18(Suppl 1):P190 Result This was a major project management endeavour but on 31 January 2016 the trial completed recruitment. In total 20,695 patients were invited to participate in the trial with a total of 1922 participants re- cruited, on target of the revised timeline and recruitment objectives, 11 months behind the original schedule. By the time recruitment closed 196 RIS had been opened across 15 regions; 54% of these were opened in just 11 months. Overall 171 RIS successfully re- cruited. In total 19% of participants were recruited from secondary care sites, 18% from pics and 63% from RIS. Conclusion This trial demonstrates the importance of monitoring recruitment, and rapidly investigating and responding to poor and unexpected patterns in recruitment. Through close monitoring and clear report- ing we were quickly able to take action to adapt the recruitment Results The mean (SD, range) number of women screened per cluster was 334 (331, 56–1384) and 356 (318, 25–1061) in the intervention and control arms respectively. The mean (SD, range) number of women recruited per cluster was 30 (30, 2–99) and 19 (15, 2–52), resulting in recruitment fractions of 9.4% (4.6%, 3.3%-18.8%) and 5.8% (5.6%, 1.3%-24%) in the intervention and control arms respectively. The per- centage of women who screened positive on EPDS and were not subsequently recruited to the trial was small and similar between arms: 4.6% (n = 22) and 7.5% (n = 19) in intervention and control arms. A scatter plot of numbers screened versus recruited revealed two clinics both with recruitment fractions twice the mean of other clinics, and one clinic that screened more than five times the mean of other clinics. All three clinics were in the intervention arm. When these three clinics are removed from calculations, mean recruitment fraction and total number recruited in the intervention arm is 7.3% (3.0%, 3.3%-12.5%) and 205. When all 29 clinics are considered, we found no marked between-arm differences in participant characteris- tics (age/education/marital status/gestational age/clinical outcomes/ pregnancy outcomes) at baseline. l g Conclusion 1 Harrison. T. W., J. Obourne et al. (2004). “Doubling the dose of inhaled corticosteroid to prevent asthma exacerbations: randomised controlled trial” Lancet 363 (9405): 271–275 Quintet RCT recruitment training increased the self-confidence of sur- geons and nurses in discussing RCTs with potential participants and self-assessed recruitment practice. Further research will examine whether they translate into improvements in informed consent and RCT recruitment rates. Background g Open studies have a high risk of bias in participant reported out- comes. However, effects on recruitment, adherence and retention are less well defined. In eczema many patients are keen to trial non- pharmacological interventions, and hold high hopes that they are successful. Therefore, they may not want to be randomised to a study with a control arm. If randomised to control, they may actively seek additional therapy and/or withdraw from the study. Waiting list control designs randomise participants to intervention or control, but offer the control group the intervention at a later date. Rationale for this design include enhancing recruitment and adherence and redu- cing differential loss to follow-up. However, there is the potential for expectation bias as both groups are offered the intervention, and thus exaggeration of the treatment effect, especially with participant reported outcomes. There is also the risk of contamination between treatment groups when the intervention is available to independ- ently source outside of the trial. This abstract considers recruitment, contamination, retention and treatment effects in two studies which compared non-pharmacological interventions to usual care, in chil- dren with eczema. One trial (SWET) examined the use of water soft- eners for 3 months. The other (CLOTHES) examined wearing silk clothing for 6 months. The interventions in both trials, can be inde- pendently sourced. Here, we propose using multiple imputation (MI) methods, which have been adopted by the clinical trials community to handle missing data problems, to estimate CACEs and causal average treatment effects (ATE). We propose three MI approaches. The first one imputes the potential outcomes directly (MI-Y), assuming the non-compliance is ignorable given the variables in the imputation model, to estimate ATEs. The other two approaches impute the principal compliance classes, which are assumed to be independent of randomized treatment and to have known marginal distribution. The outcome models use to estimate CACEs include an interaction between compliance class and random- ized treatment, and it is the coefficient of this interaction which gives us the CACEs. There are two possible ways of handling this interaction term in the imputation step. The first is passive imputation (denoted MI-C). The second is to use rejection sampling for the proposed im- puted values. This is known as "substantive model compatible" (MI-C- SMC) imputation. We modify the imputation model probabilities to ob- tain conditional distribution with the desired marginals. Background Trish Hepburn, Lucy E. Bradshaw, Alan A. Montgomery, Eleanor F. Harrison, Eleanor J. Mitchell, Kim S. Thomas University of Nottingham Correspondence: Trish Hepburn Trials 2017, 18(Suppl 1):P191 Non-compliance with the treatment assigned is a common problem in randomised controlled trials. In order to obtain unbiased estimates of the causal effect of the treatment received, sophisticated statistical methods are necessary. One popular estimand is the complier average causal effects (CACE). Approaches to estimating the CACE include Bayesian and frequentist methods for principal stratification and instrumental-variables (IV) estimators. These approaches are rarely used in practice, probably because of their perceived complexity. Methods Background Using a full- factorial simulation, we investigate the finite sample properties of MI-Y, MI-C and MI-C-SMC methods for estimating ATEs/CACEs, in terms of coverage of the 95% confidence interval (CI) and bias. We consider set- tings where the outcome is (i) normally distributed or (ii) binary, and compare them to competing procedures (IV 2-stage least squares and full Bayesian modelling) in settings where the association between treatment received and outcome is confounded. We have two settings, low or high confounding, i.e. There exists a variable X which is ((i) weakly or (ii) strongly) associated with at the probability of complying and also ((i) weakly or (ii) strongly) associated with the outcome. We considered situations where the confounder X is (i) measured or (ii) un- measured. We also motivate and illustrate the methods in practice using a real clinical trial. p y Methods The number of participants recruited, contamination between the intervention and control groups and numbers lost to follow up in each group were examined for each trial. The treatment effects in each of the studies were also observed for objective and participant reported outcomes. p Results Both trials recruited to target (310 in 25 months (SWET), 300 in 18 months (CLOTHES). Contamination was low - No control partici- pants had water softeners installed during the study period (SWET). Six control participants reported wearing silk clothing during the study period (CLOTHES). Follow-up rates were high - 96% (SWET) and 94% (CLOTHES) - and similar between groups. In both trials, there was no evidence of an intervention effect in objective out- come measures (including the primary outcome). Similar differ- ences between intervention and control groups were observed in the mean Patient Orientated Eczema Measure score - a subjective measure of symptoms (SWET-2.0 (95% CI −3.5 to −0.5); CLOTHES −2.8 (95% CI −3.9 to −1.8)). g Results In settings where the confounder is low, and measured, all MI methods perform well, but when there is unmeasured confounding MI-Y results in low coverage rate (89%). For high confounding situations, MI-C performs well when the confounder is measured, but results in biased estimates with unmeasured confounding. MI-C-SMC seems to perform well in all settings considered, as do the Bayesian methods. MI-C and MI-C-SMC ap- pear to be more efficient than 2sls methods in small sample settings. Conclusions p g y Conclusion Conclusion Although nearly twice as many women were recruited in the inter- vention arm, there was no evidence of selection bias. It appears that Page 74 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 the imbalance in numbers was due mainly to unusual recruitment in three clinics, all of which were in the intervention arm. More exten- sive feasibility work may have identified these issues. Recruitment imbalance also potentially has implications for data analysis in trials. Multiple imputation methods for estimating causal treatment effects in trials with non-compliance Karla Diazordaz, James Carpenter LSHTM Correspondence: Karla Diazordaz Trials 2017, 18(Suppl 1):P192 Multiple imputation methods for estimating causal treatment effects in trials with non-compliance Karla Diazordaz, James Carpenter LSHTM Correspondence: Karla Diazordaz Trials 2017, 18(Suppl 1):P192 Discussion Multiple imputation methods can be used to impute the unobserved compliance classes and then used these to obtain compliance ad- justed causal effects of treatment. MI methods may have the advan- tage of dealing with missing data and non-compliance seamlessly, and increased efficiency. In both trials recruitment targets were met, contamination was low, and follow-up rates were high and comparable in control and inter- vention. It is not known if this was due to the relatively short dur- ation of the studies, a compliant participant population or offering the intervention to the usual care group. The consistent intervention effect on subjective but not objective outcomes be the result of expectation bias due to the design. However, there may be precon- ceived expectations of benefit regardless of access to the interven- tion. Research should be performed in this patient population to determine whether the waiting list control design impacts on willing- ness to participate, non-adherence and withdrawal from the study. This should take into account the availability of the intervention, dur- ation of the study, and participant’s prior opinions. Estimating intervention effect in cluster randomised controlled trials with non-compliance p Charles Opondo, Katherine Halliday, Stefan Witek-McManus, Elizabeth Allen London School of Hygiene & Tropical Medicine Correspondence: Charles Opondo Trials 2017, 18(Suppl 1):P195 p Charles Opondo, Katherine Halliday, Stefan Witek-McManus, Elizabeth Allen P196 Multiple imputation for missing covariates in clinical trials with interactions Through a simulation study we examine the influence on bias, accur- acy and coverage of this assumption using real underlying temporal trends from paediatric intensive care outcomes where this assump- tion does not hold. We also examine the performance of alternative models, including models in which time effects are ignored, common linear time effects, cluster specific linear time effects and models in- cluding random time effects. We consider both mixed models and generalised estimating equations, and use a full factorial design to examine a range of scenarios including small and large numbers of clusters and steps; small and large inter-cluster correlations; and small and large cluster sizes. Soeun Kim University of Texas Health Science Center Trials 2017, 18(Suppl 1):P196 Multiple imputation (Rubin, 1978, 1996) is a widely used method for handling incomplete data, and availability of software packages that implement multiple imputation has allowed various practical applications. In randomized clinical trials, the treatment assignment is usually completely observed but other predictors may be miss- ing, and it is important to appropriately account for incomplete data. In this paper we consider an interaction model in a clinical trial setting with a missing covariate and the treatment variable, where outcome can be binary or continuous. With the interaction model, the multivariate normal assumption is no longer satisfied, and the usual implementation of multiple imputation under multi- variate normal assumption can lead to biased results. We introduce a joint model approach for imputation of missing covariates in clin- ical trials for the linear or logistic regression setting, and evaluate various approximation approaches in a simulation study. We recom- mend specific approaches that incorporates interactions in the im- putation procedure. These approaches are applied in the analysis of clinical trial data on randomized blood products for severely injured patients. P193 P193 The assumption of common secular trends across clusters in a stepped-wedge cluster randomised trial: does it matter? Hemming1, Alan Girling1, Andrew Forbes2 1University of Birmingham; 2University of Monash Correspondence: Hemming Trials 2017, 18(Suppl 1):P193 Trials 2017, 18(Suppl 1):200 Page 75 of 235 estimators and their applicability in cluster randomised trials with non-compliance. The typical framework for modeling data from a stepped-wedge cluster randomised trial (SW-CRT), the Hussey and Hughes model, assumes a common (piecewise constant) secular trend across all clusters. In some situations this may be a tenable assumption. But, in other situations, for example with clusters set across very different settings (for example countries) this assumption might be implausible. estimators and their applicability in cluster randomised trials with non-compliance. Methods Blinded estimators for the variance parameters of a SW-CRT ana- lysed using the Hussey and Hughes model are developed, and demonstrated to be unbiased in the absence of treatment and period effects. Following this derivation, complete procedures for blinded and unblinded SSRE after any time period in a SW-CRT are detailed. Explicitly, we address the case where a limited num- ber of clusters for recruitment have been set, but increased re- cruitment within a cluster is feasible. The performance of both procedures is then examined and contrasted through a simula- tion study, using a recently completed SW-CRT as motivation. A simple adjustment to more accurately control the type-I error rate is also proposed. The primary analysis was an ITT analysis, and additionally an AT and a PP analysis were carried out. We will use causal modelling to ex- plore other possible estimators of the effect of the intervention in the presence of non-compliance. The two possible estimators we consider are: (i) the complier average causal effect (CACE) which esti- mates the treatment effect among compliers and involves identifying groups of individuals with respect to their group assignment and compliance, and comparing the outcome across groups in those who would have complied with the intervention they were rando- mised to, and (ii) the average treatment effect in the treated (ATT) which estimates the average effect of treatment on those subjects who ultimately received the treatment. Background Like any trial, the ability to accurately estimate the required sample size of a stepped-wedge (SW) cluster randomised trial (CRT) depends upon the precise specification of several nuisance parameters. In practice, providing accurate estimates for these nuisance parameters may be difficult, and thus there is a risk that many SW- CRTs may be conducted with undesirable operating characteristics. Trials could be over-powered; leading to increased cost, or under-powered; increas- ing the likelihood of a false negative result. We address this issue here for cross-sectional SW- CRTs by proposing methods for blinded and unblinded sample size re-estimation (SSRE). Methods Here we explore alternative estimates of intervention effects in a cluster randomised trial in the presence of non-compliance. Background In a randomised controlled trial the effect of an intervention can be estimated by calculating the difference in outcomes between the groups. The Standard Approach uses an intention to treat analysis (ITT) where all participants are included in the group to which they were assigned, whether or not they received their allocated interven- tion. An ITT analysis reduces post-randomisation selection bias, and estimates the intervention effect under routine application, its effect- iveness. However, an ITT analysis does not estimate efficacy, the ef- fect of an intervention under ideal circumstances. Two approaches are commonly used to estimate efficacy when there is non- compliance (or non-adherence): per-protocol (PP) analysis, in which only individuals who received the intervention they were randomised to are included in the analysis, and an As-treated (AT) analysis in which individuals are analysed according to the intervention they re- ceived. Both approaches may lead to biased estimates of the treat- ment effect since randomisation is broken. Materials and methods We use data from a trial of learner treatment kits (LTKs), comprising malaria rapid diagnostic test kits (RDTs) and artemisinin-based com- bination therapy drugs (ACTS) administered by teachers, on school attendance by Malawian children. Observations were clustered within schools. p Results For our example scenario, if the two key variance parameters were under-estimated by 50%, the SSRE procedures were able to increase power over the conventional SW-CRT design by up to 26%. The per- formance of the SSRE procedures is demonstrated to be robust to the choice of re-estimation time point, whilst the proposed adjust- ment to account for the observed type-I error rate inflation is often able to control to approximately the nominal level. y Results, conclusions and future research The ITT and PP analyses provided no evidence of an effect of the intervention on school attendance. However, an AT analysis sug- gested that that children who actually used the LTKs were less likely to be absent from school. We will present the CACE and ATT esti- mates and explore the assumptions underpinning these effect Page 76 of 235 Trials 2017, 18(Suppl 1):200 Page 76 of 235 p Results The method is randomisation-based and uses only the randomised treatment group, observed event times and treatment history in order to estimate a causal treatment effect. The treatment effect, Psi, is estimated by balancing counter-factual event times (i.e. The time that would be observed if no treatment were received) between treatment groups. A g-estimation procedure is used to find the value of Psi such that a test statistic Z(Psi) = 0. Recensoring must be per- formed as censoring becomes informative on the counter-factual time scale. When baseline prevalence was low, 30% increase vs no change yielded high frequency (>99%) of statistically significant results using all methods. When baseline prevalence was high for comparisons of 30% increase or decrease vs no change, maxpb (45-69%) yielded more statistically significant results than bladj (40-63%) regardless of statistical test, with the modeling approach (80-85%) having higher frequency than chi-squared test (49-69%), Gray’s test (45-58%), Wil- coxon rank-sum test (45-66%), and t-test (40-57%). In varying the baseline prevalence (5% vs 10% vs 30% vs 50%) but maintaining the post-baseline scores linearly increasing from 55% to 80%, rate of maxpb >0 was 93% in all simulations compared to 93%, 91%, 83%, and 72% for bladj. An R package titled “Rpsftm” has been developed and is freely avail- able for download on the CRAN website. This package implements the method as described above. The main features are: Building dir- ectly on the established “Survival” package to calculate the z-statistic, and the uniroot() function to solve the estimating equation; A famil- iar formula syntax: Surv(time, status) ~ rand(arm, rx) + covariate, to represent the censored failure time, the rand(arm,rx) representing the randomised treatment arm and observed proportion of time spent on the randomised treatment, plus any adjusting covariates; Implementation of the re-censoring method, when a theoretical Conclusions censoring time is known; Auto-detection of perfect compliance in a treatment arm, with corresponding adjustment to the re-censoring; Routine output in terms of summary, and print methods; Estimates, and confidence intervals of the causal parameter; Sensitivity analysis to the model assumption of a common treatment effect, allowing the user to vary the magnitude of effect of treatment between pa- tients; Diagnostic plot to help resolve potential numerical non- convergence issues. The considered SSRE procedures can bring substantial gains in power when the underlying variance parameters are mis-specified. Though there are practical issues to consider, such as the requirement for data to be collected and stored efficiently for analysis, the proce- dures performance means researchers should consider incorporating SSRE in to future SW- CRTs when there is uncertainty over the values of the variance parameters. We provide worked examples to illustrate the use of the package and the methodology. 198 Investigating multiple imputation in cluster randomized trials Brittney Bailey, Rebecca R. Andridge, Abigail B. Shoben The Ohio State University Correspondence: Brittney Bailey Trials 2017, 18(Suppl 1):198 P200 Statistical analysis strategies for PRO-CTCAE data in oncology clinical trials: a simulation study Amylou Dueck1, Jeff A. Sloan1, Jared Foster1, Jennifer Le-Rademacher1, Gita Thanarajasingam1, Ethan Basch2 1Mayo Clinic; 2University of North Carolina Correspondence: Amylou Dueck Trials 2017, 18(Suppl 1):P200 Missing data in cluster randomized trials are often handled with parametric multiple imputation (MI), assuming multivariate normality and using random effects to incorporate clustering. Since data do not always satisfy this assumption, a nonparametric approach to MI is desirable. Predictive mean matching (PMM) is a nonparametric ap- proach where missing outcomes are imputed with observed out- comes in the data from donors that are similar to the missing cases. It is not clear how best to extend PMM to multilevel data. Two possi- bilities are to ignore clustering in the imputation model or to include fixed effects for clusters. In parametric MI, ignoring clustering in the imputation model leads to underestimation of the MI variance, while including fixed effects for clusters tends to overestimate the variance. A mixed effects imputation model can be used as the basis for matching, but this is computationally intensive and increases reliance on distributional assumptions. To simplify computation and reduce bias in the estimated variance, we investigate a weighted PMM ap- proach that incorporates both the fixed effects imputation model and the imputation model that ignores clustering. Background g Adverse events (AEs) in oncology trials have historically been re- ported by clinicians using National Cancer Institute’s (NCI’S) Common Terminology Criteria for Adverse Events (CTCAE). Traditional statistical analysis of AE data has primarily involved summary measures (e.g., maximum grade post-baseline) even though a variety of other ap- proaches exist including cumulative incidence estimation in the pres- ence of competing risks. AE data reported directly by patients using NCI’s Patient-Reported Outcomes version of the CTCAE (PRO-CTCAE) are similar in ordinal format to CTCAE data. PRO-CTCAE data may introduce statistical challenges due to high baseline symptom rates and non-ignorable missing data. h d g Methods Baseline and six post-baseline scores on a five-level ordinal scale (0 = none, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe) were simu- lated for 100 patients/arm in 1,000 two-arm trials using a multivariate ordinal distribution for combinations of baseline prevalence rates (5% vs 50%) and change over time (no change vs 30% increase vs 30% decrease). Between-arm comparisons included t-tests and Wil- coxon rank-sum tests of the maximum score post-baseline (maxpb) and a novel baseline adjustment score (bladj); chi-squared tests of the rate of maxpb or bladj >0; general linear mixed models (GLMM) of longitudinal scores; and Gray’s tests of the cumulative incidence of score >0 with and without adjustment for baseline. The GLMM mod- eled all scores allowing for random intercepts and slopes with statis- tical significance based on the arm-by-cycle interaction effect (unstructured covariance was used to account for within-patient cor- relation of scores over time). Bladj was computed for each patient as maxpb if maxpb was worse than the baseline score, or as zero if maxpb is the same or better than baseline. P199 R package to implement rank preserving structural failure time models Simon Bond1, Annabel Allison2 1Cambridge Clinical Trials Unit; 2Medical Research Council Biostatistics Unit Correspondence: Simon Bond Trials 2017, 18(Suppl 1):P199 R package to implement rank preserving structural failure time models d1 b l ll 2 1Cambridge Clinical Trials Unit; 2Medical Research Council Correspondence: Simon Bond Trials 2017, 18(Suppl 1):P199 The rank preserving structural failure time model (RPSFTM) is a method used to adjust for treatment switching in trials with survival outcomes. Treatment switching occurs when patients switch from their randomised arm to the other treatment during the study. The RPSFTM is due to Robins and Tsiatis (1991) and has been developed by White et al. (1997, 1999). P202 Structured statistical analysis plans for improved clarity of intended analysis The overall objective of this research is to transform the design and analysis of PU trials by making better use of all data collected from repeated measures of skin changes. The aim of this project is to re- view currently used PU research designs, focussing on outcome mea- surements and their analysis. Colin Everett University of Leeds Trials 2017, 18(Suppl 1):P202 Aim and objectives Aim and objectives Background Pressure ulcers (PUs) are defined as a “localized injury to the skin and/or underlying tissue usually over a bony prominence, as a result of pressure, or pressure in combination with shear” [1]. PUS are pain- ful and debilitating for patients, represent a significant cost to the NHS and are a key quality indicator for the Department of Health. Motivation Diary cards and questionnaires are frequently used to collect data in clinical trials. However, data collection can be burdensome and com- pletion may decline over time. Despite the often large volume of data, this may be reduced to summary measures for analyses. The CLOTHES trial randomised 300 children with moderate to severe eczema to standard care plus silk clothing for 6 months or standard care alone. A nested qualitative evaluation was included: 32 parents participated in focus groups or telephone interviews. Patient-reported symptoms were assessed weekly using online or paper questionnaires for 6 months and during scheduled clinic visits at baseline, 2, 4 and 6 months using the Patient Orientated Eczema Measure (POEM). The mean of participants’ weekly POEM scores was a secondary outcome measure. We explored whether the results and conclusions would have changed if only data collected at 2, 4 and 6 months were used in the analyses. PUs are categorised using an ordered categorical scale based on the appearance of skin. PU classification requires clinical judgement and misclassification can occur when undertaken by non-specialist staff, particularly for early skin changes. For PU research, investigators assess multiple skin sites for each patient at multiple time points, recording whether skin is healthy or not, and the PU classification where applic- able. During analysis these repeated measurements are often aggre- gated into a single outcome measure, defined as development of at least 1 category 2 PU; therefore many observations are not directly ana- lysed. Consequently large sample sizes are required for trials of PU pre- vention and intervention strategies. PU trials are further complicated by missing data due to administrative or patient factors and misclassifica- tion due to the judgement required for categorisation of skin changes. Methods that use all observations, including repeated assessments at multiple skin sites, such as multi-state models, have the potential to address these problems. It is important to understand how trials are currently designed and analysed in this context in order to develop recommendations for optimal designs. Conclusion Templates for the derivation of variables are proposed for how to make clear how in mathematical and programming terms an end- point is to be derived, and the analysis is to be performed. Endpoint templates include worked examples and references. Analysis model templates include types of variables (binary, categorical, continuous), expected ranges for continuous variables, or meanings of values for categorical variables, the type of model to fit, whether effects are fixed or random. Procedures for checking assumptions are listed. Strategies for dealing with potential analysis pitfalls are included, in- cluding simplifying models in the case of non-convergence, non- positive variance component for random effects and violation of modelling assumptions. Currently used methods for design and analysis of PU trials are ineffi- cient and ignore many complexities that introduce variation into the results. More efficient designs and analysis methods may reduce the numbers of patients required and be less subject to bias. Methods may generalise to other situations in which a disease process can be represented by correlated longitudinal categorical data. A review of design and analysis methods for pressure ulcer research 1 2 2 g g Lucy Bradshaw1, Trish Hepburn2, Alan A. Montgomery2, Eleanor F. Harrison2, Eleanor J. Mitchell2, Laura Howells3, Kim S. Thomas3 1University of Nottingham; 2Nottingham Clinical Trials Unit, University of Nottingham; 3Centre of Evidence Based Dermatology, University of Nottingham Isabelle Smith1, Linda Sharples2, Jane Nixon2 1University of Leeds; 2University of Leeds, Leeds Institute of Clinical Trials Research Isabelle Smith1, Linda Sharples2, Jane Nixon2 1University of Leeds; 2University of Leeds, Leeds Institute of Clinical Trials Research Correspondence: Lucy Bradshaw Trials 2017, 18(Suppl 1):P204 Correspondence: Isabelle Smith Trials 2017, 18(Suppl 1):P203 Correspondence: Isabelle Smith Trials 2017, 18(Suppl 1):P203 Correspondence: Lucy Bradshaw Trials 2017, 18(Suppl 1):P204 Reference [1] NPUAP/EPUAP/PPPIA, Prevention and Treatment of Pressure Ulcers: Clinical Practice Guideline. 2014, Cambridge Media: Osborne Park, Western Australia P204 Frequency of data collection in a randomised controlled trial for long term eczema management in children Lucy Bradshaw1, Trish Hepburn2, Alan A. Montgomery2, Eleanor F. Harrison2, Eleanor J. Mitchell2, Laura Howells3, Kim S. Thomas3 1University of Nottingham; 2Nottingham Clinical Trials Unit, University of Nottingham; 3Centre of Evidence Based Dermatology, University of Nottingham Background We will present a review of methods used in PU trials and observa- tional cohort studies including how data are collected and analysed to illustrate the extent of the problem. Key manuscripts were identi- fied through systematic reviews of published PU research. From these a pearl-growing strategy was adopted to identify other trials and large cohort studies. Finally experts in the field were approached to ensure major studies were not overlooked. Data extraction was pre-specified to include study design, frequency of assessments, as- sessor characteristics, PU definition, primary outcome including deriv- ation, analysis methods including relevant assumptions and accommodation of complications such as censoring or missing data and effect size to quantify differences in study conclusions based on analysis methods used. Summaries of methods used in PU research will be presented and critiqued for quality and information provided from a statistical perspective. g Prior to any formal analysis of data in a clinical trial, a Statistical Ana- lysis Plan (SAP) must be written, reviewed and approved. This docu- ment describes how the data analysis will be performed, lists the endpoints of interests, and defines how they will be derived. Docu- ments and descriptions are typically written in prose, meaning that clarity of the analysis intended by the statistician, and that under- stood by a reviewer depend on the writing style of the person who drafts the SAP. Assumptions made by a reviewer about the descrip- tions of the analysis or endpoint derivation may differ from that intended, but not specified. It is important to avoid instances of a derivation needing to be overruled at analysis time due to disagree- ment on what is meant by sentences both thought had clear and ob- vious meanings, or where alternative approaches are not defined upfront. Conclusions Existing statistical methods for clinician-reported AE data and PRO data are candidate methodologies for the statistical analysis of PRO- CTCAE data. The general linear mixed model approach appears to provide the most power for between-arm comparisons among the tested approaches. The novel baseline adjustment method appears to account for some but not all pre-existing symptoms. Page 77 of 235 Trials 2017, 18(Suppl 1):200 analyses. Methods For the trial analysis, the mean of participants’ Weekly scores was analysed using a linear model weighted according to the number of weekly questionnaires completed. This analysis was repeated using the scores from week 8, 16 and 24 questionnaires only and the scores collected in clinic at the same timepoints (2, 4 and 6 months). Results of the nested qualitative study were reviewed to determine whether completion of the weekly questionnaires had been identi- fied as a theme. Trials 2017, 18(Suppl 1):200 Page 78 of 235 Page 78 of 235 P207 Prediction model for developmental outcome at 2 years of age for babies born very preterm 1 2 Lesley Anne Carter, Chris Roberts 1Centre for Biostatistics, School of Health Sciences, University of Manchester, UK Correspondence: Lesley-Anne Carter Trials 2017, 18(Suppl 1):P208 Karan Vadher1, Brad Manktelow2 1Oxford Clinical Trials Research Unit; 2University of Leicester Correspondence: Karan Vadher Trials 2017, 18(Suppl 1):P207 Karan Vadher1, Brad Manktelow2 1Oxford Clinical Trials Research Unit; 2University of Leicester Correspondence: Karan Vadher Trials 2017, 18(Suppl 1):P207 To investigate treatment efficacy in randomised control trials, re- peated observations are taken on a cohort of participants and the change in response following treatment is assessed. The commit- ment required of participants to stay involved in the study, however, makes this design open to both recruitment issues and attrition. A cross-sectional design may be used in conjunction with the cohort design to protect against these problems, recruiting additional partic- ipants who only contribute once to the study, resulting in a ‘mixed’ design. P208 This abstract is not included here as it has already been published. Methods The three nominal outcomes were modelled using multinomial logis- tic regression. Missingness was investigated by complete case ana- lysis, Inverse Probability Weighting (IPW) and multiple imputation. To allow for comparison between the three methods, the same covari- ates were adjusted for in the final multinomial logistic regression out- come models. Probabilities, odds ratios, log odds and standard errors were used to compare the three different approaches. Results Several parents felt that questionnaire completion had been useful in prompting more regular use of usual treatments whilst others felt they were repeating themselves each week and this may not be helpful (Qualitative study). Results missing outcome response. An investigation into the missingness and its assumptions were also investigated as almost half the dataset had a missing outcome which this abstract will concentrate on. Subjects The difference between the two groups using all of the question- naire data in the participant mean of the weekly scores was −2.8 (95% CI −3.9 to −1.8) in favour of the intervention group, n = 147 control, n = 145 intervention). Repeating this analysis using data only from the questionnaires at weeks 8, 16 and 24 showed a dif- ference of −2.6 (95% CI −3.9 to −1.3), n = 134, n = 137 respectively, and using clinic data at 2, 4 and 6 months was −2.3 (95% CI −3.5 to −1.1), n = 141, n = 142 respectively. j The dataset is a subset of TNS and contained 2028 participants, which included babies born very preterm admitted to neonatal care between 2009 and 2010. Methods Conclusion h l The results were very similar for all three analyses and conclusions would not have changed if less data had been collected. Therefore, weekly data collection may not be needed when summary measures are used to compare groups. More frequent data collection may be useful in other circumstances for example if there is a need to iden- tify sudden flares. were use Results Missing completely at random was disregarded as the IPW missing- ness model highlighted that the deprivation area, mother’s age and mother’s ethnicity had an effect on whether the PARCA-R survey was completed. For instance, mothers aged 34+ were 3.4 times more likely to respond than mothers younger than 23 years, when control- ling for deprivation area and mother’s ethnicity. The imputation model also produced strong evidence of covariates predicting non- responders. Once the investigation of missingness had been con- ducted the same multinomial logistic regression was produced. The optimal model predicting developmental outcome contained gesta- tional age, sex of the baby and CRIB II score as well as a quadratic term for gestational age. Unsurprisingly, complete case analysis yielded very different results to the models that used IPW and mul- tiple imputation. Odds ratios and probabilities of each outcome were broadly similar with multiple imputation yielding smaller standard er- rors of the odds ratios in the multinomial logistic regression. Conclusions The process of data collection should also be considered. Frequency of data collection needs to be balanced against the potential for data collection itself to act as an intervention and influence behaviour in pragmatic trials. Further work is needed to determine the optimum frequency of data collection to capture both the chronic relapsing nature of eczema and changes in condition due to an intervention. This is planned as part of the Harmonising Outcome Measures for Eczema long term control outcome domain. Beyond maximum grade: a novel longitudinal toxicity over time (TOXT) adverse event analysis for targeted therapy trials in lymphoma IPW and multiple imputation both vary methodologically and there are a number of limitations with both methods, however, it is proven to produce very similar results and can be effective to use the data available to predict the missing outcome. It is concluded multiple im- putation is more flexible than IPW when modelling missing outcomes. y Gita Thanarajasingam, Pamela J. Atherton, Levi Pederson, Paul J. Novotny, Thomas M. Habermann, Jeff A. Sloan, Axel Grothey, Shaji Kumar, Gita Thanarajasingam, Pamela J. Atherton, Levi Pederson, Paul J. Novotny, Thomas M. Habermann, Jeff A. Sloan, Axel Grothey, Shaji Kumar, Thomas E Witzig Amylou C Dueck Gita Thanarajasingam, Pamela J. Atherton, Levi Pederson, Paul J. Novotny, Thomas M. Habermann, Jeff A. Sloan, Axel Grothey, Shaji Kumar, Thomas E. Witzig, Amylou C. Dueck Mayo Clinic Correspondence: Gita Thanarajasingam Trials 2017, 18(Suppl 1):P206 Background Well-designed clinical trials are the gold standard for evaluating healthcare interventions. It is essential for the trial methodology to be pre-specified in the protocol in order to avoid issues such as se- lective reporting of outcome measures. However, little attention has been paid to whether trialists are adequately pre-specifying the method of analysis for their primary outcome in the trial protocol, or what impact inadequate pre-specification might have on trial results. Methods We re-analysed primary clinical outcome data from the TRIGGER trial to examine the impact that differing analytical approaches could have on the trial outcome. We varied several aspects of the analysis: (a) the patient population included in the analysis; (b) the analysis model used; (c) the set of covariates included in the model; and (d) methods of handling missing data. We then conducted a review of published trial protocols to assess how well the statistical analysis ap- proach for the primary outcome was pre-specified. Results Scoring systems based on multiple components are often used in in- tensive care trials to characterise disease severity. Missing data in the overall score can be substantial due to the number of contributing components, and the problem is exacerbated if data are collected at multiple time points. A complete case analysis is prone to selection bias, and for component scores is highly inefficient. It is preferable to include individuals with incomplete data in the analysis by imputing their missing values. The imputation process should be based on plausible assumptions about the causes of the missing data and re- flect the longitudinal trajectory for each patient. We demonstrate how this is facilitated by adopting a Bayesian framework, using data from the Levosimendan for the Prevention of Acute Organ Dysfunc- tion in Sepsis (LEOPARDs) trial. Our re-analysis of TRIGGER found that the choice of statistical ana- lysis approach had a large impact on both the estimated treatment effect and p-value. Across the different analytical approaches, the es- timated odds ratio ranged from 0.40 (95% CI 0.17 to 0.91; p-value 0.03) to 1.09 (95% CI 0.56 to 2.10; p-value 0.80). It was possible to ob- tain both significant and non-significant results by varying either the patient population included, the set of covariates used in the analysis model or the method of handling missing data. Background Children who are born preterm are known to be at increased risk of a range of developmental problems. The Preterm and After (PANDA) study aims to provide information about the long term outcome of children born very preterm (less than 31 gestational weeks) admitted for acute neonatal care in the east of England. Within PANDA, the PARCA-R questionnaire is completed by parents in order to measure cognitive and language development at 2 years of age. These data are added to obstetric and neonatal data collected by The Neonatal Survey (TNS), an ongoing study of neonatal intensive care activity in the same geographic area. It includes clinical information on the child and their neonatal care as well as the developmental outcome of the child; alive with no developmental delay (DD), alive with DD and death before 2 years of age. The EQUIP cluster randomised control trial was designed to evaluate the efficacy of a training intervention for community mental health teams (CMHT), employing such a mixed design. The ‘cluster cohort’ sample provided baseline data on service users prior to randomisa- tion and follow up data at six months following baseline assessment, via face-to-face interviews. The ‘cluster cross-sectional’ sample in- volved all service users under the care of the cmhts not in the cohort sample to be sent a postal questionnaire six months after randomisa- tion. Comparison of the results of the two designs would allow exter- nal validity of the intervention to be investigated. The combined sample was intended to increase power to detect the intervention The aim of this project was to investigate developmental outcome at 2 years of age for children born very preterm. The PARCA-R survey completed by the parent was used and failure to do so led to a Page 79 of 235 Trials 2017, 18(Suppl 1):200 component nature of SOFA score. We recommend that this approach be considered more widely for informative missingness in longitu- dinal data. effect should retention rates not meet expectations. As data were only collected in the cross-sectional design at six months post ran- domisation, baseline data were missing in this sample, posing a problem for the combined analysis. Background The review of pub- lished protocols is ongoing, however preliminary results indicate that most trial protocols do not adequately pre-specify their analysis ap- proach for the primary outcome. p ( ) In the LEOPARDs trial, the primary outcome was the mean daily total Sequential Organ Failure Assessment (SOFA) score while in ICU. The total SOFA score is the sum of five components and some of these components are determined by multiple variables. Although 6% of scores were missing across components, this led to 17% of the total SOFA scores having a missing component. There was a clinical ex- pectation that measurements may not be taken if there was no change, or if the scores were normal. The assumption of a lack of change is in line with the last observation carried forward (LOCF) ap- proach. This method gives a single imputation, so does not take ac- count of the uncertainty due to the missing data, leading to over- precise estimates. Standard multiple imputation (MI) overcomes this problem, but typically assumes that the probability of a missing score does not depend on the score itself, after conditioning on observed data. This was implausible in the LEOPARDs trial because the de- cision on whether to take a measurement is informed by clinical judgement about its likely value, and so the missingness is ‘informative’. Background Two methods for overcoming this issue were considered: using baseline as response, where a joint model of baseline and response is fitted with all observed data, and the missing indicator method in which an indicator variable for the missing data is include in the model as a covariate. P210 Do trialists adequately pre-specify their statistical analysis approach? A review and re-analysis Lauren Greenberg1, Vipul Jairath2, Brennan C. Kahan1 1Queen Mary University of London; 2Department of Medicine, Epidemiology and Biostatistics, Western University Correspondence: Lauren Greenberg Trials 2017, 18(Suppl 1):P210 These two methods will be presented with a discussion of the chal- lenges encountered in the application of each to the cluster random- isation trial design of the EQUIP study. P209 Bayesian methods for informative missingness in longitudinal intensive care data Shalini Santhakumaran1, Alexina J. Mason2, Anthony C. Gordon1, Deborah Ashby1 1Imperial College London; 2London School of Hygiene and Tropical Medicine Correspondence: Shalini Santhakumaran Trials 2017, 18(Suppl 1):P209 p Conclusions The statistical analysis approach can greatly influence trial results. It is essential that the planned analytical method is pre-specified in the trial protocol in order to avoid selective analysis reporting. Background Rare cancers have complications in analysis due to limited recruit- ment, meaning the event of interest does not occur enough to ac- curately discern which treatment arm is better. Due to unclear knowledge of the best way of treating patients suffering from rare diseases, a disproportionately high number of deaths occur. p p y g We propose a method of analysing clinical trials for rare diseases when comparing two treatments already in use, which can give a good indication of which treatment arm is better, that does not re- quire sample sizes of the magnitude of conventionally-powered trials. Treatment effect modification on the additive scale is generally per- ceived to be of primary interest for explaining differential treatment response because absolute treatment effects do not depend on baseline risks which may differ between patient subgroups. For ex- ample, if age is, regardless of treatment, associated with the outcome of interest, the baseline risk will vary across age subgroups. If the relative treatment effect, say the relative risk, is found to be similar across the age subgroups, this implies variation in the absolute treat- ment effect across the subgroups. Specifically, this implies that pa- tients in the subgroup(s) with a lower baseline risk have a smaller absolute treatment compared to patients in the subgroup(s) with a higher baseline risk. Since the absolute treatment effect conveys the absolute magnitude of the treatment response, this variation will likely be of interest. Merkel cell carcinoma (MCC), a skin cancer which recorded 1515 cases in the UK in a 10-year period, is one such rare disease. Cur- rently, the main treatment method for MCC is prioritising surgery, then administering radiotherapy to eradicate remaining cancer cells. It was postulated whether reversing this treatment order would be more efficacious. This question is analogous to comparing two treat- ments in use, because patients would receive access to both radio- therapy and surgery regardless of the outcome, and there are arguably no losers. g py g It was postulated whether reversing this treatment order would be more efficacious. This question is analogous to comparing two treat- ments in use, because patients would receive access to both radio- therapy and surgery regardless of the outcome, and there are arguably no losers. P211 Effective graphical analyses of adverse events in DMC reports Allison Furey, Robin Bechhofer University of Wisconsin-Madison Correspondence: Allison Furey Trials 2017, 18(Suppl 1):P211 We simulated the survival time of patients in a two arm trial with the treatment arm as the sole pre- dictor and analysed the data using the Cox hazard model. over time and to review a large amount of information in a short amount of time. We employ various presentation styles, including graphics produced in R (bar charts, stacked bars, Kaplan-Meier plots, forest plots), and tables and listings produced in SAS; latex is used for layout and report production. In simulations of 10,000, various sample sizes and true hrs of the treatment arms were modelled, with the power to conclude efficacy using the conventional null hypothesis, and the re-definition, compared. A major challenge in AE reporting is to separate signal from noise, drawing attention to important issues while not sacrificing complete- ness of reporting. Our standard suite of AE analyses employs a “Drill down” Approach, beginning with an overall summary of Aes falling into selected categories (serious, fatal, related to treatment, leading to treatment discontinuation, etc.), graphical summaries by SOC and of most common preferred terms, followed by incidence tables of preferred terms within SOC and listings of Aes of concern. Our stand- ard displays provide visual information regarding severity as well as incidence, and highlight treatment comparisons between groups. A novel approach to analysis of clinical trials for rare cancers assuming symmetry 2 A novel approach to analysis of clinical trials f assuming symmetry Emma Wang1, Peter D. Sasieni1, Bernard V. North2 1Queen Mary University, London; 2Exploristics Ltd Correspondence: Emma Wang Trials 2017, 18(Suppl 1):P212 g y y Emma Wang1, Peter D. Sasieni1, Bernard V. North2 1Queen Mary University, London; 2Exploristics Ltd g y y Emma Wang1, Peter D. Sasieni1, Bernard V. North2 1Queen Mary University, London; 2Exploristics Ltd Correspondence: Emma Wang Trials 2017, 18(Suppl 1):P212 Predictive markers are variables that identify patient subgroups with differential response to treatment and can be useful in guiding treat- ment decisions. Practically, predictive markers are those found to moderate the relationship between treatment and an outcome. How- ever, the presence of treatment effect modification is dependent upon measurement scale of the outcome. If the absolute effect of treatment varies across patient subgroups, treatment effect modifica- tion is present on the additive scale. Alternatively, if the relative ef- fect of treatment varies across patient subgroups, treatment effect modification is present on the multiplicative scale. P211 Effective graphical analyses of adverse events in DMC reports Allison Furey, Robin Bechhofer University of Wisconsin-Madison Correspondence: Allison Furey Trials 2017, 18(Suppl 1):P211 P211 Effective graphical analyses of adverse events in DMC reports Allison Furey, Robin Bechhofer University of Wisconsin-Madison Correspondence: Allison Furey Trials 2017, 18(Suppl 1):P211 We used Bayesian Markov Chain Monte Carlo (MCMC) methods to impute missing values at a component level, based on a selection model factorisation which specifies a marginal distribution for the scores (analysis model) and a conditional distribution for the missing- ness indicators given the scores (missingness model). An autoregres- sive process was incorporated into the analysis model to take account of the longitudinal structure in the scores, and informative prior distributions specified for the parameters in the missingness model to reflect various assumptions about the missingness mechan- ism. We applied a bootstrap approach to calculate the difference be- tween treatment groups because of the non-normal distribution of the daily total SOFA scores, with a separate bootstrap sample taken at each MCMC iteration. The primary charge of a Data Monitoring Committee (DMC) is to monitor the safety of clinical trial subjects. Among the most import- ant sources of safety data is adverse events (Aes) reported by investigators. Often, the Sponsor’s statistical analysis plan for the final study ana- lysis simply indicates that Aes will be summarized by meddra system organ class (SOC) and preferred term. Lengthy tables of Aes are com- prehensive, but may overwhelm DMC members with detail and fail to highlight relevant treatment differences, important constellations of related Aes, or answer key questions regarding the severity, im- pact, or timing of events. The Statistical Data Analysis Center (SDAC) at the University of Wisconsin-Madison specializes in producing interim reports and ana- lyses for DMCs. Our reports are graphically based, allowing DMC members to easily identify differences between treatment groups or Results from the Bayesian analysis showed more uncertainty than those obtained using LOCF, whilst allowing for informative missing- ness unlike standard MI approaches. In addition, the methods applied here accommodated both bootstrap sampling and the Trials 2017, 18(Suppl 1):200 Page 80 of 235 Page 80 of 235 over time and to review a large amount of information in a short amount of time. We employ various presentation styles, including graphics produced in R (bar charts, stacked bars, Kaplan-Meier plots, forest plots), and tables and listings produced in SAS; latex is used for layout and report production. Survival analysis was conducted with a patient having the endpoint of interest if they died within 2 years. p Results In all examples simulated pertaining to MCC, using our rules leaded to substantial gains in power, sometimes even a doubling. The results of theoretical sample size equations had close concord- ance with the powers for various sample sizes observed in simulations. In all examples simulated pertaining to MCC, using our rules leaded to substantial gains in power, sometimes even a doubling. The results of theoretical sample size equations had close concord- ance with the powers for various sample sizes observed in simulations. Background g y Hypothesis testing using conventional levels of Type I and II error would require in excess of 3000 patients, which is unfeasible to re- cruit, even across countries, leading such a trial to be underpowered. We applied our new analysis method using the statistics associated with MCC. However, in clinical trials with binary and time-to-event outcomes, treatment effect modification is often assessed only on the multiplica- tive measurement scale as this corresponds to a comparison of the more commonly presented relative treatment effect measures (relative risks, odds ratios, hazard ratios) across patient subgroups. This is usually obtained from the widely used regression models for these outcome measures, i.e. The logistic regression model and the Cox proportional hazards regression model, by the inclusion of a product term between treatment and the predictor of interest. The analysis of treatment effect modification on the additive measurement scale can be less easy to Conclusion Flexibility is a key feature of our reports; analyses evolve depending on the stage of the trial as well as in response to DMC concerns, and are often tailored to characteristics of the subjects and/or treatments in the specific trial. We find graphical presentations useful, not only for aggregate data, but also for examining individual subjects – for example, to illustrate the relationship between Aes and other data (e.g., dosing, lab data). Custom graphical displays may also address, in aggregate or by subject, timing of Aes, recurrent events, or events of special interest. By restricting the probability of making a wrong decision to be 2.5%, the analysis method we have proposed is more robust than generic non-inferiority tests. The interpretation of hypothesis testing from our rule is the patient may be informed, “on the balance of probabil- ity, this treatment is better”. Our proposed analysis method means conducting clinical trials for rare diseases is worthwhile after all, potentially leading to better standard of care for patients suffering from them. This poster presents examples of innovative displays designed to re- spond to specific questions posed by the DMC, as well as our stand- ard AE presentations for DMC reports. P213 Evaluating treatment effect modification on the additive scale for the evaluation of predictive markers Antonia Marsden1, Richard Emsley2, William Dixon3, Graham Dunn1 1Centre for Biostatistics, School of Health Sciences, University of Manchester; 2Centre for Biostatistics, School of Health Sciences, University of Manchester. MRC northwest Hub for Trials Methodology Research, UK; 3Arthritis Research UK Centre for Epidemiology, Manchester Academic Health Science Centre, University of Manchester Correspondence: Antonia Marsden Trials 2017, 18(Suppl 1):P213 Methods The Type I error was redefined as probability of concluding a treat- ment was better than the other when in fact it was worse, and the minimum sample size was the sample size needed for this value of Type I error to be to 2.5%. To conclude ‘superiority’ using our rules, the upper limit of two-sided 95% confidence interval of the hazard ratio (HR) observed had to be below 1.25, and the upper limit of two-sided 50% confidence interval had to be below 1. Page 81 of 235 Page 81 of 235 Trials 2017, 18(Suppl 1):200 Background Due to major advances being made in clinical trials for prevention of cardiovascular events (including stroke and transient ischaemic at- tack, TIA), and the falling risk of recurrent events, cardiovascular pre- vention trials are increasing in size. Since the number of trials has also increased, it is becoming more difficult to recruit patients into new trials. New strategies are now needed to reduce trial sample sizes and to amplify the potential to demonstrate benefit. The inter- national Triple Antiplatelets for Reducing Dependency after Ischae- mic Stroke (TARDIS) trial assessed the safety and efficacy of intensive (combined aspirin, dipyridamole and clopidogrel) versus guideline (aspirin/dipyridamole, or clopidogrel alone) antiplatelets given for one month in patients with acute stroke or transient ischaemic attack (TIA). Data from a subgroup of the Efficacy of Nitric Oxide in Stroke trial (ENOS-early; concerning patients randomised within 6 hours of ictus, a pre-specified subgroup) and the Rapid Intervention with Glyceryl trinitrate in Hypertensive stroke Trial (RIGHT), suggest that glyceryl trinitrate (GTN), when given early, improved dependency, death, dis- ability, cognitive impairment, mood disturbance, and quality of life. However, individual outcomes do not provide a global estimate of ef- fect. Previous acute stroke trials have used global tests to assess the overall effect of treatment on a group of outcomes: NINDS and IM- AGES (the National Institute of Neurological Disorders and Stroke RT- PA trial and the Intravenous Magnesium Efficacy in Acute Stroke trial; Wald test for binary outcomes) and CARS (Cerebrolysin and Recovery After Stroke trial; Wei-Lachin test for ordinal and continuous out- comes). Transdermal GTN is a candidate treatment for ultra- and hyper-acute stroke, potentially acting through reperfusion, haemo- dynamic and cytoprotectant effects. Methods In the context of acute stroke trials, the modified Rankin scale (MRS) is often used as the primary outcome measure, due to its sensitivity to treatment effects. The MRS is a seven level ordered categorical scale (0: No symptoms, 1: No significant disability, 2: Slight disability, 3: Moderate disability, 4: Moderately severe disability, 5: Severe dis- ability, 6: Death) that assesses independence, dependency and death. The primary objective of the TARDIS trial was to assess treatment ef- fect on recurrence and severity of that recurrence at 90 days. There- fore, the primary outcome consisted of a combination a) the type of recurrent event (stroke or TIA) and b) the score from the MRS taken at three months. This produced a six level ordered categorical poly- chotomised scale with the following structure; Fatal stroke (MRS =6)/ Severe non-fatal stroke (MRS =4 or 5)/Moderate stroke (MRS =2 or 3)/Mild stroke (MRS =0 or 1)/TIA/No recurrent event. The assessment of this primary outcome measure utilised the shift approach, with the use of ordinal logistic regression analysis. Design Vascular prevention studies typically count outcomes as dichotomous events (e.g. Event vs no event) although this is inefficient statistically and gives no indication on the severity of the recurrent event. Recur- rent vascular events, such as stroke, could therefore be polychoto- mised with ordering of outcome events determined by severity. A retrospective analysis of published vascular prevention trials (includ- ing antithrombotic, antihypertensive, lipid lowering, carotid surgery, and hormone replacement therapy) suggested that polychotomised outcome measures provide information on both events and their se- verity, generate smaller numbers-needed-to-treat, and may be more efficient statistically. M h d y Methods y y p Methods The global effects of ultra- or hyper-acute administration of GTN were tested using three statistical approaches: the Hotelling T2 test (combines continuous variables), and Wei-Lachin and Wald tests. Analyses using ordinal logistic regression and multiple lin- ear regression were also performed to test the individual effects of GTN on each outcome. Raw (and dichotomised) outcome data at 90 days included telephone assessments of dependency (modi- fied Rankin Scale, MRS >2), disability (Barthel index, BI < 60), mood (short Zung depression scale, ZDS > 70), cognition (t-Mini Mental state examination, tmmse < 14) and quality of life (health utility status, HUS < 0.5, as derived from euroqol-5D-3 level). Data are odds ratio (OR), mean difference (MD), Mann–Whitney esti- mates (MW) and T2 statistic. Comparison of global statistical analyses in patients with hyper-acute stroke: assessment of randomised trials of transdermal glyceryl trinitrate, a nitric oxide donor Lisa Woodhouse1, Polly Scutt 1, Stuart Pocock2, Alan Montgomery1, Nikola Sprigg1, Philip M. Bath1 1University of Nottingham; 2London School of Hygiene & Tropical Medicine Correspondence: Lisa Woodhouse Trials 2017, 18(Suppl 1):P214 Comparison of global statistical analyses in patients with hyper-acute stroke: assessment of randomised trials of transdermal glyceryl trinitrate, a nitric oxide donor Lisa Woodhouse1, Polly Scutt 1, Stuart Pocock2, Alan Montgomery1, Nikola Sprigg1, Philip M. Bath1 1University of Nottingham; 2London School of Hygiene & Tropical Medicine Correspondence: Lisa Woodhouse Trials 2017, 18(Suppl 1):P214 Conclusions obtain in these settings, particularly for time-to-event outcomes due to the dependency on time. GTN improved global aggregates of dependency, disability, mood, cognition and quality of life data. This exploratory finding is being tested prospectively in the ongoing 850-patient RIGHT-2 trial. Though individual test results for RIGHT suggest that GTN only had a signifi- cant effect on dependency (MRS), global analysis of the data (using the Wei-Lachin test) suggested that GTN improved all outcomes. Reporting global tests adds summary information on overall treat- ment effects. Further, it may be advantageous to base the primary outcome on a global analysis since global tests are statistically more efficient; in this case, individual outcomes would be presented in pre-specified secondary analyses. The Wei-Lachin test may be pre- ferred since it allows analysis of ordinal and continuous variables; in contrast, the Wald test only analyses binary outcomes, and the Hotel- ling T2 test does not take account of direction of effect. This works aims to highlight why an analysis of treatment effect modification on the additive scale is more informative in the evalu- ation of markers predictive of differential treatment response and to present how this can be performed in practice. We propose the use of a novel measure, the Ratio of Absolute Effects (RAE) measure, as an approach for the assessment of treatment effect modification on the additive scale which can be calculated from the more commonly used multiplicative regression models used for binary and time-to- event outcomes. We suggest this measure to be particularly useful for time-to-event outcomes as it is time invariant. Also discussed is the use of alternative regression models on the additive scale (e.g. The additive hazards model) from which effect modification on the additive scale can be directly assessed. 5 Rationale for using an ordinal primary outcome in clinical trials for the prevention of recurrent stroke and transient ischaemic attack Lisa Woodhouse1, Jason P. Appleton1, Stuart Pocock2, Alan Montgomery1, Nikola Sprigg1, Philip M. Bath1 1University of Nottingham; 2London School of Hygiene & Tropical Medicine Correspondence: Lisa Woodhouse Trials 2017, 18(Suppl 1):P215 5 Rationale for using an ordinal primary outcome in clinical trials for the prevention of recurrent stroke and transient ischaemic attack Lisa Woodhouse1, Jason P. Appleton1, Stuart Pocock2, Alan Montgomery1, Nikola Sprigg1, Philip M. Bath1 p Methods Assuming log-hazard-ratio is a logistic function of continuous prog- nostic variable, the midpoint of the sigmoid curve (x_mp) would be a natural choice for the cutpoint. 100,000 survival datasets were gen- erated via Monte Carlo simulations using R language. Each simulated dataset included 200 observations (x) with exponential distribution (similar to the number of the patients and the distribution of AR- copy numbers in the trial) and log-hazard-ratio (y) as a logistic func- tion of x. Parameters of the steepness of the curve and location of the midpoint (x_mp) were randomly assigned in each run. For every simulated dataset, the best cutpoint was sought via the following it- erative steps: (i) assigning 0 to all observations below copy number x_i and 1 to all observations equal to or above copy number x_i, (ii) fitting Cox model (for x_i), (iii) using the maximum values of the sta- tistics of survival modelling including Hazard Ratio, Log-Likelihood - or Cox-Snell Pseudo-R-Squared (RSQ) -, Concordance Index, Wald- test, and Log-Rank-test as indicators (correlative measures) of the cutpoint, (iv) calculating the difference between the cutpoints sug- gested by each correlative measure and the true cutpoint (x_mp). Altogether, six sets of 100,000 differences along with their medians and interquartile ranges (IQR) were estimated. The statistical measure associated with the smallest absolute median and IQR was chosen as the best correlative measure. The chosen measure was used in the trial data to determine the optimal cutpoint for AR-copy number. We also used bootstrapping to increase reliability of the estimated cut- point in the trial data. Institutions will be randomized to implement the intervention versus usual care. Randomization will be stratified by institution characteris- tics (3 factors: institutional volume, thoracic surgery fellowship train- ing program, dedicated general thoracic surgeon present). In order to randomize all institutions at the same time, a run-in phase will be implemented to allow for sites to obtain institutional and regulatory approvals. In addition, objectives of the run-in phase are to provide a more accurate assessment of local accrual and preliminary estimates of outcomes. The primary objective of this study is to compare the 3- year disease free survival (DFS) among patients at institutions ran- domized to implement the intervention to those randomized to usual care. The secondary objective is to compare the frequency of patient up-staging (from cn0/1 to pn1/2/3) following surgical resec- tion among patients receiving intervention to those receiving usual care. ( Results 312 patients (GTN 168, no GTN 144) were randomised within 6 hours of ictus into ENOS-early (n = 273) and RIGHT (n = 39). GTN improved certain individual and global outcomes for both the ENOS-early and RIGHT trials respectively: Individual tests MRS: OR 0.55, (p = 0.0055); 0.27, (p = 0.0306) BI: MD 13.5, (p = 0.0029); 25.4, (p = 0.0724) ZDS: MD −10.3, (p = 0.0013); −14.3, (p = 0.0631) tmmse: MD 3.5, (p = 0.0007); 4.3, (p = 0.1151) HUS: MD 0.09, (p = 0.0753); 0.21, (p = 0.0618) Global tests Hotelling T2: T2 24.91, (p = 0.0087); 9.85, (p = 0.1763) Wei-Lachin: MW 0.64, (p = 0.0018); 0.73, (p = 0.0301) Wald: OR 0.52, (p = 0.0011); 0.38, (p = 0.0826). Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 82 of 235 Page 82 of 235 Page 82 of 235 p Results Median and IQR of the differences between true cutpoint (x_mp) and the the copy numbers indicated by the highest values of Hazard Ratio, Concordance Index, Wald test, Log-Rank, RSQ, and Log- Likelihood were −13.39 (45.38), −3.13 (5.32), −3.10 (3.60), −2.82 (3.43), −2.06 (3.24), and −2.06 (3.24), respectively. Consistent results were also observed using simulated AR-copy numbers with normal distri- bution. Thus, Log-Likelihood (or interchangeably RSQ) was chosen as the recommended correlative measure and was used to determine 30;22(18) Results The study design is based on a design with 80% power to detect a 50% improvement in DFS (HR =0.67) at the 1-sided 0.025 level. We assume uniform accrual and an average accrual rate of 15 patients/ site/year. Under independence, the total sample would be 568. Ac- counting for within institution correlation, the total accrual is 670 pa- tients (an inflation of 18%), accrued over 2 years with 3 years of follow-up. p Conclusion Among various statistical measures of survival, Log-Likelihood is the best correlative measure for estimating optimal cutpoint of a con- tinuous prognostic variable with normal or exponential distribution. Wald and Log-Rank tests are slightly less reliable and Hazard Ratio is the least reliable correlative measure. P218 SWOG s1700: an institutional cluster-randomized trial of a surgical lymph node specimen collection kit in the cooperative group setting Jieling Miao, Yingqi Zhao, Jim Moon, Mary W. Redman SWOG Statistics and Data Management Center, Fred Hutchinson Cancer Research Center Correspondence: Jieling Miao l l Background g Approximately 60,000 patients annually undergo resection for non- small cell lung cancer (NSCLC) in the US. Most of them will not achieve long-term survivorship and the status of nodal involvement is the most powerful determinant of prognosis. Accurate pathologic nodal staging requires the combination of surgical dissection of the appropriate hilar and mediastinal lymph nodes and thorough patho- logic examination of lymph modes present within the lung resection specimen. S1700 or SILENT (Strategies to Improve Lymph Node Examination of Non-Small Cell Lung Tumors), a trial proposed by SWOG, is designed to evaluate a lymph node specimen collection kit. It is anticipated that this simple intervention on how the surgeon does his/her lymph node sampling, will improve the accuracy of pathologic nodal staging of resected lung cancer. It was determined that a cluster randomized trial (CRT) design is necessary to address this question. Conduct of a CRT is rarely done (to almost never) in the Cooperative Groups within the US. Methods p Methods Given feasibility considerations, the planned goal is to limit par- ticipation to 40 institutions (20 randomized to implement the inter- vention and 20 to continue with usual care). Given historical data, it is estimated that the intraclass correlation coefficient is 0.01. Sample size calculations were based on Xie & Waksman. (Stat Med. 2003 Sep 30;22(18):2835–46). Log-likelihood is the best correlative measure to estimate the cutpoint for a continuous prognostic variable: a Monte Carlo simulation study Jieling Miao, Yingqi Zhao, Jim Moon, Mary W. Redman SWOG Statistics and Data Management Center, Fred Hutchinson Cancer Research Center y Mansour Sharabiani, Clare Peckitt, Gerhardt Attard The Royal Marsden NHS Trust Correspondence: Mansour Sharabiani Trials 2017, 18(Suppl 1):P216 y Mansour Sharabiani, Clare Peckitt, Gerhardt Attard The Royal Marsden NHS Trust Correspondence: Mansour Sharabiani Trials 2017, 18(Suppl 1):P216 Correspondence: Jieling Miao Trials 2017, 18(Suppl 1):P218 Discussion the optimal AR-copy number cutpoint for stratification of CRPC patients. The TARDIS trial was the first vascular prevention trial to assess pro- spectively both recurrence and its severity, rather than recurrence alone. This novel approach both increases statistical power through comparing the difference in the distribution across the whole scale of severity between the treatments, and allows the effect of treat- ment on severity to be assessed. Such an approach can reduce trial sample size and ultimately costs, whilst improving statistical effi- ciency and amplifying the potential to demonstrate a treatment ef- fect. Data will be presented once the main findings have been presented in late 2016. p Conclusion Background Stratification of patients into high- and low-risk categories using a cutpoint for a continuous prognostic variable has important applica- tions in clinical decision making. Different approaches including bio- logical determination, median value, and clustering as well as using correlative measures such as logrank test, minimum p-value, hazard ratio, and log-likelihood have been used to determine the cutpoint. Here we try to choose the most reliable correlative measure using Monte Carlo simulation. We also apply the chosen measure to bio- logical data (androgen receptor [AR] gene copy number) from castration-resistant-prostate-cancer (CRPC) patients where it is as- sumed, based on previous studies, that AR-gain (higher number of copies of AR) patients have higher hazard rates of survival than AR- Normal patients. Discussion These findings suggest that phase I trials are generally safe, however there are important opportunities to improve the design, conduct and dissemination of these studies. Methodological gaps exist which should be addressed when planning phase I trials, particularly for dose escalation studies. Much greater transparency through the public registration and dissemination of findings from phase I trials is needed to improve the safety and conduct of future studies. Background There is a couple of issues in practice. One of issues is that it de- pends on the distribution of propensity score. The score is usually es- timated by logistic regression. However, it is not easy to assume prior to commencing the study. Another one is that some factor which has association with treatment selection but is not correlated with endpoint decrease the precision of confidence interval for the estimate of treatment effect. As result, it leads to decreasing statis- tical power of test as previous report warned. However, identifying these factors to be excluded would contradict the nature of propen- sity score analysis which collects data not to miss confounding fac- tors as much as possible. Furthermore simple stratified analysis, ex Cox regression, is enough if it is possible to identify these factors in advance. p Discussion In an era of increasing costs for cancer care, low-cost and relatively sim- ple interventions such as the one being evaluated in SILENT are very valuable. Careful consideration of design and implementation can lead to a valuable resource and address an important yet simple question. Trials 2017, 18(Suppl 1):200 Page 83 of 235 Page 83 of 235 popularity of propensity score rapidly increases its application in pro- spective observational studies with time-to-endpoint in various areas including cancer or cardiovascular disease and some would expect it as an alternative of confirmatory trials. However, a limited number of papers have discussed sample size calculation. We proposed practical sample size re-estimation in mid-course of the study. The approach provides not only statistical power but also the incorporating with in- terim analysis which have to adjust type I error. An assessment of the design and dissemination of phase I clinical trials: where can we improve? Bethan Copsey1, Ayodele Odutayo2, Jonathan Cook2, Susan Dutton2, Douglas Altman2, Sally Hopewell2 1University of Oxford; 2Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford Correspondence: Bethan Copsey Trials 2017, 18(Suppl 1):P220 Objective To explain, demonstrate and compare methods for evaluating perso- nalised treatment recommendations using a standard, two-arm, par- allel randomised controlled trial. p p y Naoki Ishizuka1, Takeharu Yamanaka2, Noriko Tanaka3 1Cancer Institute Hospital; 2Yokohama City University; 3National Center for Global Health and Medicine Correspondence: Naoki Ishizuka Trials 2017, 18(Suppl 1):P221 Results The operational characteristics concerning the statistical power for various scenarios in which there is no correlation between the factor of treatment choice and endpoint were examined by the simulation study. The result guaranteed the statistical power as planned. Conclusions After a median 2.7 years since completion, only 3 of the 32 fully com- pleted phase I trials have been published and only 10 of these 32 tri- als have a publicly accessible trial registry entry. None of the trials with SAEs have been published. Our approach keeps the statistical power without any assumption of propensity score including the distribution and the correlation be- tween the endpoint and the factors of the treatment choice. Methods We assume the situation that one would assess the new treatment compared to the standard one. Calculate the sample size tenta- tively if one assumes alpha level, power and an effect size delta. If time to event is a primary endpoint, expected number of events is determined by the method of Schoenfeld and the variation. Esti- mate propensity score when the sample size or the number of events reaches tentative sample size or expected number of events. Use stratified logistic or stratified Cox model to estimate the param- eter of the effect size. Calculate the inflation coefficient - Which is defined as follows = (Observed Standard Error)^2/(1/(_1 × _2 × D)) where _1,_2 are the fractions of each treatment group and D is the tentative expected number events for time to event. =(Observed Standard Error)^2/(Assumed Variance) for binary endpoint. Calcu- late the target sample size or the number of events as a product of inflation coefficient - and tentative sample size or the number of events. Do the interim analysis ad Information time as 1 if one would like to plan. If the interim analysis is not significant or one has not done it, do the final analysis. Of the 426 HRA-approved trial protocols, 54 were phase I trials (17 oncology; 37 non-oncology). Forty-five (83%) were industry funded and 17 (31%) were first-in-human studies. All trials were registered in a trial registry, although registry details were publicly available for only 21; as per EU regulations. Across the included studies there were 869 participants; the median sample size was 27 (interquartile range 18 to 41). Of the first-in-human studies, 13 specified an observation period be- tween administration of the study drug to the first and subsequent participants. Only one study provided justification for this observa- tion period. Thirteen first-in-human studies used biological agents but only 5 of 13 used the MABEL (minimum anticipated biological ef- fect level) for calculating the starting dose or justified not doing so. Of the 54 phase I trials, 32 have been completed and 24 submitted CSRs to the HRA as of April 2016. No deaths occurred but 11 SAEs were reported, of which 3 were deemed potentially related to the study treatment. All treatment-related SAEs occurred in non- oncology trials. Methods A representative sample (n = 426) of clinical trial protocols that re- ceived ethical approval by the UK Health Research Authority (HRA) in 2012. We extracted details related to study design and methods from the protocols on phase I studies. Additionally, information on serious adverse events (SAEs) from submitted clinical study reports (CSRs) and searched for publications (by April 2016) of the completed trials was collated. Findings were narratively summarised. Background Phase I trials involve the early testing of investigational medicines in humans in order to assess their safety, tolerability and pharmacokin- etics. Questionable design and conduct of phase I trials has led to long-term morbidity and mortality. There is limited information pub- licly available regarding how these trials are conducted, monitored and disseminated. A systematic methodological review of the ethical submission for phase I trials was carried out to address this gap. Methods Evaluating personalised treatment recommendations using randomised controlled trials Evaluating personalised treatment recommendations using randomised controlled trials Matthias Pierce, Richard Emsley Matthias Pierce, Richard Emsley University of Manchester Correspondence: Matthias Pierce Trials 2017, 18(Suppl 1):P222 Background The modern paradigm of stratified medicine (also termed persona- lised or precision medicine) seeks to move beyond a one-size-fits all approach, that treats patient populations as a whole, towards one that identifies patient strata with different disease pathways or re- sponses to treatment. A major aspect of stratified medicine is to pro- vide personalised treatment recommendations (PTR’S): an algorithm Background Sample size must be determined when one start any prospective study regardless of it is intervention or observational. The recent Trials 2017, 18(Suppl 1):200 Page 84 of 235 Page 84 of 235 biomarker-driven sub-studies in response to the approval of im- munotherapies in our study population. As of November 3, 2016, 4 sub-studies have been closed to accrual, 1 new non-match sub-study has been activated, 1 new biomarker-driven sub-study is expected to open to accrual by the end of 2016, 1 new non-match sub-study for immune-therapy (IO) exposed patients is expected to activate in the first quarter of 2017, and an additional biomarker-driven sub-study is expected to be activated mid-2017. The anticipated study schema is included below. that recommends treatment based on the patient’s predicted treat- ment response using biomarkers, a patient’s measureable characteris- tics collected at clinical visit. A PTR may be constructed using a single biomarker, or using multiple biomarkers. After estimating a PTR, the next step is to assess whether the expected outcome under a PTR improves on the expected outcome under an alternative policy that recommends treatment based on the patient’s predicted treat- ment response using biomarkers, a patient’s measureable characteris- tics collected at clinical visit. A PTR may be constructed using a single biomarker, or using multiple biomarkers. After estimating a PTR, the next step is to assess whether the expected outcome under a PTR improves on the expected outcome under an alternative policy – one where either everybody receives the treatment or everybody receives the control condition. The evaluation of a PTR differs from the evaluation of prognostic or diagnostic models because, for any individual, the object of inference (whether a subject benefited from treatment) remains unobserved. This is because the individual treat- ment effect cannot be separated from prognostic effects. Therefore standard methods of model evaluation, for example ROC-curve ana- lysis, are inappropriate in this context. The Lung-MAP trial is a continually evolving study. The study team continues to evaluate new biomarker/investigational therapy pairs, including immunotherapy drugs and biomarkers, and combinations of therapies. y , Methods This presentation will cover two methods for evaluating a PTR using a standard, two-armed randomised controlled trial. The first, termed the inverse probability weighting (IPW) approach, uses a weighted average of the outcome in those lucky to have been randomised to the treatment they were recommended under the PTR. The second is an augmented version of the IPW (AIPW), developed using semi- parametric theory, that borrows information from a regression model for the outcome under treatment or control, to establish a more effi- cient estimator. Monte-Carlo simulations are used to compare the statistical properties of the IPW and AIPW methods using a range of data generating scenarios. These methods will be demonstrated with application to data from a randomised controlled trial for Chronic Fa- tigue Syndrome Patients, using the user-written Stata command ptr.ado. Inference for these parameters will also be discussed. Methods In order to explore the issue of performance bias in depth, a narrow clinical field (appendicitis) was selected. Rcts evaluating at least one surgical intervention (defined as procedures that cut a patient's tis- sues, involving the use of a sterile environment, anaesthesia, antisep- tic conditions, surgical instruments, and suturing or stapling) for patients with appendicitis were identified. Because there is no formal tool for assessing performance bias, information from existing litera- ture relating to various aspects of performance bias was used to guide data extraction: i) blinding (Cochrane Risk of Bias tool), ii) standardisation (CONSORT-NPT and SPIRIT statements). An inductive approach was used, whereby an initial extraction form was used and where new themes relating to performance bias were identified, the form was modified to incorporate these and all trials reviewed using the new form. Design, conduct, and analysis of a master protocol within an evolving landscape of standard of care: the lung-map trial Mary Redman, James Moon, Shannon McDonough, Jieling Miao, Katie Griffin, Michael LeBlanc Fred Hutchinson Cancer Research Center Correspondence: Mary Redman Trials 2017, 18(Suppl 1):P223 The Lung- MAP trial (Lung Cancer Master Protocol), launched in 2014, is an umbrella protocol to evaluate targeted therapies in bio- marker selected patients for previously-treated stage IV or recurrent non-small cell lung cancer. It is the first precision medicine trial launched with the support of the National Cancer Institute in the United States. Moreover, Lung-MAP is designed as a pathway for FDA approval of investigational therapies that successfully meet study objectives. Beyond blinding: a systematic review to explore performance bias in surgical RCTs Beyond blinding: a systematic review to explore performance bias in surgical RCTs Natalie Blencowe, Barry G. Main, Jane M. Blazeby University of Bristol Correspondence: Natalie Blencowe Trials 2017, 18(Suppl 1):P224 Background g Performance bias arises from unintended deviations from the intended intervention, comparator or co-interventions that occur differentially by allocated group. Conventionally, it can be re- duced through blinding of healthcare providers and patients; however, this represents a major challenge in surgical settings and other strategies are therefore required. Standardisation of surgical intervention and co-interventions, and monitoring adher- ence to these standards, represents one solution for reducing performance bias. The aim of this study, therefore, was to sys- tematically explore the issue of performance bias in randomised controlled trials in surgery, to inform the design and delivery of future studies. Simulations demonstrate that the AIPW method is consistently shown to be more efficient, even when the parametric model for the outcome used in the AIPW procedure is misspecified. C l i The evaluation of a PTR is qualitatively different from the evaluation of a model used for diagnosis or prognosis. There are two methods available for establishing whether the outcome under a PTR is an im- provement (or not) on an alternative policy where everybody is given the treatment/control conditions. These methods are easily imple- mented in standard statistical software; for example using our user- written Stata command ptr.ado. Of the two methods, the AIPW is demonstrably more efficient than the IPW. Background Conduct of such a study requires a substantial amount of effort and on-going attention beyond the conduct of a stand- alone clinical trial. This presentation will provide an overview of the current status of Lung-MAP, both active and closed studies, discuss some lessons learned in the conduct of these so-called platform trials, and a view into the future of Lung-MAP. Discussion This study will assess the quality of reporting of RCTs conducted in KSA given the increasing number of RCTs being conducted in the re- gion and the limited data in the literature regarding the quality of RCTs reporting conducted. Findings achieved from this STUDY might help in identifying CURRENT strengths and gaps that may impact the Good Clinical Practice in the clinical setting in KSA. This abstract is not included here as it has already been published. Network meta-analysis of antiembolic interventions: adjustment for confounders 1 1 1 Nada Alsowaida1, Doaa Bintaleb2, Hadeel Alkofide3, Hisham Aljadhey4, Tariq Alhawassi5 Nada Alsowaida1, Doaa Bintaleb2, Hadeel Alkofide3, Hisham Aljadhey4, Tariq Alhawassi5 1 1Medication Safety Research Chair, Pharmacy services, King Saud University Medical City, Riyadh, Saudi Arabia; 2Investigational Drugs and Research Unit, King Saud University Medical City, Riyadh, Saudi Arabia; 3College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; 4Saudi Food and Drug Authority, Medication Safety Research Chair, Riyadh, Saudi Arabia; 5Medication Safety Research Chair, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Background The goal of this study was to compare confounding effect of patient population characteristics on the comparative effectiveness of indi- vidual antiembolic interventions in non-valvular atrial fibrillation (AF): novel oral anticoagulants (NOACs) (apixaban, dabigatran, edoxaban, rivaroxaban), vitamin K antagonists (VKA), aspirin, and the Watchman device. Background Randomized controlled trials (RCTs) are considered the gold standard to assess the efficacy and safety of new treatment interventions and compare conventional therapies. RCTs are used to support decision- making, and guidelines recommendations. However, despite their clinical importance, RCTs have some limitations as they are at high risk for bias, can over and/or underestimate treatment interventions, which limit their generalizability. It's estimated that poor quality trials have led to 30% - 40% overestimation of the treatment. Therefore, the quality of reported RCTs is still questionable and multiple studies have concluded that RCTs are yet hindered by several limitations making risk-benefit assessment, which is an essential element for RCTs quality, a challenge in certain medical conditions for healthcare professionals. With the largely emerging data and new treatments that required pharmaceutical companies to do more RCTs, the need for assessing the quality of RCT becomes increasingly important. The Consolidated Standards of Reporting Trails (CONSORT) statement is a tool designed to assess the quality of RCTs reported and significantly improve the quality of RCTs. To our knowledge there is no current data in the literature regarding the quality of RCTs conducted in Saudi Arabia (KSA). Given the increasing number of RCTs being con- ducted in the region, it is essential to gain an understanding on the quality of reporting of these RCTs, which might impact future regula- tions for conducting such studies in the country. Results 45 rcts met the inclusion criteria. Six compared surgical and non- surgical treatments, and 39 compared different surgical ap- proaches (open versus laparoscopic surgery, n = 35; laparoscopic versus single-port surgery, n = 4). In the six RCTs comparing surgi- cal and non-surgical treatments, blinding of participants was not undertaken and there was no information relating to healthcare professionals or trial personnel. In the 39 comparing different sur- gical procedures, information about blinding was rarely reported. Eight, seven, and five studies reported that blinding of partici- pants, healthcare professionals and trial personnel was attempted, respectively. Just one RCT reported that the success of blinding was evaluated. Data extraction and analysis is ongoing and fur- ther results (relating to standardisation) will be available for pres- entation at the conference. Lung-MAP activated with 4 biomarker-driven sub-studies and one sub-study for patients with no matching biomarkers; all sub-studies were randomized with docetaxel as the control in 4 of 5 sub-studies. While the standard of care (docetaxel) had been unchanged for de- cades in this patient population, within the first year of the study, the Checkmate 017 trial (Brahmer NEJM 2015), demonstrating that nivolumab is superior to docetaxel in this patient, changed the treat- ment paradigm for this population. p g p p In December 2015, a major revision of the trial was implemented with modifications to the patient population and design of the Trials 2017, 18(Suppl 1):200 Page 85 of 235 Page 85 of 235 Methods We performed network meta-analysis of randomized clinical trials (RCTs) that enrolled 200 patients with non-valvular AF, mean or me- dian follow-up Six months, with published reports in the English lan- guage. NOAC phase II studies were excluded. Placebo/control arm received either placebo or no treatment. All-cause mortality served as the primary outcome. Results of unadjusted and adjusted meta- regression analyses were compared. The following confounders were included, one-by-one: time in therapeutic range (TTR), CHADS2 score, mean/median duration of follow-up, mean age, the percentage of males, the percentage of VKA-naïve, the percentage of secondary prevention patients. Objective To assess the reporting quality of RCTs conducted in KSA from 2005 and above using the CONSORT tool. p Results A total of 21 RCTs (96,017 non-valvular AF patients; median age 72y; 65% males; median follow-up 1.7y). In unadjusted analysis, in com- parison to placebo/control, use of aspirin (OR 0.82 (95%CI 0.68-0.99)), VKA (OR 0.69 (95%CI 0.57-0.85)), apixaban (OR 0.62 (95%CI 0.50- 0.78)), dabigatran (OR 0.62 (95%CI 0.50-0.78), edoxaban (OR 0.62 (95%CI 0.50-0.77), rivaroxaban (OR 0.58 (95%CI 0.44-0.77)), and the Watchman device (OR 0.47 (95%CI 0.25-0.88)) significantly reduced all-cause mortality. Apixaban (OR 0.89 (95%CI 0.80-0.99)), dabigatran (OR 0.90 (95%CI 0.82-0.99)), and edoxaban (OR 0.89 (95%CI 0.82- 0.96)) reduced risk of all-cause death as compared to VKA. Life- saving effect of Watchman device and NOACs was supported not only by 95% confidence intervals (CIS) but also, importantly, by 95% probability intervals (PRIs). However, 95% PRI for aspirin crossed the ‘no effect’ line, indicating that life-saving effect of aspirin might not be confirmed in future RCTs if ever conducted. After adjustment for Conclusion conducted. An attempt to identify unpublished data by searching clinical trial registries, through clinicaltrails.gov, and the Saudi Food and Drug Administration (SFDA) registry will be conducted. The search strategy will contain a combination of mesh terms and key- words relevant to the study design. Identified RCTs will be exported to Endnote X7 to check and remove any duplication. All titles and abstracts of identified RCTs will be screened by two investigators for potential relevance. Reference lists of potential studies, systematic re- views and meta-analysis will be also reviewed manually to identify relevant original RCTs. Search will be limited to RCTs either phase II, III and IV, published in 2005 and above in both English and Arabic language. Studies conducted in KSA as part of international multicen- ter RCTs, non-therapeutical RCTs will be excluded. The protocol of this study was submitted for publication to the International pro- spective register of systematic reviews PROSPERO. Preliminary results from this study indicate that surgical RCTs are likely to be at high risk of performance bias. Although blinding of surgeons performing operations was not possible in this clinical area, blinding of patients, other healthcare professionals and trial personnel was plausible yet rarely undertaken. This may be because existing guidance is difficult to apply in a surgical setting. A potential solution would be to improve the process of quality assurance in rcts, by i) clearly defining interventions and co-interventions, ii) standar- dising their delivery, and iii) careful monitoring and reporting of ad- herence to these standards. Further work is required to explore how this might be achieved in surgical RCTs. 5 Network metanalysis benchmarking the technological development of implantable medical devices Catherine Klersy, Valeria Scotti, Luigia Scudeller, Chiara Rebuffi, Carmine Tinelli, Annalisa De Silvestri IRCCS Fondazione Policlinico san Matteo Correspondence: Catherine Klersy Trials 2017, 18(Suppl 1):P225 Network metanalysis benchmarking the technological development of implantable medical devices Catherine Klersy, Valeria Scotti, Luigia Scudeller, Chiara Rebuffi, Carmine Tinelli, Annalisa De Silvestri IRCCS Fondazione Policlinico san Matteo Correspondence: Catherine Klersy Trials 2017, 18(Suppl 1):P225 Network metanalysis benchmarking the technological development of implantable medical devices Background g Online registration of trial protocols has been implemented to sup- port transparency and good clinical practice for the conduct of a trial. One aspect is to ensure that the primary outcome is pre-specified prior to any data being collected and interim analysis performed. This is to discourage the outcomes being selectively chosen for reporting based on significant p-values. We aimed to examine the status of randomised clinical trials (RCT) whose primary outcome changed between protocol registration and published paper, and to quantify the impact of this change on the resulting treatment effect estimates. To investigate agreement between generic evidence and detailed trial assessment, we asked three assessors with access to summary trial descriptions to rank pairs of trials from 30 sampled meta- analyses according to severity of bias. We compared the assessor rankings to rankings based on a bias model fitted to the sampled meta-analyses. Analyses were performed for biases associated with sequence generation, allocation concealment and blinding. Subse- quently, we explored methods for bias adjustment based on bias dis- tributions derived from generic evidence, detailed trial assessment or combinations of the two. Generic distributions were derived from a hierarchical model fitted to 64 meta-analyses from the Cochrane Database of Systematic Reviews. Opinion-based distributions were averaged across 12 assessors who read summary information on each trial in a new meta-analysis, and independently gave their opin- ions on bias. We developed three different approaches to combine generic evidence with detailed trial assessment. The first method sta- tistically combines the generic and opinion-based bias distributions. In two alternative methods, assessors are provided with generic bias distributions and summary trial information, and asked to give their opinion on where in the distribution the particular trial might lie (nu- merically or by selecting broad areas of the distribution). In two case study meta-analyses, we adjusted for bias according to the set of dis- tributions derived using each of the three approaches. p Results Among 29,749 searched articles (Medline: 28,810, EMBASE: 939), 1,488 articles were selected in this study. Of the 487 eligible tri- als, 63 (12.9%) published articles were reported with no or an un- clear description of primary outcome. 21(4.3%) studies were registered with no or an unclear description of primary outcome. 75 (15.4%) trials were registered after the completion of the study. Among the remaining trials with primary outcome clearly registered and reported, 29.0% (95/328) showed some discrepan- cies in primary outcome between trial registration and published article. Further excluding 33 trials due to uncalculated data, there were 295 trials that could be included in the bias assessment and we found a clearly larger intervention effect (pooled ratio of odds ratios 0.79 (95% confidence interval 0.68 to 0.91), p = 0.0012) among trials with changed primary outcome compared to Good agreement was observed between data-based and opinion- based approaches to ranking pairs of trials according to risk of bias. Among the assessor opinions judging that one trial was more biased, the proportion that agreed with the ranking based on evidence- based fitted biases was highest for allocation concealment (79%) and blinding (79%) and lowest for sequence generation (59%). In an ex- ample meta-analysis, bias-adjustment based on generic evidence had the effect of shifting the intervention odds ratio towards the null by 28%, and between-trial variance reduced substantially by 56%. Ex- pert opinions have been obtained recently and the final bias adjust- ment results based on these are pending. p g Discussion Background Systematic reviews of randomised controlled trials provide the best evidence on the benefits and harms of healthcare interventions. However, trials within meta-analyses are often affected by varying amounts of internal bias caused by methodological flaws. Currently, there is no consensus over how to make allowance for biases in meta-analysis. Two methods for adjustment for within-trial biases in meta-analysis have recently been proposed. The first uses empirical (generic) evidence on the magnitude of biases observed in a large collection of meta-analyses; the second uses expert opinion informed by detailed assessment of the potential biases affecting each trial. The objectives of this research are to investigate the extent to which these two approaches agree, and to explore how they could be inte- grated in order to gain the advantages of both. Tao Chen , Rui Qin , Duolao Wang , Victoria Cornelius 1Tropic Clinical Trial Unit; 2Department of Health Education; 3Imperial Clinical Trials Unit Correspondence: Tao Chen Trials 2017, 18(Suppl 1):P230 Correspondence: Tao Chen Trials 2017, 18(Suppl 1):P230 Method Electronic search of the following databases: Cochrane Central Regis- ter of Controlled Trials (CENTRAL), EMBASE, MEDLINE via Ovid will be Page 86 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 RCT population characteristics (TTR, duration of follow-up, and CHADS2), no antiembolic intervention was statistically significantly better than placebo/control, and there was no significant difference between VKA and NOACs, or other antiembolic interventions. Conclu- sion: Adjusted meta-regression analysis allows to study confounding effects of RCT population characteristics on results of network meta- analysis. adjustment, informed by both available generic evidence and elicited opinion. Choice of integrated approach may be based on the prefer- ences of the systematic review authors. P229 Placebo response is not decreased by enrichment trial designs in randomized controlled trials of triptan medications in the paediatric age group Lawrence Richer, Ben Vandermeer, Lisa Hartling University of Alberta Correspondence: Lawrence Richer Trials 2017, 18(Suppl 1):P229 Placebo response is not decreased by enrichment trial designs in randomized controlled trials of triptan medications in the paediatric age group paediatric age group Lawrence Richer, Ben Vandermeer, Lisa Hartling University of Alberta Correspondence: Lawrence Richer Trials 2017, 18(Suppl 1):P229 p g g p Lawrence Richer, Ben Vandermeer, Lisa Hartling University of Alberta Correspondence: Lawrence Richer Trials 2017, 18(Suppl 1):P229 P228 Adjusting trial results for biases in meta-analysis: combining generic evidence on bias with detailed trial assessment Kirsty Rhodes1, Rebecca M. Turner1, Jelena Savovic2, Roy Elbers2, Hayley Jones2, David Mawdsley2, Jonathan AC. Sterne2, Julian PT. Higgins2 1MRC Biostatistics Unit; 2University of Bristol Correspondence: Kirsty Rhodes Trials 2017, 18(Suppl 1):P228 This abstract is not included here as it has already been published. P230 Influence of primary outcome change on treatment effect estimates in clinical trials: meta-epidemiological study Tao Chen1, Rui Qin2, Duolao Wang1, Victoria Cornelius3 1Tropic Clinical Trial Unit; 2Department of Health Education; 3Imperial Clinical Trials Unit Correspondence: Tao Chen Trials 2017, 18(Suppl 1):P230 Method We searched registered RCT from Medline and EMBASE between 2011 and 2015 and randomly selected 5% of searched articles for each year. Articles were excluded if they are not RCT or trials with multiple primary outcomes. For each included trial, we collected in- formation on the primary outcome reported in the article and in the registered protocol. Trials were classified as having a changed pri- mary outcome if there was an inconsistency between the registered and published outcome. Additional information on effect size, type of outcome, type of study design, post-randomisation exclusions were extracted. For consistency, we inverted the effect estimates where necessary so that each trial indicated an odds ratio less than 1 (where the active group has more favourable result than the control group). The relative odds ratio (that is, the summary odds ratio for trials with a primary outcome change divided by those without) was calculated and a value less than 1 indicated larger treatment effects in trials with changed primary outcome compared to trials whose pri- mary outcome was the same between the protocol and final publication. Conclusion Trials that deviated from the originally registered outcome showed larger intervention effects than trials whose primary outcome was unaltered from the original protocol registration. This highlights the important role of trial registration prior to the initiation of trial and the need for clear specification of the primary outcome. The AIDS Malignancy Consortium (AMC) is a National Cancer Institute supported multicenter clinical trials group founded in 1995 to sup- port innovative trials for AIDS-related cancers. In 2010 the AMC ex- panded operations internationally and opened 4 sites located in sub- Saharan African countries with a high prevalence of HIV. The goal of this expansion was to build a cancer clinical trials network in sub- Saharan Africa (SSA) that was capable of conducting contextually appropriate therapeutic and prevention trials in a variety of HIV- associated cancers and contributing to the AMC’S scientific agenda. The AMC Operations and Data Management Center (AMC ODMC) provides data management and site management support for both domestic and international AMC trials. Over the past 7 years, the AMC ODMC has supported 3 trials in SSA and identified a number challenges to trial implementation and activation. P231 Reducing under- and over-triage in motor vehicle crashes using an injury-based approach Jennifer Talton1, Ashley A. Weaver2, Ryan T. Barnard2, Samantha L. Schoell2, Joel D. Stitzel2 1Wake Forest School of Medicine; 2Virginia Tech-Wake Forest University Center for Injury Biomechanics Correspondence: Jennifer Talton Trials 2017, 18(Suppl 1):P231 Reducing under- and over-triage in motor vehicle crashes using an injury-based approach 1Wake Forest School of Medicine; 2Virginia Tech-Wake Forest University Center for Injury Biomechanics Correspondence: Jennifer Talton Trials 2017, 18(Suppl 1):P231 Advanced Automatic Crash Notification (AACN) systems aim to re- duce both over- and under-triage from motor vehicle crashes (MVC) by using vehicle telemetry data to predict risk of serious injury and thus aiding first responders in the triage decision making process. Reducing under-triage (UT) translates into transporting severely in- jured occupants to a level-I or II trauma center (TC) and reducing over-triage (OT) means transporting occupants with lesser injuries to a non-trauma center (non-TC). Treating more severely injured occu- pants initially at tcs reduces death and disability, and treating occu- pants with minor injuries at non-tcs leads to better hospital resource utilization and decreased healthcare costs. Conclusion Seonaidh Cotton, Karen Innes, Joanna Kaniewska, Mark Forrest, Graham Devereux University of Aberdeen Correspondence: Seonaidh Cotton Trials 2017, 18(Suppl 1):P233 Conclusion The key challenges the AMC ODMC faced in implementing these trials included identifying research priorities, developing multicenter trials that are appropriate across a diverse group of trials sites, con- ducting clinical research trials within the public healthcare system, in- adequate infrastructure, availability of qualified staff, and identifying and addressing site logistical barriers such as drug and supply needs. Furthermore, the importance of supporting capacity-building activ- ities such as training of health care staff at the research sites is part of the AMC’S mandate in SSA and requires additional site manage- ment support. Currently, there are 2 open trials and 4 trials in ex- pected to open within the next 18 months across 7 sites in Sub- Saharan Africa, including sites in Zimbabwe, Uganda, Kenya, Malawi, Tanzania and South Africa. Site management lessons learned from these trials may be applicable to other international trials and par- ticularly relevant to those designed for implementation in developing countries where both human and material resources may be limited. In order to estimate the need for transport to a TC or non-TC, current AACN systems model the risk of severely injured occupants using injury severity scores (ISS) as the outcome. ISS are a well- known measure based on the Abbreviated Injury Scale (AIS) coding lexicon where occupants with ISS > = 16 indicate severe injuries re- quiring treatment at a TC, while ISS < 16 may be treated at a non- TC. Our group has developed an AACN algorithm, the Occupant Transportation Decision Algorithm (OTDA), using an injury-based approach rather than AIS severity alone. We have identified three facets of injury that contribute to need for treatment at a TC: sever- ity, time sensitivity and predictability. Severity is a measure of an injury’s mortality, time sensitivity quantifies its urgency and predict- ability is its likelihood of being missed upon evaluation by first re- sponders at the scene. These three components are then jointly optimized to create a list of 240 injuries, each with a yes/no indica- tor of being on the Target Injury List (TIL). We believe that the TIL gives a better picture of the extent of injury severity and need of treatment at a TC or non-TC. Efficiencies in multi-centre RCTs - What lessons can be learned from a trial with more than 100 recruitment sites? Background The OTDA was implemented using data from National Automotive Sampling System-Crashworthiness Data System 2000–2011, which in- cluded 38,970 cases. The OTDA uses multivariable logistic regression to predict the risk of an occupant sustaining an injury on the TIL for specified crash conditions. In addition to using an injury-based ap- proach for modeling the risk of severely injury occupants, another novel feature of the OTDA compared to other AACN systems is that the OTDA uses a genetic algorithm to optimize each of the compo- nents and determines the risk threshold for the decision to transport to a TC or non-TC. The goal of the optimization was to minimize UT and OT, ideally producing UT rates < 5% and OT rates < 50% as rec- ommended by the American College of Surgeons (ACS). We have recently completed recruitment to a large multicentre trial with recruitment sites in both primary and secondary care. Opening a recruitment site is a substantial amount of work: it requires ap- provals to be in place; a site agreement to be signed by all parties; site initiation/training; copies of CVs, GCP certificates and a com- pleted delegation log to be returned to the study office; for the site to have received a site file and study documentation; and in this study, an estimate from the site of number eligible patients to be invited. As in previous studies, spreadsheets were maintained to log informa- tion about contacts, documents returned, CVs and GCP certificates, such that a green-light form could be populated for sign off prior to opening the site. However, given the number of recruitment sites in- volved, this logging of information was very time-consuming. We reviewed our processes, and, where possible, have implemented al- ternative processes that are likely to generate efficiencies in recruit- ment site set-up. These are described below. Results of the OTDA produced UT rates ranging from 3-16% depend- ing on the crash mode and OT rates meeting the ACS 50% recom- mendation. The OTDA also showed improved UT rates compared to other AACN algorithms in literature. We believe the OTDA will aid emergency personnel to make the correct triage decision for an oc- cupant after a MVC. With nation-wide implementation, we estimate a potential benefit of improved triage decision-making for 165,000 oc- cupants annually. Discussion Adjustment for biases is useful in meta-analyses synthesizing all available evidence. We recommend an integrated approach to bias Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 87 of 235 trials whose primary outcome was the same. The results were consistent after adjustments for type of outcome, type of study design, post-randomisation exclusions, and variance of log odds ratio (0.79 (0.69 to 0.92), p = 0.0019). Challenges in implementing and managing clinical trials in developing countries: lessons learned by the national cancer institute’s (NCI) AIDS malignancy consortium (AMC) Megan Wirth, Dikla Blumberg, Kimberly Mosby-Griffin, Don Vena Emmes Corporation Correspondence: Megan Wirth Trials 2017, 18(Suppl 1):P232 Challenges in implementing and managing clinical trials in developing countries: lessons learned by the national cancer institute’s (NCI) AIDS malignancy consortium (AMC) Megan Wirth, Dikla Blumberg, Kimberly Mosby-Griffin, Don Vena Emmes Corporation Correspondence: Megan Wirth Trials 2017, 18(Suppl 1):P232 Conclusion Sites opened In total, 175 recruitment sites were identified (36 secondary care sites and 139 primary care sites). 141 sites were opened to recruitment Page 88 of 235 Trials 2017, 18(Suppl 1):200 Page 88 of 235 (36 secondary care sites and 105 primary care sites). Of the 36 sec- ondary care sites opened to recruitment, 31 recruited and 5 did not. Of the 105 primary care sites opened to recruitment, 86 recruited participants and 19 did not. The review of reasons for suspension identified five main themes: drug/intervention issues (drug/intervention safety issues, drug/inter- vention supply issues); trial evaluation (futility, review of trial/changes to protocol); funding (funding issues); recruitment (primarily slow ac- crual, but some cases of rapid accrual); and running of the trial (oper- ational issues, staffing, wider organisational issues). The proportions of trials suspended/stopped for each of these reasons differed be- tween the two trial registers. 19% of those suspended on clinical- trials.gov had been suspended because of drug/intervention safety issues compared to 6% of ISRCTN. The proportions suspended/ stopped for the other reasons are: trial evaluation 29% in clinical- trials.gov vs 18% in ISRCTN; funding 17% vs 31%; recruitment (20% vs 36%), running of the trial (15% vs 9%). There were a number of reasons why sites did not open to recruit- ment. The most common reason was that the site did not return documentation to the study office (CVs, GCPs certificates, delegation log, site agreement). A few sites actively withdrew from the study be- fore being green-lighted due to staff changeover or perceived lack of eligible patients. Once opened, some sites failed to recruit any patients to the study. Reasons for this included staff changeover and lack of eligible pa- tients, competing priorities and eligible patients who did not agree to take part. Background Background Randomised controlled trials (RCTs) of therapy interventions are be- coming increasingly common, and provide a series of challenges. Here we discuss our experience from the PD COMM Pilot (A Pilot Randomised Controlled Trial Of Lee Silverman Voice Treatment Ver- sus Standard NHS Speech And Language Therapy Versus Control In Parkinson’s Disease) trial. Problems with speech or voice are common with people with Parkinson’s (PWP). Miller (2006) noted how changes in communication led to increased physical and mental demands during conversation, an increased reliance on family members and/ or carers, an increased likelihood of reduced participation and social withdrawal. Two types of speech and language (SLT) therapy are available to PWP: NHS SLT an individually tailored intervention of ~6- 8 sessions per local practice and Lee Silverman Voice Treatment (LSVT) a structured set of 16 sessions over 4 weeks focussed on vol- ume. There is little evidence that either is effective. PD COMM Pilot examined the feasibility of a full scale trial, and to optimise the de- sign. Intervention-based issues and solutions are discussed below. Recruitment g Seonaidh Cotton, Chloe Brooks, Lynda Constable University of Aberdeen Correspondence: Seonaidh Cotton Trials 2017, 18(Suppl 1):P234 A reflection on the management of a trial of speech and language therapy 1 1 1 1 1 y Caroline Rick1, Carl E. Clarke1, Natalie Ives1, Smitaa Patel1, Rebecca Woolley1, Francis Dowling2, Lauren Genders1, Christina H. Smith3, Marian C. Brady4, Keith Wheatley1 1University of Birmingham; 2Cambridge University; 3University College, London; 4Glasgow Caledonian University 1University of Birmingham; 2Cambridge University; 3University College, London; 4Glasgow Caledonian University Correspondence: Caroline Rick Trials 2017, 18(Suppl 1):P235 ; g y Correspondence: Caroline Rick Trials 2017, 18(Suppl 1):P235 y Correspondence: Caroline Rick Trials 2017, 18(Suppl 1):P235 Background Within our trials unit two trials have recently been suspended, and then restarted. While there is a fairly mature literature on early ter- mination of studies, there is a paucity of literature about the tempor- ary suspension of studies. y We aimed to document reasons for trial suspensions using data avail- able on publically available registers of clinical trials (clinicaltrials.gov and ISRCTN). Lessons learned The observation that there are differences in the relative importance of reasons why trials are suspended/stopped may reflect the types of trials registered on the two registries. We identified potential for efficiencies in terms of logging informa- tion about sites and staff. Minimal information was already logged onto the study website (to register a site to enable randomisation and collection of study data and to maintain appropriate website ac- cess for site staff). For future studies, the website template has been amended to include: (i) an additional web form to log information about the site, including approvals in place, progress of site agree- ment, etc.; and (ii) a web form to log information about site staff, in- cluding CVs received, date of GCP training, along with the facility to upload CVs/GCP certificates onto the website. In addition, the web- site template facilitates the upload and storage of local documents. h l l k l l ff b We identified potential for efficiencies in terms of logging informa- tion about sites and staff. Minimal information was already logged onto the study website (to register a site to enable randomisation and collection of study data and to maintain appropriate website ac- cess for site staff). For future studies, the website template has been amended to include: (i) an additional web form to log information about the site, including approvals in place, progress of site agree- ment, etc.; and (ii) a web form to log information about site staff, in- cluding CVs received, date of GCP training, along with the facility to upload CVs/GCP certificates onto the website. In addition, the web- site template facilitates the upload and storage of local documents. Having such systems in place is likely to generate trial efficiencies: be time-saving for trial office staff, recording information in a single place and allow the green-light forms to be generated automatically; have capacity to run regular reports (for example progress reports on site set-up); and generate notifications for, for example renewal of GCP training. A number of those registered as ‘suspended’ on clinicaltrials.gov appeared to have been terminated early (with no intention of restarting) or completed rather than suspended. More guidance for those maintaining records on trial registries may aid the consistency of recording. p y Background Since tracking approvals is intended to protect participant data and ensure data was obtained and released to the DCC in an ethical manner consistent with regulatory oversight, we identified ways to innovate and simplify processes. Historically, tracking has been done on paper involving complex filing systems; however, technological advancements expanded options for executing this responsibility. Methods Clinicaltrials.gov define a ‘suspended’ study as a ‘study that has stopped recruiting or enrolling participants early, but may start again’. We searched clinicaltrials.gov for interventional studies which had their recruitment status recorded as ‘suspended': the search was run on 29 June 2016. ISRCTN do not have an equivalent term for sus- pended trials. The closest term is ‘stopped’ which includes studies that have never started along with those that have stopped prema- turely. We searched ISRCTN for interventional studies which had their status recorded as ‘stopped’: the search was run between 18 July and 12 August 2016. as the intervention was dependent on speech and language therapists (salts) being available to start therapy within 4–6 weeks, bottlenecks occurred e.g. School holidays, staff turnover. Good com- munication between the research nurses and salts was vital and sites were allowed to pause recruitment if salts were unable to start ther- apy within the trial timelines. While this slowed recruitment, 95% of 59 participants had received therapy by the 3 month primary end- point. Staffing: There were a number of potential issues 1. Did the level of experience of staff treating participants in the NHS and LSVT arms differ? 2. Does the beliefs of the salts? Regarding patient suit- ability for interventions or treatment preference impact the results? 3. Limited research experience of many salts. 17 therapists only saw 1 participant, 11 saw only participants in 1 arm and 8 saw partici- pants in both. The trial provided a supportive network for salts to ex- change information. These potential issues will be examined in more detail in the substantive trial where an in-depth process evaluation will also be performed. For each suspended trial, a code was assigned to each trial to classify the reason for the suspension. The coding framework was developed inductively and continually refined during the process of coding. Results 837 trials registered on clinicaltrials.gov had their recruitment status designated as suspended. 403 trials registered on ISRCTN were re- corded as ‘stopped’. It was not possible to identify the reason for sus- pension/stopping for 40% of those recorded as suspended on clinicaltrials.gov and 8% of those recorded as stopped on ISRCTN: either no reason was given, or the reason was not clear or ambiguous. Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 89 of 235 Results Ninety-one potential RBM tools were identified and 24 were eligible for inclusion. These tools were published between 2000 and 2015. Eight tools were paper based or electronic questionnaires and 16 op- erated as Service as a System (SAAS). Risk associated with the investi- gational medicinal product (IMP), phase of the clinical trial and study population were examined by all tools and suitable mitigation guid- ance through on-site and centralised monitoring was provided. Conclusion Intervention Tools and processes for tracking IRB approvals as a dcc for large multi-center clinical research networks Jenna Gabrio, Jeanette O. Auman, Lindsay M. Parlberg, Margaret M. Crawford, Kristin Zaterka-Baxter RTI International Correspondence: Jenna Gabrio Trials 2017, 18(Suppl 1):P237 Does the content of the interventions change over time? The trial kept treatment records and has explored the dose and content. The numbers of participants randomised to each treatment arm at individual sites were too small to test for changes over time, however this will be examined in the substantive trial. Logistic is- sues: Frequently the Trust providing the intervention was differ- ent to the recruiting site. This produced a number of issues e.g. Recognition including SFT funding is associated with recruitment not the treatment site. Further, the catchment areas of the Trusts may only partially overlap. In some cases, the only resolution was for sites to only recruit from a subset of potential participants dependent on treatment sites’ catchment area. Communication, support and recognition of different perspectives and priorities has built a research group that will form the basis of the sub- stantive trial: 10 of the 11 pilot sites were happy to participate in the substantive trial. multi-center clinical research networks Jenna Gabrio, Jeanette O. Auman, Lindsay M. Parlberg, Margaret M. Crawford, Kristin Zaterka-Baxter RTI International Correspondence: Jenna Gabrio Trials 2017, 18(Suppl 1):P237 p Results The development and implementation of a database increased ef- ficiencies both for ccs and DCC s. The processes reduced the vol- ume of email regarding IRB approvals sent to ccs. Individual emails to ccs notifying them an approval is about to expire are no longer necessary. Instead, a single monthly email is sent to all ccs indicating updated IRB reports have been posted and should be reviewed. Automated highlighting of approvals that will expire soon has also reduced the burden on the DCC coordinators and minimized the likelihood of oversight. The creation of a central database and formalized procedures have streamlined internal regulatory processes for DCC staff. If questions arise about an ap- proval for a specific CC, DCC staff can access the database to look up information needed. RBM tools for clinical trials are a relatively new, their features and use varies widely and they continue to evolve. This makes it difficult to identify the “best” RBM technique or tool. For example, equivalence testing is required to determine if RBM strategies directed by paper based and SAAS based RBM tools are comparable. Such research could be embedded within multi-centre clinical trials and conducted as a SWAT (Study within a Trial). Risk based monitoring (RBM) tools for clinical trials: a systematic review Caroline Hurley1, Frances Shiely2, Patricia Kearney2, Mike Clarke3, Joseph Eustace4, Evelyn Flanagan4, Jessica Power3 1University College Cork, Ireland; 2Department of Epidemiology and Public Health; 3Centre for Public Health; 4Health Research Board -Clinical Research Facility 1. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Integrated Addendum to ICH E6 (R1): Guideline for Good Clinical Practice, 2015. Available from: http://www.ich.org/fileadmin/Public_Web_Site/ ICH_Products/Guidelines/Efficacy/E6/E6_R2__Addendum_Step2.pdf. 2. Organisation for Economic Co-operation and Development (OECD). OECD Recommendation on the Governance of Clinical Trials, 2013 Methods Review of published and grey literature using a detailed search- strategy and cross-checking of reference lists. This review included academic and commercial instruments that met the Organisation for Economic Co-operation and Development (OECD) classification of RBM tools. Methods Programmers developed an in-house Microsoft Access database used to track receipt of IRB approvals for numerous studies from multiple ccs. The custom database has capacity to monitor approval expir- ation dates for an unlimited number of studies and ccs, and it can generate automated reports displaying information on all docu- mented approvals. The system has functionality to produce reports organized by individual protocol and/or CC, as well as the ability to highlight IRB approvals that must be renewed within the next three months. Coordinators formalized communication procedures for col- lecting updated approvals and informing ccs of the status of infor- mation currently on file. We established a central email account to which ccs submit updated documentation. Upon receipt of docu- mentation a DCC coordinator acknowledges delivery, files documen- tation, and enters updated information into the database. Monthly, a DCC coordinator generates automated individual center IRB reports and posts them to the research network private website. Ccs receive an email notification from the DCC and can then access their center reports through the website. Based on these reports ccs determine what documentation must be sent to the DCC to keep their records up to date. Correspondence: Caroline Hurley Trials 2017, 18(Suppl 1):P236 Correspondence: Caroline Hurley Trials 2017, 18(Suppl 1):P236 Objective A primary responsibility of Data Coordinating Centers (DCC) for multi-center research networks is tracking individual center IRB ap- provals. In networks with high numbers of studies and clinical cen- ters (CCs) the amount of documentation can be overwhelming and burdensome to manage. A team of DCC programmers and coordina- tors developed simple electronic tools and processes to fulfill this responsibility. Background In November 2016, the Integrated Addendum to ICH-GCP E6 (R2) will advise trial sponsors to develop a risk-based approach to clinical trial monitoring. This new process is commonly known as risk based mon- itoring (RBM). To date, a variety of tools have been developed to guide RBM. However, a gold standard approach does not exist. This review aims to identify and examine RBM tools. 1. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Integrated Addendum to ICH E6 (R1): Guideline for Good Clinical Practice, 2015. Available from: http://www.ich.org/fileadmin/Public_Web_Site/ ICH_Products/Guidelines/Efficacy/E6/E6_R2__Addendum_Step2.pdf. Background l l Keele Clinical Trials Unit (CTU), based within the Faculty of Medicine and Health Sciences at Keele University, is a UK Clinical Research Collaboration registered CTU specialising in the develop- ment and delivery of large multicentre clinical trials testing treatments and health services, as well as conducting large epi- demiological studies in primary and secondary care settings. Keele CTU supports the design, delivery and analysis of research studies. Keele CTU works closely with the Patient and Public Involvement and Engagement (PPIE) Team located within the Institute for Primary Care and Health Sciences at Keele University. The PPIE Team have a Research User Group (RUG) which consists of people with experience of, or carers of close relatives with, long-term con- ditions. The CTU Project Management team are pivotal in ensuring the success of research studies and work closely with the RUG to achieve this. p Results The RUG are involved in a variety of activities including assisting with grant application, intervention development, document de- sign, ethical approval, development of recruitment and retention strategies, patient simulation, quality assurance, study monitoring and dissemination of findings. To highlight the contribution and impact of the RUG two specific examples are presented in this abstract. The RUG played an active role in developing a ‘usual care’ leaflet for a trial of an intervention for hand osteoarthritis. Their contribution made the leaflet clear, practical and acceptable to patients. The RUG provided valuable ideas about how to ap- proach patients in a GP waiting room to enrol them into a study which involved video-recording a GP consultation. They made realistic and patient-centred suggestions for how this could be achieved ethically. References Although an initial investment is needed to design a database, de- velopment and formalization of these processes have resulted in significant time and cost savings throughout the organization’s ten- ure as a DCC. The flexible nature of an Access database makes it an efficient and suitable solution for tracking a growing number of studies in research networks that may have a fluid composition of centers over time. Trials 2017, 18(Suppl 1):200 Page 90 of 235 P239 A good use of time and money? A study of how trial data collection effort is distributed across different categories of data Gordon Fernie1, Katie Banister1, Suzanne Breeman1, Lynda Constable1, Anne Duncan1, Heidi Gardner1, Kirsteen Goodman2, Doris Lanz3, Alison McDonald1, Emma Ogburn4 1University of Aberdeen; 2Glasgow Caledonian University; 3Queen Mary University Of London; 4University of Oxford Correspondence: Gordon Fernie Trials 2017, 18(Suppl 1):P239 The role and impact of patient and public involvement and engagement (PPIE) in clinical research: perspectives from Keele CTU project management The role and impact of patient and public involvement and engagement (PPIE) in clinical research: perspectives from Keele CTU project management 1 1 1 2 A good use of time and money? A study of how trial data collection effort is distributed across different categories of data Gordon Fernie1, Katie Banister1, Suzanne Breeman1, Lynda Constable1, Anne Duncan1, Heidi Gardner1, Kirsteen Goodman2, Doris Lanz3, Alison McDonald1, Emma Ogburn4 1 2 3 Helen Myers1, Sarah A. Lawton1, Stefannie Garvin1, Steven Blackburn2 1Keele Clinical Trials Unit; 2Institute for Primary Care and Health Sciences Correspondence: Helen Myers Trials 2017, 18(Suppl 1):P238 Helen Myers1, Sarah A. Lawton1, Stefannie Garvin1, Steven Blackburn2 1Keele Clinical Trials Unit; 2Institute for Primary Care and Health Sciences Correspondence: Helen Myers Trials 2017, 18(Suppl 1):P238 Helen Myers1, Sarah A. Lawton1, Stefannie Garvin1, Steven Blackburn2 1Keele Clinical Trials Unit; 2Institute for Primary Care and Health Sciences Correspondence: Helen Myers Trials 2017, 18(Suppl 1):P238 g 1University of Aberdeen; 2Glasgow Caledonian University; 3Queen Mary University Of London; 4University of Oxford Correspondence: Gordon Fernie Trials 2017, 18(Suppl 1):P239 Methods We have developed a list of 16 data categories (e.g. Partici- pant identification, eligibility, demographics, health economics and safety data), along with guidance on what each category might contain. A standard operating procedure describes how to go through a trial’s data collection forms to categorise each collected data item. Data categorisation is done independently in pairs, one person having in-depth knowledge of the trial, the other independent of the trial. Any disagreement is re- solved through discussion, with the rest of the project team being brought in if necessary. Current work has focused on piloting our materials and method with three trials run from three different UK Trials Units. We will extend this work to fur- ther trials run from more Trials Units prior to the SCT/ICTMC conference. Background Data collection consumes a substantial portion of the resources used in any randomised trial. In addition to the participant identifier, the most important data collected is the primary out- come; it drives the sample size calculation and should be the main focus of the research effort. Most trials also collect sec- ondary outcomes to supplement the primary. These additional data are often collected to monitor safety, maintain quality and ensure regulatory and data management requirements are ful- filled. Many trials also collect outcome data not listed in the trial protocol. Adding ancillary and exploratory data collection can result in a substantial portion of a trial’s limited resources, in time, money and participant burden, being devoted to collecting data that are not directly linked to answering the research question. The cost of this is not trivial: a large US study of drug trials estimated that non-core data collection costs $3.7 billion annually. As part of the Trial Forge initiative (a systematic approach to making trials more efficient) here we describe our categorisation of the distribution of data collection effort in a range of trials. This abstract provides examples of the role and impact of PPIE in the conduct of research and presents perspectives from the Project Management team on PPIE contribution to the delivery of research studies. Results Our preliminary results suggest that trial teams spend more time collecting data than they do collecting outcomes: some- times less than 50% of the data collected is linked to primary and secondary outcomes. The largest single category is almost always secondary outcomes, which range between 28% and 52% in the three trials categorised to date. Primary outcome data ranges from 1.2% to 14.2%. Safety and regulatory data accounted for between 1.1% and 13%. In one of the three tri- als 2,530 data items were collected, 78.8% of which were mandatory. y The overall impact of the RUG involvement is captured in the follow- ing quotes from the Project Management team: “Valuable team members”, “Enhance research relevance”, “Unique contributions and viewpoints”, “Patient perspectives”, “Essential role”. Methods The RUG plays an essential role in each stage of research design and delivery, helping to ensure that the research is ethical and acceptable to research participants. We asked the Project Management team for examples of the ways in which involvement of the RUG had benefit- ted the studies they managed, and for their perceptions of the impact the RUG had on research. Responses were collated and orga- nised thematically to provide a description of PPIE contribution and its impact. Context OCTET was a randomised controlled trial investigating the effectiveness of low intensity interventions for the treatment of obsessive-compulsive disorder (OCD). Two trial managers coordinated the study. This included managing and coordinating personnel working across a variety of roles within the study - research assistants, clinical practitioners, site leads, and independent committee members. Learning from the OCTET trial - exploring acceptability of clinical trials management h d l1 d h ll l 2 g Catherine Arundel1, Judith L. Gellatly2 1University of York; 2University of Manchester Correspondence: Catherine Arundel Trials 2017, 18(Suppl 1):P241 Catherine Arundel1, Judith L. Gellatly2 1University of York; 2University of Manchester Correspondence: Catherine Arundel Trials 2017, 18(Suppl 1):P241 Background The ending of a clinical trial may be challenging, particularly if staff are required to withdraw the investigated treatment(s); however, this aspect of trial work is surprisingly under-researched. To address this gap, we explored the experiences of staff involved in closing out a trial which entailed withdrawal of treatment (insulin pumps) from some patients. Conclusion Organisation, openness, and positivity are crucial for executing a trial successfully, whilst clear and focused processes and resources are es- sential in monitoring and controlling the progress of a trial. Trial managers should therefore consider developing these elements when setting up a study. There is however, always room for improve- ment and the continued sharing of effective techniques will help to further evolve efficient trial management. p Conclusions p Methods Workshops, questionnaires and semi-structured interviews were used to explore acceptability of trials management methods with a spe- cific focus on the execution and monitoring of the study. Members of the OCTET Trial research team were asked to comment, both posi- tively and negatively, on their experience of the management, proce- dures, training, and their overall involvement in the trial. 9 members participated in the workshop, 10 completed a questionnaire and 20 were interviewed as part of qualitative work for the main OCTET study. Data was collected and analysed using thematic analysis, with the key phases of this approach adhered to. y Conclusions Our early results suggest that a substantial proportion of trial data is not outcome data. Primary outcomes accounted for less than 15% of all data collected; secondary outcomes were at least 3 times as many but in two trials represented over 20 times as much. Should the remaining trials in our study follow this pattern then, given the expense of collecting, storing and cleaning data, it suggests trialists should have an increased awareness of the burden and costs associated with adding data items to data collection forms. Regulators and others should bear in mind the burden their requirements may place on trial teams. Well managed, high quality research can provide evidence for best practice in diagnosis, treatment, management and prognosis to improve outcomes for patients. RUG involvement in research design and delivery forms an integral role in the pathway which provides the best evidence for both funders and clinicians, and contributes to the best care for patients. The Project Manage- ment team greatly value the views, opinions and suggestions made by the RUG. The personal experiences of those living with, or supporting those with, the research condition of interest, strengthens study design and greatly enhances the research rele- vance for the public. Trials 2017, 18(Suppl 1):200 Page 91 of 235 Page 91 of 235 benefit of others. For the wider research team, the key focus will always be on the execution and delivery of a study. We therefore evaluated the acceptability of clinical trials management, focusing on study execution and monitoring, as implemented in the NIHR HTA funded Obsessive Compulsive Treatment Efficacy Trial (OCTET). P240 Staff experiences of closing out a clinical trial involving withdrawal of treatment: qualitative study David White1, Julia Lawton2, David Rankin2, Jackie Elliott1, Carolin Taylor3, Cindy Cooper1, Simon Heller1, Nina Hallowell4 1University of Sheffield; 2University of Edinburgh; 3Sheffield Teaching Hospitals NHS Foundation Trust; 4University of Oxford Correspondence: David White Trials 2017, 18(Suppl 1):P240 Results Staff described a number of ethical and emotional challenges at close-out, many of which had been unforeseen when the trial began. A key challenge for staff was that, while patients gave their agreement to participate on the understanding that pump treat- ment could be withdrawn, they often found themselves benefiting from this regimen in ways they could not have foreseen. Hence, as the trial progressed, patients became increasingly anxious about withdrawal of treatment. This situation forced staff to consider whether the consent patients had given at the outset remained valid; it also presented them with a dilemma at close-out because many of those who had wanted to remain on a pump did not meet the clinical criteria required for post-trial funding. When deciding whether to withdraw treatment, staff not only had to take funding pressures and patient distress into account, they also found them- selves caught between an ethic of Hippocratic individualism and one of utilitarianism. These conflicting pressures and ethical consid- erations resulted in staff decision-making varying across the sites, an issue which some described as a further source of ethical un- ease. Staff concluded that, had there been more advanced planning and discussion, and greater accountability to an ethics committee, some of the challenges they had confronted at closeout could have been lessened or even prevented. Staff experiences of closing out a clinical trial involving withdrawal of treatment: qualitative study David White1, Julia Lawton2, David Rankin2, Jackie Elliott1, Carolin Taylor3, Cindy Cooper1, Simon Heller1, Nina Hallowell4 1 2 3 David White1, Julia Lawton2, David Rankin2, Jackie Elliott1, Carolin Taylor3, Cindy Cooper1, Simon Heller1, Nina Hallowell4 1 2 3 Background Conducting research can be a time consuming, difficult and challen- ging process. Guidance and pragmatic advice focusing on randomised controlled trial conduct are available but do not necessarily constitute comprehensive guidance. Standardised trial management tools, have previously outlined key elements constituting a successful trial as a method of ensuring good practice in research trials: initiation, planning, execution, monitoring, and analysis. Despite existing tools and guid- ance, lessons are also frequently learnt during the development and conduct of trials however rarely are these experiences shared for the Conclusions Th ki Recruitment estimates provided by sites - are they consistent with observed accrual? Christy Toms Alexa Gillman Clare Cruickshank Shama Hassan Emma Hall Recruitment estimates provided by sites - are they consistent with observed accrual? Christy Toms, Alexa Gillman, Clare Cruickshank, Shama Hassan, Emma Hall, Claire Snowdon, Judith Bliss, Rebecca Lewis The Institute of Cancer Research Clinical Trials & Statistics Unit (ICR-CTSU) Correspondence: Christy Toms Trials 2017, 18(Suppl 1):P242 The same kinds of ethical issues which may vex staff at the begin- ning of a trial (e.g. Patients having unrealistic expectations of trial participation; staff experiencing conflicts between research and clinical roles) may re-present themselves at the end. To safeguard the wellbeing of staff and patients, greater planning, coordination and ethical oversight should go into the close-out of trials involving withdrawal of treatment(s). Background Background Integral to the development of new trial proposals and a key consid- eration for funders is the assessment of feasibility of recruiting the planned sample size. Estimated accrual figures provided by sites are usually based on local clinical experience of the relevant patient population, data from internal audits of number of patients seen and previous experience of recruitment to trials. These estimates are used during trial development to inform trial design and plan recruitment timelines and as such have a substantial impact on the funding sup- port requested. We aimed to assess how close actual recruitment to- tals were to the estimates provided at the funding application stage to determine if evidence-based correction factors could be defined. Methods y p Results Six key themes associated with study execution and monitoring were identified within the data: support; communication; processes; re- sources; training and ethos. Clear and open communication and enthu- siasm and accessibility of the trial managers and Chief Investigator were noted across all themes as an important facet of the successful running of the trial. Clear resources and training materials were also found to be crucial in helping staff to work within the trial setting how- ever constructive suggestions were also made for improvement. Conclusion p Methods Interviews were conducted with n = 22 staff, recruited from seven trial sites. Data were analysed thematically. Methods Six oncology trials covering a range of disease sites and treatment modalities were selected from the ICR-CTSU portfolio. Individual sites’ estimated annual recruitment was compared with the average an- nual accrual observed. The proportion of sites which failed to open following initial expression of interest at funding application stage and number of sites which opened which were not included in the original funding application were also reviewed. Page 92 of 235 Trials 2017, 18(Suppl 1):200 Page 92 of 235 Conclusion The use of CPRD data enabled site recruitment efforts to be concen- trated on those practices with eligible patients close to the research site locations. The provision of CRPD data to pre-screen for patients meeting the study eligibility criteria reduced the amount of work required from GPs. There were however a significant number of screen-failures detected by GPs that were not covered by the search criteria, suggesting further improvements can be made to the search criteria to make this process more efficient. Through CPRD data we were able to successfully screen and recruit patients with COPD from GP practices within central London to participate in research over a 6 month period. This is an effective and novel method of using EHRs to screen and recruit participants for research. P243 P243 Using electronic healthcare records (I) to screen, locate and recruit participants from primary care Maimoona Hashmi1, Kirin Sultana1, Elizabeth Moore2, Jennifer Quint2, Mark Wright1 1Clinical Practice Research Datalink (Centre of the MHRA); 2Imperial College London Correspondence: Maimoona Hashmi Trials 2017, 18(Suppl 1):P243 Clinical Trials Units (CTUs) oversee a large resource of data and linked samples generated from clinical trials and have a duty to facilitate responsible sharing of these collections with the wider research com- munity. Sharing has the potential to improve scientific and medical knowledge, improve and validate research methods, encourage col- laboration and reduce duplication of effort. Sharing must take into consideration the scientific integrity of the original trial and the pro- posed research, the terms of the consent with which tissue and data were collected, relevant governance and regulatory requirements and the terms and conditions of the sponsors and funders of the original trial. Results One hundred & twenty two sites were on the funding applications of the six trials reviewed, representing 82 centres in total, some listed for >1 trial. Sites estimated they would recruit a total of 446 patients per annum. Of those which opened, only 7/77 (9%) exceeded their recruitment estimates. 8/77 sites (10%) recruited 0-40% less than predicted, 28/77 sites (36%) 40-80% less and 31 sites (40%) 80-100% less. Three sites did not provide recruitment estimates. Median percentage reduction between predicted and actual recruitment per site was 74% (inter quartile range 91 to 44). 45/122 of sites did not proceed to open the respective trial (37%). Of the sites which partici- pated in each trial, 48% were not originally included on the funding applications. These sites contributed an average of 17% of target ac- crual of the six trials. Over all trials, average observed annual accrual was 66% of that estimated by sites. Eighty-one practices were approached of which 24 (29%) con- sented to participate, resulting in a pre-screened list of 904 pa- tients. There were 314 screen-failures (35%) of which 55% were “unable/unsuitable” to participate in the study for reasons such as housebound, dementia and other co-morbidities; a further 29% of screen-failures were excluded for reasons associated with COPD diagnoses and exacerbations, lastly, 16% were either transferred out or deceased. 590 patients were invited of which 209 responded: 141/209 (67%) declined to participate and 61/209 (29%) agreed to participate. The main reasons for declining were: study too demanding (43%); not interested (14%); currently facing health issues (15%). Background Conduct of clinical studies traditionally involves study teams ap- proaching clinicians to screen and find potential study participants. This can be both time-consuming and labour intensive for clinicians and researchers. Leveraging Electronic Healthcare Records (I) as a re- source to locate and screen eligible study participants is often under- utilised but significantly reduces pre-screening activities. An example of the advantage of this method is illustrated in the following study investigating the association between air pollution and COPD exacer- bations using portable air monitors and symptom diaries. Using I data within the Clinical Practice Research Datalink (CPRD), we screened for eligible patients in primary care practices, based on the protocol inclusion/exclusion criteria. CPRD data are comprised of con- tinually provided anonymised UK electronic primary care records to enable clinical studies into improving public health. Clinical trials are conducted to provide a precise unbiased estimate of effect to inform the next trial and influence clinical practice. It’s imperative that the integrity of the trial is maintained until the pri- mary research questions have been answered. When considering a request for access to a specific trial cohort, CTUs consider: the clinical importance of the hypothesis; whether the hypothesis requires access to the specific trial collection; whether the relevant data or tis- sue are held and are of sufficient quality and quantity; the statistical validity of the proposed research; whether the proposed research is validation or discovery; whether sharing would compromise the col- lection or reporting of the original trial; and whether there are oppor- tunities for collaboration. Conclusion Potential participating sites substantially overestimate accrual at the funding application stage. This has consequences for trial develop- ment as it impacts assessments of trial feasibility and planned re- cruitment period. Sites which express interest and then fail to open can also skew the recruitment estimates; however this appears to be mitigated to a certain extent by those sites which do not provide ex- pressions of interest at funding application stage but proceed to open the trial at a later date. Estimating projected trial accrual is challenging for sites and trials units. Options for improving recruit- ment estimations, including use of national electronic health records and documenting provenance of recruitment estimates (e.g. Local audits), should be considered. Our data suggests feasibility of accrual should be routinely reassessed per site once funding approval is confirmed. P244 Clinical trial data and tissue: considerations for responsible sharing Claire Snowdon, Emma Hall, Judith Bliss The Institute of Cancer Research Correspondence: Claire Snowdon Trials 2017, 18(Suppl 1):P244 P243 Methods There is a wealth of legislation and supporting codes of practice con- cerning the appropriate use of tissue and data collected from clinical trial participants. At the heart of this is the need to ensure appropriate informed consent and to protect participant confidentiality. The use of data and samples is limited by the scope of the consent and conditions of approval under they were originally collected. Data and tissue may be used without explicit consent if it is fully anonymised and the re- search has been approved by a research ethics committee. However fully anonymising data may be difficult to achieve and may even re- duce the utility of the collection for the intended purpose. Ethics com- mittees now accept the need to seek broad and enduring consent for future use of data and tissue. However advances in technologies and changes in societal expectations can make this difficult to achieve. The CPRD I database was interrogated to create a pre-screened list of patients located in practices close to the research sites in central London. The search engine used a study-specific validated codelist and algorithm. Using diagnostic codes alone, this algorithm had a Positive Predictive Value (PPV) of 86.5% which is improved slightly by including use of antibiotics and oral corticosteroids in previous 12 months and spirometry; spirometry was not used as a screening criterion within the I but was subsequently performed at the research site. Patients were excluded if they were either current smokers or aged < 35 years old. Participating GPs were provided with a pre-screened list from which to identify and select suitable patients to receive information about the study. Potential participants could contact the research team directly to be enrolled. Trials 2017, 18(Suppl 1):200 Page 93 of 235 Page 93 of 235 Tissue and data collected within clinical trials are of a high quality. They are collected mostly prospectively in a systematic and unbiased fashion and are well curated and documented. They are a precious resource and represent a considerable investment from those involved in the original trial including the clinical trial participants, investigators, CTU, trial oversight committees, funders and the sponsor. Their opinions, terms and conditions must be taken into account when considering proposals for data sharing. This can be managed through formal access policies, processes and agreements. q Conclusions Key factors contributing to statistical rigour, in a trials context, are discussed. These findings support the importance of 'inter-disciplin- ary' teamwork. Increased understanding of each other’s roles and more transparency in communication between statisticians, CDM members and healthcare professionals, is of critical importance to determining and communicating the clinical relevance of statistically significant findings. Optimising trial recruitment with well-designed screening log: experiences from the ROCS study 1 2 3 4 Correspondence: Marina Zaki Trials 2017, 18(Suppl 1):P246 Methods of ethics committees Joerg Hasford University of Munich Trials 2017, 18(Suppl 1):P245 The Cochrane Library Databases, Google Scholar, pubmed and Web of Science were explored using P(Population), E(Exposure) and O(Outcomes) search terms, with no restriction on years. Snowballing yielded Grey literature and international guidelines. Results University of Munich Trials 2017, 18(Suppl 1):P245 Background g Macleod et al. Criticized in 2014 that there is too much avoidable waste and too little value in biomedical research and identified sev- eral relevant issues (Lancet 2014:383:101–104). The literature discussed roles, responsibilities and rights of statisticians, but also of pis and CDM members - where the aforementioned trial statistical aspects are often a joint effort. Key barriers and facilitators to statistical rigour in trials were then identified from the literature. Objective Objective Similarly, statisticians have a responsibility to be knowledgeable about their therapeutic field of research, to be up-to-date with novel statistical approaches and have a firm understanding of trial method- ology. Statisticians also have a responsibility to ensure final study re- ports are a 'fair reflection' of trial findings. Some authors also call for statisticians to be recognized as ‘full-collaborators’ in the decision- making aspects of trials, and maybe even as a ‘co-investigator’. 'Barriers' to trial statistical rigour include: lack of availability to statis- tical expertise, timing and workloads and not adhering to regula- tions. Facilitators include: understanding clear roles of statisticians and CDM members in the oversight of certain procedures, adequate resources, qualifications and experience. l g y g 'Barriers' to trial statistical rigour include: lack of availability to statis- tical expertise, timing and workloads and not adhering to regula- tions. Facilitators include: understanding clear roles of statisticians and CDM members in the oversight of certain procedures, adequate resources, qualifications and experience. Ethics Committees can play an important role in improving the qual- ity of clinical trials re substance, content and methods. It seems that Ethics Committees are not yet sufficiently aware of their responsibil- ities in this context. In addition there should be no Ethics Committee any more without sufficient biostatistical expertise. P247 Optimising trial recruitment with well-designed screening log: experiences from the ROCS study Martina Svobodova1, Lisette Nixon2, Jane Blazeby3, Anthony Byrne4, Dougal Adamson5 1Cardiff University; 2Cardiff University, Centre for Trials Research; 3Centre for Surgical Research, School of Social and Community Medicine, University of Bristol; 4Cardiff University School of Medicine, Marie Curie Palliative Care Research Centre; 5Tayside Cancer Centre, Ward 32, Ninewells Hospital Correspondence: Martina Svobodova Trials 2017, 18(Suppl 1):P247 P245 Clinical trials: increasing value, reducing waste – the potential role of ethics committees Methods Factors relating to statistics and clinical data management (CDM) how- ever, are crucial to the planning, design, conduct, monitoring, analyses and reporting of trials. Reliable results from statistical analyses are im- perative to ensure confidence in the clinical interpretation of treat- ments. Some key trial statistical and CDM aspects include: choosing trial design, variables and outcomes, building databases, planning and implementing randomization schedules, sample size calculations, statis- tical monitoring and quality control, maintaining accurate statistical documentation, Source Data Verification, interim and statistical analysis and translating statistical results into clinically meaningful findings. One key aspect to ensuring this ‘statistical rigour’, is having competent and enthusiastic inter-disciplinary ‘Trialists’ - most notably: trial statisticians, data managers and principal investigators (PIs). g There is a balancing act between data sharing on one hand and pro- tection of the collection and those who contributed to the collection on the other. However sharing can and should be achieved ethically, legally and with scientific probity with appropriate considerations and controls. The objectives were to: 1) Develop an understanding of roles and re- sponsibilities of statisticians, PIs and CDM team members in order to 2) Better understand the barriers and facilitators to statistical rigour in clinical trials. Objective As in the European Union all clinical trials have to be reviewed and approved by a competent Ethics Committee prior to the start, it is time to check whether Ethics Committees can play a role re reducing waste and increasing value of clinical trials. A number of authors raise concerns of statisticians only being con- sulted after data collection - for analyses and reporting. It is strongly recommended however, to have skilled statisticians involved in the design and implementation, which may prevent statistical pitfalls during the trial. Errors in trial design, conduct and analysis may intro- duce bias and affect patient safety. Macleod et al., e.g. State that more than 50% of the studies are de- signed without reference to systematic reviews of already existing evidence. As experimentation and research with humans is ethically only legitimate if the knowledge is not yet available and is at the same time definitely needed, a systematic review should be an absolutely essential part of each application dossier sent to an Ethics Committee. Unfortunately, even the recently passed European Clinical Trial Regulation 536/2014 which specifies the content of the application dossier in Annex I does not require the submission of such a systematic review. Another relevant issue is according to Macleod et al. That adequate steps to reduce bias are not taken in more that 50% of the studies and that there are still too many trials with inaedequate statistical power. Examples and explanations for these flaws and the ethical problems involved will be presented. Conclusions Authors emphasize the importance of statistician involvement in 'teaching and learning'. They teach and inform colleagues about important statistical information and interpretation of trial results. Similarly, statisticians have a responsibility to be knowledgeable about their therapeutic field of research, to be up-to-date with novel statistical approaches and have a firm understanding of trial method- ology. Statisticians also have a responsibility to ensure final study re- ports are a 'fair reflection' of trial findings. Some authors also call for statisticians to be recognized as ‘full-collaborators’ in the decision- making aspects of trials, and maybe even as a ‘co-investigator’. Authors emphasize the importance of statistician involvement in 'teaching and learning'. They teach and inform colleagues about important statistical information and interpretation of trial results. Barriers and facilitators to statistical rigour in clinical trials - emerging themes from the literature Marina Zaki1, Eilish McAuliffe2, Marie Galligan3 1 Marina Zaki1, Eilish McAuliffe2, Marie Galligan3 1School of Nursing, Midwifery and Health Systems, University College Dublin & Health Research Board - Trials Methodology Research Network; 2School of Nursing, Midwifery and Health Systems, University College Dublin; 3School of Medicine and Medical Sciences, University College Dublin Background g Rigorous clinical trial methodology is dependent on a number of fac- tors, including but not limited to: appropriate team communication, funding and compliance with ethical, legal and regulatory frameworks. Correspondence: Martina Svobodova Trials 2017, 18(Suppl 1):P247 Trials 2017, 18(Suppl 1):200 Page 94 of 235 Page 94 of 235 Objectives TIDIER id TIDIER guidance is an extension of CONSORT 2010 and SPIRIT 2013 statements and aims to improve intervention reporting [1]. Our ob- jective was to use tidier to identify and reduce the uncertainties about the design of a complex behaviour change intervention prior to a feasibility randomised controlled trial to reduce obesity in men. Background Correspondence: Garry Meakin Trials 2017, 18(Suppl 1):P248 Correspondence: Garry Meakin Trials 2017, 18(Suppl 1):P248 Opportunities and pitfalls encountered when using the template for intervention description and replication (TIDIER) to develop a complex intervention to reduce obesity in men dd 1 h b k 1 d l2 Mock activation of a pandemic influenza clinical trial: testing for rapid recruitment Garry Meakin1, Clare Brittain1, Lelia Duley1, Wei Shen Lim2 1Nottingham Clinical Trials Unit; 2Nottingham University Hospitals NHS Trust Correspondence: Garry Meakin Trials 2017, 18(Suppl 1):P248 Mock activation of a pandemic influenza clinical trial: testing for rapid recruitment 1 1 1 2 Garry Meakin1, Clare Brittain1, Lelia Duley1, Wei Shen Lim2 1Nottingham Clinical Trials Unit; 2Nottingham University Hospitals NHS Trust Garry Meakin1, Clare Brittain1, Lelia Duley1, Wei Shen Lim2 1Nottingham Clinical Trials Unit; 2Nottingham University Hospitals NHS Trust Methods ROCS study screening logs were initially designed as a two-stage paper form. The first form included all patients requiring stent insertion, and then those potentially eligible were copied across onto a 2nd stage screening form. Research nurses were involved in the re-design of the screening logs at regular face to face ROCS Nurses Meetings. Modifica- tions were made to criteria and subsequently the two forms were com- bined onto one Excel sheet. Nurses were advised to include all patients receiving an oesophageal stent for palliative reasons. Reasons for de- clining the study and for ineligibility could be selected from drop-down options. Completion electronically allowed less writing and nurses could email back the results weekly. Summary data was presented to the ROCS TMG regularly. Derby Royal Hospital was chosen as the target site given its close proximity to the coordinating centre and established links with the trial team. The site was alerted to activation via a “Declaration of Activation” letter which provided detailed information on the actions required to receive the “green light” for recruitment of a “patient” (volunteer) to commence. The mock activation allowed for the assessment of: 1) Investigational Medicinal Product (IMP) manufacture, labelling and supply proce- dures 2) training material for local investigators and site staff on trial processes, 3) data management processes, 4) channels of communi- cation between the coordinating centre and the mock activation site and 5) the recruitment pathway. ) Results After implementation, feedback from nurses was very positive, with 100% return rate, following reminders in some cases. The ongoing personal contact with individual sites improved their engagement with the study. Early in the trial, screening logs clearly demonstrated to the funders that the predicted number of patients requiring stent insertion was correct, and that 63% acceptance rate of the trial was above the 50% initially predicted. The reason for lower than expected recruitment was due to low eligibility of 26% against the original esti- mate of 70%. The main reason for ineligibility was patients identified only after the stent was inserted; 14% of patients were ineligible owing to this reason. The TMG provided this as evidence to funder and sponsor as reason to change the trial design. Since the change to allow patients to be recruited after stent 55% of patients have been rando- mised post stent. A change was also made to the histology eligibility criterion, but the effect of this has not been seen yet as it was only implemented recently. Screening logs also highlighted low acceptance rates in some centres, which allowed the trial team to provide more ad- vice and support to these sites. One site improved acceptance rate from 35 to 50% following additional support. C l i The site was mock activated on 15th September 2015, with recruitment “green light” issued within 4 weeks, in conformity with trial targets. The mock activation of the trial provided reassurance to the trial team that trial processes and procedures were adequate for successful site activa- tion and recruitment, it also helped to highlight a number of potential areas in which trial processes could be improved. As a result changes are being made to the trial including minor amendments to the CRF and third party contracts, the streamlining of IT processes and increasing staff resource; this will help to further build trial resilience in the event of a pandemic, and reduce the burden of any queries generated by unclear processes at activation. An evaluation of the costs of conducting this mock activation will also be reported. Conclusion Comprehensive screening log data can be collected. This is useful to track proportions of incident patients that are eligible and randomised. Data also provide information about non-eligibility and non-participation to feedback to centres, the funders and TMG. Commitment of the study centres played a key role to in the screening data return. This was easier to achieve through direct en- gagement at the regular investigator meetings and acting accord- ingly on participating centres feedback. p Discussion Mock activation allowed trial processes to be tested and problems addressed before actual patient recruitment. Such activation may have wider relevance for streamlining trials where rapid recruitment is critical, or anticipated to be complex. Although mock activation has cost implications in time and resources, the investment may be worthwhile if it improves recruitment and trial conduct, improving trial efficiency. about the de to a feasibility Background Conducting a clinical trial during a public health emergency creates novel challenges to successful execution and requires an innovative approach to trial design. The ASAP trial (Adjuvant Steroids in Adults with Pandemic Influenza) has been set-up in advance of an influenza pandemic ready to be rapidly activated in such an event. All ap- provals have been obtained, and documents and materials necessary Opportunities and pitfalls encountered when using the template for intervention description and replication (TIDIER) to develop a complex intervention to reduce obesity in men Pat Hoddinott1, Stephan U. Dombrowski1, Marjon van der Pol2, Frank Kee3,Mark Grindle1, Cindy Gray4, Alison Avenell2, Michelle McKinley3, on behalf of the research team 1University of Stirling; 2University of Aberdeen; 3Queens University Belfast; 4University of Glasgow Trials 2017, 18(Suppl 1):P249 Opportunities and pitfalls encountered when using the template for intervention description and replication (TIDIER) to develop a complex intervention to reduce obesity in men Pat Hoddinott1, Stephan U. Dombrowski1, Marjon van der Pol2, Frank Kee3,Mark Grindle1, Cindy Gray4, Alison Avenell2, Michelle McKinley3, on behalf of the research team 1University of Stirling; 2University of Aberdeen; 3Queens University Belfast; 4University of Glasgow Trials 2017, 18(Suppl 1):P249 Background for the conduct of the trial have been prepared. Upon activation, the trial needs to recruit the first participant within 4 weeks, and to complete recruitment of 2200 participants at approximately 40 sites within the first pandemic wave of approximately 6 weeks. g Many RC trials struggle to reach their pre-specified sample size. Screen- ing logs offer an indication of eligibility and why patients do not enter the trials as well as providing required figures for the CONSORT dia- gram. The ROCS (Radiotherapy after Oesophageal Cancer Stenting) study is a pragmatic RCT of external beam radiotherapy in addition to stent versus stent alone in patients clinically assessed as requiring stent insertion for relief of dysphagia caused by oesophageal cancer. Aim Even with the best of planning, unexpected barriers and issues affecting recruitment and trial conduct often occur; this is particularly likely to be true of a trial to be conducted during an influenza pan- demic. As recruitment to ASAP must be completed within the first wave of the pandemic, it is important that key trial process have been tested, and adjusted if necessary, prior to activation of the trial and that the site activation plan is realistic and deliverable. We there- fore conducted and evaluated a mock site activation. Methods Re-design a screening log to provide data to optimise study design and recruitment. Use of routine databases to aid the design of multicentre surgical trials with length-of-stay as the primary outcome Olympia Papachristofi Linda Sharples Use of routine databases to aid the design of multicentre surgical trials with length-of-stay as the primary outcome Olympia Papachristofi, Linda Sharples London School of Hygiene and Tropical Medicine Correspondence: Olympia Papachristofi Trials 2017, 18(Suppl 1):P251 Results In Stage 1 a number of substantive changes to the design of the intervention were made, including the addition of a physiotherapy referral pathway and rapid access to suitable medications for neuro- pathic pain. Running the intervention in Stage 2 found that the as- sessment clinic was acceptable to patients and highlighted the need for some changes to the clinic processes, including the need for add- itional self-report screening tools and standardised radiographs. This work also informed development of a comprehensive training pack- age for Extended Scope Practitioners who would deliver the inter- vention during the trial. Stage 3 found that stakeholders understood the intervention and could see how the intervention would affect the nature of their own work. They were aware of the proposed ben- efits of the intervention for patients and were keen to engage with the new practices. With little literature on how best to develop successful complex inter- ventions that eventually translate into health service implementation, tidier guidance for reporting interventions provides a useful starting point. However, prospective development of guidance on intervention development may be preferable to retrospective approaches. Our study begins to systematically address the uncertainties and deci- sions involved to develop a complex intervention. This is necessary so that more interventions proceed to become successful trials, are implemented into policy and practice and have impact on health care outcomes. Background Complex surgical interventions are an indispensable part of modern healthcare and there is increasing recognition that novel procedures should undergo the same rigorous evaluation as other non-invasive treatments. However, the multi-component nature of surgery compli- cates evaluation. For instance, surgical procedures are delivered by multidisciplinary teams and thus their outcome may vary due to pa- tient characteristics, skill of the operators and the environment within which they are conducted. Recognition and accommodation of this variation is important in order to design adequately powered related trials. The duration of postoperative Length-Of-Stay (LOS) (or incidence of prolonged hospitalisation) has been the focus of many surgical trials as it is a principal driver of surgical costs, and acts as a surrogate for a range of post-operative complications. Therefore it is Correspondence: Vikki Wylde Trials 2017, 18(Suppl 1):P250 Results Some intervention features had no evidence to inform a decision, so we undertook a primary survey, qualitative research and PPI. Other intervention features e.g. Behaviour change components had inform- ative data from studies of varying quality which generated hypotheses. A team decision was made about whether further primary research data was required or whether PPI and expert opinion would suffice. Some intervention features, particularly those relating to sustainability (e.g. Website, text messages) and future implementation (who delivers), had a strong underpinning logic which was considered sufficient for the team to make a decision. Tidier helped to focus on the decisions to be made. Limitations became apparent in relation to the intervention context, delivery [3] and how the loose categories for intervention features could be interpreted differently. Pragmatic decisions were sometimes required due to limits in funding, time and staff availability. Conclusions p Conclusions We have undertaken a comprehensive programme of research to refine the design and development of a complex intervention prior to evaluation in a randomised trial. Our study provides an example of the methods that can be used to address key questions within intervention design in a relatively tight timeframe. The next stage is to evaluate the clinical and cost-effectiveness of the intervention in a definitive multi-centre randomised trial, which will include an internal pilot phase. Methods Three stages of work were undertaken over a 12 month period. To develop the intervention, the first stage involved consensus ques- tionnaires with 22 health professionals about the appropriateness of individual components within a draft care pathway intervention. Mean appropriateness ratings were calculated and discussed at meetings with 18 healthcare professionals. To refine delivery of the intervention and assess whether it was acceptable to patients, Stage 2 involved scrutiny of the trial intervention with 10 patients who attended an assessment clinic. Stage 3 involved 10 health pro- fessional stakeholders to evaluate their views about implementa- tion potential of the intervention using a questionnaire based on the nomad instrument. Methods We considered the literature on i) complex intervention development methods ii) behaviour change theory (psychological and economic); iii) systematic review evidence about weight loss; iv) health inequalities; v) the acceptability to men and the public for similar interventions. With Public Patient Involvement (PPI) and expert opinion, these sources were used to populate the tidier checklist. We then decided how to fill the gaps as robustly as possible in order to produce a replicable interven- tion manual. y Background Intervention development is seldom reported and can be viewed as a black box [2]. Many interventions either do not result in a successful trial or are not implemented and therefore do not impact on health outcomes. This contributes to considerable research waste. Carefully Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 95 of 235 Page 95 of 235 designed interventions that are desirable, acceptable, feasible and sustainable are required. In our study, intervention engagement and reach are crucial because the prevalence of obesity in men is high, men infrequently engage with weight loss interventions and there are considerable health inequality consequences. designed interventions that are desirable, acceptable, feasible and sustainable are required. In our study, intervention engagement and reach are crucial because the prevalence of obesity in men is high, men infrequently engage with weight loss interventions and there are considerable health inequality consequences. developed a complex intervention comprising a novel assessment clinic and onward referral pathway for patients reporting moderate-severe pain at 3 months after knee replacement. The initial development of the intervention was informed by a systematic review, survey of NHS service provision, qualitative work with health professionals, consensus meetings with pain experts and patient and public involvement activ- ities. The aim of this work was to refine the design and delivery of this intervention before evaluation in a randomised trial, in keeping with the Medical Research Council’s recommendations for complex interven- tion development. developed a complex intervention comprising a novel assessment clinic and onward referral pathway for patients reporting moderate-severe pain at 3 months after knee replacement. The initial development of the intervention was informed by a systematic review, survey of NHS service provision, qualitative work with health professionals, consensus meetings with pain experts and patient and public involvement activ- ities. The aim of this work was to refine the design and delivery of this intervention before evaluation in a randomised trial, in keeping with the Medical Research Council’s recommendations for complex interven- tion development. Development of a complex intervention for patients with chronic pain after knee replacement 1 2 2 1 Vikki Wylde1, Nicholas Howells2, Wendy Bertram2, Andrew Moore1, Julie Bruce3, Candy McCabe4, Ashley Blom2, Jane Dennis1, Amanda Burston1, Rachael Gooberman-Hill1 1 2 3 Vikki Wylde1, Nicholas Howells2, Wendy Bertram2, Andrew Moore1, Julie Bruce3, Candy McCabe4, Ashley Blom2, Jane Dennis1, Amanda Burston1, Rachael Gooberman-Hill1 1University of Bristol; 2North Bristol NHS Trust; 3University of Warwick; 4University of West of England 1University of Bristol; 2North Bristol NHS Trust; 3University of Warwick; 4University of West of England References 1. Hoffman TC et al. Better reporting of interventions: template for intervention description and replication (tidier) checklist and guide. BMJ 2014;348:g1687. 1. Hoffman TC et al. Better reporting of interventions: template for intervention description and replication (tidier) checklist and guide. BMJ 2014;348:g1687. 2. Hoddinott P. A new era for intervention development studies. Pilot and Feasibility Studies. 2015; 1:36. 3. Dombrowski S, et al. Form of delivery as a key “active ingredient” In behaviour change interventions. 2016. B J Health Psychology (in press). P252 P252 Process evaluation for the PREPARE-ABC study: context mapping, pinchpoints and implications for implementation and theoretical fidelity Jamie Murdoch1, Anna Varley1, Jane McCulloch1, John Saxton2, Erika Sims1, Jennifer Wilkinson1, Megan Jones1, Juliet High1, Allan Clark1, Sue Stirling Process evaluation for the PREPARE-ABC study: context mapping, pinchpoints and implications for implementation and theoretical fidelity 1 1 1 2 p g Conclusions The value of context mapping is that we can predict areas of vulner- ability prior to intervention delivery, then make recommendations for adapting flexible elements of the intervention during implementa- tion. In addition, we can target and observe key pinchpoints as they are enacted, thereby offering opportunities for exposing the ‘active ingredients’ of interventions in action and providing insights into im- plementation and theoretical fidelity. An application of the methods in cardiac surgery, one of the most expensive yet widely used surgery types, is presented using a cohort of more than 100,000 consecutive case series patients from ten UK specialist centres. The implications of the results for the design of re- lated trials are also discussed. Process evaluation for the PREPARE-ABC study: context mapping, pinchpoints and implications for implementation and theoretical fidelity 1 1 1 2 Jamie Murdoch1, Anna Varley1, Jane McCulloch1, John Saxton2, Erika Sims1, Jennifer Wilkinson1, Megan Jones1, Juliet High1, Allan Clark1, Sue Stirling 1 2 Jamie Murdoch , Anna Varley , Jane McCulloch , John Saxton , Erika Sims1, Jennifer Wilkinson1, Megan Jones1, Juliet High1, Allan Clark1, Sue Stirling 2 1University of East Anglia; 2Northumbria University Correspondence: Jamie Murdoch Trials 2017, 18(Suppl 1):P252 1University of East Anglia; 2Northumbria University Correspondence: Jamie Murdoch Trials 2017, 18(Suppl 1):P252 p Results Data collection is ongoing at the time of submission. We will present findings from our current evaluation of standard care for patients pre and post-surgery for colorectal cancer, conducted prior to main trial recruitment. We will discuss what recommendations can be made from these findings for improving main trial implementation, using qualitative field notes from observations of pre and post-surgery con- sultations, and quantitative and qualitative data obtained through a telephone scoping exercise conducted at all colorectal units partici- pating in the study. l Background Process evaluations assess the implementation and sustainability of healthcare interventions within clinical trials, offering explanations for observed effects of trial findings and specifying the circumstances under which interventions are likely to succeed or fail. Such evalua- tions are particularly needed in trials of complex interventions which contain multiple interacting components. However, while theoretical models are available for evaluating intervention delivery within spe- cific contexts, there is a need to translate conceptualisations of con- text into analytical tools which enable the dynamic relationship between context and intervention implementation to be captured and understood. The aim of this article is to present the different types that have been detailed in the literature which aim to assess multiple out- comes in clinical trials, which are considered to be equally important in the assessment of the treatment effect. They include (i) co- primary outcomes and (ii) composite outcomes. We outline the challenges faced in the sample size calculation and the statistical analysis of co- primary outcomes, given different distributions and approaches of analysis. We also illustrate example of clinical trials where co-primary outcomes have shown treatment efficacy, which is not evident with a single primary outcome. For a composite outcome, we summarise the challenges faced in the analysis and reporting and interpretation of results. In addition, we illustrate the pitfalls and strengths of these approaches. g p Methods We start by exploring the variation between surgeons and anaesthetists separately, whilst adjusting for patient heterogeneity, using hierarchical (random effects) models. In order to estimate the contribution of differ- ent components of care and their interactions to variation in outcomes, a series of hierarchical models with cross-classifications is employed. Using the two most influential providers in the surgical treatment path- way, the surgeon and anaesthetist, for illustration, we show that key components of surgery do not necessarily follow a strict hierarchy e.g. Patients are nested within surgeons nested within centres, but surgeons are not nested within anaesthetists. We extend the proposed models to accommodate an additional Centre level in the hierarchy which introduces further variation due to infrastructure and policy differences. Potential drivers of between-centre variation are further examined through the incorporation of random coefficients. As there may be multiple components that contribute to extended LOS, we demonstrate how we can identify those which can be more effectively manipulated in order to standardise practice in trials. We examine the LOS both as continuous outcome, appropriately addressing its non- normality, and as a binary outcome (prolonged hospital stay). Results Aims This study aims to demonstrate how routine databases can be used to explore variation induced by patients, provider and centre practices in LOS outcomes, in order to inform surgical trial design and estimate key design parameters. h d Conclusions h l Ranjit Lall, Chen Ji The University of Warwick Correspondence: Ranjit Lall Trials 2017, 18(Suppl 1):P253 High-quality routine databases can be used to identify sources of variation in surgical care and outcomes. The resulting outputs can then be used to inform surgical trial design and analysis to ensure the robust and efficient analysis of intervention effects. For many decades, the randomised controlled clinical trial has been the gold standard of conducting research studies in health care. Its design and aims orientate around proving or disproving hypotheses based on the efficacy or safety of an intervention powered for a single primary outcome measure. However, in the awake of many medical techniques and devices, that reveal the complexity and depth of a disease, treat- ments such as complex interventions, are often assessed to obtain a comprehensive picture of these multiple manifestations. In order to support this, a single end-point will not provide sufficient information that is adequate in treatment assessment. The MRC complex interven- tion framework guidance (2008) states “Identifying a single primary out- come may not make best use of the data; a range of measures will be needed, and unintended consequences picked up where possible.” The choice of more than one primary outcome measure seems to be per- fectly plausible from a clinical view point, but statistically it presents many complexities. Background Over 70,000 primary total knee replacements are performed annually in the NHS. People choose to undergo knee replacement with the hope that surgery will improve their pain, but approximately 20% of people who have primary total knee replacement experience chronic pain afterwards. Our research has demonstrated that current UK NHS service provision for people with chronic pain after knee replacement is patchy and inconsistent. This reflects an absence of evidence about effective interventions and highlights the need to develop and evaluate inter- ventions to address chronic pain after knee replacement. We have Page 96 of 235 Page 96 of 235 Trials 2017, 18(Suppl 1):200 Page 96 of 235 important to understand how and why this outcome varies, so that rec- ommendations for trial design and analysis can be made. Traditionally, surgical trial design suffered from a lack of detailed multicentre data. The current availability of high-quality, routinely collected administra- tive databases allows us to explore current practice and outcomes, in order to inform trial design in this context. important to understand how and why this outcome varies, so that rec- ommendations for trial design and analysis can be made. Traditionally, surgical trial design suffered from a lack of detailed multicentre data. The current availability of high-quality, routinely collected administra- tive databases allows us to explore current practice and outcomes, in order to inform trial design in this context. technique involves: 1) prospectively mapping contextual features likely to affect intervention delivery; 2) using the mapping exercise to identify likely pinchpoints in delivery; and 3) analysing imple- mentation at the pre-identified pinchpoints during delivery. As an example, we will present ongoing work from PREPARE-ABC - a randomised controlled trial of suportive Exercise Programmes for Accelerating recovery after major abdominal Cancer surgery. PREPARE-ABC, funded by the NIHR, sponsored by Norfolk and Norwich University Hospitals NHS Foundation Trust and co- ordinated by the Norwich Clinical Trials Unit, University of East Anglia, is recruiting 20 hospitals and 1146 patients in the UK requir- ing surgery for colorectal cancer. Patients are randomised to one of three arms: hospital based supervised exercise; home based sup- ported exercise; or treatment as usual. Exploring automated free SMS from email in clinical trials Amarnath Vijayarangan Emmes Services Pvt Ltd. Trials 2017, 18(Suppl 1):P254 Short Message Service (SMS) is one of the basic functionality avail- able in all types of mobile phones. Research had shown that almost all SMS are getting read as soon as they reach. It is the easiest route to anyone to be notified as it does not require computer/internet. It is reliable and secured in all the situations as it is completely moni- tored and controlled by the mobile service providers. One might be surprised to know that SMS can be sent from Email to mobile num- ber at free of cost. Every mobile number is uniquely attached to an email address with the domain chosen by the mobile carriers. Every- one will be interested to make use of a feature if it is available at free and at the same time reliable. The proposed approach is exploring and automating the feature of sending SMS to mobile phones from Email to serve the following various activities in clinical trials. 1. Reporting/Notifying the stake holders about importance occurrences of events 2. Notifying the programming team about the current pro- gram status. These two activities are completely SAS data driven. In clinical trials, SAS is one of the widely used software for the data management, analysis and reporting since the clinical trial datasets are often available in SAS format. The analysis reports (Tables, List- ings & Figures) are generated using SAS programming language for the FDA submission. SAS programming language based automation is implemented for these two activities. 3. Engaging Study partici- pants 4. Reminder notifications to the project team on various activ- ities. These two activities are completely Microsoft Excel data driven. Excel is one of the Microsoft applications always available in all the computers and VBA is the language of Excel application. Using Excel VBA programming these tasks are automated. The data based automations are always developed using at least one programming language. In order to send the n number of SMS from Email without any manual intervention, we are making use of SAS and Excel VBA programming languages. The SMS can be triggered from Email at a scheduled interval or whenever certain criterion is met. Even though the whole process can be automated using only SAS programming language, we have come up with Excel VBA based automation as well since SAS is very expensive software and hence cannot be made to be available for all the project team members. g Conclusion Conclusion DMs at ICR-CTSU typically work on a combination of trials which may include both paper and EDC, requiring excellent time manage- ment skills and flexibility. Attention to detail, investigational skills and effective communication remain crucial, however the transition to EDC trials requires development of additional competencies and technical expertise in order to support DMs to produce and review programmed listings. P256 Using central statistical monitoring to drive quality into clinical trials Erik Doffagne Cluepoints Trials 2017, 18(Suppl 1):P256 Erik Doffagne Cluepoints Trials 2017, 18(Suppl 1):P256 Following encouragements from FDA guidance and the recent ICH E6 Addendum, many organizations are adopting RBM (Risk-based Monitoring). There is no single solution since RBM usually relies on a combination of on-site monitoring visits and central monitoring methods. CSM (Central statistical Monitoring) can play a major role in the RBM strategy in detecting investigational sites with atypical pat- terns in the collected data. Electronic data capture; changing data management at ICR-CTSU Charlotte Friend, Lisa Jeffs, Deborah Alawo, Leona Batten, Joanna Illambas, Judith Bliss, Emma Hall, Claire Snowdon, Alexa Gillman, Rebecca Lewis The aim of this presentation is to share lessons learned and best practice in terms of integration of CSM within the clinical operation processes. In particular, emphasis will be given on how the outcomes from CSM can be utilized in order to drive on-site monitoring efforts and in identifying areas with potential risk. The Institute of Cancer Research Correspondence: Charlotte Friend Trials 2017, 18(Suppl 1):P255 The Institute of Cancer Research Correspondence: Charlotte Friend Trials 2017, 18(Suppl 1):P255 Exploring automated free SMS from email in clinical trials Amarnath Vijayarangan Emmes Services Pvt Ltd. Trials 2017, 18(Suppl 1):P254 As Excel VBA is available in everyone’s computer the VBA based proposed approach can be utilized for various activities even if SAS is not available. This proposed approach has wide range of cost effective applications which can be quickly leveraged to perform various activities depend- ing on the study requirement. Methods In this paper we propose an alternative approach to the design, im- plementation and analysis of process evaluations for complex health interventions through a process of ‘context mapping.’ This innovative Page 97 of 235 Page 97 of 235 Page 97 of 235 Trials 2017, 18(Suppl 1):200 checks are performed on all CRFs when the DMs at ICR-CTSU transcribe data into the database. In-built database validations flag discrepancies to DMs s during data entry. Resulting queries are docu- mented, printed and sent to sites periodically and several months can pass before query resolution occurs. Data entry of different forms at any one time means trends in errors in data reporting are not al- ways easily identified. In EDC trials, participating sites enter data into an EDC system. Database validations highlight potential discrepan- cies to sites at the time of data entry so that they can correct issues immediately, reducing the number of data queries required. DMs at ICR-CTSU receive daily automated emails listing newly completed electronic CRFs from the database tracking system. Manual review of specific forms is performed as required, for example, on trial entry forms and important safety and endpoint data. Only one form per patient can be open in the database for review at a time therefore DMs also use advanced data review software to programme checks which identify data discrepancies across forms for all trial partici- pants. DMs run programmed checks at a frequency determined by their priority, to systematically identify potential discrepancies. Data are reviewed in context, and queries are raised electronically. These are immediately available for sites to review and provide a response. Trends in data entry errors can be readily identified during review of the programmed checks. This allows specific data entry guidance tar- geting common errors to be provided to sites sooner and changes to the database or CRFs can be considered by the ICR-CTSU trial team earlier. Sites are therefore less likely to make the same errors during future data entry. P254 Exploring automated free SMS from email in clinical trials Amarnath Vijayarangan Emmes Services Pvt Ltd. Trials 2017, 18(Suppl 1):P254 g for all? for all? Liz Tremain1, Elaine Williams1, Tom Walley, CBE2 1NIHR Evaluation, Trials and Studies Coordinating Centre; 2University of Liverpool Correspondence: Liz Tremain Trials 2017, 18(Suppl 1):P257 The sharing and re-use of data for further hypothesis generation, interrogation and analysis is now universally recognised as a key principle in research. Furthermore there is acceptance that data gen- erated by public funding, through participation of patients and the P257 Managed access to NIHR-funded research data: an opportunity Managed access to NIHR-funded research data: an opportunity for all? Liz Tremain1, Elaine Williams1, Tom Walley, CBE2 1NIHR Evaluation, Trials and Studies Coordinating Centre; 2University of Liverpool Correspondence: Liz Tremain Trials 2017, 18(Suppl 1):P257 g ICR-CTSU introduced Electronic Data Capture (EDC) in 2012; this neces- sitated a revision of data management systems and processes. In trials using paper case report forms (CRFs) –“paper trials” – data managers (DMs) at ICR-CTSU manually check all paper CRFs during data entry. With the introduction of EDC, they now perform manual checks and additional programmed checks using data review software. Here we describe how data management has changed as a result of the transi- tion from paper to EDC trials. Implementation For paper trials, CRFs are received by ICR-CTSU and are entered onto the database by DMs. CRFs are stored chronologically in a patient folder, and are available for review at the time of data entry. Manual Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 98 of 235 public, should be put to maximum use by the research community and, whenever possible, translated to deliver patient benefit. The National Institute for Health Research (NIHR) is a major public funder of research in the UK, and is committed to transparency. The NIHR position on data sharing and access is an important factor support- ing this, with its standard research contract containing a clause re- garding data release for many years, and the NIHR Journals Library requiring a specific data sharing statement since 2015 for all its open access publications. Mechanisms and processes for sharing data have recently been subject to a great deal of global debate and dis- cussion. Although a consensus has now largely been achieved on the “Why” Aspect, early activity has been stalled to a certain extent by an inability to address the “How”, and provide suitable and af- fordable repositories to permit data sharing, discoverability and ac- cess. Against the backdrop of recent international developments, NIHR has reviewed its requirements and processes on access to data to ensure that NIHR activity was appropriately reflected and that funded researchers were supported. A number of initiatives fo- cused on or related to data sharing have been developed which have all built on the iom/NIH report ‘Sharing Clinical Trial Data’ published in 2015. Key areas include the publication of the ICJME proposal on data sharing following publication, proposals from the MRCT Center at Harvard for ‘Vivli’ (as a centre for Global Clinical Re- search Data), and the ongoing development of the Clinical Study Data Request (CSDR) system. In light of these developments, the NIHR is developing a revised position on the sharing of Anon- ymised Individual Participant Data (IPD) generated by NIHR-funded research and a managed-access system to support this. This seeks to build upon and strengthen NIHR activity in this area and initially includes; Confirmation that anonymised datasets from NIHR funded research should be available for further analysis wherever possible. NIHR data will be released via a ‘Managed Access’ System, subject to data use agreement. NIHR protocols should contain a ‘Data Shar- ing Plan’ which will be publically available. Implementation NIHR publications must include a data sharing statement/access link which clearly explain how data can be requested. The NIHR is aware of the demands placed on researchers in this area, and the need to retain a focus on this area. As a result it is noted that NIHR requirements and sup- port will need to evolve as the wider data agenda develops. Objectives Rosie Harris, Lauren J. Scott, Chris A. Rogers Clinical Trials and Evaluation Unit Correspondence: Rosie Harris Trials 2017, 18(Suppl 1):P258 Rosie Harris, Lauren J. Scott, Chris A. Rogers Clinical Trials and Evaluation Unit Correspondence: Rosie Harris Trials 2017, 18(Suppl 1):P258 To explore the feasibility of conducting a clinical trial of CHM within a primary care setting with particular reference to recruitment, refer- ral patterns, compliance, drop out rates, the relevance of outcomes measures, the QOL of participants, adverse effects, and differences between standardised/individualised remedies. To compare outcomes of duration and severity of acute UTIs, rates of re-infection, measuring acute and prophylactic antibiotic use, and evaluating long-term changes. These preliminary data may be used to inform a future, adequately powered, definitive study. Discussion Writing the code to produce the tables is straight forward to do. The program has been successfully used to produce data completeness reports in several multicentre RCTs. It has significantly reduced the time needed to prepare reports for study meetings and independent oversight committees and has removed the risk of transcription errors. The reports produced have been consistently well received. The program can easily be used for reporting on other key aspects of trial conduct not just completeness of data. Efficient routine cen- tral monitoring of trial conduct can serve to highlight issues early and help minimise risks to a trial. Background Efficient and timely monitoring of data throughout the conduct of randomised controlled trials (RCTs) is essential to ensure high quality data and robust results; monitoring may include sum- maries of recruitment, data completeness and data queries. Here we focus on efficient methods for monitoring complete- ness of trial data. Variations in the time herbs were taken will be explored with the outcomes as there is no definitive length of time recognised - it will vary from 4 to 16 weeks. Background Chinese herbal medicine (CHM) has a history of treating the symptoms of UTIs for >2000 years. In the UK UTIs are the commonest bacterial infection presented by women within Primary care. RUTIs have a signifi- cant negative effect on QOL, impact hugely on health care costs from outpatient visits, diagnostics and prescriptions. Current treatment of RUTIs relies heavily on antibiotics. Methods We have developed a Stata program that enables the user to simply and effectively monitor data completeness rates. The program allows the user to look at the overall completeness of case report forms (CRFs) or at the completeness of individual data fields. To use the program, three variables must be specified: the variable to be sum- marised; an indicator for the subjects to be included in the summary; and the variable by which the completeness of the variable of inter- est is to be grouped. The program also handles conditional data (i.e. Where the requirement for a response is conditional on the answer to a preceding question). The program directly outputs the results to Microsoft Excel, where they can be further manipulated if required. The generated output contains the number and percentage of en- tries with data present and the number and percentage of entries with data missing, by group and overall. A pragmatic, double blinded randomised controlled feasibility study in- volving 4 groups of 20 women, using standardised or individualised CHM for RUTIs in Primary care and traditional care. Women with a history of RUTIs will be identified by medical record searches and in- vited to participate. Within the Wessex, Western and Peninsula regions allocation will be to the standardised arm of the trial (Primary care). Women from London and Hove will be allocated to individualised CHM treatment. MHRA approval was needed for the standardised arm but not the individualised arm. Results An example of the basic output (for one group). In this example, CRF A1 is present for 121 study participants in group 1 (95.3%) and miss- ing for 6 participants in this group (4.7%). g p p g p Discussion y Method y Method Methods P259 P259 Chinese herbal medicine (CHM) for recurrent UTIs in women, a feasibility trial Kim Harman1, George Lewith2, Felicity Bishop3, Beth Stuart 4, Andrew Flower2 1University of Southampton; 2University of Southampton, Complementary and Alternative Health; 3University of Southampton, Faculty of Social, Human and Mathematical Sciences; 4University of Southampton, Primary Care & Population Sciences Correspondence: Kim Harman Trials 2017, 18(Suppl 1):P259 p Disclaimer This work was supported by the National Institute for Health Research (NIHR) Health Technology Assessment (HTA) programme and by the NIHR Bristol Biomedical Research Unit in Cardiovascular Disease at University Hospitals Bristol NHS Foundation Trust. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. P261 P261 Value of a sister observational cohort alongside a randomised controlled trial with an internal feasibility phase Janet Dunn1, Andrea Marshall1, Maria Ramirez1, Andy Evans2, Peter Donnelly3 1Warwick Clinical Trials Unit, University of Warwick; 2Ninewells Medical School; 3South Devon Healthcare NHS Foundation Trust Correspondence: Janet Dunn Trials 2017, 18(Suppl 1):P261 One option, suggested in the literature, is to use the implied sample size for the maximal negative correlation between the two tests, thus, giving the largest possible sample size. However, this overly conservative technique is highly likely to be wasteful of resources and unnecessarily burdensome on trial participants - as the trial is likely to be overpowered and recruit many more participants than needed. A more accurate estimate of the sample size can be deter- mined at a planned interim analysis point where the sample size is re-estimated - thereby incorporating an internal pilot study into the trial design, with the intention of producing an accurate estimate of the correlation between the tests into the trial. Observational cohorts alongside randomised controlled clinical trials can be very informative. They enable an assessment of the number of patients that do not want to be randomised and reasons for non- participation in the randomised controlled trial (RCT). They also allow investigation of what is standard practice at each site. They can be ex- tremely useful in hard to recruit trials to gain information about the outcomes of these patients in standard clinical practice. Mammo-50 is a multi-centre RCT of different mammographic surveillance schedules for breast cancer patients aged 50 years or older at diagnosis. A total of 5000 patients are randomised to annual surveillance versus 2-yearly for conservation surgery and 3-yearly for mastectomy patients. There was a 24 month pre-planned internal feasibility study assessing recruitment, acceptability to be randomised and logistical endpoints which in- cluded a sister observational cohort. The aim of the cohort was to assess standard practice for non-randomised patients in terms of information given to patients, type of follow-up at each centre and frequency of mammographic surveillance. During the 24 month feasibility phase of the trial, 1354 patients were enrolled into the study; 936 (69%) patients choosing to participate in the RCT and 418 (31%) patients were recruited into the sister observational cohort study. The main reason for not going into the RCT but being part of the observational cohort was patient choice. A qualitative feasibility study to inform fluids in shock (FISH) - a pilot randomised controlled trial of fluid bolus therapy in septic shock P262 Internal pilot sample size re-estimation in paired comparative diagnostic accuracy trials with a binary response Gareth McCray1, Andrew Titman2, Paula Ghaneh3, Gillian Lancaster1 1Keele University; 2Lancaster University; 3University of Liverpool Correspondence: Gareth McCray Trials 2017, 18(Suppl 1):P262 pilot randomised controlled trial of fluid bolus therapy in septic shock Caitlin O'Hara1, David Inwald2, Ruth Canter3, Paul Mouncey3, Mark D. Lyttle4, Simon Nadel2, Mark Peters5, Kerry Woolfall1 1The University of Liverpool; 2St Mary’s Hospital, Imperial College Healthcare NHS Trust; 3Intensive care national audit & research centre (ICNARC); 4Emergency Department, Bristol Royal Hospital for Children; 5Institute of Child Health, University College London Correspondence: Caitlin O'Hara Trials 2017, 18(Suppl 1):P260 P261 Patients wanted to remain on their standard mammographic surveillance and didn’t want the possibility of changing regardless of whether the standard was more or less frequent. Cohort patients have simi- lar baseline patient characteristics to those entering the RCT, although the cohort did contain slightly more patients who had undergone mastectomy for their breast surgery. The feasibility phase demonstrated that the trial was acceptable by patients and clinicians but the ratio of patients entering the cohort compared to the RCT increased over the duration of the feasibility phase. The cohort demonstrated that standard practice regarding mammo- graphic surveillance and follow-up is highly varied across sites. In Methods This paper discusses a sample size estimation and re-estimation method based on the maximum likelihood estimates, under an im- plied multinomial model, of the observed values of correlation be- tween the two tests and, if required, prevalence, at a planned interim. The method is illustrated by comparing the accuracy of two procedures for the detection of pancreatic cancer, one procedure using the standard battery of tests, and the other using the standard battery with the addition of a PET/CT scan all relative to the gold standard of a cell biopsy. Simulation of the proposed method are also conducted to determine robustness in various conditions. Results The results show that the type I error rate of the overall experiment is stable using our suggested method and that the type II error rate is close to or above nominal. Furthermore, the instances in which the type II error rate is above nominal are in the situations where the lowest sample size is required, meaning a lower impact on the actual number of participants recruited. Results Recruitment is challenging and varies greatly by region. Total recruit- ment to date for the standardised arm n = 26, better in Peninsula than anywhere else, for the individual arm total recruitment n = 21, more participants found in Brighton & Hove by Kent, Surrey and Page 99 of 235 Trials 2017, 18(Suppl 1):200 Page 99 of 235 Page 99 of 235 Sussex CRN compared to two London CRNs. Numbers finishing to date are in the standardised arm n = 1 with n = 5 loss to follow up and n = 1 withdrawal and in the individualised arm n = 7 with n = 2 lost to follow up. conclusion the observational cohort can provide valuable informa- tion about a population of patients that are not willing to partici- pate in a RCT. The purpose of the cohort should be clear and informative. Recruiting into a sister observational cohort may be seen by sites as an easier option and thus detract from recruiting patients into the main RCT. It may be advantageous to close the observational cohort at a time when sufficient numbers are recruited and the aims of the cohort fulfilled. Mammo-50 demonstrated the strength of a sister observational cohort alongside the RCT, especially within the internal feasibility stage, leading to the success of the full RCT. We have yet to analyse the full data for recruits including diaries for QOL data. We will be interviewing both NHS staff and participants for their views on CHM and the processes involved. The trial finishes recruitment at the end of October 2016 as the herb expiry date is the end of November 2016. Background The sample size required to power a trial to a nominal level in a paired comparative diagnostic accuracy trial, i.e. Trials in which the diagnostic accuracy of two testing procedures are compared relative to a gold standard, depends on the correlation between the two diagnostic tests being compared. The lower the correlation between the tests the higher the sample size required, the higher the correlation between the tests the lower the sample size required. A priori, we usually do not know the correlation between the two tests and thus cannot determine the exact sample size. Furthermore, the correlation between two tests is a quantity for which 1) it is difficult to make an accurate intuitive esti- mate and, 2) it is unlikely estimates exist in the literature, particularly if one of the tests is new, as is very likely to be the case. Trials 2017, 18(Suppl 1):P260 This abstract is not included here as it has already been published. Results Of the 186941 trials in clinicaltrials.gov, 1567 (0.8%) were studying one rare condition only and with prevalence information from Orphadata. There were 19 trials studying disease with prevalence <1/ 1,000,000, 126 trials with 1-9/1,000,000, 791 trials with 1-9/100,000 and 631 trials with 1-5/10,000. Of these, 1160 were described as phase 2 trials. All the covariates described above except type of lead sponsor were significant and thus, were included in the regression model. The estimated mean sample sizes in phase 2 trials were simi- lar across all prevalence classes after adjusting for other covariates; 15.7 (95% CI, 8.7-28.1), 26.2 (16.1-42.6), 33.8 (22.1-51.7) and 35.6 (23.3-54.3) for prevalence <1/1,000,000, 1-9/1,000,000, 1-9/100,000 and 1-5/10,000, respectively. Phase 3 trials of rarer diseases (<1/ 1,000,000 and 1-9/1,000,000) had similar size (estimated mean, 19.2, 6.9-53.2 and 33.1, 18.6-58.9, respectively) to those in phase 2 but were statistically significant lower than the slightly less rare diseases (1-9/100,000 and 1-5/10,000) trials (estimated mean, 75.3, 48.2-117.6 and 77.7, 49.6-121.8, respectively). We plan to recruit a total of n = 150 patients (75 patients in each arm) to the INTERIM trial in 18 months. It is anticipated that approxi- mately 90 PFS events will be observed with a minimum of 9 months follow up. The INTERIM trial proposal has received funding from the UK RFPB NIHR programme. The concept of predictive power will provide us with confidence to conclude whether the follow-on phase III trial is justified, while it is easily implemented in practice. p Conclusion Conclusion We recommend a paired comparative diagnostic accuracy trial which used an internal pilot study to re-estimate the sample size at the in- terim. This design would use a maximum likelihood estimate, under a multinomial model, of the correlation between the two tests being compared for diagnostic accuracy, in order to more effectively estimate the number of participants required to power the trial to at least the nominal level. Page 100 of 235 Trials 2017, 18(Suppl 1):200 Page 100 of 235 P263 How does prevalence affect the size of clinical trials for treatments of rare diseases? Siew Wan Hee1, Adrian Willis1, Catrin Tudur Smith2, Simon Day3, Frank Miller4, Jason Madan1, Martin Posch5, Sarah Zohar6, Nigel Stallard1 1University of Warwick; 2University of Liverpool; 3Clinical Trials Consulting and Training Limited; 4Stockholm University; 5Medical University of Vienna; 6INSERM Correspondence: Siew Wan Hee Trials 2017, 18(Suppl 1):P263 interventional trials is low, around 34%. Many novel trial designs, espe- cially using adaptive and Bayesian methods, have been developed to help reduce costs and improve success rate of such trials. These novel techniques, however, are not always easily implemented in practice. We propose to apply the predictive power concept in the design of feasibility studies. interventional trials is low, around 34%. Many novel trial designs, espe- cially using adaptive and Bayesian methods, have been developed to help reduce costs and improve success rate of such trials. These novel techniques, however, are not always easily implemented in practice. We propose to apply the predictive power concept in the design of feasibility studies. , Conclusions O d h Conclusions g 1University of Bristol; 2Bangor University; 3University of Oxford; 4PPI Representative Our study shows that there was association between prevalence and the size of phase 3 trials with rarest diseases trials noticeably smaller than the less rare diseases trials. However, prevalence was not associ- ated with the size of phase 2. Correspondence: Joanna Thorn Trials 2017, 18(Suppl 1):P265 How does prevalence affect the size of clinical trials for treatments of rare diseases? 2 3 Siew Wan Hee1, Adrian Willis1, Catrin Tudur Smith2, Simon Day3, Frank Miller4, Jason Madan1, Martin Posch5, Sarah Zohar6, Nigel Stallard1 1University of Warwick; 2University of Liverpool; 3Clinical Trials Consulting and Training Limited; 4Stockholm University; 5Medical University of Vienna; 6INSERM y BRAF + MEK inhibitor drugs are effective treatment for patients with BRAF mutant melanoma, but benefit is limited by secondary resist- ance and toxicity. Clinical case reports and animal models suggest intermittent dosing may delay onset of resistance and reduce side- effects. The INTERIM trial is an open label two-arm randomised phase II feasibility trial designed to investigate the intermittent BRAF + MEK inhibitor drug dosing. Eligible patients will be randomly allocated to either the intermittent or the standard continuous dosing arm. Since there are concerns whether patients and their doctors will accept allocation to less than standard treatment, the overall objective of the study is to determine the acceptability of randomisation and compliance of patients allocated intermittent BRAF + MEK inhibitor drug dosing. The study will also investigate the probability of a suc- cessful follow-on phase III trial, measured using the predictive power. The definitive phase III trial will be powered to test the hypothesis that intermittent dosing will prolong progression-free survival (PFS) compared with standard continuous dosing. It will need ~1000 pa- tients (846 PFS events) to detect a hazard ratio (HR) = 0.80 with a 5% significance level and 90% power. The log(HR) is normally distrib- uted, and therefore the predictive power could be easily calculated. Assuming a prior of no difference and 1 PFS event, that is log(HR) ~ N(0, 4), a significance level 5% and 90 PFS events observed in the feasiblity study: Correspondence: Siew Wan Hee Trials 2017, 18(Suppl 1):P263 Core items for a standardised resource-use measure (ISRUM): expert Delphi consensus survey 1 2 1 1 Joanna Thorn1, Colin Ridyard2, Ruth Riley1, Sara Brookes1, Dyfrig Hughes2, Sarah Wordsworth3, Sian M. Noble1, Gail Thornton4, William Hollingworth1 P264 P264 Predictive power in feasibility study design: implementation in the interim melanoma cancer trial Wendi Qian1, Andrea Machin1, Pippa Corrie2 1Cambridge Clinical Trials Unit - Cancer Theme; 2Cambridge Cancer Centre, Cambridge University Hospitals NHS Foundation Trust Correspondence: Wendi Qian Trials 2017, 18(Suppl 1):P264 Wendi Qian1, Andrea Machin1, Pippa Corrie2 1Cambridge Clinical Trials Unit - Cancer Theme; 2Cambridge Cancer Centre, Cambridge University Hospitals NHS Foundation Trust Correspondence: Wendi Qian Trials 2017, 18(Suppl 1):P264 Wendi Qian1, Andrea Machin1, Pippa Corrie2 1Cambridge Clinical Trials Unit - Cancer Theme; 2Cambridge Cancer Centre, Cambridge University Hospitals NHS Foundation Trust Correspondence: Wendi Qian Trials 2017, 18(Suppl 1):P264 Background Economic evaluations are commonly conducted within randomised controlled trials in order to assess value for money by measuring both the costs and the outcomes associated with a particular intervention. However, resource-use measurement by patient recall in economic evaluations is characterised by inconsistency and a lack of validation. A well validated standardised resource-use measure could increase data quality, improve comparability between studies and reduce research burden on patients and health economists. Aim Background Clinical trials are typically designed based on the classical frequentist framework constrained to some pre-specified type I and II error rates. Depending on the targeted effect size, the sample size required in such designs range from hundreds to thousands. Trials for rare diseases with prevalence 1/2000 or fewer may find it challenging to recruit patients to trials of large size. In this work, we examine the re- lationship between prevalence and other factors with the size of interventional phase 2 and 3 trials conducted in the US and/or EU. Methods We downloaded all trials from Aggregate Analysis of clinialtrials.gov (AACT) in May 2016 and identified rare disease trials by matching mesh terms in AACT with those in Orphadata. Actual sample sizes of com- pleted trials or anticipated sizes of non-completed trials were used for analysis. We investigated effects of trials’ characteristics such as: inclu- sion criteria (e.g. Gender, age), intervention model (e.g. Factorial design, single arm), lead sponsor type (e.g. Industry, US Federal Agency), trial location, number of countries involved in the trial, year that enrolment to the protocol began, number of interventions in the trial, whether or not the trial had a data monitoring committee and whether or not the intervention studied in the trial was FDA regulated on sample size. The effect of prevalence on sample size was tested adjusting for phase, interaction between prevalence and phase, and all other significant covariates. If the observed HR = 0.80 (an improvement in median PFS from 12 to 15 months) in the phase II feasibility trial, the predictive power to re- ject the null hypothesis of no PFS benefit of intermittent dosing schedule under the planned phase III trial (with 846 PFS events) is 72%. If the observed HR = 0.75 (an improvement in median PFS from 12 to 16 months) in the phase II trial, the predictive power to reject the null hypothesis of no PFS benefit of intermittent dosing schedule under the planned phase III trial (with 846 PFS events) is 81%. Aim To identify a minimum set of core items of resource use that should be included in a standardised instrument for health economic evalu- ations conducted in the United Kingdom (UK). Cancer clinical trials are expensive in terms of running costs and time taken to complete, while the success rate of phase III cancer Trials 2017, 18(Suppl 1):200 Page 101 of 235 Methods Knowledge of the clinical pharmacological properties of drugs in development evolves throughout the drug development process. Such properties include the absorption, distribution, metabolism and excretion of the drug (pharmacokinetics) as well as its effect on the body (pharmacodynamics), such as disease progression or biomarker activity. Pharmacometric models can be deployed to de- scribe these processes based on available data and simulation can be used to both evaluate efficacy and safety in differing popula- tions under different conditions to assess the impact of disease, background medication or adherence. These estimates can further be used as inputs to pharmacoeconomic models which compare the costs and benefits of the hypothetical drug to those of existing therapies. This has the added value of, going beyond efficacy, and identifying what properties are most likely to result in a drug being deemed cost-effective. The content of 59 questionnaires in the Database of Instruments for Resource-Use Measurement (www.dirum.org) was analysed to generate a list of 60 resource-use items relative to an NHS and personal social services perspective (e.g. Visits to healthcare profes- sionals in the community; prescribed medications). An electronic Delphi survey was developed, and health economists with experi- ence of conducting economic evaluations in the UK were recruited through personal approaches and a general request to the Health Economists Study Group mailing list (a professional group based in, though not restricted to, the UK). Respondents were asked to rate items on a scale of 1 to 9 according to the importance of the item in a generic context, and were encouraged to comment on their choices or add additional items. Responses were used to identify items considered less important according to predefined consensus criteria. A second round was developed, in which feedback from round 1 (median responses to each item and summarised com- ments) was presented to respondents, alongside a reminder of their own scores. An individualised email was sent to each participant from round 1, and round 2 participants were asked to re-rate items. A final item selection meeting of the project team and a represen- tative of the participants was held to discuss the results of the Delphi. Identifying the optimal clinical pharmacological properties of potential future medicines using clinical trial simulation Daniel Hill-McManus1, Scott Marshall2, Steven Lane3, Elena Soto2, Dyfrig Hughes1 y g g 1Bangor University; 2Global Pharmacometrics Pfizer Ltd; 3University of Liverpool Correspondence: Yuanyuan Kong Trials 2017, 18(Suppl 1):P268 Correspondence: Yuanyuan Kong Trials 2017, 18(Suppl 1):P268 Correspondence: Daniel Hill-McManus Trials 2017, 18(Suppl 1):P266 Demographic and baseline characteristics of patients in china registry of hepatitis B (CR-HEPB) 1 2 3 4 g y p Yuanyuan Kong1, Lai Wei2, Jinlin Hou3, Zhongping Duan4, Hui Zhuang5, Hong You1, Jidong Jia1, the CR-hepb study group 1Beijing Friendship Hospital, Capital Medical University; National Clinical Research Center for Digestive Disease; 2Peking University People’s Hospital; 3Nanfang Hospital, Southern Medical University; 4Beijing Youan Hospital, Capital Medical University; 5Peking University Health Science Center standard Results The results reveal the optimal combinations of clinical pharmacological properties for any future uricosuric medicine and therefore provide the target characteristics to drive future drug discovery. The results of the economic modelling show the probability that new drugs will be cost- effective for a given willingness-to-pay threshold based on the impact of these properties on the expected efficacy and safety. The findings could serve as a guide of the potential for future innovation in the area of uricosuric treatments for gout. Methods This study investigates the potential for using modelling and simulation to inform drug discovery and development using a case study of urate- lowering therapies (ULTs) used in the long term management of gout. Gout is characterised by a reliable biomarker, serum uric acid (UA) con- centration, which is also used as a primary endpoint in clinical trials. Serum UA concentrations are well correlated with gout symptoms and can also be predicted using pharmacometric models. This case study focusses on estimating the desired clinical pharmacological properties of uricosuric drugs, which stimulate the renal excretion of UA thus low- ering the serum concentrations. Successful uricosuric drugs must bal- ance the risks resulting from the renal excretion of large amounts of UA against the benefits of reduced serum concentrations, while being forgiving to poor adherence which is common for ULTs. The linkage to economic modelling comparing the hypothetical drug with current standard ULT is used to provide estimates of cost-effectiveness. Results Conclusions The apparent consensus on which items are important to trialists in a generic context suggests that a standardised instrument for core items is feasible, provided it is developed with the flexibility to add ‘bolt-on’ modules. Although this work was conducted in the context of RCTs within the UK, in principle, the items identified are generalizable to other jurisdictions and study designs. P268 P268 Demographic and baseline characteristics of patients in china registry of hepatitis B (CR-HEPB) Yuanyuan Kong1, Lai Wei2, Jinlin Hou3, Zhongping Duan4, Hui Zhuang5, Hong You1, Jidong Jia1, the CR-hepb study group 1Beijing Friendship Hospital, Capital Medical University; National Clinical Research Center for Digestive Disease; 2Peking University People’s Hospital; 3Nanfang Hospital, Southern Medical University; 4Beijing Youan Hospital, Capital Medical University; 5Peking University Health Science Center p Results 45 health economists with wide-ranging trial experience completed round 1. Following application of the consensus criteria, items such as length of outpatient appointments (median = 3) and email/text communications with healthcare professionals (median = 4) were considered less important. The list of 60 items was reduced to 34 items for the second round; no new items were added. 42 respon- dents completed round 2 (93.3% of the original respondents), and the list of items considered important converged towards a shorter list where there is greater consensus about importance (including hospital outpatient appointments (median = 9) and appointments at GP surgeries (median = 9)). Following the meeting to discuss the re- sults, a list of 10 core items was identified with further items identi- fied as suitable for development as ‘bolt-on’ modules. Background Type 1 diabetes is associated with cardiovascular disease (CVD) and the electrocardiogram (ECG) is the most accessible test for screening and detection of subclinical myocardial infarction and CVD. The Dia- betes Control and Complications Trial (DCCT) and its follow-up study, the Epidemiology of Diabetes Interventions and Complications (EDIC) is currently following 1,214 of the 1,441 originally randomized partici- pants (94% of the surviving). During the 30 years of follow-up, con- tinual efforts were made to modernize systems and data processing to improve efficiency and reduce cost. Sena Jawad, Daniel Gray, Neena Modi, Chris Gale Imperial College London Correspondence: Sena Jawad Trials 2017, 18(Suppl 1):P269 Methods We searched the four most cited general medical journals (New Eng- land Journal of Medicine, The Lancet, BMJ and The Journal of the American Medical Association) and extracted neonatal clinical trials from 2006 onwards with no restriction on the disease area or treatment type. The following data items were extracted from each identified paper by two independent reviewers: baseline characteristics, items used in stratification or minimisation, and items used to adjust the pri- mary outcome. Data items were combined where clinically appropriate. NNRD data completeness was examined over the period January to June 2015 for infants <32 weeks gestational age in England, Scotland and Wales. Missing was defined as an empty field or an invalid value. This review was pre-registered (PROSPERO: CRD42016046138). Results p Methods Participants were administered a resting 12-lead ECG at each annual visit during DCCT/EDIC. Paper tracings and accompanying paper tracking forms were sent from the 27 clinics to the Data Coordinating Center (DCC), then forwarded in monthly batches to the Central ECG Reading Center (CERC) for analysis. In 2012 the paper tracking form was replaced with direct entry into a proprietary data management system. In 2014, the study implemented the use of digital ECG re- cordings and transmission. Unlike paper ECGs, digital scans automat- ically provide hundreds of waveform measurements and can be stored indefinitely without the risk of deterioration. Digital ECGs are transmitted directly to the CERC via analogue phone lines. Site coor- dinators were centrally trained on the use of the new digital ECG ma- chines and how to transmit the recorded ECGs electronically to the CERC. Conclusions In this largest registry for HBV patients in China mainland, middle- age men were predominant and less than half of all patients were HBEAG positive. NAS were the most commonly used but nearly half of the patients did not choose the recommended high potent and less resistant agents. Long-term follow-up and judicial evaluation of the antiviral efficacy and clinical outcomes would provide valuable information for decision-making in clinical and public health sector. Background With the exception of maternal ethnicity, none of the commonly reported data items were missing in more than 10% of babies in the NNRD. Di i reported data item (96%). Sex (89%) and birth weight (89%) were also common items reported. Antenatal steroids were reported in 25 (86%) pre-term studies and 1 (8.3%) term study. Mother’s ethnicity and mul- tiple births were reported in 50% of all studies but the latter was pre- sented in 72% (21) of pre-term studies and 8% (1) of term studies. Gestational age, the most reported data item, was complete for 99.3% of infants in the NNRD. With the exception of maternal ethnicity, none of the commonly reported data items were missing in more than 10% of babies in the NNRD. and database were developed, maintained and managed by an infor- mation technology service provider and professional statistical and methodological terms As of Oct 31, 2016, 38 tertiary or secondary hospitals across China had participated in this registry, and totally122,987 patients and 486,976 visits were recorded. Among them 61.9% were men; the average age at inclusion was 44.3 years, with 67.7% of them being between 30 and 60 years old. Approximately 6.7% were initially diagnosed with immune tolerance phase of HBV infection, 68.5% with chronic hepatitis B, 14% with cirrhosis (compensated 6.3%; de- compensated 7.7%), 1.8% with HCC, and 9% with others (including acute-on-chronic failure and recovery phase of HBV infection). Forty seven percent of the patients were positive for HBEAG and 74.1% were positive for HBV DNA. Among 859 patients with liver biopsy the necroinflammation score G0-G4 were seen in 2.6%, 30.5%, 36.2%, 25.6% and 5.1%, respecdtively; and fibrosis stage S0-S4 were seen in 12.5%, 34.8%, 28.9%, 18.3%, and 5.5%, respectively. Totally, 33,533 (27.3%) patients had therapeutic information, among them 10.6% receiving conventional or pegylated interferon-alpha and 88.7% re- ceiving nucleos(t)ide analogues (NAS). The split out of the NAS showed that entecavir accounted for 51.2%, lamivudine for 18.8%, adefovir for 16.1%, telbivudine for 12.5% and tenofovir (approved but reimbursable for HBV therapy until recently) for 1.4%. Conclusions Discussion We show that there are a limited number of data elements which are common to recent influential neonatal clinical trials. Such essential data elements can be used to focus the measurement and improvement of data quality in preparation for point of care trials. The NNRD has low rates of missing data for these core data elements. This supports the use of routinely collected NNRD baseline data in UK neonatal clinical tri- als. Other key requirements are to establish national and international consensus for common outcomes for neonatal trials and assess the quality of corresponding NNRD data. Background Chronic infection of hepatitis B virus (HBV) is the predominant cause of cirrhosis and hepatocellular carcinoma (HCC) in China. However, the real-world data on the clinical and treatment profile is still lack- ing. Therefore, we initiated a nation-wide, hospital-based registry sys- tem, China Registry of Hepatitis B (CR-HepB) in June 30, 2012. Methods g Modelling and simulation are increasingly being used to assist in planning and decision making during drug development. This in- cludes clinical trial simulation which has been applied to optimise the design of drug trials; such as calculating sample sizes, determin- ing the optimal dosing regimen or estimating the impact of protocol deviations. This requires constructing a mathematical representation of the trial processes and makes use of data on the population of interest and likely effects of the interventions being studied. These methods are typically applied to a specific compound in clinical de- velopment to support decision-making and trial design in subse- quent phases of development. Another potential application of modelling and simulation, however, is in informing drug discovery by identifying the theoretical properties of a drug which would maxi- mise the desired trial outcomes. This on-line registry system is owned by China Foundation for Hepatitis Prevention and Control and academically supported by Chinese Society of Hepatology. The criteria for inclusion to this registry was any adult patients with HBSAG-positivity for than 6 months, meeting the diagnos- tic criteria for various stages of chronic HBV infection and consent to re- ceive follow-up per clinical practice guidelines (check HBVDNA, biochemistry, alpha fetoprotein and ultrasonography at least every 6 months). The decision on treatment was at discretion of the patients and the clinicians based on standard of care. The information system Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 102 of 235 Page 102 of 235 Page 102 of 235 reported data item (96%). Sex (89%) and birth weight (89%) were also common items reported. Antenatal steroids were reported in 25 (86%) pre-term studies and 1 (8.3%) term study. Mother’s ethnicity and mul- tiple births were reported in 50% of all studies but the latter was pre- sented in 72% (21) of pre-term studies and 8% (1) of term studies. Gestational age, the most reported data item, was complete for 99.3% of infants in the NNRD. Background Point-of-care registry trials extract data from existing Electronic Pa- tient Records (EPR). Poor accuracy and completeness of EPR data are key barriers to the success of such trials. Neonatal care in the United Kingdom has a well-developed EPR database, the National Neonatal Research Database (NNRD). We hypothesised that within neonatal clinical trials there are a subset of common data items reported across important trials, and aimed to assess the completeness of these items in the NNRD. q y Conclusion High impact neonatal clinical trials share core data elements. In the UK these data can be obtained reliably from the NNRD, a well-established EPR-derived national database, supporting its use for point-of-care registry trials. P270 Transition from paper to digital electrocardiograms (ECGs) after 30 years of follow-up in the DCCT/EDIC study Jye-Yu Backlund1, Barbara H. Braffett1, Catherine L. Martin2, Wanyu Hsu1, Lisa Diminick1, Susan Hensley3, Charles Campbell3, Yabing Li3, Elsayed Z. Soliman3, The DCCT/EDIC Research Group1 1George Washington University; 2University of Michigan; 3Epidemiological Cardiology Research Center (EPICARE) Correspondence: Jye-Yu Backlund Trials 2017, 18(Suppl 1):P270 Transition from paper to digital electrocardiograms (ECGs) after 30 years of follow-up in the DCCT/EDIC study Jye-Yu Backlund1, Barbara H. Braffett1, Catherine L. Martin2, Wanyu Hsu1, Lisa Diminick1, Susan Hensley3, Charles Campbell3, Yabing Li3, Elsayed Z. Soliman3, The DCCT/EDIC Research Group1 1George Washington University; 2University of Michigan; 3Epidemiological Cardiology Research Center (EPICARE) Correspondence: Jye-Yu Backlund Trials 2017, 18(Suppl 1):P270 Building a generic drug supply management system for ECRF systems - Challenges and experience gained 1 1 2 Robbie Wilson , Sharon Kean , Elizabeth Douglas 1Robertson Centre for Biostatistics; 2R&D, NHS Greater Glasgow & Clyde Correspondence: Robbie Wilson Trials 2017, 18(Suppl 1):P271 Methods Common DSM components we provide include batch management, order and receipt management (both manual and triggered), post- receipt status updates and summary reporting tools. The processes behind each component can vary slightly or significantly based on the logistical challenges identified when reviewing a trial protocol and after discussion with the trial management team and most importantly the lead pharmacist or their equivalent. Although the basic functionality appears to be the same the changes are usually identified ‘under the hood’ and we will look to identify the different types of changes that we have had to put in place from system-to-system. Developing sys- tems like this as part of our Clinical Trials Unit (CTU) has allowed for gradual refinement of functionality. However, we have faced increasing challenges when integrating with third-party systems and working to an external set of Standard Operating Procedures. These differences will be considered and compared. The advantages of simple analytics in EPRO environments Dionne Russell, Jonathan R. Gibb, Sharon Kean Robertson Centre for Biostatistics Correspondence: Dionne Russell Trials 2017, 18(Suppl 1):P273 Background g For many years Google have offered companies a range of tools to pro- vide analytics on web sites. These tools can be used to track and report on where or when the visitor to the site is having a bad experience and does not complete a transaction or chooses to leave the site. Companies use these tools to identify problem pages and further enhance the users experience with a view to boosting sales and revenue. Clinical trials have become less reliant on paper based systems and more reliant on elec- tronic data capture (EDC) and electronic patient reported outcomes (EPRO). This type of technology enables upfront validation of data, which in turn should provide a cleaner data flow and should lead to less data management queries. If however there is a problem with the design of the system it may result in either partial completion or non-completion this will lead to missing key data in any self-completion questionnaires in the study. This is particularly dangerous in EPRO scenarios where there is often less feedback from participants and there is usually the option to skip most questions. The problem of questionnaire abandonment in EPRO is somewhat similar to the problem of basket abandonment in ecommerce websites in that it can be difficult to know what has caused the abandonment and they both lead to significant loss. Methods Conclusion We will discuss the different scenarios faced when developing previous DSM systems including why we had to modify the standard platform and how we adapted it. We will also discuss how this experience can be used to produce a generic system that is suitable to adapt to differ- ent use cases as required. The difference between open-label and double-blind DSM systems will be considered as this may be the reason for two separate systems instead of just one. The feasibility of a ‘truly adaptable’ system will be considered after this discussion. Building a new EDC in 2016 Jonathan Gibb, Sharon Kean Robertson Centre for Biostatistics Correspondence: Jonathan Gibb Trials 2017, 18(Suppl 1):P272 Building a new EDC in 2016 Jonathan Gibb, Sharon Kean Robertson Centre for Biostatistics Correspondence: Jonathan Gibb Trials 2017, 18(Suppl 1):P272 To improve the user experience of EDC and EPRO and ultimately in- crease the completion rate, a range of existing approaches from mainstream web analytics and observational usability evaluations sessions were combined and embedded within the EDC and EPRO environments. y Conclusion The authors will present the major design decisions faced during the creation of a new EDC, the new features introduced for the different staff roles involved with the EDC usage lifecycle, including: extensible programming language and platform support, inter- nationalisation, multiple means of API access, online/offline version- ing system. Background As our Centre moves towards a standardised in-house platform for ECRF development we are taking steps to modify supporting processes (such as randomisation, pharmacovigilance and document manage- ment) into ‘generic’ components to build into this system. Another such component is the Drug Supply Management (DSM) tool suite which has been critical in previous systems to ensure allocation and monitor- ing of supplies across trial sites is accurate and efficient. Experience of developing custom versions of the DSM components has thrown up a number of challenges; these in turn have allowed refinement of functionality across multiple distinct system deployments. Identifying the ‘best’ features from each system will allow us to move towards the creation of a generic system. Results Implementation of digital ECG recordings substantially reduced the time between ECG acquisition and receipt by the CERC, with 49% of the ECG’s transmitted on the day of the clinical visit (mean 14 ± 18 days). The time from ECG acquisition to generation of the ECG clin- ical report was reduced by 52 days (120 ± 39 and 68 ± 21 days for paper and digital ECGs, respectively). Furthermore, the DCC received ECG results from the CERC 51 days sooner (108 ± 40 days and 57 ± 21 days for paper and digital ECG, respectively). There was no reduction We identified 46 studies involving 33,276 infants and 132 data items; 29 studies on pre-term babies, 12 on term babies and 5 studies where either age was unrestricted or undisclosed. Thirty seven trials (80%) were multicentre trials. Gestational age was the most commonly Page 103 of 235 Trials 2017, 18(Suppl 1):200 Page 103 of 235 in ECG quality. The cost of purchasing new ECG machines capable of digital transmission was offset and balanced by the reduction in the cost of processing and reading paper ECG’s over time. Conclusion to collect and collate the data must be usable and provide reprodu- cibility potentially for decades after the trial has finished. This cul- ture of long lived projects and gradual organic evolutionary change in software development has many advantages. Ideally best of breed features and solutions remain and weaker elements are re- placed. However legacy artefacts are left over from all evolutionary approaches. In geography there are ox bow lakes, in human anat- omy there is the tailbone and in jeans the small inner pocket was originally used to hold a pocket watch. In popular culture, the tele- vision show “Lost” featured a character that lived in an under- ground bunker and types a series of mysterious numbers into a computer every 108 minutes or there would be undisclosed “disas- trous consequences”. While a fictitious plot device, there are nu- merous similar occurrences in real business software and processes where the knowledge exchange process is simply - just enter a zero in that field, we don’t know why, but it doesn’t work if you don’t. Results When faced with the large amount of expertise gathered from past EDC development in a CTU, the large number of processes involved - the reason for which may or may not be understood, the risk of mistaking valid important elements for legacy artefacts and com- batting the phrase “that’s how it’s always been done”, creating a new EDC system is difficult. to collect and collate the data must be usable and provide reprodu- cibility potentially for decades after the trial has finished. This cul- ture of long lived projects and gradual organic evolutionary change in software development has many advantages. Ideally best of breed features and solutions remain and weaker elements are re- placed. However legacy artefacts are left over from all evolutionary approaches. In geography there are ox bow lakes, in human anat- omy there is the tailbone and in jeans the small inner pocket was originally used to hold a pocket watch. In popular culture, the tele- vision show “Lost” featured a character that lived in an under- ground bunker and types a series of mysterious numbers into a computer every 108 minutes or there would be undisclosed “disas- trous consequences”. While a fictitious plot device, there are nu- merous similar occurrences in real business software and processes where the knowledge exchange process is simply - just enter a zero in that field, we don’t know why, but it doesn’t work if you don’t. When faced with the large amount of expertise gathered from past EDC development in a CTU, the large number of processes involved - the reason for which may or may not be understood, the risk of mistaking valid important elements for legacy artefacts and com- batting the phrase “that’s how it’s always been done”, creating a new EDC system is difficult. Digital ECGs have simplified and enhanced the processing of ECGs between the reading center, coordinating center, and clinical sites by reducing the processing time by 43% with less effort and cost. P271 Building a generic drug supply management system for ECRF systems - Challenges and experience gained Robbie Wilson1, Sharon Kean1, Elizabeth Douglas2 1Robertson Centre for Biostatistics; 2R&D, NHS Greater Glasgow & Clyde Correspondence: Robbie Wilson Trials 2017, 18(Suppl 1):P271 Background g Compared to most software development projects, clinical trial electronic data capture (EDC) systems have a very long shelf life. It is not uncommon for clinical trials to last ten years. Even when tri- als themselves may be comparatively short lived, the systems used We will demonstrate the significant advantages this approach pre- sents when combined and how it can be used to identify and resolve issues when they arise rather than discovering than facing incom- plete datasets at the end of the collection period. Trials 2017, 18(Suppl 1):200 Page 104 of 235 Page 104 of 235 resources for programmer support to create such tools can greatly benefit from REDCAP’s features, which are continually being advanced. Piping is a capability within redcap that allows inserting values previ- ously entered by the user into a survey’s text. This feature enables the generation of dynamic questions that appear to be customized for a specific individual. P274 The paperless paradigm: transitioning to direct participant data entry in the search for diabetes in youth study Julia Robertson1, Leora Henkin1, Ken Wilson1, Jerry Barnes1, John Hepler1, Catherine Pihoker2, Beth Loots2, Amy K. Mottle3, Lynne Wagenknecht1, Ralph D'Agostino Jr.1 1Wake Forest School of Medicine; 2Seattle Children's Research Institute; 3University of North Carolina at Chapel Hill Correspondence: Julia Robertson Trials 2017, 18(Suppl 1):P274 p g 1Wake Forest School of Medicine; 2Seattle Children's Research Institute; 3University of North Carolina at Chapel Hill Correspondence: Julia Robertson Trials 2017, 18(Suppl 1):P274 p The EPHIM (electronic Patient Health Information Management) Pro- ject is a research study with the aim of understanding the factors that facilitate and limit use of patient portal systems by older, lower- income adults. Documenting human-technology interaction is a key component of the project. To assess the participants’ interactions with technology, we asked them about their e-mail use, and if they had any of the following in their home: stationary computer, laptop computer, tablet, smartphone, or internet. These items were to be asked in a baseline questionnaire and in six follow-up questionnaires spanning 12 months. While we wanted to follow any changes in the status of these items, given the age of the participants and relatively short time frame across visits, we decided it was unlikely there would be many changes in the technology available in participants’ homes during the two months between interviews. Background Thus, in order to prevent discrepancies in responses across time we used a combination of the piping feature and branching logic within redcap to remind the par- ticipants how they answered previously. For example, if a participant answered that he/she had a laptop computer in his/her home in the baseline questionnaire, at the next visit the question would read, “When we spoke last time you indicated that you do have a laptop computer in your home. Is this still correct?” The response to this yes/no question was stored and used within the next follow-up visit questionnaire which would have the same wording (“When we spoke last time you indicated that you? Is this still correct?”). This feature was enabled using a series of conditional statement code similar to syntax used within Excel. Web-based, direct data entry by research participant populations is becoming more common. In a long running research study, transi- tioning away from paper-based data entry provided opportunities and highlighted challenges. SEARCH for Diabetes in Youth (SEARCH) is an on-going, large, na- tional multi-center and population-based epidemiological study of youth with diabetes. Initiated in 2000, SEARCH was designed to esti- mate the prevalence, incidence and clinical presentation of diabetes in youth age < 20 years, by age, sex, race/ethnicity and diabetes type. More than 25,000 youth have been enumerated as part of the regis- try study; 3000 are enrolled in the longitudinal cohort study. These individuals represent diverse racial and ethnic backgrounds helping SEARCH determine the extent of diabetes in the community and its impact on different populations. SEARCH has 5 clinical centers lo- cated in South Carolina, Ohio, Colorado, California, and Washington. Funding is provided by the Centers for Disease and Prevention (CDC) and the National Institute of Diabetes and Digestive and Kidney Dis- eases (NIDDK). The cohort study sample, followed longitudinally for 16 years, is char- acterized using a variety of age specific surveys and a physical exam in which complications and laboratory measures are assessed. For the present SEARCH visit, the study took the opportunity to broaden the method of data collection to allow participants the choice of using a web-based direct data entry approach or to have data collected on paper forms. To facilitate this transition, data entry forms were carefully evaluated, re-organized and updated to better accommodate direct data entry. Background The goals are to facilitate a positive end-user experience, ensure high quality data capture, increase re- sponse rates and decrease clinic staff burden. In addition, because of funding limitations, approximately half of the past participants were invited to participate only in the online surveys (e.g., were not of- fered an in person visit). To date, response rates are on track to meet study goals. In its simplest form, the piping feature within redcap allows for a more personal correspondence with participants and increases efficiency in the data collection process. We have highlighted a more advanced example of the piping feature which, when used in combination with branching logic, can be used to customize data collection and help pre- vent discrepancies across time. P276 Using dedicated study laptops to enhance data quality, participant safety, and trial efficiency: experience from a large-scale, international, randomized clinical trial Michelle Nunn, John Nolan, Simon Gilbert, Alex Baxter, Bob Goodenough, Mike Lay, Louise Bowman, Martin Landray Clinical Trial Service Unit (CTSU) and MRC Population Health Research Unit, Nuffield Department of Population Health, University of Oxford Correspondence: Michelle Nunn Trials 2017, 18(Suppl 1):P276 Some of the challenges of adding the option of participant direct data entry include creating an intuitive, seamless data entry flow, encouraging participants to answer all questions and to complete all modules, ensuring invalid or out of range answers are corrected and that the participant understands overall navigation and the meaning of individual questions and facilitating consistency in future analyses after questionnaire updates and re-organization. This poster will further explore the challenges encountered, both in planning and programmatic implementation, and will describe the advantages and outcomes both for clinic staff and study participants. Using REDCAP’s piping feature to aid in longitudinal data collection Jennifer Talton, Phillip Summers, Kathryn Melius, Thomas A. Arcury Wake Forest School of Medicine Correspondence: Jennifer Talton Trials 2017, 18(Suppl 1):P275 Background Background The REVEAL study is a streamlined randomized controlled trial (RCT) investigating the effects of adding the CETP inhibitor anacetrapib to LDL-lowering treatment with atorvastatin among 30 000 study participants at high risk of vascular disease. It is coordinated by the CTSU at the University of Oxford (the trial Sponsor) with 6 Regional Coordinating Centres responsible for the conduct of the trial in 431 sites in 10 countries. Laptops are provided to all sites so that participant visit data can be captured directly into the electronic Case Report Form (ECRF) using software specifically designed for this purpose by CTSU. The laptop database therefore contains the source data. M h d P275 Using REDCAP’s piping feature to aid in longitudinal data collection Jennifer Talton, Phillip Summers, Kathryn Melius, Thomas A. Arcury Wake Forest School of Medicine Correspondence: Jennifer Talton Trials 2017, 18(Suppl 1):P275 Methods REDCAP (Research Electronic Data Capture) is a secure, web-based application designed to support data capture for research studies and is free to non-profit organizations who join the redcap Consortium. Its capabilities include the building of custom databases and surveys (that supports online or offline data capture); it also has data manage- ment features, such as participant scheduling calendars. Projects which do not need highly specialized data collection tools or have the Features of the laptop system used include: Consistency in laptop setup by utilising a Windows 7 image which also allows for regional variations where required. Remote central management of laptops by CTSU, including user accounts and passwords plus distribution of software updates. Timely, secure and regular transfer of data between the laptops and the designated FTP & HTTPS servers via a reliable internet connection. Robust laptop & data security achieved Trials 2017, 18(Suppl 1):200 Page 105 of 235 Page 105 of 235 through a combination of data encryption, whole-disk-encryption, passwords, ‘Locked down’ user accounts and staff training. Partici- pant identifiable data is retained on the laptop but only transferred to CTSU if permitted by local data protection laws. Discussion dissemination of a clinical trial as a journal publication. By inputting in- formation about the process, the status of the manuscript is transpar- ent. Finally, the system needed to provide an easy way for investigators and other staff to show the effects that their completed clinical re- search has had on the overall scientific community. dissemination of a clinical trial as a journal publication. By inputting in- formation about the process, the status of the manuscript is transpar- ent. Finally, the system needed to provide an easy way for investigators and other staff to show the effects that their completed clinical re- search has had on the overall scientific community. y These challenges were addressed by looking at a number of offline tracking systems that had failed to meet the needs of various clin- ical networks, and finding ways to improve upon them. An extensi- ble.NET module was developed to store and display all relevant data for publications tracking. Methods and results There were 80 CPRD general practices randomised. The trial interven- tions include a webinar and decision support tools that are delivered into general practice systems. In addition, intervention trial arm prac- tices are sent monthly-updated reports on their prescribing of antibi- otics for respiratory tract infections. During the 12 month intervention period, electronic health records data are analysed from each month’s CPRD release to provide detailed feedback on the number of RTI consultations, number of antibiotic prescriptions and proportion of con- sultations with antibiotics prescribed. Prescribing-report templates were designed through qualitative research with non-trial practices. An R program was written to extract, analyse and summarise electronic health records data for intervention trial arm practices. The “reporters” package in R was used to create personalised “Pdf” reports, based on the templates, for each practice. The Global Network’s (GN) clinical trials produce rich data sets to in- form investigators about treatments to prevent, diagnose, or treat human diseases. From these datasets, the clinical investigators pub- lish results from their preplanned investigation to inform colleagues of the outcomes of the clinical trial. These same datasets can be used for secondary analyses to leverage the information available. All of these publications enrich the understanding of a disease state and possible therapies as well as serve as measurable deliverables, demonstrating productivity to the funding source. Therefore, we needed a system to track 1) data requests (for secondary analyses), 2) pre-publication development (includes all primary and secondary reports using a clinical trial dataset) and 3) post-publication track- ing (citation counts, journal impact factor and author h-index). Background Antibiotic resistance is a growing problem that transcends national boundaries. 80% of all antibiotic used in the UK are prescribed within primary care, and up to 50% of prescriptions may be unnecessary. In- terventions are needed that can be delivered to prescribers at low cost. Objectives This study utilises electronic health record data (EHR) on antibiotic prescribing for specific indications to provide feedback to prescribers. This intervention is being evaluated in a cluster randomised trial. P277 Tracking data requests from initiation to publication through the use of a web-enabled database system Michael Ham1, Elizabeth M. McClure1, Kevin A. Wilson1, Edward A. Liechty2, Norman Goco1, Lori Schwarze1, Carolyn M. Petrie Huitema1 1RTI International; 2Indiana University Correspondence: Michael Ham Trials 2017, 18(Suppl 1):P277 This study utilises electronic health record data (EHR) on antibiotic prescribing for specific indications to provide feedback to prescribers. This intervention is being evaluated in a cluster randomised trial. Study setting h d l b d d h l l y Conclusion p g p y Dorota Juszczyk, Judith Charlton, Martin C. Gulliford, the REDUCE Trial Research Group Dorota Juszczyk, Judith Charlton, Martin C. Gulliford, the REDUCE Trial Research Group In comparison to paper-based clinical trials, the use of carefully pro- grammed and managed study laptops has significant benefits for participant safety, data quality and regulatory compliance. How- ever, it is probable that in the future web-browser based systems (including tablet and smartphone implementations) will become the norm, with advantages in terms of cost, convenience, familiar- ity, and usability. However, the lessons learned from delivering a large-scale, laptop-based, direct data entry system will be valuable if the potential of these newer approaches is to be realised in fu- ture clinical trials. King’s College London Correspondence: Dorota Juszczyk Trials 2017, 18(Suppl 1):P278 Methods Web services were employed to con- nect the system to popular publication data stores such as The United States National Library of Medicine’s PubMed database, Thomson Reuters’s Web of Science database, and the International DOI Foundation’s digital object identifier (DOI) to retrieve and store accurate information about publications at any point in the creation process. The presentation will discuss the different technical chal- lenges of this approach and how they were resolved. Pros and cons of the approach will also be discussed along with methods used to report information out of the system. Using laptops in REVEAL offers a highly effective and user-friendly method of collecting good quality participant data with significant advantages over paper-based systems. Careful scripting of the user interface enhances compliance with the study protocol and proce- dures, and prompts any necessary actions related to participant safety. Data entry (which can be done offline) directly into an ECRF provides immediate validation of data and a full audit trail, whilst site investigators retain access to their participants? Data held lo- cally and securely both during and after the trial. The use of a ‘thick client’ rather than a web-browser provides resilience against inter- net outages whilst enabling timely data management across multiple countries and time zones. Potential drawbacks include reli- ance on a good internet connection for regular data transfer (ideally at least daily), central study databases not being updated in real time (although this is much less of a problem than systems that rely on transcription of data from paper forms), and restric- tions on moving participant identifiable data out of some countries. In addition the provision of laptops can be expensive and resource intense requiring remote support and maintenance over many years. Tracking data requests from initiation to publication through the use of a web-enabled database system Michael Ham1, Elizabeth M. McClure1, Kevin A. Wilson1, Edward A. Liechty2, Norman Goco1, Lori Schwarze1, Carolyn M. Petrie Huitema1 1RTI International; 2Indiana University Correspondence: Michael Ham Trials 2017, 18(Suppl 1):P277 The study is currently being conducted in the Clinical Practice Re- search Datalink (CPRD). Methods and results Background ASCEND is a randomised 2x2 factorial study of aspirin versus placebo, and of omega-3 fatty acid supplementation versus placebo, for pri- mary prevention of cardiovascular events in people with diabetes. The study used a mail-based approach to identify and randomise 15,000 people with diabetes. The project is now approaching its final follow-up stage. In the course of the study, the vast majority of data collection was done via paper designed to be processed with Optical Character Recognition software. In previous paper based studies, double data entry was used, which meant sending paper forms to a commercial company for data entry and finding and resolving any data or keying discrepancies in-house (often with the necessity to see the paper form). For ASCEND, a more streamlined and cost ef- fective approach has been developed. pp Methods A third party OCR software, FORMS, from Readsoft (now Lexmark) was identified as the best tool to scan, review and save form content for fur- ther use in the study. The software gives a rich selection of ways the process can be customised. An option to review different parts of the form by users with different roles was required, e.g. Lists of medication or medical events to be reviewed and coded by medical staff, the rest of the form to be reviewed by administration staff. Using the FORMS' API, extra validations and background calculations were performed and bespoke dialogues were added. The dialogues made it possible to save handwritten medical events and medications as Read codes. Another required outcome was a digital archive of form images with links to relevant participants for viewing at any later point (instead of pulling a form out of the paper archive). P281 Integration of patients, caregivers and healthcare providers into a hypertension management research priorities process Simon Bacon1, Nadia Khan2, Hypertension Canada Priorities Setting Process Group3 1Concordia University & CIUSSS-NIM HSCM; 2University of British Columbia; 3Hypertension Canada Correspondence: Simon Bacon Trials 2017, 18(Suppl 1):P281 1Concordia University & CIUSSS-NIM HSCM; 2University of British Columbia; 3Hypertension Canada Correspondence: Simon Bacon Trials 2017, 18(Suppl 1):P281 p Results Study paper forms were designed to fit FORMS requirements and printed in a professional printing house using specific Pantone colours, as we wanted to drop-out some elements during scanning. Once the form stationery was available, we could start a long process of imple- mentation of our own validation rules into the, unfamiliar at that time, FORMS processing flow. Later on frequent maintenance work was needed. The trial was extended in length and size and additional stocks of form stationery had to be ordered. The set-up had to be tuned each time a new printout of a known form was to be used. R l There were 386 respondents who submitted 598 questions (after exclusions). The majority of respondents were patients or caregivers (78%). In addition, 29% lived in rural areas, 78% were aged 50–80 years and 75% were women. The 598 questions were distilled down to 42 unique questions and from this list, the top 10 research ques- tions prioritized included: determining the combinations of healthy lifestyle modifications to reduce the need for antihypertensive medications, stress management interventions, evaluating treat- ment strategies based on out-of-office blood pressure compared with conventional (office) blood pressure; education tools and tech- nologies to improve patient motivation and health behavior change, management strategies for ethnic groups, evaluating nat- ural and alternative treatments and the optimal role of different healthcare providers and caregivers in supporting patients with hypertension. In total, about 340,000 forms of seven types (with multiple printouts in each type) were processed by a team not larger than 4 administra- tive and 2 medical staff and this task was only one of their many du- ties. Coding of medication and medical events reported was done from the form image, while with a double data entry system it was possible only after the keyed data was transferred into the database. Conclusions Methods Using the James Lind Alliance approach, a national web based sur- vey asked patients, care givers and health care providers to submit their unanswered questions on hypertension management. This questionnaire was distributed through a variety of partner organisa- tions to try and reach as wide a population as possible. Questions already answered from randomized controlled trial evidence were removed. A second questionnaire containing 42 distilled unique questions were distributed in a 2nd round for feedback and rank- ing. An in-person priority setting meeting of patient, caregivers and healthcare providers then ranked the final top 10 research priorities. Conclusions As well as informing the design of the cluster trial and outcome evaluation, electronic health records were used as a key component of the trial intervention. The automated system for writing feedback reports was time-consuming to establish, but then worked efficiently. The most time consuming elements of the report production were those that could not be automated, such as updating the meta-files and adjusting the template design and content. Since the GN is a collaborative network, comprised of several sites lo- cated across the U.S and in other countries, the tracking system had to be centrally accessible. To accomplish this, we chose to allow access through our website. We developed this tracking tool such that the in- vestigators could submit concepts, requesting data from a completed clinical trial, in an easy and trackable way. By collecting additional infor- mation about the concept, all stakeholders could access the informa- tion and understand the current status of the progress. The system was also developed to track primary manuscript development as the first Trial Registration and Funding: NIHR HTA Trial registration: ISRCTN95232781 Trials 2017, 18(Suppl 1):200 Page 106 of 235 This abstract is not included here as it has already been published. This abstract is not included here as it has already been published. P279 Using optical character recognition for processing paper forms in a large randomised clinical trial Aleksandra Murawska, Jolyon Cox, Jane Booth, Jill Barton, Kevin Murphy, Youcef Mostefaiv, Michael Lay, Louise Bowman, Jane Armitage Clinical Trial Service Unit (CTSU) and MRC Population Health Research Unit Correspondence: Aleksandra Murawska Trials 2017, 18(Suppl 1):P279 P279 Using optical character recognition for processing paper forms in a large randomised clinical trial P281 Integration of patients, caregivers and healthcare providers into a hypertension management research priorities process Simon Bacon1, Nadia Khan2, Hypertension Canada Priorities Setting Process Group3 1Concordia University & CIUSSS-NIM HSCM; 2University of British Columbia; 3Hypertension Canada Correspondence: Simon Bacon Trials 2017, 18(Suppl 1):P281 Background Despite the tremendous strides in hypertension management over the last 4 decades, blood pressure remains poorly controlled in 35- 50% of patients with hypertension. This research agenda has gener- ally been led by the scientific community and pharmaceutical indus- try. Integrating patients and other key stakeholders as co-builders in the development of research priorities may offer a new strategy to increase the direct relevance and applicability of research to ultim- ately close the current gaps in hypertension management. Objective We aimed to identify the 10 most important research priorities for hypertension management across all stakeholder groups: patients, caregivers and healthcare providers (family physicians, nurses, phar- macists and dieticians). This presentation will discuss the methodology, costs and benefits of this approach. h d Methods Setting: This multicentre pragmatic 2-arm randomised controlled trial (RCT) was conducted in 24 trauma centres in the UK. Partici- pants: All adult patients presenting with an open lower limb frac- ture, with a Gustilo and Anderson (G&A) grade 2/3, were eligible for inclusion. Initial wound management in Emergency Departments involved removal of gross contamination, followed by surgical de- bridement under anesthesia, where contaminated tissue is removed and the open fracture is washed out. The fracture is then immobi- lized using internal or external fixation. Interventions: Participants were then randomised to treatment allocation at the end of sur- gery, to either usual wound care or to negative pressure wound therapy. – Ensuring compatibility of research questions. – Ensuring compatibility of research questions. – Developing a mutually acceptable protocol. – Identifying and being explicit about potential bias. – Developing solutions for potential bias. – Keeping abreast of market forces. Methods R l Regular meetings were arranged with Zimmer Biomet, with updates on progress and feedback on issues could be discussed. The trial was devel- oped as a study providing evidence for current practice variations. Previ- ously within industry, surgical implant-based research has been limited and undertaken essentially for marketing purposes. ALLIKAT moved the focus to providing robust evidence for the use of new implants. We also ensured the protocol supported the funder’s objective to comply with the Beyond Compliance programme, which was set up by the BOA (British Orthopaedic Association) to monitor safety of new products. Working together allowed us to manage the inevitable product ?A3B2 show $132#?>design modifications, so that the study was not jeopardised or over-shadowed by market pressures from within the company. As a pragmatic study within the NHS it also needed to be patient-focused. This was achieved by utilising patient reported out- come measures, rather than subjective clinical reports and ensuring pa- tients were not referred to as ‘subjects’ In study related documents. Discussion yp Conclusions These research priorities can be used to guide researchers and funding bodies on hypertension management research considered most relevant to patients, caregivers and healthcare providers. Given the challenging nature of these priorities, consideration of what kinds of innovative clinical trial methodologies to answer these questions is also needed. The choice of the OCR system to process the ASCEND form over the traditionally used double data entry proved to be more effi- cient, although the path to a working system was strenuous and full of pitfalls. P280 A toolkit for patient and public involvement in a clinical trials unit: an update & next steps Heather Bagley1, Paula Williamson1, Carrol Gamble1, Nicola Harman1, Hannah Short1, Priya Francis1, Helen Hickey1, Bridget Young1, Kerry Woolfall1, Delia Muir2 1University of Liverpool; 2University of Leeds Correspondence: Heather Bagley Trials 2017, 18(Suppl 1):P280 Building a relationship with industry - An academic’s experience working with Zimmer Biomet C hl C 1 R h l D 1 D id B d1 Ji Xi 2 A d P i 1 g Cushla Cooper1, Rachel Dorman1, David Beard1, Jing Xie2, Andrew Price1 1University of Oxford; 2Zimmer Biomet Correspondence: Cushla Cooper Trials 2017, 18(Suppl 1):P282 Trials 2017, 18(Suppl 1):200 Page 107 of 235 Trials 2017, 18(Suppl 1):200 Background ALLIKAT compares the clinical and patient reported outcomes of the new bi-cruciate retaining knee system (Vanguard XP) to the current single cruciate-retaining knee system (Vanguard CR). It is sponsored and managed by the Surgical Intervention Trials Unit (SITU) at the University of Oxford and funded by Zimmer-Biomet. As a key stake- holder in the trial, the needs of Zimmer Biomet, with its product-based approach, had to be carefully considered alongside an academic assur- ance of protocol integrity (Goldenberg et al., 2011). Challenges identi- fied in our industry collaboration include: Discussion A multi-disciplinary collaborative team have been involved in the de- sign and set-up of the study and have produced a protocol that con- siders academic, patient and industry needs, which is practicable and will provide evidence for proof of principle of a new marketable de- vice to be used in clinical practice. Maintaining a patient focus and promoting evidence-based health care has been key to working with industry and the open communication dialogue has been helpful in achieving objectives. There has been a corollary advantage in the in- creased network links with industry, orthopaedic consultants and NHS Trusts around the UK and a greater understanding of the needs of these different parties. Outcomes – Ensuring the study is patient-focused. The primary outcome is the Disability Rating Index (DRI) score at one year. Secondary outcomes include incidence of deep infec- tion of the limb, quality of life and postoperative complications. Superficial and Deep SSI was assessed at six weeks after surgical repair of open fracture. Research associates (ras) observed wounds at six weeks and completed data collection forms. Clinical and patient-reported criteria were used, based upon the Centers for Disease Control diagnostic criteria (CDC, 2015). Photography: Digital cameras were used to capture a one or more images of trial wounds at six weeks postoperatively; no upper limit on num- ber of images was applied. A protocol was developed for ras. Photographs were taken in a variety of clinical settings whereby light conditions were optimised to reduce glare. A 15-cm ruler with clear millimeter divisions was placed next to the wound, with participant identification number. Images were returned to and stored by Warwick CTU. – Agreeing resource allocation and ownership for data collection – Agreeing resource allocation and ownership for data collection Background Investigator Initiated Trials (IITs) are academic sponsored trials funded by industry. IITs expand product knowledge and are often seen to provide more robust data in delivering evidence based practice. They provide evi- dence for clinicians, funders and industries about what happens in the real world (Suvarna, 2012) and can be used to show proof of principle. Our academic unit is currently collaborating with industry (Zimmer Bio- met) on the All Ligaments Left in Knee Arthroplasty Trial (ALLIKAT). We report the challenges and benefits of the collaboration in a device based trial and how a mutually acceptable study is being delivered. Patients who sustain open lower limb fractures are at high risk of postoperative surgical site infection (SSI); rates of up to 27% are re- ported for deep SSI. The type of dressing applied after initial de- bridement may influence postoperative wound infection and healing. The aim of WOLLF is to investigate the effectiveness and cost-effectiveness of standard postoperative wound dressings with negative pressure wound therapy in adult patients undergoing sur- gical management of open lower limb fracture. We supplemented wound assessment data collection with digital photographic im- ages of surgical wounds at six weeks postoperatively to investigate the feasibility and utility of this methodology to aid the diagnosis of wound healing and SSI. Results A total of 1515 images were obtained from 358 participants (mean 4.2 images per participant). Two assessors independently judged photographs; an experienced tissue viability nurse and se- nior researcher with clinical background. A third independent clinician acted as final arbiter where there was lack of agreement. Assessors were blinded to treatment allocation. Wounds were judged for healing and visual evidence of infection, without knowledge of clinical and patient-reported criteria recorded by RAs. We report inter-rater and intra-rater reliability (kappa values) for agreement between clinical assessors and compare rates of healing and SSI with and without photographic data. This poster will present our experience of the feasibility of supplementing standard data collection methods by the addition of a 2- dimensional image to aid the accurate diagnosis of postoperative infection in a large pragmatic trial. j Methods A cohort of 1102 patients having coronary artery bypass grafting in four trials was assembled. Data on 11 adverse events, recorded consistently across trials, were analysed. For each event, the timing was compared between patients with and without other adverse events. For the sub- group with multiple events, the timing of other events experienced relative to a target event (before, on the same day as, or after the target event; each event considered as the target in turn) was summarised. Sequential pattern mining techniques were used to identify common sequences of events in patients with at least two adverse events. Results Correspondence: Lucy Bradshaw Trials 2017, 18(Suppl 1):P285 P283 Using photographic images to aid wound assessment within a randomised controlled trial of standard wound management versus negative pressure wound therapy: UK WOLLF trial Julie Bruce1, Amy Verdun2, Sonia Davis3, Juul Achten3, Nick Parsons3, Susie Hennings3, Matt Costa4 1University of Warwick; 2University Hospitals Coventry & Warwickshire; 3Warwick Clinical Trials Unit; 4Nuffield Orthopaedic Centre, University of Oxford Using photographic images to aid wound assessment within a randomised controlled trial of standard wound management versus negative pressure wound therapy: UK WOLLF trial Julie Bruce1, Amy Verdun2, Sonia Davis3, Juul Achten3, Nick Parsons3, Susie Hennings3, Matt Costa4 1University of Warwick; 2University Hospitals Coventry & Warwickshire; 3Warwick Clinical Trials Unit; 4Nuffield Orthopaedic Centre, University of Oxford Using photographic images to aid wound assessment within a randomised controlled trial of standard wound management versus negative pressure wound therapy: UK WOLLF trial Dementia outcomes after addition of proxy-based assessments fo deceased or proxy-dependent participants Sarah Gaussoin, Dan P. Beavers, Mark A. Espeland, Katelyn R. Garcia, Beverly M. Snively, Sally A. Shumaker 1Wake Forest School of Medicine Correspondence: Sarah Gaussoin Trials 2017, 18(Suppl 1):P284 Dementia outcomes after addition of proxy based assessments f deceased or proxy-dependent participants Sarah Gaussoin, Dan P. Beavers, Mark A. Espeland, Katelyn R. Garcia, Beverly M. Snively, Sally A. Shumaker 1Wake Forest School of Medicine Correspondence: Sarah Gaussoin Trials 2017, 18(Suppl 1):P284 Correspondence: Sarah Gaussoin Trials 2017, 18(Suppl 1):P284 Correspondence: Sara Trials 2017, 18(Suppl 1): Correspondence: Julie Bruce Trials 2017, 18(Suppl 1):P283 This abstract is not included here as it has already been published. Trials 2017, 18(Suppl 1):200 Page 108 of 235 sequenc Results Common adverse events were supraventricular tachycardia/atrial fibrillation (SVT/AF) (341/1102, 31%) and suspected infections (312/ 1102, 28%); half of those with both complications (62/124) had the SVT/AF prior to infection. Reoperation occurred less frequently (56/ 1102, 5%), and typically occurred alongside other events (44/56, 79%). Where reoperation and reintubation both occurred, they typic- ally happened on the same day whereas, when reoperation and sus- pected infection both occurred, reoperation usually preceded the infection. Trends were less apparent for rarer complications, but pa- tients who had both a myocardial infarction (MI) and SVT/AF (10/13 of those who had an MI) all had the MI first. As part of the preparation for independent adjudication of cranial ultrasound scans in a perinatal trial (Cord pilot trial), we measured the intra- and inter-observer agreement of interpretation of cranial ultrasound scans by expert adjudicators. Background Variation in ascertainment of diagnosis based on radiological im- aging has implications for randomised trials that report outcome based on radiological diagnosis. This variation is particularly import- ant for neonatal trials where diagnosis of intraventricular haemor- rhage (IVH) and its severity based on cranial ultrasound scan are important outcomes. Methods Eight experienced neonatologists or radiologists with expertise in cranial ultrasound scans completed standardised training from a paediatric neuroradiologist. To measure intra- and inter-observer agreement each trained assessor rated the same 64 anonymised cranial ultrasound scan images; comprising two sets of 32 scans. The second set was a duplicate of the first with order and anonymi- sation code changed. These scans were prepared by the neuroradi- ologist. Six different diagnoses (IVH grades 1 to 4, periventricular leukimalacia (PVL) and ventriculomegaly) were represented at least five times in the 32 scans, with some scans showing more than one target pathology and at least five with no abnormality. Assessors were unaware of the duplicates. Results are presented for any IVH, mild/moderate IVH (grade 1 or 2), severe IVH (grade 3 or 4), PVL and ventriculomegaly. The multiple rater Kappa statistic was used to assess inter observer agreement. The intra-observer agreement was assessed by calculating the intra-observer Kappa statistic be- tween the two sets of scans for each adjudicator. Most events occurred early during the post-operative stay, and timings were similar between patients with one or multiple events. Exceptions to this were reoperation, which happened later in pa- tients with multiple events (although most reoperations were still within one day of surgery) and gastrointestinal complications (which tended to occur later in patients with multiple events). Unlike other events, tracheostomy happened at any time during the post-operative stay but, when this occurred, it was typically the final event reported. Twelve frequently occurring “event sequences” were identified, two of which included three events. 5% of patients with two or more events had SVT/AF and suspected infections following a reoperation; 25% of patients who needed reoperation went on to have SVT/AF and suspected infections. Also, suspected infections and reintubation were followed by a tracheostomy in 6% of after. Discussion Results Inter-observer agreement for severe IVH was substantial (multiple rater Kappa 0.62), fair for mild/moderate IVH and all IVH (multiple rater Kappa 0.26 and 0.37 respectively), moderate for ventriculome- galy (0.59) and poor for periventricular leukomalacia (multiple rater Kappa 0.11). Intra observer agreement was substantial for severe IVH (mean Kappa 0.74) and ventriculomegaly (mean Kappa 0.78), fair to moderate for mild/moderate IVH and all IVH (mean Kappa 0.44 and 0.53 respectively) and moderate for PVL (mean Kappa 0.46). l Conclusions This study has shown that there is considerable variation in the clas- sification of scan results between individual assessors and between the same assessor on repeated review. This suggests that for peri- natal and neonatal trials it is important to use standardised criteria and centralised adjudication with a consensus process for interpret- ing cranial ultrasound scans. This has implications for other trials where radiological images are used to assess outcome. P287 Development of the standardising measures in arm rehabilitation trials for stroke (SMART) toolbox (work in progress) Julie Duncan Millar1, Myzoon Ali1, Frederike van Wijck2, Alex Pollock1 1Nursing Midwifery and Allied Health Professions Research Unit, Glasgow Caledonian University; 2School of Health and Life Sciences, Glasgow Caledonian University P287 Development of the standardising measures in arm rehabilitation trials for stroke (SMART) toolbox (work in progress) Julie Duncan Millar1, Myzoon Ali1, Frederike van Wijck2, Alex Pollock1 1Nursing Midwifery and Allied Health Professions Research Unit, Glasgow Caledonian University; 2School of Health and Life Sciences, Glasgow Caledonian University Discussion The collection of comprehensive adverse event data across trials has allowed us to identify temporal relationships between different events. Some patterns were expected while others were less so. Some events are complications (e.g. MI), while others represent actions taken as a re- sult of complication(s) occurring (e.g. Reoperation or reintubation). Working with clinicians, we are using these observations to inform dis- cussions about which events to include in a composite outcome de- scribing “post-operative course” and the relative importance of events. Intra and inter-observer agreement in the interpretation of preterm neonatal cranial ultrasound scans After cardiac surgery, post-operative adverse events are common and many patients experience several events. Some are frequent and less severe, others are rare but serious. Most trials are under-powered to de- tect differences in specific adverse events and event rates are often de- scribed but not formally compared. We have examined the timing and ordering of adverse events to inform the development of a composite objective measure of “recovery” for use in cardiac surgery trials. Methods Lucy Bradshaw1, Jon Dorling2, Lelia Duley3, Lindsay Armstrong-Buisseret3, Joe Fawke4, Bernard Schoonakker5, Eleanor Mitchell3, Rob Dineen6 1University of Nottingham; 2Early Life Research Group, University of Nottingham; 3Nottingham Clinical Trials Unit, University of Nottingham; 4Leicester Neonatal Service, University Hospitals Leicester NHS Trust; 5Nottingham Neonatal Service, Nottingham University NHS Trust; 6Division of Clinical Neuroscience, University of Nottingham 8 Development of the standardising measures in arm rehabilitation trials for stroke (SMART) toolbox (work in progress) 1 1 2 1 Development of the standardising measures in arm rehabilitation trials for stroke (SMART) toolbox (work in progress) 1 1 2 1 ( ) ( p g ) Julie Duncan Millar1, Myzoon Ali1, Frederike van Wijck2, Alex Pollock1 1Nursing Midwifery and Allied Health Professions Research Unit, Glasgow Caledonian University; 2School of Health and Life Sciences, Glasgow Caledonian University y Correspondence: Julie Duncan Millar Trials 2017, 18(Suppl 1):P287 Patterns of adverse events after cardiac surgery Rachel Maishman1, Barnaby C. Reeves1, Gianni D. Angelini2, Chris A. Rogers1 1University of Bristol Clinical Trials and Evaluation Unit; 2Bristol Heart Institute, University of Bristol Correspondence: Rachel Maishman Trials 2017, 18(Suppl 1):P286 Patterns of adverse events after cardiac surgery Rachel Maishman1, Barnaby C. Reeves1, Gianni D. Angelini2, Chris A. Rogers1 1University of Bristol Clinical Trials and Evaluation Unit; 2Bristol Heart Institute, University of Bristol Correspondence: Rachel Maishman Trials 2017, 18(Suppl 1):P286 Background Stroke is the leading cause of complex disability worldwide and up to 77% of stroke survivors experience impairments in arm function. Rehabilitation of arm function is a research priority for stroke survivors, Background Trials 2017, 18(Suppl 1):200 Page 109 of 235 carers and healthcare professionals. Randomised controlled trials (RCTs) of arm rehabilitation measure numerous outcomes (e.g. Strength, pain, ability to use arm) hindering comparisons and synthesis of trial data for efficacy analyses to inform clinical practice. However, arm function is a complex concept and a variety of outcomes and measurement tools may be warranted. Therefore, we aim to develop consensus recom- mendations on a toolbox of key outcome measures for use in arm re- habilitation RCTs. Reaching a consensus on causes of pain: use of an expert independent panel to determine a reference standard J D i l 1 K i T ki di 1 L Middl 1 L P Matthew Nankivell1, Paula Mulvenna2, Rachael Barton3, Corinne Faivre-Finn4, Paula Wilson5, Elaine McColl6, Barbara Moore7, Iona Brisbane8, David Ardron9, Richard Stephens10 1 Khalid Khan2, Jonathan Deeks1 1University of Birmingham; 2Queen Mary University of London Correspondence: Jane Daniels Trials 2017, 18(Suppl 1):P289 p 1MRC Clinical Trials Unit at UCL AND MRC London Hub for Trials Methodology Research; 2Northern Centre for Cancer Care; 3Queen’s Centre for Oncology and Haematology; 4Institute of Cancer Sciences; 5Bristol Haematology and Oncology Centre; 6Newcastle Clinical Trials Unit and Institute of Health and Society; 7Wales Cancer Research Network; 8The Beatson West of Scotland Cancer Centre; 9Patient representative; 10MRC Clinical Trials Unit at UCL (retired) Correspondence: Matthew Nankivell Method Phase 1: systematically explore trial data within a Cochrane Overview of arm rehabilitation RCTs, extracting data on assessment tool use. Tools must be clearly defined and reproducible to be considered as an outcome measure in phase 2 and 3. p Methods QUARTZ randomised 538 patients to receive either WBRT, or supportive care alone, with 407 carers also agreeing to participate. Here we com- pare the baseline responses to the EQ-5D-3 L questionnaire of the pa- tients and carers. The trial’s primary outcome measure of quality adjusted life years (QALYs) was also calculated separately from patient and carer data, and the results compared. Phase 2: using nominal group technique (NGT) identify and agree on outcomes relevant to life after stroke with arm impairment. Eight NGTs will be undertaken with stroke survivors and carers, and eight NGTs with healthcare professionals experienced in arm function rehabilita- tion. This will be supplemented by eight semi-structured interviews with stroke survivors and carers. Data will be analysed using content analysis. Outcome measures identified (Phase 1) will be linked with out- comes from Phase 2 followed by systematic exploration of outcome measures psychometric properties. Results Overall levels of agreement between patient and carer responses to the EQ-5D at baseline were 82% for mobility, 79% for self-care, 71% for usual activities, 78% for pain/discomfort, and 66% for anxiety/ depression. For anxiety/depression, carers reported more problems than patients in 25% of cases, with 9% reporting fewer problems. For the other questions carers reported more problems as often as they reported fewer: mobility 9% vs 8%; self-care 11% vs 11%; usual activities 15% vs 15%; pain/discomfort 13% vs 9%. Overall levels of agreement between patient and carer responses to the EQ-5D at baseline were 82% for mobility, 79% for self-care, 71% for usual activities, 78% for pain/discomfort, and 66% for anxiety/ depression. For anxiety/depression, carers reported more problems than patients in 25% of cases, with 9% reporting fewer problems. For the other questions carers reported more problems as often as they reported fewer: mobility 9% vs 8%; self-care 11% vs 11%; usual activities 15% vs 15%; pain/discomfort 13% vs 9%. QALYs were calculated for the 397 patients where both patient and carer data were available. The average QALY was slightly higher using patient data (45.3 days) than carer data (39.0 days). When assessing the treatment effect, the difference in average QALY (95% CI) was 3.2 days (−13.1, 7.4) when calculated from patient responses, and 5.3 days (−15.4, 3.9) from carer responses. Conclusions Phase 3: edelphi to achieve consensus amongst stroke arm rehabilitation researchers on important and feasible outcome measures from phase 2. A final consensus meeting with stakeholders (stroke survivors, carers, re- searchers, trialists, and healthcare professionals) will determine which out- come measures will be recommended as part of the SMART toolbox. Results p QALYs were calculated for the 397 patients where both patient and carer data were available. The average QALY was slightly higher using patient data (45.3 days) than carer data (39.0 days). When assessing the treatment effect, the difference in average QALY (95% CI) was 3.2 days (−13.1, 7.4) when calculated from patient responses, and 5.3 days (−15.4, 3.9) from carer responses. C l i Phase 1: We extracted data from 254 RCTs; 208 assessment tools were identified of which 146 met the criteria of reproducible out- come measure. The Fugl-Meyer (arm function section) was used most frequently (79/254 (31%) RCTs). 120/208 (58%) outcome measures were only used in one RCT. Background Clinical trials requiring patient reported data involving patients with multiple symptoms and/or a poor prognosis are often considered challenging. There is concern about over-burdening trial participants, either through more regular follow-up or by using longer question- naires. One possible solution is to obtain data about the patient in- directly by asking their carer. The QUARTZ trial assessed the use of whole brain radiotherapy (WBRT) in patients with inoperable brain metastases from non-small cell lung cancer. This is a very poor prognosis group, and patients can experience rapid changes in condition, which necessitated fre- quent data collection. As the trial focused on quality of life, patients were asked to complete the EQ-5D questionnaire on a weekly basis. At the same time, their carer was asked to complete the same ques- tionnaire from the point of view of the patient, so that the potential use of proxy scores could be assessed. Objectives y Conclusions The level of agreement between patients and carers was reason- ably high on most questions. The agreement was lowest for the question about anxiety and depression, with carers tending to re- port more problems compared to the patient’s own assessment. The level of agreement seen means it may be reasonable to use the carer response in some situations where it is not appropriate to ask the patient directly. Caution is advised though as even for the question on mobility there was disagreement in 18% of cases. The difference in the analysis of the trial’s primary endpoint was minor and did not change the main conclusion of the study. Therefore within the confines of a clinical trial, it could be appro- priate to use these proxy assessments to assess treatment effects. QUARTZ is one of few trials in this setting, and further studies looking at the use of proxy responses in poor prognosis popula- tions are warranted. Phase 2: 43 stroke survivors and carers, and 58 health professionals par- ticipated in the NGT sessions. Ten stroke survivors and carers participated in eight interviews. Data analysis will be completed by January 2017. C l i Phase 1 highlighted wide variation and lack of consistency in use of arm function outcome measures in RCTs. Consensus recommendations that account for psychometric properties, and the perspective of stroke survivors, carers, and healthcare professionals, will enable valid, reliable and meaningful measurement in future RCTs of arm rehabilitation. Therefore, recommendations for priority outcome measures that meas- ure important outcomes are warranted. By agreeing on a toolbox of key outcome measures for inclusion, subsequent RCTs’ outputs will en- hance comparability of RCT results and facilitate comprehensive meta- analyses of the effectiveness of interventions. quartz trial Matthew Nankivell1, Paula Mulvenna2, Rachael Barton3, Corinne Faivre-Finn4 Paula Wilson5, Elaine McColl6, Barbara Moore7, Iona Brisbane8, David Ardron9, Richard Stephens10 1MRC Clinical Trials Unit at UCL AND MRC London Hub for Trials Methodology Research; 2Northern Centre for Cancer Care; 3Queen’s Centre for Oncology and Haematology; 4Institute of Cancer Sciences; 5Bristol Haematology and Oncology Centre; 6Newcastle Clinical Trials Unit and Institute of Health and Society; 7Wales Cancer Research Network; 8The Beatson West of Scotland Cancer Centre; 9Patient representative; 10MRC Clinical Trials Unit at UCL (retired) Correspondence: Matthew Nankivell Trials 2017, 18(Suppl 1):P288 Objectives Describe current outcome measures used in arm rehabilitation RCTs and their psychometric properties. Identify outcomes important to stroke sur- vivors with arm function problems, their carers and healthcare profes- sionals. Produce final consensus recommendations to support selection of outcome measures for use in future arm rehabilitation RCTs. Results The EIP could produce the same diagnosis with summary data as for complete data, although the EIP preferred the complete data, or used in conjunction with the summary report, where cases were considered complex. Sometimes it was felt necessary to select two or more conditions where they could equally be the cause of pain, although the EIP member was required to be at least 50% certain. The panel were in excellent agreement in identifying deep infiltrating endometriosis and endometrioma of the ovary as causes of CPP, and in good agreement for superficial peritoneal endometriosis and ade- nomyosis when the MRI reports were used in the reference standard. Lower levels of agreement were noted for deciding that adhesions, fibroids and pelvic inflammatory disease were the cause of pelvic pain. The EIP failed to demonstrate adequate reliability in determin- ing whether any of the non-gynaecological causes were the cause of CPP. Conclusion There is little evidence on how panels should be convened, how information should be presented or what the best methods for consensus are and the MEDAL study highlights many potential challenges. 98 unique outcomes were identified from 182 studies. The outcomes were mapped to 37 outcome domains in Filter 2.0. Outcomes were most common in the domains of the structure of the intestine, defecation functions, pain in the stomach and abdomen and quality of life. Disease activity indices and QOL questionnaires were used extensively in the included studies. Their coverage of the Crohn’s Filter framework domains is dependent upon the combinations in which they are used. Five hundred ninety-three unique AEs were identified in the trials, which mapped to 46 outcome domains in Filter 2.0. AEs were most common in domains of infections, sensations of pain, the structure of the intestine, pain in the stomach and abdomen and sensations associated with the digestive system. Conclusions and recommendations There is large variation in the outcomes and AEs reported across RCTs for CD, which supports the need for a core outcome set to be developed. – Using an existing conceptual framework to organise outcomes data into domains and assessing their importance by frequency of reporting is a pragmatic solution in lieu of a consensus approach. – Using an existing conceptual framework to organise outcomes data into domains and assessing their importance by frequency of reporting is a pragmatic solution in lieu of a consensus approach. Background g Crohn’s disease (CD) is a chronic condition causing digestive tract inflammation. Treatments may be used in combination and include corticosteroids, immunosuppressives, 5asas, biologics and antibiotics. All carry risks of adverse events (AEs). Case study y The MEDAL study asked whether magnetic resonance imaging (MRI) could replace or triage the use of laparoscopy in establishing gynae- cological diagnosis among women with chronic pelvic pain (CPP). The primary analysis assessed the accuracy of the MRI for identifying the condition(s) causing CPP, using an EIP. The panel consisted of 15 Consultant Gynaecologists, not involved in the study recruitment, with each meeting involving three members. The reference diagnosis was made in two stages. 1. Using patient history and reported symp- toms, clinical examination, ultrasound, laparoscopy and follow-up 2. As above with MRI scan report For both stages, each member indi- vidually recorded the condition(s) for which they were >50% certain were the cause of pain, prior to a group discussion to achieve a final consensus diagnosis for the reference standard. To assess the reliabil- ity of the EIP diagnoses we considered the agreement between the three individual ratings, using Kappa, made by the panel members prior to any group discussion. We faced several challenges: 1. The quantity of information to assimilate and use of summary or complete data 2. Eliciting a binary response, when there are degrees of uncer- tainty 3. Multiple potential causes of pain which are not directly corre- lated with the extent of pathology observed 4. Avoiding incorporation bias from knowledge of MRI results 5. Laparoscopy cannot identify some target conditions, only MRI can 6. Potential dual purpose of lapar- oscopy as diagnostic and therapeutic. The study aim was to identify the important outcomes and AEs resulting from treatments for CD. The study objectives were to: – Perform a systematic review of trials, extracting the reported outcomes and AEs for CD. – Classify the outcomes and AEs into a conceptual framework proposed for developing COSs (OMERACT Filter 2.03, in such a way as to rank their importance. Identify longer term AEs from the Summary of Product – Identify longer term AEs from the Summary of Product Characteristics (SPCs) 2 for the identified treatments. Characteristics (SPCs) 2 for the identified treatments. Results – Making use of AEs data is complicated as they may reflect disease process, failure or side effects of drugs and unrelated events. – Researchers should make decisions on the use of disease activity indices and QOL questionnaires by considering the outcomes that will be captured. The mapping conducted here will aid this. – Further recommendations will follow when the work is concluded (analysis of withdrawals and SPC data). – Further recommendations will follow when the work is concluded (analysis of withdrawals and SPC data). This abstract is not included here as it has already been published. Outcome measures in clinical trials of traditional Chinese medicine for stable angina pectoris Junhua Zhang1, Dongmei Xing2, Mingyan Zhang1 1Tianjin University of Traditional Chinese Medicine; 2The first teaching hospital of Henan University of TCM Correspondence: Junhua Zhang Trials 2017, 18(Suppl 1):P292 Outcome measures in clinical trials of traditional Chinese medicine for stable angina pectoris 1 2 1 Junhua Zhang1, Dongmei Xing2, Mingyan Zhang1 1Tianjin University of Traditional Chinese Medicine; 2The first teaching hospital of Henan University of TCM Correspondence: Junhua Zhang Trials 2017, 18(Suppl 1):P292 Methods MEDLINE MEDLINE, EMBASE, CENTRAL and CINAHL were searched. The inclusion criteria were: 1. Randomised controlled trials, 2. Adult patients with CD, 3. All interventions to treat CD and its complications. AEs were standardised using the Medical Dictionary for Regulatory Activities (MEDDRA)5 terminology. Outcome and AEs were classified into domains using the ICF, the Wilson and Cleary model of health- related quality of life (QOL)6 and other domains recommended by OMERACT. The domains were used to populate the Filter 2.0 frame- work, creating the ‘Crohn’s Filter’. Background In a test evaluation study, where there are a number of target condi- tions to be considered, not all of which have a perfect reference stand- ard, there is a risk of partial or differential verification of the underlying causes, with the inherent bias. One approach is to use an expert inde- pendent panel (EIP) to determine the presence or absence of the target Correspondence: Matthew N Trials 2017, 18(Suppl 1):P288 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 110 of 235 Page 110 of 235 condition based on several sources of information, ideally using a pre- determined algorithm. condition based on several sources of information, ideally using a pre- determined algorithm. The most important outcomes and AEs are not well defined, espe- cially long term. There is no CD core outcome set (COS). One is pro- posed for Inflammatory Bowel Disease (IBD) using the International Classification of Functioning, Disability and Health (ICF) 1. py Results py g p Results Outcome measures in clinical trials of traditional Chinese medicine for stable angina pectoris Junhua Zhang1, Dongmei Xing2, Mingyan Zhang1 1Tianjin University of Traditional Chinese Medicine; 2The first teaching hospital of Henan University of TCM Correspondence: Junhua Zhang Trials 2017, 18(Suppl 1):P292 Objectives To analyze the outcome measures in clinical trials of Traditional Chinese Medicine (TCM) for stable angina pectoris and provide data to develop core outcome sets (COS) for clinical trials of TCM. Page 111 of 235 Page 111 of 235 Trials 2017, 18(Suppl 1):200 Methods agreement level was achieved in 78 (83.4%) of procedural steps. For example, it is mandatory to place transanal purse-string suture with small bites (78.6%) to achieve a tight closure and stable pneumorec- tum, and begin transanal TME with circumferential full thickness muco- sal dissection (92.9%). A Competency assessment tool (CAT) of TATME was developed incorporating the mandatory steps and quality from the standardisation process. Three Chinese electronic biomedical literature databases (sinomed, CNKI and Wanfang) were searched to identify randomized clinical trials (RCTs) of TCM for stable angina pectoris published in 2010. RCTs evaluated the effectiveness of TCM for stable angina pectoris were considered to be eligible. There was no limitation to the type of TCM intervention and control. All reported outcomes and related information of included trials were extracted. Two authors screened literatures and extracted information dependently. Res lts Standardisation and competency assessment of transanal total mesorectal excision (TATME) for rectal cancers: surgical quality assurance in the context of a randomised controlled trial Ali T i S ll M li G H Standardisation and competency assessment of transanal total mesorectal excision (TATME) for rectal cancers: surgical quality assurance in the context of a randomised controlled trial Alice Tsai, Stella Mavroveli, George Hanna Imperial College London Correspondence: Alice Tsai Trials 2017, 18(Suppl 1):P293 assurance in the context of a randomised controlled trial Alice Tsai, Stella Mavroveli, George Hanna Imperial College London Correspondence: Alice Tsai Trials 2017, 18(Suppl 1):P293 Alice Tsai, Stella Mavroveli, George Hanna Imperial College London Correspondence: Alice Tsai Trials 2017, 18(Suppl 1):P293 , , Imperial College London Correspondence: Alice Tsai Trials 2017, 18(Suppl 1):P293 Correspondence: Kirsty Cockle Trials 2017, 18(Suppl 1):P294 Correspondence: Kirsty Cockle Trials 2017, 18(Suppl 1):P294 Conclusion The EAGLE study: improving the wellbeing of men by evaluating and addressing the gastrointestinal late effects of radical treatment for prostate cancer Kirsty Cockle1, Ann Muls2, Jervoise Andreyev2, John Green3, Sam Ahmedzai4, Catherine Ferguson5, Lesley Smith6, Sara Pickett7, Damian Farnell8, Susan Campbell1 1Marie Curie Palliative Care Research Centre, Cardiff University; 2The Royal Marsden NHS Foundation Trust; 3Cardiff and Vale University Health Board; 4Academic Unit of Supportive Care, Department of Oncology, University of Sheffield; 5University of Sheffield Medical School; 6Macmillan Cancer Support; 7Swansea Centre for Health Economics, Swansea University; 8School of Dentistry, Cardiff University The EAGLE study: improving the wellbeing of men by evaluating and addressing the gastrointestinal late effects of radical treatment for prostate cancer Kirsty Cockle1, Ann Muls2, Jervoise Andreyev2, John Green3, Sam Ahmedzai4, Catherine Ferguson5, Lesley Smith6, Sara Pickett7, Damian Farnell8, Susan Campbell1 1Marie Curie Palliative Care Research Centre, Cardiff University; 2The Royal Marsden NHS Foundation Trust; 3Cardiff and Vale University Health Board; 4Academic Unit of Supportive Care, Department of Oncology, University of Sheffield; 5University of Sheffield Medical School; 6Macmillan Cancer Support; 7Swansea Centre for Health Economics, Swansea University; 8School of Dentistry, Cardiff University P293 Standardisation and competency assessment of transanal total mesorectal excision (TATME) for rectal cancers: surgical quality assurance in the context of a randomised controlled trial Alice Tsai, Stella Mavroveli, George Hanna Imperial College London Correspondence: Alice Tsai Trials 2017, 18(Suppl 1):P293 Conclusion Standardisation of techniques is crucial in a RCT in order to assess sur- gical competency for pre-trial recruitment and monitor compliance dur- ing the trial period. We have developed an objective and robust methodology to perform standardisation, which was used in develop- ing an operation manual and a competency assessment tool (CAT) for SQA in an international multicentre RCT. Validation of TATME CAT will be performed to ensure usability, reliability and validity. Finally ninety-four RCTs (with 9111 subjects) were included for ana- lyses. There were totally 79 outcome measures were reported in all included RCTs. The ten mostly reported outcomes were efficacy rate of ECG, efficacy rate of angina pectoris, efficacy rate of TCM syndrome, fasting lipid profile, withdrawal rate of nitroglycerin, total scores of TCM syndrome, rate of clinical efficacy, nitroglycerin consumption, hemodynamic indexes, endothelin, efficacy rate of in- dividual symptoms of TCM, frequency of angina attack. There were also several significant problems about outcomes in the included trials: (1) Significant heterogeneity of outcomes. The number of out- comes ranged from 1 to 21 with an average number of 5. For a same outcome, the evaluation points and detecting methods varied (2) Continuous data were arbitrarily transformed into ranked data. More than 90% of the trials reported the efficacy of outcome mea- sures with percentages. (3) Only a few trials reported the outcomes associated advantages and characteristic of TCM. (4) Outcomes were randomly selected and reported with bias. y y y The CAT will be used in video analysis during the trial entry process. Surgeons that wish to participate will be invited to submit 2 unedited full-length videos for transanal TME and 1 for laparoscopic TME. There should be at least one male case for TATME; this is due to the fact that pelvic dissection in a male patient is usually more challenging. Both the abdominal and transanal components should be recorded and submit- ted. Two assessors approved by the expert panel will assess the videos independently using CAT. Aim Prostate cancer survival is improving, in the UK it has tripled in the last 40 years. Radiotherapy is an effective treatment with the aim of cure, however, many patients experience a change in bowel habit, such as urgency and incontinence, developing months to years after comple- tion of treatment. Evidence suggests that management of these symp- toms by a gastroenterologist can be effective when following a detailed investigative and treatment algorithm and an equivalent level of care can be given by a trained nurse. The EAGLE study focuses on the early identification and management of radiotherapy-induced bowel symptoms, with the aim of improving the quality of life of pros- tate cancer survivors and their partners and/or families. M th d Transanal total mesorectal excision (TATME) is a new innovative tech- nique and theoretically allows more precise dissection with lower rates of incomplete mesorectum and involved circumferential resection margins comparing to laparoscopic TME. An international multicentre randomised controlled trial (RCT) comparing the two operations has been designed. Studies have shown the importance of standardisation of interventions in surgical rcts, however it is poorly conducted. The aim is to standardise the surgical steps and quality of tatme and develop a competency assessment tool (CAT) for Surgical Quality Assurance (SQA) of the trial. Conclusions Inappropriate outcomes with wrong evaluating points and methods will impaired the value of clinical trial. Establishing a core outcome set (COS) of TCM for stable angina are warranted. Recruitment and participation in the stem cell trial of recovery enhancement-3 (stems-3): experiences of stroke survivors and their carer’s Claire Diver1, Rebecca O'Connor2, Nikola Sprigg3, Louise Connell4, Marion Walker5, Philip Bath3 Method Method Successful evidence-based healthcare interventions are often challen- ging to embed into local clinical practice. In this effectiveness- implementation hybrid study, a multi-component intervention consist- ing of: a simple screening tool to identify men with late effects of radio- therapy in oncology follow-up clinics; rapid referral to a specialised gastroenterology service; and enhanced algorithm-led assessment lead- ing to targeted advice and treatment, was implemented in three UK centres. Mixed method approaches to data collection and analyses are used to assess the effectiveness of the intervention and the implemen- tation strategy at baseline, six month and twelve month intervals. Implementation outcome variables such as, acceptability to patients and healthcare professionals, large scale adoption and sustainability are addressed via longitudinal semi-structured interviews, alongside the introduction of possible solutions to facilitate local implementation. A control group of prostate cancer survivors with radiotherapy-induced bowel symptoms from another local health board are followed up for twelve months to assess the cost effectiveness of the intervention. A robust 4-round Delphi methodology was applied with peer- nominated international expert consultants in TATME. Semi-structured interviews were conducted in Delphi round 1 where surgeons gave de- tails on how they perform TATME. Interviews were transcribed and ana- lysed using a qualitative analysis software. The result was used for hierarchical task analysis (HTA) to identify the key stages and steps. The HTA result was subsequently used in Round 2, 3 and 4 as question- naires, which contained all the variations and were distributed to the same group of experts. Each step was as rated mandatory, optional or prohibited. The steps that have reached 70% agreement were used to develop a competency assessment tool and operation manual for SQA. Result The 4 rounds of Delphi achieved 96.4% of response rate. Three main phases were identified: peri-operative, abdominal phase and transanal phase. Four main stages were identified within the transanal phase: (i) Transanal platform set up, (ii) Purse-sting placement, (iii) TME dissec- tion, and (iv) Specimen extraction and anastomosis. A seventy percent Trials 2017, 18(Suppl 1):200 Page 112 of 235 Page 112 of 235 Patients’ experience in adaptive oncology clinical trials: trial acceptability, feasibility, efficiency, processes and outcomes Annmarie Nelson, Mirella Longo Annmarie Nelson, Mirella Longo Cardiff University Correspondence: Annmarie Nelson Trials 2017, 18(Suppl 1):P295 g Discussion Trials with adaptive designs attempt are intended to be efficient and streamlined, by modifying parameters, as appropriate, usually based on objective outcomes. The addition of participant experience as a sec- ondary outcome measure allows for contextual factors and participant preference to be included in decisions to adapt, therefore increasing the likelihood of trial completion, and adoption and compliance with interventions in a real world setting..Patient experience of the interven- tion as a secondary outcome, combined with real time reporting of pa- tient experience of trial processes can provide both immediate benefit to trial processes, and a further understanding of the acceptability and effectiveness of the intervention when aligned with the primary and secondary trial outcomes. Background Qualitative studies of participant experience embedded within oncol- ogy ctimps are rarely undertaken but can usefully explain why recruit- ment is slow, or why participants are unlikely to comply with the intervention in a real world setting, or why they might decline to enter or withdraw from the trial. Previous studies undertaken by the Marie Curie Research Centre, using patient experience as a secondary trial outcome, have: Highlighted patient preferences for treatment in a non- inferiority trial, Exposed issues of clinical equipoise in two feasibility trials that failed to recruit, Supported strategies for recruitment in a primary care trial, Explored participant’s understanding of complex trial processes in a stratified trial of personalised therapies, Articulated the potential reasons that patients decline surgical trials. Additionally, par- ticipant interview data may be used in real time for presentation to TMG and TSC meetings to allow timely protocol amendments in order to improve recruitment and retention of participants. This concurrent approach is being successfully used in an HTA funded, ongoing clinical trial (ROCS: NCT01915693), and has enabled protocol amendments, such as timing of recruitment and assessments, that have improved re- cruitment and improved data capture. g Many stroke survivors have residual disability, even after discharge from post-hospital community rehabilitation. Advancements in acute stroke management suggest a role for the use of pharmacological agents in enhancing the neurological response to rehabilitation therapy. STEMS-3 (ISRCTN16714730) was a feasibility, 2x2 factorial randomised controlled trial of granulocyte colony stimulating factor (G-CSF) and/or physiother- apy at least 6 months after stroke. Recruitment and retention to clinical trials can be influenced by participant views, research design, context- ual and environmental factors. It is reported stroke survivors believe continued physiotherapy will be beneficial, but it is not known whether these beliefs influence participation in clinical trials of rehabilitation. Evidence suggests willingness to participate in drug trials is directly re- lated to the presence of an illness the drug is designed to improve. It is not known if this is true amongst stroke survivors or when drug ther- apy is offered in conjunction with rehabilitation interventions. Family members and carers frequently support older patients in decision mak- ing about participation in clinical trials; this has not been studied amongst stroke survivors. The aim of this study was to explore recruit- ment and participation in STEMS-3 from the perspective of participat- ing stroke survivors and their carers. Aims An integrated qualitative component within an adaptive design trial can inform the primary and secondary trial objectives in the recruit- ment and testing phases of the trial and, for the purposes of an adaptive MAMS design, the stop/keep criteria per arm by assessing, for example: The feasibility of patients; Recruitment to the trial by examining their experience of consent and recruitment, including reasons for declining participation; Participants’ motivation to accept randomisation to different interventions; Potential improvements to recruitment processes; Participants’ understanding and experience of each trial arm; Participants’ experience and tolerability of toxicities; Participants’ attitudes to the value of predicted treatment benefit. Methods Conclusion This study evaluates the effectiveness of an enhanced assessment and treatment service, spanning oncology and gastroenterology de- partments, in improving the outcome of prostate cancer survivors post-radiotherapy. The factors needed for successful implementation into local practice, which are essential for new initiatives in health- care settings, will act as examples of best practice for a network of centres of excellence in this field. Recruitment and participation in the stem cell trial of recovery enhancement-3 (stems-3): experiences of stroke survivors and their carer’s Recruitment and participation in the stem cell trial of recovery enhancement-3 (stems-3): experiences of stroke survivors and their carer’s Claire Diver1, Rebecca O'Connor2, Nikola Sprigg3, Louise Connell4, Marion Walker5, Philip Bath3 1School of Health Sciences; 2Nottingham University Business School, University of Nottingham; 3Stroke Trials Unit, University of Nottingham; 4Allied Health Research Unit, University of Central Lancashire; 5Division of Rehabilitation and Ageing, University of Nottingham Correspondence: Claire Diver Trials 2017, 18(Suppl 1):P296 Results strategies may be used according to the context and sensitivity of topic and the quality of the data. Our previous trials have utilised Thematic Analysis, Framework Analysis, and Interpretative Phenom- enological Analysis, amongst others. Discussion The screening tool used in oncology to identify men suffering from the late effects of radiotherapy has been validated against the Gastrointestinal Symptoms Rating Scale (GSRS). Qualitative interview data from patients suggests that the screening tool is quick and easy to understand, addresses symptoms that are of most concern to pa- tients, and facilitates open discussion of their bowel symptoms with healthcare professionals. Final results will measure the effect of the new service in terms of acceptability to staff and patients, quality of life improvements, symptom control and cost effectiveness. Qualita- tive interview data from healthcare professionals across the three UK sites will address how each team is working towards sustainability of the service. g Method Q l Qualitative interview study underpinned by theoretical perspectives of pragmatism and critical reflection using a purposive sampling strategy to identify stroke survivors who had been recruited to the trial and their carers. Semi-structured interviews were conducted, recorded and tran- scribed verbatim. Thematic analysis using an iterative and emergent approach was adopted. Results 16 participants (11 stroke survivors, 5 carers) were recruited from 60 participants in STEMS-3. 3 themes were identified: personal, practical and procedural. Decisions to participate were influenced by: opportunity for further treatment, potential bene- fits to others, practical factors including treatment and assessment loca- tion; side effects were considered worth the risk. Information given to stroke survivors prior to consent was perceived as too long and difficult to comprehend, and did not influence participation decisions. Carers supported decision making and in contrast to participants, valued de- tailed information. The principles of randomisation and blinding were understood by participants, and participants thought the trial team would not give them a treatment that was ineffective or harmful. There Qualitative data sets can be analysed for common themes in relation to real-time participant experience of the trial processes and treat- ment protocols. The analysis takes into account spontaneously re- ported participant experience, which reflects idiosyncratic attitudes and personal contexts, to enable patient reported outcomes supple- mentary to the stopping criteria in an adaptive design and main trial outcomes. A range of qualitative analytic frameworks and sampling Trials 2017, 18(Suppl 1):200 Page 113 of 235 Page 113 of 235 Mum 137: I think for us it’s probably gonna be the activity one, rather than the graded, umm, exercise, because, err, we’ve been doing the pacing, it’s been quite good, so that’s the one I’d probably lean against but I’d like to know a bit more about them both. Conclusions Mum 137: I think for us it’s probably gonna be the activity one, rather than the graded, umm, exercise, because, err, we’ve been doing the pacing, it’s been quite good, so that’s the one I’d probably lean against but I’d like to know a bit more about them both. Conclusions were concerns trial recruitment was used to facilitate discharge from existing services and trial completion was considered the ‘end of the road’; this was most marked for those that received no active intervention. Outcome measure assessments were often the first time anxiety, depression and personal relationships were discussed and heard by the carers. P297 Treatment preference and recruitment to a paediatric randomised controlled trial: managed activity graded exercise in teenagers and pre-adolescents (MAGENTA) Treatment preference and recruitment to a paediatric randomised controlled trial: managed activity graded exercise in teenagers and pre-adolescents (MAGENTA) p Lucy Beasant, Nicola Mills, Esther Crawley University of Bristol Correspondence: Lucy Beasant Trials 2017, 18(Suppl 1):P297 Methods PRECIS-2 ratings were collected at two annual in-person meetings of the Pragmatic Trials Collaborative Project. After a presentation and brief training at the first meeting (Time 1), Principle Investigators (PIs) rated their trials as originally designed (prior to trial initiation). After a refresher session at the second annual meeting (Time 2), PIs again rated their trials on current status on each domain, without access to their ratings from the first meeting. Qualitative telephone interviews were conducted with each PI subsequent to the meeting (summer 2016) to review the domains with a rating change; domains that were stable (no change in rating) were not discussed. The interview protocol included questions about the PI’s experience with and per- ception of the value of the PRECIS-2 tool. Interviews were recorded and transcribed. Background The intended purpose of the Pragmatic-Explanatory Continuum Indi- cator Summary (PRECIS-2) tool is to help trialists consider the impact design choices across nine domains have on the applicability of their results in the clinical setting: from “1” Very explanatory (intervention is tested under ideal conditions) to “5” Very pragmatic (intervention is tested under usual conditions). Added value of increased adoption of the tool is the emergence of a framework for investigators to com- municate trial design decisions. PRECIS-2 is currently being used in a National Institutes of Health (NIH), National Heart, Lung, and Blood Institute (NHLBI) Pragmatic Trials Collaborative Project to explore whether a rating adjustment from the planning phase to the first year of implementation in a group of low-cost, pragmatic trials re- flects true shifts in trial design. p Methods Recruiters were offered one-to-one training and feedback on four separate occasions as the feasibility trial progressed over a period of 12 months [8]. Recruitment discussions were recorded, tran- scribed and analysed thematically using techniques of constant comparison [9]. Background & Objective Effective recruitment and retention in randomised controlled trials (RCTs) is hugely important. Reduced patient recruitment rates may prevent trial completion [1] and post randomisation dropout leads to the loss of statistical power [2]. Recruitment to an RCT can be af- fected if patients hold a preference for one of the treatments offered in the trial [3]. Qualitative research methodology embedded in adult RCTs has demonstrated that acknowledging and addressing patient concerns about preference can enhance the acceptability of random- isation and improve informed consent [4–6]. There is limited evidence on whether or not preferences are expressed by young patients and their parents during recruitment to paediatric trials, which have the added complexity and interplay between patient, parent and health professional equipoise [7]. The aim of the current research was to investigate whether effective communication approaches used to discuss treatment preference in adult trials were also effective in paediatric RCTs. g Method Q l Communication strategies and approaches used to train recruiters in adult trials can be used in a paediatric setting with young patients (aged 10-17 yrs) and their parents, but care should be taken to explore reasons underlying both patient and parent preferences. Providing re- cruiters with training and guidance can ensure families are more fully informed about treatment options at recruitment, but further investiga- tion of the effect on retention is required. Discussion This study has highlighted how trial design results in multiple factors that impact on stroke survivors and their carers’ decisions to take part in clinical trials and their subsequent experiences. Feasibility and acceptability studies are essential in the evaluation and development of trials of complex interventions. They should include a qualitative investigation of study design that focusses on the procedural, prac- tical and personal factors that impact upon individuals’ decisions to participate, and remain, in clinical trials. For trials involving stroke survivors consideration should be given to incorporating the views of caregivers who support stroke survivors. P298 Framing the conversation: use of PRECIS-2 ratings to advance understanding of pragmatic trial design domains Paula Darby Lipman1, Kirsty Loudon2, Leanora Dluzak1, Rachael Moloney 3, Donna Messner3, Catherine M. Stoney4 1Westat; 2Stirling University; 3Center for Medical Technology Policy; 4National Heart, Lung, and Blood Institute, National Institutes of Health Correspondence: Paula Darby Lipman Trials 2017, 18(Suppl 1):P298 Background Challenge 1 - Accommodating diverse populations: Rational MCC accommodates biological diversity including presence of an inte- grated virus, immune dysfunction, immune infiltration and somatic mutations and multiple management pathways driven by clinical variation, uncertainty and opinion. This has been achieved through relaxed eligibility criteria and a focus on one question early in the treatment pathway. An inbuilt 3-year feasibility study allows adapta- tions to be made based on current data resulting in a more homoge- neous population. In randomised control trials where outcomes are collected via ques- tionnaire, maximising the response rate is paramount. Loss to follow- up reduces the effective sample size and can place added assump- tions on analysis, potentially leading to bias and compromising the validity of results. SIFT (ISRCTN76463425) is a multicentre randomised controlled trial of a feeding intervention in very preterm or very low birthweight infants (gestational age at birth <32 weeks; birth weight <1500 g) in neonatal units in the United Kingdom and Ireland. Primary outcome is the proportion of infants surviving without moderate or se- vere disability at 24 months of age corrected for prematurity. This is assessed via a questionnaire sent directly to parents asking for informa- tion on their infant’s health and development. Challenge 2 - Design for rare populations: A conventional trial design would require >3000 patients to prove superiority beyond reason- able doubt. Instead, using a Bayesian probabilistic approach, ob- served data from 250 patients can still be informative to guide the decision-making process; providing clinicians and patients with prob- abilities that either treatment out-performs the other in reducing risk of loco-regional failure; aiding individual decisions. Having confirmed address details and survival status, the SIFT Trial Coordinating Centre sent participants’ parents a card for their child’s second birthday and a thank-you note reminding them of the questionnaire and encouraging them to update their contact details. Staff then posted a questionnaire to the parent (including a sticker set for their child) 17 days before the child’s age-corrected second birthday. If there was no response, another copy of the questionnaire plus reminder letter was posted on their age- corrected birthday. Two weeks later, another copy of the question- naire was posted, and two weeks after that, SIFT staff attempted to call parents about the questionnaire. Challenge 3 - Methodology for aggressive cancers: Rapidly progres- sing diseases leave little time for screening. Results Adapting trial design for challenging populations: how the rational MCC trial was designed to account for a rare, aggressive cancer in an elderly and diverse population Rachel Blundred1, Christina Yap2, Sarah Pirrie2, Sarah Bowden2, Oliver Cassell3, Carie Corner4, Catherine Harwood5, Pat Lawton6, Clair Mcgarr7, Ian Zealley8 1University of Birmingham; 2Cancer Research UK Clinical Trials Unit, University of Birmingham; 3Oxford University Hospitals NHS Trust; 4East and North Herts NHS Trust; 5Queen Mary University of London; 6Nottingham University Hospital NHS Trust; 7University Hospitals Birmingham NHS Foundation Trust; 8NHS Tayside C d R h l Bl d d Adapting trial design for challenging populations: how the rational MCC trial was designed to account for a rare, aggressive cancer in an elderly and diverse population Rachel Blundred1, Christina Yap2, Sarah Pirrie2, Sarah Bowden2, Oliver Cassell3, Carie Corner4, Catherine Harwood5, Pat Lawton6, Clair Mcgarr7, Ian Zealley8 P300 Using a patient registry to conduct non-clinic engaged studies Stephanie Dubose, Nicole C. Foster, Kellee M. Miller Jaeb Center for Health Research Correspondence: Stephanie Dubose Trials 2017, 18(Suppl 1):P300 P300 Using a patient registry to conduct non-clinic engaged studies Stephanie Dubose, Nicole C. Foster, Kellee M. Miller Jaeb Center for Health Research Correspondence: Stephanie Dubose Trials 2017, 18(Suppl 1):P300 1University of Birmingham; 2Cancer Research UK Clinical Trials Unit, University of Birmingham; 3Oxford University Hospitals NHS Trust; 4East and North Herts NHS Trust; 5Queen Mary University of London; 6Nottingham University Hospital NHS Trust; 7University Hospitals Birmingham NHS Foundation Trust; 8NHS Tayside This abstract is not included here as it has already been published. Correspondence: Rachel Blundred Trials 2017, 18(Suppl 1):P299 Correspondence: Rachel Blundred Trials 2017, 18(Suppl 1):P299 Increasing follow-up response among parents of very preterm infants: personalised contact, external promotion, and web-based questionnaires People with rare cancers need evidence-based treatment. Trials require large samples to disprove no difference in treatment effect. Rare cancers, like commoner malignancies, can be diverse affecting popula- tion selection, stratification and applicability of trials. The aggressive skin cancer, Merkel cell carcinoma (MCC) affects <300, predominantly elderly, UK patients annually. Standard management with surgery and/ or radiotherapy for loco-regional MCC is based on retrospective data. Rational MCC aims to compare surgery and radiotherapy as first defini- tive treatment for MCC in control of loco-regional disease. The chal- lenges encountered in designing this trial can be applied to many disease areas. Madeleine Hurd1, Louise Linsell1, Ed Juszczak1, Oliver Hewer1, Ursula Bowler1, Samantha Johnson2, Jon Dorling3, all on behalf of 'The SIFT Collaborative Group 1University of Oxford; 2University of Leicester; 3University of Nottingham Correspondence: Madeleine Hurd Trials 2017, 18(Suppl 1):P301 Background Rational MCC has min- imal screening requirements and collects most baseline data from routine investigations. In addition, sites are required to hit Protocol- defined treatment target times that mirror optimal clinical practice, ensuring that randomisation does not produce a delay to front line treatments. p q Follow-up began in August 2015. By February 2016, response rate remained low: approximately 50%. Objective Challenge 4 - Overcoming clinical preconceptions: It can be difficult for clinicians to consider alternative options when one treatment is used more commonly. Rational MCC has an observational arm mir- roring the interventional arm, which patients can enter if they are unsuitable/unwilling to be randomised. This produces prospective data even if no patients are randomised. Sites will be provided with a checklist to guide SSMDT discussions to facilitate consideration of treatment options. To outline measures taken to increase follow-up response rate during the trial and their efficacy. Results Thematic analysis revealed: 1. Acceptance of preference at face value without exploration; this resulted in families declining the trial with little understanding of the reasons that underpin preference: Five PIs completed paired ratings for Time 1 and Time 2 for 9 PRECIS-2 domains, resulting in a total of 45 paired ratings. Of these, over half (N = 24) demonstrated changes in ratings from Time 1 to Time 2, and each trial had changes on at least three domains (range “3” To “5”). Domains with the fewest changes were Recruitment Path, Primary Outcome, and Primary Analysis; and those with the most were Eligibility, Organization, and Flexibility of Adherence. Some do- mains were rated as more pragmatic at Time 2 (11/24 domains with higher rating), and others as more explanatory (13/24). Qualitative analysis of the interview data identified three reasons for a rating change: true change in design; change in interpretation of the do- main; and misunderstanding of the PRECIS-2 domain. All PIs agreed that the tool is useful during the planning phase of trial design, and most have used the tool in subsequent work including proposals and protocols. Mum 140: if we were given the graded exercise, I think he would prob- ably withdraw Recruiter: I think, if you feel very strongly that you would want one treatment rather than the other, I think it’s better not to be in the study…[patient declined trial]. 2. Identification and exploration of preference; in-depth discussion about reasons for preference allowed families to make a more in- formed decision about study participation: y p p Patient 3: It’s the exercise one I don’t really like the sound of Patient 3: Its the exercise one I don t really like the sound of Recruiter: graded exercise therapy doesn’t mean that you’ve got to go and do, sort of a jog round the block, or anything like that, umm what it’s doing is monitoring your activity and helping you to build that up. We’re trying to sort out a better treatment for children in the future…some of our patients when we’re talking about exercise, umm, exercise for them is brushing their hair[patient accepted randomisation]. Conclusion The NIH Pragmatic Trials Collaborative Project demonstrates how the PRECIS-2 tool can frame a conversation around initial trial design 3. Results ‘We’ Or ‘I’; parents often expressed preference on their child’s behalf: Page 114 of 235 Page 114 of 235 Trials 2017, 18(Suppl 1):200 Page 114 of 235 choices and provide clarity regarding where these decisions fall along the pragmatic-explanatory continuum. In addition to advan- cing our understanding of pragmatic trial design, discussions around application of the tool can be used to reflect on drivers of genuine adjustment to trial design as well as to help to clarify misunderstand- ings about the definitions of the tool domains which could be ad- dressed through training. choices and provide clarity regarding where these decisions fall along the pragmatic-explanatory continuum. In addition to advan- cing our understanding of pragmatic trial design, discussions around application of the tool can be used to reflect on drivers of genuine adjustment to trial design as well as to help to clarify misunderstand- ings about the definitions of the tool domains which could be ad- dressed through training. that reflect the information required at different time points. Large font versions of documents are available and information will also be provided in DVD format Randomised trials are expensive and failure to recruit wastes funding. The observational arm produces a bank of biological and clinical data even if patients are not randomised. Additional questions will be reviewed during the feasibility study to further maximise value. Data from the feasibility phase will be used to guide operational adaptations to ensure the Protocol continues to be informed by best practice The Rational MCC trial is funded by the Efficacy and Mechanism Evaluation (EME) Programme, an MRC and NIHR partnership. that reflect the information required at different time points. Large font versions of documents are available and information will also be provided in DVD format Randomised trials are expensive and failure to recruit wastes funding. The observational arm produces a bank of biological and clinical data even if patients are not randomised. Additional questions will be reviewed during the feasibility study to further maximise value. Data from the feasibility phase will be used to guide operational adaptations to ensure the Protocol continues to be informed by best practice The Rational MCC trial is funded by the Efficacy and Mechanism Evaluation (EME) Programme, an MRC and NIHR partnership. Methods h f ll The following adaptations were made to the follow-up process: 1) One week prior to sending the first questionnaire, the SIFT Trial Coordinating Centre telephoned parents to confirm address details and inform them that the questionnaire was to be dispatched. Parents were also telephoned within a day of the second reminder being posted (1 February 2016 onwards). Challenge 5 - Working with elderly populations: It is important to ad- dress potential barriers in communication when working with elderly populations. Rational MCC patient literature has been reviewed by PPI representatives and multiple documents have been produced Page 115 of 235 Trials 2017, 18(Suppl 1):200 Page 115 of 235 2) A link to an electronic version of the questionnaire was sent to parents with a valid email address or mobile number within a day of posting the first paper version (26 February 2016 onwards). audio-recorded consultation data to understand recruitment chal- lenges. Phase II involved tailored interventions to improve recruit- ment, including components such as trial-specific feedback/training and suggestions to clarify patient-facing documentation. Data pro- vided by clinical centres were aggregated and displayed graphically to compare targets with achievements over time, in relation to eligi- bility assessment and numbers approached, consented, and rando- mised. QRI intervention-components were also plotted over time. Results 3) Parents were contacted by text message as well as or instead of by telephone at second reminder stage (29 March 2016 onwards); charity for premature and sick babies BLISS promoted the follow-up via Facebook and Twitter (5 April 2016). 4) Posters publicising follow-up were sent to recruiting sites for dis- play, accompanied by a list of local participants with their response status (20 June 2016 onwards). Recruitment in the initial periods of these RCTs rarely hit targets (Phase I of the QRI), with the exception of one trial where recruiters had previously received QRI training. Recruitment rates moved closer to targets after implementation of QRI interventions (Phase II), with the scale and patterns of improvement varying across RCTs. A sharp increase in recruitment was evident in an RCT following a feedback/ training session attended by all centres, whereas smaller improve- ments were seen in a multi-centre trial where a subset of centres attended sessions. Other evidence of recruitment improvement in- cluded centres moving from ‘no recruitment’ over weeks/months, to recruiting first participants after feedback (four RCTs). Generating student recruiters for randomised trials in surgery (GRANULE): a one-day course to empower a cohort of future surgical trialists 2 g p y Leila Rooshenas1, Kerry Avery1, Daisy Elliott1, Sangeetha Paramasivan1, Alba Realpe2, Caroline Wilson1, Jenny L Donovan1, on behalf of the quintet study group 1 2 g y Leila Rooshenas1, Kerry Avery1, Daisy Elliott1, Sangeetha Paramasivan1, Alba Realpe2, Caroline Wilson1, Jenny L Donovan1, on behalf of the quintet study group 1 2 g James Glasbey1, Dmitri Nepogodiev1, Jane Blazeby2, Dion G. Morton1, Nicola Fearnhead3, Simon Bach1, Thomas Pinkney1, Aneel A. Bhangu1 1University of Birmingham; 2University of Bristol; 3University of Cambridge James Glasbey1, Dmitri Nepogodiev1, Jane Blazeby2, Dion G. Morton1, Nicola Fearnhead3, Simon Bach1, Thomas Pinkney1, Aneel A. Bhangu1 1University of Birmingham; 2University of Bristol; 3University of Cambridge 1University of Bristol; 2University of Warwick Correspondence: Leila Rooshenas Trials 2017, 18(Suppl 1):P303 Correspondence: James Glasbey Trials 2017, 18(Suppl 1):P304 Correspondence: James Glasbey Trials 2017, 18(Suppl 1):P304 p p Conclusion These preliminary observational data provide some evidence to show an association between QRI interventions and improved recruitment in very challenging RCTs. The QRI is increasingly appreciated by RCT in- vestigators and funders. Formal evaluation of the QRI is itself challen- ging, and will need to carefully consider appropriate markers of success that include recruitment rates and levels of informed consent. y Correspondence: Adwoa Hughes-Morley Trials 2017, 18(Suppl 1):P302 y Correspondence: Adwoa Hughes-Morley Trials 2017, 18(Suppl 1):P302 This abstract is not included here as it has already been published. An intervention to optimise recruitment in challenging RCTs: implementation of the 'quintet recruitment intervention' across eight pilot/feasibility RCTs Generating student recruiters for randomised trials in surgery (GRANULE): a one-day course to empower a cohort of future surgical trialists James Glasbey1, Dmitri Nepogodiev1, Jane Blazeby2, Dion G. Morton1, Nicola Fearnhead3, Simon Bach1, Thomas Pinkney1, Aneel A. Bhangu1 1University of Birmingham; 2University of Bristol; 3University of Cambridge Correspondence: James Glasbey Trials 2017, 18(Suppl 1):P304 Background g Surgical trials are complex; patient populations are heterogeneous, interventions are difficult to standardise, and outcome measures are difficult to assess. Fewer patients are recruited to trials in surgery and anaesthesia than any other medical specialties. There is an urgent need to train a cohort of future surgical trialists, equipped with skills to convey clinical equipoise and appropriately recruit patients into well-designed randomised studies. Methods h f ll Despite their challenging nature, four pilot/feasibility RCTs recruited to target and progressed to main trials, and two are currently recruiting well. Two feasibility studies did not progress to main RCTs: one, because the QRI enabled a nuanced understanding of equipoise issues previously unbeknown to the trial management group, which were discussed during a feedback session and found to be unsurmountable; and the other, because a main trial was not feasible due to insufficient inci- dent cases of eligible patients. Recruiters exposed to QRI training in the latter trial went on to successfully recruit to a subsequent RCT with integrated QRI, which consistently recruited above target des- pite anticipated difficulties. C l i Learning lessons from trial decliners about improving recruitment: qualitative study 1 2 3 4 Adwoa Hughes-Morley1, Bridget Young2, Roelie J. Hempel3, Ian T. Russell4, Waquas Waheed5, Peter Bower2 Adwoa Hughes-Morley1, Bridget Young2, Roelie J. Hempel3, Ian T. Russell4, Waquas Waheed5, Peter Bower2 1University of Manchester and University of York; 2MRC North West Hub for Trials Methodology Research, University of Liverpool; 3School of Psychology, University of Southampton; 4Swansea University Medical School, Swansea University; 5NIHR School for Primary Care Research, University of Manchester Background Randomised controlled trials (RCTs) are the most rigorous approach for evaluating health care interventions, but many fail to recruit to target, requiring costly extensions. Anticipation of recruitment challenges can also deter investigators and funders from tackling important, potentially practice-changing clinical questions. Devel- oping interventions to optimise recruitment is a priority in trials methodology research, but few interventions are transferrable across RCTs. The quintet Recruitment Intervention (QRI) aims to op- timise recruitment in ‘difficult’ RCTs, whilst safeguarding informed consent. The intervention comprises in-depth investigation of re- cruitment obstacles (Phase I), followed by implementation of strat- egies to address recruitment challenges as the trial proceeds (Phase II). Having integrated the QRI in eight rcts anticipated by funders/trial investigators to encounter recruitment challenges, we undertook a preliminary evaluation of the QRI’s implementation. Methods Results Response rate prior to all interventions was 51.0%. On 30 September 2016 it was 68.0%, a significant increase. The response rate also in- creased (non-significantly) after implementation of each new initiative. SIFT intends to initiate an incentives programme to further increase the rate (subject to ethical approval). Conclusion Contacting parents prior to dispatching questionnaire; reminder to parents via text message; availability of questionnaire electronically; and promotion via hospitals or charities are approaches which, when combined, can significantly increase follow-up rate. g Method A national, one-day course (GRANULE) was conducted with support from Birmingham Clinical Trials Unit, and the Bristol Medical Research Council-funded conduct-II hub. Undergraduate medical students were competitively selected from a national UK student research group (Stu- dent Audit & Research in Surgery). Completion of Good Clinical Practice (GCP) certification was required prior to commencement. GRANULE course content was framed around active trials within colo- rectal and oesophagogastric surgery, with a faculty of chief investiga- tors and active trial recruiters. The course featured; 1. A series of interactive lectures to give clinical context (STAR-TREC (Birmingham), ACCURE-UK (Birmingham) and By-Band-Sleeve (Bristol)); 2. Structured, A national, one-day course (GRANULE) was conducted with support from Birmingham Clinical Trials Unit, and the Bristol Medical Research Council-funded conduct-II hub. Undergraduate medical students were competitively selected from a national UK student research group (Stu- dent Audit & Research in Surgery). Completion of Good Clinical Practice (GCP) certification was required prior to commencement. GRANULE course content was framed around active trials within colo- rectal and oesophagogastric surgery, with a faculty of chief investiga- tors and active trial recruiters. The course featured; 1. A series of interactive lectures to give clinical context (STAR-TREC (Birmingham), ACCURE-UK (Birmingham) and By-Band-Sleeve (Bristol)); 2. Structured, The QRI was integrated into the recruitment processes of eight ‘diffi- cult’ feasibility/pilot RCTs that addressed controversial issues or compared very different treatment arms (including ‘no treatment’). Phase I of the QRI involved collecting and analysing interview and Page 116 of 235 Trials 2017, 18(Suppl 1):200 Page 116 of 235 Correspondence: Gwenllian Moody Trials 2017, 18(Suppl 1):P305 Correspondence: Gwenllian Moody Trials 2017, 18(Suppl 1):P305 Missing data in clinical trials is common and can reduce trial effi- ciency and bias the estimate of treatment effect. Higher levels of missing data have been associated with specific study areas (e.g. Mental health, substance abuse), longer lengths of follow up, trials with more than two arms, and poor allocation concealment. How- ever, little is known about the prevalence of different causes of miss- ing data. Analysis of the reported reasons for missing data within published literature could provide valuable insights to inform the de- velopment of effective strategies to mitigate the problem. Methods Correspondence: Gwenllian Moody Trials 2017, 18(Suppl 1):P305 Correspondence: Gwenllian Moody Trials 2017, 18(Suppl 1):P305 Discussion Feedback from the GRANULE course demonstrated that undergradu- ate medical students in the UK can be trained to confidently recruit patients to high-quality clinical trials. As an early-years intervention, this course has the capacity to change the culture of trials recruit- ment within surgery at a junior level. Course delegates can access ac- tive networks of postgraduate, trainee-led surgical research networks in the UK to foster new mentoring relationships and actively recruit patients into portfolio trials. Further course iterations are planned for Spring 2017 (UK) and September 2017 (Germany). Managing the challenge of recruiting foster carers into a trial of a supportive group-based social care intervention: lessons learned from the confidence in care trial 1 1 1 Gwenllian Moody1, Lucy Brookes-Howell1, Rebecca Cannings-John1, Sue Channon1, Elinor Coulman1, Mandy Lau1, Alyson Rees2, Jeremy Segrott3, Jonathan Scourfield2, Michael Robling1 1South East Wales Trials Unit (SEWTU), Centre for Trials Research, Cardiff University; 2Children’s Social Care Research and Development Centre (CASCADE), Cardiff University; 3South East Wales Trials Unit (SEWTU), Centre for Trials Research & Centre for the Development and Evaluation of Complex Interventions for Public Health Improvement (decipher), Cardiff University Conclusion Recruitment to the CIC trial has improved since the above amend- ments were made and we expect to reach our recruitment target. Modifying the allocation ratio increases the target sample but we considered on balance that this made the study both more likely to achieve clinically viable groups and was more acceptable to pro- viders. However, the impact of remaining sub-optimal group size on fidelity will need to be determined. Similarly, the quality of communi- cation about the trial to carers and also local providers will be assessed in our process evaluation. One pilot site did not continue into the trial phase but has enabled an on-going carer contact group to provide timely input to both our recruitment and retention strat- egy. Recruiting participants potentially well in advance of group on- set introduces, as well as avoids some problems which will be reviewed in our presentation. Results f Since the changes were fully implemented after the pilot and the first main recruitment wave, the average number of carers recruited to each site increased from 9.17 (5.83 intervention: 3.33 control) to14.25 (9.25 intervention: 5.00 control). While group sizes have in- creased, it remains difficult to reach target group size. Most consent- ing participants are now sourced via the direct approach from providers (as opposed to the ongoing open approach letter). During the second main recruitment wave, 85.09% of consented participants were sourced directly from providers, whereas the remaining 14.91% were self-nominated. Twenty-four delegates (10 female, 14 male) were invited to complete the course. All 24 completed GCP certification prior to attendance, and completed feedback (response rate = 100.0%). After course completion, participants felt more confident in their understanding of clinical equipoise (P < 0.001), ability to communicate clinical equi- poise effectively (P < 0.001), ability to communicate risks and benefits of each trial intervention (P < 0.001) and gain consent for participa- tion in trials (P = 0.004). In free-text analysis, the simulated patient interactions, and the high facilitator: student ratio were highly com- mended. Lessons learnt included reducing the complexity of included trial interventions, and giving extended time for single delegates’ simu- lated patient interactions. P305 P306 Reported reasons for missing data Anna Kearney1, Anna Rosala-Hallas2, Naomi Bacon2, Anne Daykin3, Alison J. Heawood3, Athene Lane3, Jane Blazeby3, Mike Clarke4, Paula R. Williamson1, Carrol Gamble1 1North West Hub for Trials Methodology Research/University of Liverpool; 2Clinical Trials Research Centre/University of Liverpool; 3conduct-II Hub for Trials Methodology Research/University of Bristol; 4Centre for Public Health, Queen’s University of Belfast Correspondence: Anna Kearney Trials 2017, 18(Suppl 1):P306 Methods small group discussions surrounding challenges in trial recruitment; 3. Simulated patient recruitment, with formative assessment of actor interactions. Confidence and experience in trials recruitment was col- lected pre- and post-intervention using paired, self-reported, 15-point online tools. 5-point Likert scales were used to quantify confidence across four domains (understanding of equipoise, ability to communi- cate equipoise, communicating risks and benefits of research involve- ment, gaining consent for participation in trials). A mix of positive and negatively weighted statements were used to minimise acquiescence bias. Changes in perceived confidence were tested to a 5% level of sig- nificance using McNemar’s paired chi-squared test. Free text feedback underwent thematic analysis. We piloted at two sites prior to the first main recruitment wave, and further optimised recruitment strategy in initial main trial waves. Strat- egies introduced both preceded and followed the piloting phase. These included 1) supplementing postal approaches to carers from their re- spective Local Authority (LA) or Independent Fostering Provider (IFP) by asking LAs and IFPs to select eligible carers from their databases for direct invitation, 2) increasing lead time from approaching provider to group initiation to up to four months, 3) modifying the allocation ratio from 1:1 to 2:1 (intervention:control) to increase the chances of obtain- ing the required training group size, 5) revising participant materials to better align with the expectations of foster carers. Results Acknowledgements h l h k With special thanks to the Bowel Disease Research Foundation. Background The Confidence in Care (CIC) trial is evaluating Fostering Changes (FC), a training programme for foster and kinship carers which aims to increase their skills and coping strategies and improve their rela- tionship with their child. The CIC consortium of third-sector providers train groups of 12 carers in both trial and non-trial settings. Groups are timed to commence with each new school term, which is then reflected by waves of recruitment to the trial. Previous evaluations have found filling groups to be challenging, both Pallett et al. (2002) and Briskman et al. (2012) ran groups with as few as six carers, which impacts fidelity. FC requires carers to attend twelve 3-hour weekly sessions, a considerable time commitment and possibly a recruit- ment barrier. Randomised controlled trials in social care settings are also uncommon compared to public and clinical health and in two pilot sites we tailored, tested and revised our recruitment strategy. p Methods Two cohorts were identified: A search of Medline (Ovid) for rando- mised trials published within the four major medical journals in 2013; National Institute of Health Research Health Technology Assessment Programme (NIHR HTA) monographs published between 2009 and 2014. Parallel, two arm randomised control trials were included. Early phase trials and pilot studies were excluded. Data on the number of patients randomised, analysed, excluded and imputed was independ- ently abstracted by two authors along with reported reasons and trial Page 117 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 117 of 235 characteristics. Missing data was defined as the number of randomised patients who did not contribute primary outcome data to the analysis either because it was not available or because the data was excluded. Results characteristics. Missing data was defined as the number of randomised patients who did not contribute primary outcome data to the analysis either because it was not available or because the data was excluded. Results retention challenges and implemented strategies at trial design. Pa- tient initiated withdrawal was the most common cause of missing data. CTUs routinely used newsletters, timeline of participant visits, and telephone reminders to mitigate missing data. CTUs reported evaluating 36 of the 59 strategies presented using nested studies or a comparison of retention before and after implementation. How- ever, some frequently used strategies such as site initiation training have had no research to inform practice. retention challenges and implemented strategies at trial design. Pa- tient initiated withdrawal was the most common cause of missing data. CTUs routinely used newsletters, timeline of participant visits, and telephone reminders to mitigate missing data. CTUs reported evaluating 36 of the 59 strategies presented using nested studies or a comparison of retention before and after implementation. How- ever, some frequently used strategies such as site initiation training have had no research to inform practice. In 7/166 (4%) journal articles and 2/36 (6%) HTA monographs it was not possible to determine the levels of missing data. 141/ 159 (89%) and 34/34 (100%) trials had missing primary outcome data with median [IQR] levels of missing data of 5.5% [1.5, 11.0] and 11% [2.9, 19.8]. 35 CTUs (74%) participated in the Delphi survey of which 34 (97%) completed both rounds. Pre-defined consensus was reached on seven topics. Optimising the recruitment of underrepresented ethnic minority patients to telehealth diabetes trials: examining the role of language and research reporting practices 2 3 3 Talia Isaacs1, Daniel Hunt2, Danielle Ward3, Leila Rooshas3, Louisa Edwards3 1University College London; 2University of Nottingham; 3University of Bristol Correspondence: Talia Isaacs Trials 2017, 18(Suppl 1):P308 Background Randomised controlled trials (RCTs) are often considered the gold standard of health intervention research. However, most RCTs under- recruit ethnic minority patients, potentially jeopardising the external validity of their findings. One recruitment challenge relates to asses- sing whether patients have sufficient language proficiency to provide informed consent and engage with the intervention. However, little is known about how trial recruiters assess potential participants' language proficiency. Using diabetes telehealth intervention RCTs as a case study, we investigated the proportion of published trials that include language proficiency as part of their inclusion criteria, includ- ing how and why they do so. A secondary objective was to explore any links between the inclusion of language-related eligibility criteria and the proportion of ethnic minorities recruited. M h d Identifying effective retention strategies: a research agenda Anna Kearney1, Anne Daykin3, Alison J. Heawood3, Athene Lane3, Jane Blazeby3, Mike Clarke4, Paula R. Williamson1, Carrol Gamble1 1North West Hub for Trials Methodology Research/University of Liverpool; 2Clinical Trials Research Centre/University of Liverpool; 3conduct-II Hub for Trials Methodology Research/University of Bristol; 4Centre for Public Health, Queen’s University of Belfast Correspondence: Anna Kearney Trials 2017, 18(Suppl 1):P307 p Methods Six retention strategies met consensus that further re- search was of critical importance: site initiation training; frequency of patient contact during a trial; the use of routinely collected data; the frequency and timing of reminders; triggered site training and the length of time needed to complete questionnaires. In contrast, 82% reached consensus that research into the effectiveness of Christmas cards for site staff was of low importance. The impact of the missing data within the journal cohort meant that 454 months were wasted across 126 trials recruiting patients that did not contribute outcome data (median [IQR] per trial: 1.5 [0.7, 3.2]). Trials reported multiple reasons for missing data: patient withdrawals (64%, n = 141); patients lost to follow up (60%); investigator exclu- sions (53%) and failure of clinical staff to measure the outcome (45%). 91/159 (57%) imputed data and 91 (57%) trials excluded randomised patients with protocol deviations or missing data from the analysis population. 41 (26%) trials used both approaches. Abstraction of the levels of missing data was challenging. CONSORT statements were often misleading around imputation of data and did not clearly report the number of outcomes known. The survey of current practices demonstrates a variety of strategies are being used to mitigate missing data but with little evidence to support their use. This Delphi survey has identified a consensus of re- search priorities to be evaluated. P308 P308 Optimising the recruitment of underrepresented ethnic minority patients to telehealth diabetes trials: examining the role of language and research reporting practices Talia Isaacs1, Daniel Hunt2, Danielle Ward3, Leila Rooshas3, Louisa Edwards3 1University College London; 2University of Nottingham; 3University of Bristol Correspondence: Talia Isaacs Trials 2017, 18(Suppl 1):P308 Results Of 3358 records identified in the search, 79 articles consisting of 58 distinct RCTs were included in the review. Half of the included RCTs (29/58) referred to patients’ language proficiency as an eligibility cri- terion. However, there were no common procedures across RCTs to determine if patients had the requisite language ability to participate. Whereas some studies listed different combinations of language skills as being necessary (speaking, listening, reading, writing), others re- ferred to patients’ need to be native speakers. In two RCTs, there was a mismatch between the telehealth medium used and the language skills cited (e.g., telephone intervention requires writing but not speaking ability), whereas four underspecified the language skills re- quired (e.g., speaking but not reading ability stated as necessary for a telephone and computer-text intervention). g g g Responses from the current practice survey were used to inform a subsequent two round Delphi survey with CTUs to gain consensus around research priorities to assess the effectiveness of missing data interventions. The 29 RCTs that referred to language as a patient eligibility criterion tended to be larger-scale, recruiting nearly 1.7 times the total number of recruited patients, compared to the 29 RCTs that made no reference to language at all. Twenty-one RCTs in the former group and 17 in the latter provided ethnicity information and Conclusion The percentage of missing data within the leading journals was on average lower than expected. However, a comparison with the HTA monographs suggests this may reflect a difficulty publishing RCT’s with missing data in the top journals. Patient withdrawal and loss to follow up were the leading causes of attrition, although the failure of researchers to measure the outcome in retained patients was cited in nearly half of the trials. Reporting missing data was often inadequate. We would recommend stricter adherence to the CONSORT flow diagram and suggest revisions to ensure that the flow diagram could be standalone with additional categories to allow distinction between the numbers analysed and the numbers for whom the outcomes were known and imputed. Background Identifying strategies to minimise missing data was the second highest methodological research priority in a Delphi survey of the Directors of UK Clinical Trial Units (CTUs). However, a Cochrane Methodology Re- view of nested randomised studies of missing data strategies shows a substantial evidence gap. The review demonstrates an emphasis on im- proving questionnaire response rates and an absence of evidence that address the full range of causes of missing data. In addition, published case studies frequently describe the use of strategies which have not been evaluated. Our aim was to assess current retention practices within the UK and identify priorities for future research to evaluate the effectiveness of strategies to reduce attrition. Methods: 75 Chief Investi- gators of National Institute of Health Research Health Technology Assessment (NIHR HTA) funded parallel randomised control trials start- ing between 2009 and 2012 and 47 UK Clinical Trial Units (CTUs) were surveyed to identify what approaches and strategies were used to miti- gate missing data in trial design and conduct. A systematic review was conducted on telehealth intervention RCTs that focused on type 2 diabetes and excluded ethnically-targeted studies. Two reviewers independently conducted abstract and full- text screening, risk of bias assessment, and data extraction. Results Reference 1. Reinert, C., et al., Quantitative and qualitative analysis of study-related patient information sheets in randomised neuro-oncology phase III-trials. Eur J Cancer, 2014. 50(1): p. 150–8. P310 Reducing missing data in palliative care randomised controlled trials: a mixed-methods study Jamilla Hussain1, Martin Bland2, Miriam J. Johnson3, David C. Currow4, Ian R. White5 1Hull York Medical School, University of York; 2Health Sciences Department, University of York; 3SEDA, University of Hull; 4Palliative and Supportive Services, Flinders University; 5MRC Biostatistics Unit, University of Cambridge Correspondence: Jamilla Hussain Trials 2017, 18(Suppl 1):P310 Background A central tenet of recruitment to clinical studies is that participants take part freely, armed with full information about the study. There has been little research into how the appearance of the information may affect recruitment. A study of Patient Information Leaflets (PILs) concluded that PILs need ‘to be well structured and designed in an appealing manner’. These aspects have not yet gained sufficient at- tention [1]. In the case of paper information leaflets, production of a high quality attractive leaflet is possible, but may require specialist software, and incur extra costs for colour printing. Without the evi- dence of benefit, the additional resources may not be justified. Results Results After 5 months, we have sent out 436 invitation letters and have 182 consented participants. Consent rates for each PIL are: A - 68/181 (38%) B - 76/180 (42%) C - 76/180 (42%) An unexpected finding is that consent rates are much lower across the study than was antici- pated. We are currently investigating possible reasons for this. If the current trend continues we will review the implications for the sam- ple size and power of the study. Reducing missing data in palliative care randomised controlled trials: a mixed-methods study P309 Does appearance matter? A study within a study Lucy Culliford, Rachel Brierley, Jonathan Betts, Jenny Lamb, Rachel Maishman, Barney Reeves1, Chris Rogers1 1University of Bristol Correspondence: Lucy Culliford Trials 2017, 18(Suppl 1):P309 Does appearance matter? A study within a study Lucy Culliford, Rachel Brierley, Jonathan Betts, Jenny Lamb, Rachel Maishman, Barney Reeves1, Chris Rogers1 1University of Bristol Correspondence: Lucy Culliford Trials 2017, 18(Suppl 1):P309 y Jamilla Hussain1, Martin Bland2, Miriam J. Johnson3, David C. Currow4, Ian R. White5 1Hull York Medical School, University of York; 2Health Sciences Department, University of York; 3SEDA, University of Hull; 4Palliative and Supportive Services, Flinders University; 5MRC Biostatistics Unit, University of Cambridge Correspondence: Jamilla Hussain Trials 2017, 18(Suppl 1):P310 1Hull York Medical School, University of York; 2Health Sciences Department, University of York; 3SEDA, University of Hull; 4Palliative and Supportive Services, Flinders University; 5MRC Biostatistics Unit, University of Cambridge p Methods To investigate if the appearance of pils affects recruitment, we chose to embed a randomised controlled trial (RCT) within the Outcome Moni- toring After Cardiac Surgery (OMACS) study. OMACS uses routine NHS data alongside participant questionnaires, and consent is sought by post at 3 months post-surgery. OMACS was chosen as the ‘host’ study as around 120 patients are approached for participation per month, allowing evidence to be collected quickly. Participants are randomised to receive one of 3 PILs: a tri-fold coloured leaflet produced using a graphic design package, indesign, (PIL A), a coloured A4 sheet pro- duced in Microsoft Word (PIL B), and a standard A4 black and white sheet (PIL C). Both coloured leaflets are printed professionally. The information contained in each leaflet is identical and participants do not know about the randomised element of OMACS. The sample size is 1590 which, assuming a consent rate of 70% (based on a previous simi- lar postal questionnaire study that achieved this (personal communica- tion)), will provide 90% power to detect a 10% difference in consent rate between any pair of PIL formats, with an overall significance level of 5%. (i) Trial-level factors: systematic review and meta-regression of pri- mary outcome MD in 108 palliative care trials; (ii) Participant and site-level factors: multi-level cross-classified modelling using individ- ual participant-level data (IPD) from 10 multi-site trials; (iii) Identifica- tion and exploration of factors in more depth: thematic analysis of interviews with 27 research personnel and participants. Results (i) Systematic review: MD was associated with increasing numbers of questions/tests requested (odds ratio (OR) 1.19 per-doubling, 95%CI 1.05, 1.35) and longer study duration (OR 1.09 per-doubling, 95%CI 1.02, 1.17). (ii) IPD: At the participant-level (n = 1,846), MD was associ- ated with baseline missingness (OR 17.19, 95%CI 8.55, 34.53) and poorer Karnofsky Performance Status (10-unit increase: OR 0.78, 95%CI 0.70, 0.87); at the site-level (n = 35), MD at the end of follow- up was associated with sites that randomised a greater number of participants (per 10-randomisations: OR 1.08, 95%CI 1.01, 1.16) and with fewer research personnel (4 personnel compared to 1: OR 0.07, 95%CI 0.01, 0.84). (iii) Interviews: themes included “attention-to-detail vs. attention-to-person”, “clinical vs. research-role tension”, and “be- yond GCP training”. Results 50/75 (66%) Chief Investigators and 33/47 (70%) CTUs completed the current practice surveys. 78% of Chief Investigators were aware of Trials 2017, 18(Suppl 1):200 Page 118 of 235 Page 118 of 235 recruited a median of 24.6% versus 18.0% ethnic minority patients as a proportion of the total sample respectively. The RCTs that included language proficiency as an eligibility criterion recruited a greater pro- portion of ethnic minority participants (37.8% of all recruited partici- pants) compared with those that did not (13.9%). Conclusions recruited a median of 24.6% versus 18.0% ethnic minority patients as a proportion of the total sample respectively. The RCTs that included language proficiency as an eligibility criterion recruited a greater pro- portion of ethnic minority participants (37.8% of all recruited partici- pants) compared with those that did not (13.9%). Conclusions There are a number of differences between OMACS and the previous study which may explain the difference in response rates. Previously, the timing of the approach for consent is different (1 year versus 3 months) and patient packs were simpler with fewer documents than used in the OMAC study. In attempt to cater for participants’ preferences, OMACS invites participants to elect for alternative re- sponse methods (e.g. Postal versus internet questionnaire) and also allows them to opt out of some aspects of the study. The previous study did not include this diversity of options. Conclusion Approaches for assessing patients' language proficiency were found to be inconsistent in the context of diabetes telehealth intervention RCTs. Studies referring to language in patient screening might report more on ethnicity because some ethnic minorities are also linguistic minorities. Or it may be that these studies are more robust in terms of research reporting and sample size. There was no evidence that reference to language screening is associated with lower participation from hard-to-reach groups, although the soundness and consistency of individual inclusion/exclusion decisions on language grounds could not be ascertained. Future research should focus on developing and validating a language assessment tool that could be consistently applied across RCTs to screen patients’ language proficiency during recruitment. At this early stage, no formal conclusions can be drawn as to the ef- fect of the appearance of the PIL. One year results with a formal comparison between the rates of each group will be presented. Background To reduce the risk missing data (MD) pose to the power, precision and validity of trial findings, MD should not only be handled appro- priately at the analysis stage, but more importantly potentially revers- ible MD risk factors must be identified and modified at the design and conduct stage. This mixed-methods study used palliative care tri- als, where MD due to death and disease progression are expected, to explore the association between primary outcome MD and partici- pant, trial site and trial-level MD risk factors. y Conclusion S f Spouses of research participants may be a resource for studies re- quiring a comparison group with similar demographic characteris- tics. Potential obstacles to spouse participation, such as participant refusal to allow contact spouse, distance/travel, and illness/disabil- ity need to be considered. Clearly defined eligibility criteria, recruit- ment strategies and testing procedures are needed to ensure valid comparisons between groups. Standardized evaluations by trained staff may yield stronger results compared to the use of published comparison groups. Challenges in the analysis of a randomised controlled trial with retrospective consenting: the RAPIDO study 1 1 2 2 Challenges in the analysis of a randomised controlled trial with retrospective consenting: the RAPIDO study Helena Smartt1, Katie Pike1, Rosy Reynolds2, Margaret Stoddart2, Chris A. Rogers1, Alasdair MacGowan2 1University of Bristol; 2North Bristol NHS Trust Correspondence: Helena Smartt Trials 2017, 18(Suppl 1):P312 p g y Helena Smartt1, Katie Pike1, Rosy Reynolds2, Margaret Stoddart2, Chris A. Rogers1, Alasdair MacGowan2 1University of Bristol; 2North Bristol NHS Trust Correspondence: Helena Smartt Trials 2017, 18(Suppl 1):P312 Background Use of spouses for the control group was based on unique factors. Most spouses were familiar with the DCCT/EDIC study and staff by virtue of their partner’s long-term participation and frequent accom- paniment to study visits. Most important, the spousal group was expected to be similarly distributed in age, race and socioeconomic status to the DCCT/EDIC cohort. Additionally, practical efficiencies in recruitment, scheduling, and travel were expected. p Objective To determine the feasibility and comparability of using randomly se- lected spouses of surviving DCCT/EDIC participants as a non-diabetic control group. P313 Imaging endpoint eligibility in oncology trials: what works and what doesn’t D id R i P313 Imaging endpoint eligibility in oncology trials: what works and what doesn’t d what doesn t David Raunig ICON Clinical Research Correspondence: David Raunig Trials 2017, 18(Suppl 1):P313 David Raunig ICON Clinical Research Correspondence: David Raunig Trials 2017, 18(Suppl 1):P313 Introduction RAPIDO is a multi-centre RCT comparing the effect on mortality of con- ventional versus rapid diagnostic pathways for suspected blood stream infections in hospitalised patients. In the conventional pathway, infect- ive micro-organisms in a blood sample are identified within 3–5 days, whereas the rapid diagnosis pathway takes 1 hour or less. Identifying the infective micro-organisms then allows an appropriate antibiotic to be chosen for treatment. It has been suggested that earlier antibiotic therapy could improve patient outcomes including 28 day mortality (primary outcome), length of hospital stay and time to resolution of fever (secondary outcomes). Due to the time-sensitive nature of this study, participants were consented retrospectively; where patients had left the hospital before consent was obtained, postal consent was sought. Hearing impairment is common among the general public >50 years of age however the relationship between type 1 diabetes (T1D) and hearing impairment in this age group is not well-studied. To examine this question, the Diabetes Control and Complications Trial/Epidemi- ology of Diabetes Interventions and Complications (DCCT/EDIC) Hear- ing Study is examining the prevalence of hearing impairment among a well-phenotyped T1D cohort (mean age 55 years). g Results A total of 8628 patients were randomised across 7 UK centres, 6692 of which were found to be eligible for the study. Consent was obtained before hospital discharge for 2606 (39%) patients and postal consent was successfully sought for a further 521 (8%). 1142 (17%) declined. Of the remainder, 1341 (20%) died before consent could be sought and consent was not obtained for 1082 (16%) survivors. The research ap- provals granted for the study allowed only very limited data to be retained and used for this latter group. By definition their survival status was known (allowing analysis of the primary outcome) but secondary outcome data were missing not at random. Pre-specified sensitivity analyses were undertaken to estimate the bias associated with not hav- ing data on up to 33% of the study population. The results of these analyses and their impact on the study conclusions will be discussed. Discussion y Conclusion There is the potential to reduce MD in palliative care trials by modify- ing the factors associated with MD identified from this study. Further development of the theoretical framework is required, prior to devel- oping an intervention to reduce MD that will be tested within trials. Page 119 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 *In accordance with the submission guidance for the Sylvan Green Travel Award the word count has been limited to 300. Background Studies in which patient consent cannot be obtained prospectively represent a particular methodological challenge. Approval can be sought to include patients in a randomised study without their con- sent, but every effort must be made to seek informed consent when the patients has recovered sufficient capacity or to seek consent from a consultee if this doesn’t occur. We describe our experience of seeking retrospective consent in the context of the RAPIDO trial, and the implications for the study analyses. 1George Washington University; 2University of California, San Diego; 3National Institute on Deafness and Other Communication Disorders; 4University of Toronto; 5National Institute of Diabetes and Digestive and 6 7 1George Washington University; 2University of California, San Diego; 3National Institute on Deafness and Other Communication Disorders; 4University of Toronto; 5National Institute of Diabetes and Digestive and Kidney diseasesniddk; 6University of Wisconsin; 7Episense, University of Wisconsin; 8Case Western Reserve University; 9University of Iowa University of Toronto; National Institute of Diabetes and Digestive and Kidney diseasesniddk; 6University of Wisconsin; 7Episense, University of Wisconsin; 8Case Western Reserve University; 9University of Iowa Correspondence: Barbara Braffett Trials 2017, 18(Suppl 1):P311 Use of a non-diabetic spouse control group in the DCCT/EDIC hearing study 1 2 1 3 Barbara Braffett1, Gayle Lorenzi2, Xiaoyu Gao1, Kathy Bainbridge3, Annette Barnie4, Catherine Cowie5, Karen Cruickshanks6, Dayna Dalton7, Rose Gubitosi-Klug8, John Kramer9 Barbara Braffett , Gayle Lorenzi , Xiaoyu Gao , Kathy Bainbridge , Annette Barnie4, Catherine Cowie5, Karen Cruickshanks6, Dayna Dalton7, Rose Gubitosi-Klug8, John Kramer9 1George Washington University; 2University of California, San Diego; 3National Institute on Deafness and Other Communication Disorders; 4University of Toronto; 5National Institute of Diabetes and Digestive and Kidney diseasesniddk; 6University of Wisconsin; 7Episense, University of Wisconsin; 8Case Western Reserve University; 9University of Iowa Correspondence: Barbara Braffett Methods Of h Of the total of 875 spouses, 510 were randomly identified for screen- ing. Enrollment of 270 spouses would provide 90% power to detect a clinically significant difference in hearing impairment between the EDIC surviving cohort and controls. Permission from the EDIC partici- pant was needed prior to contacting his/her spouse. Spouses with known diabetes, or illness/disability that precluded travel to the clin- ical center were excluded. A self-administered hearing assessment, brief medical history, physical measurements, hba1c and audiometry were performed on consenting participants and spouses. All data col- lection methods and equipment were standardized and consistent with DCCT/EDIC methods. Testing was performed by trained and study-certified personnel. All audiograms were scored centrally. Results analyses and Discussion The proportion of patients for whom consent was not obtained was higher than had been predicted when the study was designed. The requirement to obtain consent to use data collected in a trial after the intervention is complete and when no further participant in- volvement is required needs to be challenged. Disclaimer RAPIDO was funded as part of an NIHR Programme Grant for Applied Re- search. The views expressed are those of the authors and not neces- sarily those of the NHS, the NIHR or the Department of Health. Of the 510 spouses identified, 39 (7.7%) were ineligible, 97 (19%) were not approached (due to participant request, distance, illness/ disability, work demands, marital discord), and 88 (17.3%) were approached but declined (work demands, travel, illness/disability, dis- interest). A total of 289 spouses and 1150 (86.7% of surviving, 94.5% of active) EDIC participants were evaluated. Spouses determined to have diabetes based on hba1c (n = 5) were excluded from the ana- lyses. The spousal group was similar in age, race, education, smoking status and systolic blood pressure. P313 Imaging endpoint eligibility in oncology trials: what works and what doesn’t D id R i Methods of approaching general practices for trial participant information: feasibility cluster-randomised trial nested within a large multi-centre cluster-randomised controlled cross-over trial Denise Forshaw1, Chris Sutton1 There is limited evidence as to the time taken by general practices to respond to data requests for individual patients as research partici- pants. In stroke trials, as patient mortality in the first 12 months is high, it is common practice to ascertain participant status (dead/ alive) before contacting for follow-up; primarily to avoid emotional distress to relatives. GPs are usually informed that a patient is partici- pating in a trial via a letter sent directly from the admitting NHS Trust. The letter details that they will be contacted at a given time (dictated by data collection time-points) to ascertain patient status and verify address and contact details. We have observed consider- able variation in the time taken to reply to requests and significant variation in how practices deal with such requests. Some practices are happy to give the details over the phone following basic checks with the researcher, some practices ask for a copy of the consent form and some practices ask for a covering request by letter to be sent via fax, with varying degrees of success in eventually getting the information requested. Although numbers lost or delayed sub- stantially are relatively low, they may still have an impact on loss of valid outcome data and are resource-intensive but important. It is therefore important to identify the approach that elicits the best re- sponse from practices to inform the design of future studies. If checking status is too costly or ineffective, then studies will adopt a pragmatic model of contacting participants directly, which may not be in the best interests of the patient or their families. We have therefore designed a feasibility cluster RCT, nested within a larger trial, using 12 sites and 4 different methods of approach. 1. Tele- phone contact first and then Fax if requested using the letter format already in use 2. Telephone contact first and then Fax using a new letter format reminding them that they have already received con- firmation of consent from their secondary care provider 3. Fax con- tact first using the letter format already in use and then Telephone contact if no response 4. Fax contact first using the new letter format reminding them that they have already received confirmation of con- sent from their secondary care provider Three sites were randomised to each of these methods, with all general practices from which a Methods Ethics approval was obtained to allow full informed consent or verbal assent (using a brief information sheet) followed by full written con- sent at a later date. The brief information sheet is used when the therapeutic time window is short and the use of full written consent would inhibit recruitment into the trial. Where patients lacked cap- acity, approval was obtained to enrol them with permission from a relative, carer or friend acting as legal representative. If no one was available to act as a legal representative, permission could be ob- tained if two clinicians (one unconnected with the trial) agreed to enrol the patient. Permission from legal representatives could be given using a full information sheet, or the brief information followed by full written consent. Background There is almost nothing that will destroy a relationship with a site than disagreement on the eligibility of a patient. Oncology studies are particularly vulnerable to this problem because site investigators are very interested in saving their patients’ lives and may inadvert- ently be biased toward inclusion. For example, disease free survival Page 120 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 120 of 235 site’s participants were recruited being allocated to that method. To reduce differences in initial approach the same member of staff was used for all requests. We have collected details of the amount of extra information or processes requested by the practices and the time interval from request to provision of the information (or to ter- mination of our request). Analysis will be descriptive and inform the feasibility and design of a full nested trial of these methods. Feasibil- ity questions include whether there is the potential to detect an im- portant difference on a key outcome and whether it is feasible to design a trial for which randomisation is by secondary care provider rather than general practice. requires that a patient have no detectable disease at baseline. If eligi- bility review done by the site is then sent to central review for effi- cacy imaging analysis, there is a small but real percentage of patients that will be determined to have had disease at baseline. These patients will, in the final analysis of risk, will be deleted from the analysis since they would have progressed at baseline, an infinite risk. Another example is the requirement in RECIST to have measur- able lesions to determine response. Site determination of measurable often does not coincide with the independent reader’s assessment of measurable. Central confirmation of eligibility decreases the risk of these patients being included but only if a certain amount of due diligence is paid to the method of confirmation. P315 Brief consent forms enable rapid enrolment in acute clinical trials: data from the on-going tranexamic acid for hyperacute primary intracerebral haemorrhage (TICH-2) study p data from the on-going tranexamic acid for hyperacute primary intracerebral haemorrhage (TICH-2) study Katie Flaherty, Lelia Duley, Zhe Law, Philip M. Bath, Nikola Sprigg University of Nottingham Correspondence: Katie Flaherty Trials 2017, 18(Suppl 1):P315 Background Background Intracerebral haemorrhage (ICH) is often complicated by haematoma expansion (HE) with devastating consequences. The NIHR HTA TICH-2 study is a randomised controlled clinical trial that is testing whether tranexamic acid arrests HE and improves outcome. Typically, stroke treatments have greater efficacy if given early and so delays should be avoided. Obtaining consent in the emergency situation is difficult since many stroke patients lack capacity to consent and relatives are often not present. P314 Methods of approaching general practices for trial participant information: feasibility cluster-randomised trial nested within a large multi-centre cluster-randomised controlled cross-over trial Denise Forshaw1, Chris Sutton1 1University of Central Lancashire Correspondence: Denise Forshaw Trials 2017, 18(Suppl 1):P314 P314 Methods of approaching general practices for trial participant information: feasibility cluster-randomised trial nested within a large multi-centre cluster-randomised controlled cross-over trial Denise Forshaw1, Chris Sutton1 P314 Methods of approaching general practices for trial participant information: feasibility cluster-randomised trial nested within a large multi-centre cluster-randomised controlled cross-over trial Denise Forshaw1, Chris Sutton1 1University of Central Lancashire Correspondence: Denise Forshaw Trials 2017, 18(Suppl 1):P314 g Methods Methods to assess eligibility are site alone, site + central, central alone and site with central confirmation. Each method will be reviewed and case studies as well as simulations will show the risks and benefits of each. Case Studies Several anonymized case studies will be used to demonstrate the effects of each of the methods. Additionally, parame- ters derived from these case studies will be used to simulate clinical trials under different hazard ratios to demonstrate the impact of in- appropriate eligibility on the final results. P315 Brief consent forms enable rapid enrolment in acute clinical trials: data from the on-going tranexamic acid for hyperacute primary intracerebral haemorrhage (TICH-2) study Katie Flaherty, Lelia Duley, Zhe Law, Philip M. Bath, Nikola Sprigg University of Nottingham Correspondence: Katie Flaherty Trials 2017, 18(Suppl 1):P315 y Results y Results Of 1682 patients enrolled, 387 (23%) gave full informed consent and 201 (12%) gave brief verbal assent. Many patients lacked capacity (65%) and were enrolled after proxy consent from a legal representa- tive; full informed relative 720 (43%), brief relative 255 (15%), inde- pendent physician 119 (7%). The mean (SD) time from stroke onset to recruitment (in hours) for patients enrolled with full consent were 3.8 (1.6) for patient and 4.0 (1.7) for relative consent; this went down to 3.4 (1.7) for brief patient and 3.6 (1.5) for brief relative. The quick- est consent group was independent physician, with an average time to recruitment of 3.2 (1.5) hours. Use of a range of methods for consent enabled rapid enrollment. Participants unable to consent had dysphasia and higher stroke se- verity. Thirty nine participants who gave verbal assent died before full written consent could be obtained, and two participants declined to give further consent and later withdrew from the day 90 follow- up; no participants who used brief consent were lost to follow-up. Conclusion References 1. Parmar et al. Lancet, 2014; 384(9940):283–234. 2. Chalder, Goldsmith et al. Lancet Psychiatry, 2015; 2(2):141–152. 3. Fritz and mackinnon. Psychol Sci, 2007; 18(3):233–239. 1. Parmar et al. Lancet, 2014; 384(9940):283–234. 2. Chalder, Goldsmith et al. Lancet Psychiatry, 2015; 2(2):141–152. 3. Fritz and mackinnon. Psychol Sci, 2007; 18(3):233–239. Background g The GASP study (Groups for Alcohol Misusing Short-terms Pris- oners) is a randomised trial of an intervention to improve partici- pants’ sense of control and motivation to make changes. Men were allocated to the control arm (standard prison regimen) or a programme of nine group sessions over three weeks facilitated by an experienced clinical psychologist and psychology assistant. Clus- tering by group session is therefore present only in the intervention arm. There were 8 facilitators in total, 5 psychologists and 3 assis- tants, different pairs of facilitators ran the groups during the study. Further clustering is therefore present by facilitator which varied over the course of the study. Results All group programmes for the GASP study have now been com- pleted and longer term follow-up data collection and data cleaning is underway. Two hundred and thirty eight men were randomised on a 1:1 basis and there were 15 intervention group programmes (cycles) completed over 2 years. The total sample size target was 120 for the primary analysis. Primary outcome LCB data is available for 68 in the intervention arm and 61 in the control arm. Results of the statistical analysis of the primary outcome will be presented discussing any advantages or disadvantages of the analytic strategy employed. Results The multiple arms/explanatory design combination provided both practical and statistical advantages. From the practical point of view: A) the results from several two arm trials were obtained from one trial, B) an explanatory design meant this was true for important sec- ondary analyses as well, C) this particular design allowed for compari- sons between active treatments and with the specialised medical care comparison arm. One statistical advantage was increased power. This may have been especially important for the mediation analysis, as there is often low power to detect mediated effects [3] and some useful methods for studying mediation suffer from lower precision. For example, there was 25% gain in the precision of the parameter estimate for the mediator-outcome relationship in the full four arm dataset as compared to the two arm subset. Another statistical feature was the ability to test whether the mediator-outcome effect (conceptual theory) was similar across treatment arms (action theories). This assumption held, and making this assumption allowed for further precision gains. Objectives To compare strategies for the analysis of clustered data where clus- tering by group and facilitator is only present in the intervention arm. up; no partici Conclusion Abbreviated information sheets supporting verbal assent and proxy consent can ensure patients are enrolled rapidly into emergency clinical trials. The use of brief consent or proxy consent did not lead to large numbers of withdrawals or losses to follow-up, thus the use Page 121 of 235 Trials 2017, 18(Suppl 1):200 of two stage/or proxy consent for emergency clinical trials should be considered. ( p Methods Longitudinal structural equation models (SEM) for mediation were applied to two-arm subsets and the overall four-arm trial dataset to study longitudinal mediation of the effects of the PACE trial treat- ments. Fear avoidance (FA) was used as an example mediator and physical functioning (PF) as an example outcome [2]. A single model was fitted to the dataset in each case with the pertinent contrasts obtained. Treatment by mediator interaction terms were used to as- sess differences in mediator-outcome effects (conceptual theories) for different treatments. Informal comparisons of the standard errors were used to assess precision. Methods Recruitment and then randomisation to the intervention and control arms was carried out in small blocks over the course of the study due to a limitation on the maximum size of the group sessions. The primary outcome, Locus of Control of Behaviour (LCB), was collected for all men prior to randomisation and at the end of the group ses- sions in the intervention arm and an equivalent time point in the control arm. One analysis strategy could therefore involve the cre- ation of control clusters contemporaneously equivalent to the inter- vention group clusters. A second strategy would be to create clusters of sample size one in the control arm and is the current standard strategy for trials of this design. A third strategy would be to ran- domly create control arm clusters of equivalent size, and variation in size, to the intervention arm clusters, the ‘artificial cluster method’. A forth strategy would be to group all the control men into a single control arm cluster. The primary analysis is a two level general linear model adjusted by baseline LCB. Secondary analysis will include the additional level of facilitator in a three level model if the ICC warrants its inclusion. Analysis strategies for two-level clustering in one arm of a randomised group therapy trial set in Cardiff prison Rebecca Playle, Michael Robling, Rachel McNamara, Yvonne Moriarty, Zoe Meredith, Hannah John-Evans, Pamela Taylor Cardiff University Correspondence: Rebecca Playle Trials 2017, 18(Suppl 1):P318 Analysis strategies for two-level clustering in one arm of a randomised group therapy trial set in Cardiff prison Rebecca Playle, Michael Robling, Rachel McNamara, Yvonne Moriarty, Zoe Meredith, Hannah John-Evans, Pamela Taylor Cardiff University Correspondence: Rebecca Playle Trials 2017, 18(Suppl 1):P318 y q y Kimberley Goldsmith1, Peter White2, Trudie Chalder1, Michael Sharpe3, Andrew Pickles1 Andrew Pickles 1King’s College London; 2Centre for Psychiatry, Wolfson Institute of Preventive Medicine, Barts and the London School of Medicine, Queen Mary University; 3Psychological Medicine Research, Department of Psychiatry, University of Oxford Correspondence: Kimberley Goldsmith Trials 2017, 18(Suppl 1):P317 1King’s College London; 2Centre for Psychiatry, Wolfson Institute of Preventive Medicine, Barts and the London School of Medicine, Queen Mary University; 3Psychological Medicine Research, Department of Psychiatry, University of Oxford Correspondence: Kimberley Goldsmith Trials 2017, 18(Suppl 1):P317 1King’s College London; 2Centre for Psychiatry, Wolfson Institute of Preventive Medicine, Barts and the London School of Medicine, Queen Mary University; 3Psychological Medicine Research, Department of Psychiatry University of Oxford Background Designing explanatory trials to answer additional questions such as how and for whom treatments work should be a priority for im- proving trial efficiency. Multiple arm trials are also more efficient, as they provide more information about treatments over a shorter time span [1]. We studied the benefits of multiple trial arms and ex- planatory design using the Pacing, Graded Activity, and Cognitive Behaviour Therapy: A Randomised Evaluation (PACE) trial as an ex- ample. This trial studied three complex therapies and a specialised medical care comparison arm for the treatment of chronic fatigue syndrome. The study of how the treatments worked - mediation analysis - was built into the trial design. In terms of mediation, one interest was whether different treatments with some disparate components might vary in mechanism. In other words, might the effects of different treatments on a mediator (a paths or action the- ories) be associated with different mediator-outcome relationships (b paths or conceptual theories)? Results p Results FU data was more complete for self-reported FU patients. No significant difference in event rate was observed for first DFS event. When looking at event types separately, death rates for self-reported patients were significantly lower compared to conventional patients (HR = 0.31, 95% CI = 0.17-0.72). Although not statistically significant DR rates were lower for self-reported FU (HR = 0.62, 95% CI = 0.22-1.72) and LR rates were higher for self-reported FU (HR = 3.39, 95% CI = 0.72-15.87). Conclusions In the fall of 2015, it was noted that within-cluster recruitment was slower than expected, so the decision was made to increase the number of clusters to 40. In Spring 2016, available baseline data was used to estimate the intra-cluster correlation coefficient (ICC) in order to gauge whether the initial assumption of a 0.02 ICC was correct. With an updated baseline ICC of 0.007 (95% CI: 0.0001-0.433) the ne- cessary sample size decreased to 908 students. While favorable, this left the study team with the following choice: a) assume the updated ICC was closer to truth and proceed with the lower, more favorable sample size; or b) assume the original ICC and continue with the more conservative sample size of 1300. Given the instability of the ICC estimate, the latter decision was made, but it raised the question of whether previous cluster RCTs in adolescent medicine may have benefited from sample size re-estimation using baseline ICC. Within REACT self-reported follow up is a suitable alternative to col- lect data for the primary endpoint of DFS. However for accurate reporting of secondary endpoints a more robust method for report- ing of deaths needs to be considered. Further research is also re- quired into whether patients reliably report the correct type of relapse. When reviewing impact of FU methods which can change it is important to use a landmark analysis or other methods to reduce the risk of bias if it’s possible for events to occur before the method can change (e.g. If the method can’t change until treatment is complete). For multi-event outcomes it is also important to analyse separately by event type in case the overall result masks a difference between event types. This is particularly an issue when secondary endpoints use single events. Sensitivity analysis assessing the impact of patient self-reported follow up in the react trial Holly Tovey1, Judith Bliss1, Charles Coombes2, Gunter von Minckwitz3, Jan Steffen3, Lucy Kilburn1 1The Institute of Cancer Research; 2Imperial College London; 3German Breast Group Correspondence: Holly Tovey Trials 2017, 18(Suppl 1):P319 Good QC goes far beyond just reviewing individual results and should also consider monitoring data throughout the course of a study. In particular, QC is essential when supporting a Data Monitor- ing Committee. Given the nature of interim and incomplete data, in- herent challenges exist when it comes to generation of DMC reports. Many of the usual practices associated with quality control need to be adapted to accommodate the repetitive nature of DMC review on accumulating data that may have outstanding queries. Correspondence: Holly Tovey Trials 2017, 18(Suppl 1):P319 p Methods A univariate Cox-model was fitted for time to first DFS event with FU method as a time-varying covariate; observations were split at the time a patient consented to self-reported FU unless an event occurred prior to this. It was assumed that following consent patients could not revert back to conventional FU. The model was repeated excluding the first 2 years of FU and a landmark analysis was also carried out looking at 0–2 years, 2–5 years and 5 years + separately. This was done to reduce bias from early events; patients cannot switch to self-reported FU until they have completed treatment (usually at 2 years) so events prior to this could only occur on conventional FU. For each analysis the hazard ratio for FU type and event rates in each group were calculated. Ana- lysis was repeated separately for each type of DFS event; local relapse (LR), distant relapse (DR) and death. The Coaching Boys into Men (CBIM) Middle School study is a cluster- randomized trial of a middle school gender violence prevention pro- gram. The primary goal is to examine the effectiveness of a program for the primary prevention of adolescent relationship abuse (ARA) and sexual violence among middle school sports teams in Western Pennsyl- vania. Initially, 26 middle schools were randomized to receive either a) the CBIM intervention, which trains athletic coaches by providing con- crete strategies for discussing sexual violence as well as how to re- spond to disrespectful behaviors, or b) control (standard coaching). According to initial sample planning assumptions, this would yield 1980 students and provide 80% power to detect meaningful differ- ences in the primary outcome, positive bystander behavior. p Conclusions The prison environment is a constantly changing and challenging en- vironment for research. Prisoner numbers, prisoner mix, prison trans- fers, staff to prisoner ratios, access to services and researcher access to prisoners varied during the course of the group cycles, therefore creating contemporaneously equivalent control arm clusters may be preferable to the standard strategy in this trial. A discussion of any bias that may be introduced or controlled for by any one of these methods will also be addressed. Where indicated and/or sensible, designing trials to answer explana- tory questions using a multiple arm design will be more efficient, and provide more statistical power as well as a rich source of infor- mation about treatments. Such designs should maximise efficiency for primary outcome comparisons, and provide important informa- tion about secondary questions of interest across multiple treatments within a single study. Trials 2017, 18(Suppl 1):200 Page 122 of 235 Page 122 of 235 function of regulatory requirements, industry standards, and corporate philosophies. However, no one should underestimate the importance of independent, thoughtful consideration of relevance and impact at each step in the process from data collection through analysis. function of regulatory requirements, industry standards, and corporate philosophies. However, no one should underestimate the importance of independent, thoughtful consideration of relevance and impact at each step in the process from data collection through analysis. P319 Sensitivity analysis assessing the impact of patient self-reported follow up in the react trial Holly Tovey1, Judith Bliss1, Charles Coombes2, Gunter von Minckwitz3, Jan Steffen3, Lucy Kilburn1 1The Institute of Cancer Research; 2Imperial College London; 3German Breast Group Correspondence: Holly Tovey Trials 2017, 18(Suppl 1):P319 Background REACT is a phase III, multicentre trial of celecoxib vs placebo in primary breast cancer patients in the UK and Germany. Patients receive blinded treatment for 2 years and are followed up every 6 months, then annu- ally up to 10 years for Disease Free Survival (DFS). This creates burden on the site and the patient. For some sites it has been necessary to move towards self-reported follow up (FU) via questionnaires to reduce the burden. Not all sites have taken up the opportunity and it is not an option in the UK. There was concern within the trial committees that self-reported FU could result in a loss of data or accuracy, producing bias in the principal analysis. It was agreed that the validity of self- reported FU compared to conventional centre-based methods should be retrospectively assessed within the trial. g y g This presentation will explore adaptations to a typically rigid QC process that are necessary when reviewing interim/incomplete data. Such adaptations focus on a risk-based approach to QC to ensure that a DMC can make informed decisions with more confidence in the data and programming. P321 The impact of sample size re-estimation using baseline ICC in cluster randomized trials: 3 case studies Kaleab Abebe1, Kelley A. Jones1, Elizabeth Miller1, Daniel J. Tancredi2 1University of Pittsburgh; 2University of California Davis Correspondence: Kaleab Abebe Trials 2017, 18(Suppl 1):P321 An evaluation of statistical methods for predicting timelines for reaching target number of events in clinical trials with time-to-event endpoints 1 2 2 3 Emma Clark1, Hans-Joachim Helms2, Sven Stanzel2, Fan Xia3 1Roche Products Ltd; 2Hoffmann-La Roche Ltd; 3Roche Product Development Correspondence: Emma Clark Trials 2017, 18(Suppl 1):P323 Results g In this talk, we will review sample size re-estimation methods for cluster RCTs and describe three completed studies: CBIM High School, SHARP (School Health Center Healthy Adolescent Relationships Program), and ARCHES (Addressing Reproductive Coercion in Health Settings). After providing an overview of the study designs and primary outcomes, we will discuss the initial sample size calculations with the assumed ICC as well as the final ICC at the end of study. Additionally, we will highlight the impact of post-hoc sample size re-estimation methods on the tar- get sample size as well as the primary results. DMC report production: considering a risk-based approach to quality control DMC report production: considering a risk-based approach to quality control Amber Randall, Bill Coar Axio Research Correspondence: Amber Randall Trials 2017, 18(Suppl 1):P320 DMC report production: considering a risk-based approach to quality control Amber Randall, Bill Coar Axio Research Correspondence: Amber Randall Trials 2017, 18(Suppl 1):P320 quality control Amber Randall, Bill Coar Axio Research Correspondence: Amber Randall Trials 2017, 18(Suppl 1):P320 y Amber Randall, Bill Coar Axio Research Correspondence: Amber Randall Trials 2017, 18(Suppl 1):P320 Kukatharmini Tharmaratnam, Thomas Jaki Lancaster University Correspondence: Kukatharmini Tharmaratnam Trials 2017, 18(Suppl 1):P322 Quality control is fundamental to ensuring both correct results and sound interpretation of clinical trial data. Most QC procedures are a Page 123 of 235 Trials 2017, 18(Suppl 1):200 Page 123 of 235 Background been reached. At the planning stage of such studies, the number of events required for statistical analysis and predictions of the ex- pected date when this target number of events will be reached, are typically based on protocol assumptions and conducted by use of a simple parametric model. A blinded re-evaluation of these predic- tions is recommended to obtain more accurate predictions as the trial progresses and events accumulate. Different statistical ap- proaches have been proposed in the literature for making such predictions, including parametric approaches assuming smooth underlying survival functions, nonparametric approaches and hy- brid methods applying a non-parametric model where data are available, complemented with a parametric tail for regions where no data are yet available. Factors such as study design and ratio of number of events in relation to sample size can impact the model estimates derived from the various statistical methods, thereby making the choice of the optimal prediction method for a particular study a key decision which can influence the reliability of the pre- dictions. We report results obtained from a systematic comparison of the different methods via simulation studies. The point estimates of the predicted analysis times and number of events, along with their variability as measured by confidence interval, are investi- gated under varying study scenarios and findings are discussed. Keywords: time-to-event, event prediction, parametric, hybrid. g Individual populations within a research study are typically heteroge- neous. Characteristics such as genetics, disease etiology and severity vary between individuals and potentially affect the response to treat- ment. Treatment effectiveness is, however, typically assessed using the average treatment effect or at most treatment effect within (prespeci- fied) subgroups. Recently, developed approaches allow researchers to predict an individual's response to treatment allowing individualized treatment instead of relying on averages from a group or subgroup. Lamont et al. (2016) de ne Predicted Individual Treatment Effects (PITE) and introduce the framework of PITE. The objective of this work is to propose, derive and evaluate prediction interval for PITE. The PITE can be estimated utilizing multiple imputation to obtain treatment effect estimates on a patient level. Based on this approach we develop a method to compute prediction intervals on an individ- ual patient level. To ensure adequate estimate of the variability which is required to obtain such intervals, we investigate different model selection methods. Objective To compare ML, MI and IPW approaches for handling missing longi- tudinal proms data in RCTs. P323 P323 An evaluation of statistical methods for predicting timelines for reaching target number of events in clinical trials with time-to-even endpoints Emma Clark1, Hans-Joachim Helms2, Sven Stanzel2, Fan Xia3 1Roche Products Ltd; 2Hoffmann-La Roche Ltd; 3Roche Product Development Correspondence: Emma Clark Trials 2017, 18(Suppl 1):P323 Background Missing data are a potential source of bias in the results of randomised controlled trials (RCTs), which can have a negative impact on guidance derived from them, and ultimately patient care. However, missing data are generally unavoidable in clinical research, particularly in patient re- ported outcome measures (PROMs). For longitudinally collected out- comes, often only a small subset of participant will have complete data for all relevant time points. Multilevel mixed-effects linear regression models are commonly used to analyse longitudinal data. A number of methods are available to handle missing data in such analyses, includ- ing maximum likelihood (ML), multiple imputation (MI) and inverse probability weighting (IPW). Direct comparisons of such methods for missing proms data in RCT settings are needed to ensure the bias intro- duced in such analyses is minimised. Results We used continuous response variable and binary covariates to fit re- gression model in all simulation studies. The simulation results show that, using no variable selection leads to an under estimation of the variability and hence under coverage. But the prediction intervals achieve good coverage when we use variable selection methods stepbic or Lasso. Lasso variable selection method works better even with small sample sizes. We have considered different set of selected variables used to get the PITE, those are separately selected variables from each arm, union of selected variables from both arm and the selected variables from joint model. Our simulation results indicates all of these sets are perform well. In practice, the variables selected from joint model would be more reasonable to use because we will have new patients from any of the arms. We considered uncorre- lated, perfectly correlated and partially correlated responses in the simulation studies. Our proposed approach performs well with all of these correlation structure. To illustrate proposed method, we used PRO-ACT data (http://nctu.partners.org/proact) in ALS clinical trials. We used patients with equal number of individuals (n = 1500) from the placebo and active treatments group. We used the response vari- able ALSFRS slope as used in Kuffner et al.(2015) compute from re- peated measure of ALSFRS score (ALS Functional Rating Scale) and covariates are several baseline information, Age, Gender, Ethnicity, etc.… for each patient. We applied lasso regression and selected the variables Gender, Age, Race, onset-delta and onset-site to get good PITE for each individuals. We calculate 95% prediction intervals for each patients. The estimated PITE and its intervals are reasonably good. Discussion: Our proposed approach to find the prediction interval for PITE performs well in the simulation studies and real data example. We could use other type of response variables and covari- ates in the model to estimate PITE and also we may use interaction models and more complex mixed models to get PITE in future work. A comparison of statistical approaches for analysing missing longitudinal patient reported outcome data in randomised controlled trials Ines Rombach1, Alastair Gray1, Crispin Jenkinson2, Oliver Rivero-Arias3 1Health Economics Research Centre, University of Oxford; 2Health Services Research Unit, University of Oxford; 3National Perinatal Epidemiology Unit, University of Oxford Correspondence: Ines Rombach Trials 2017, 18(Suppl 1):P325 tudinal pro Methods Kuffner et al. (2015), Crowdsourced analysis of clinical trial data to predict amyotrophic lateral sclerosis progression. Nature Biotechnology, 33:51–57. References Kuffner et al. (2015), Crowdsourced analysis of clinical trial data to predict amyotrophic lateral sclerosis progression. Nature Biotechnology, 33:51–57. Lamont et al. (2016), Identification of predicted individual treatment effects Methods Real-life missing data following missing at random patterns were simulated within the follow-up of an RCT using the Oxford Knee Score. Datasets of sample sizes ranging from 100 to 1,000 with miss- ing proms outcome data in 10% to 40% of participants were simu- lated. Both intermittently missing data and monotone missing data patterns were considered. Missing data was addressed using ML, MI and IPW. Performance of the different approaches was assessed by the bias introduced in the treatment coefficients from multilevel mixed-effects linear regression models obtained for 1000 simulations. Root mean square errors (RMSE) and mean absolute errors (MAE) were used as performance parameters. Lamont et al. (2016), Identification of predicted individual treatment effects (PITE) in randomized clinical trials Statistical Methods in Medical Research Lamont et al. (2016), Identification of predicted individual treatment effects (PITE) in randomized clinical trials. Statistical Methods in Medical Research. (PITE) in randomized clinical trials. Statistical Methods in Medical Research. P327 T cells in type 1 diabetes: dilfrequency study James Howlett1, Adrian Mander1, Simon Bond2, Frank Waldron-Lynch3 1MRC Biostatistics Unit Hub for Trials Methodology Research; 2Cambridge Clinical Trials Unit, Cambridge University Hospitals NHS Foundation Trust; 3Division of Experimental Medicine & h f d f b d Background In this paper, we compare different methods of calculating sample sizes for comparing two independent means of a continuous outcome with baseline and post-intervention measurements of a randomised con- trolled trial (RCT). Sample size calculations typically use published re- sults from similar trials, and we illustrate the different methods using published results from the MOSAIC trial. Methods Thirty-six participants with T1D were administered repeat doses of alde- sleukin with the aim of establishing the optimal dose-frequency to de- liver drug to increase Tregs, CD25 (Subunit of the IL-2 receptor) expression on Tregs, whilst minimising the increase in Teffs. There was an initial learning phase with six pairs of participants, each pair receiv- ing one of six preassigned dose-frequencies from 0.09-0.47x106 IU/m2 and 2–14 days, in order to model the dose-frequency response. At the first interim analysis following the learning phase, the target increases (30% Treg, 25% CD25, 0% Teff) for each of the endpoints were selected by the dose frequency committee. The subsequent 3 groups of 8 par- ticipants were administered dose-frequencies based on the results from statistical analyses of all data from previous groups. When allocating treatment regimens, consideration was given to the probability of the predicted increases fall within the target ranges as well as the distance the predicted increases are from the targets for each dose-frequency. Results Comparing different ways of calculating sample size for a randomised controlled trial using baseline and post-intervention measurements yp q y y James Howlett1, Adrian Mander1, Simon Bond2, Frank Waldron-Lynch3 1MRC Biostatistics Unit Hub for Trials Methodology Research; 2Cambridge Clinical Trials Unit, Cambridge University Hospitals NHS Foundation Trust; 3Division of Experimental Medicine & Lei Clifton1, Jacqueline Birks1, David A. Clifton2 1Centre for Statistics in Medicine (CSM), Oxford University; 2Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford Correspondence: Lei Clifton Trials 2017, 18(Suppl 1):P327 Correspondence: Lei Clifton Trials 2017, 18(Suppl 1):P327 Results Non-convergence issues were observed for the IPW approach for small sample sizes. Complex MI models needed to be simplified to obtain valid results for combinations of small sample sizes and large proportions of missing data. Bias in the treatment coefficient in- creased both with decreasing sample size and increasing proportions In clinical trials with time-to-event outcomes, interim or final analyses are often planned after a pre-defined target number of events has Trials 2017, 18(Suppl 1):200 Page 124 of 235 Page 124 of 235 of missing data. MI and ML performed similarly when similar variables were included in both the imputation and analysis model, and when the imputation model was restricted to baseline variables. However, MI was less biased than ML when additional post-randomisation data were used in the imputation model. Both approaches were less biased when follow-up data was missing intermittently compared to monotone missing data scenarios due to drop-out. IPW introduced more bias in the model results than both ML and MI across all sample size and miss- ing data scenarios. ANCOVA. The correlation between the post-intervention and baseline measure is likely to reduce as the duration prolongs; therefore the duration between the post-intervention and baseline need to be taken into account For example, the MOSAIC trial reported the SF-36 energy/vitality score at 6 months, for which we have derived r = 0.7. If the post-treatment time point of this measure in the planned RCT is at 12 months, then we will need to reduce the value of r for the sample size calculation. The resulting sample sizes are shown by the sensitivity analysis in this paper. Background In type 1 diabetes (T1D) there is a deficiency in the interleukin 2 (IL-2) pathway leading to a loss of regulatory T cell (Treg) function. Dilfre- quency aims to determine the optimal dosing regimen of ultra-low dose recombinant IL-2 (aldesleukin) to improve Treg function while limiting the activation of CD4 T effector cells (Teff) in participants with T1D. Conclusions CO ll Conclusions MI can offer benefits over ML for handling missing longitudinal proms data when additional post-randomisation information is available. For RCTs with sample sizes up to 1000, the use of IPW is not recommended to handle missing data. The findings also demonstrate the importance of minimising missing data and continued data collection beyond missed appointments to inform the analysis and imputation models. The results presented in this presentation focus on missing at random mechanisms, and sensitivity analyses to investigate the effect of other missing data mechanisms remains imperative. ANCOVA allows efficient sample size calculation by utilising the cor- relation between the baseline and post-intervention measurements; however, one must be aware of its implications and consider factors such as duration of the intervention. In comparison, using a t-test produces a more conservative (i.e. Larger) sample size than using ANCOVA. In the situation when sample size is calculated by a t-test instead of ANCOVA, sample size using change score from baseline can be smaller or larger than that using post-intervention score alone, depending on the value of the correlation coefficient r. The choice of the outcome measure should be driven by clinical knowledge instead of a mere pur- suit of small sample size. p Methods The methods we discuss are suitable for sample size calculation using a continuous outcome measure. Suppose the primary continuous outcome measure is Y, with Y_0 and Y_1 denoting Y at baseline and post-intervention, respectively. Let r denote the correlation coefficient between Y_0 and Y_1. We discuss the following two factors: 1. The choice of the primary outcome measure: post-intervention measure Y_1 vs. Change from baseline (i.e. Y_1-Y_0). 2. The choice of statistical methods for sample size calculation for two independent means: t-test without using r vs. Analysis of covari- ance (ANCOVA) using r. We show how to use the Variance Sum Law to derive r between Y_0 and Y_1, and then how to use the derived r to calculate sample size by ANCOVA. We discuss the assumptions of the ANCOVA method and its implications for the trial design. Improving precision by adjusting for prognostic baseline variables in randomized trials with binary outcomes, without regression model assumptions We advocate reporting the standard error (SE) of the mean change between the baseline and post-intervention measurements, as did the MOSAIC publication. It provides insight into the correlation be- tween the baseline and post-intervention measurements, and therefore allows the sample size to be calculated and compared in different ways. p Jon Steingrimsson, Daniel F. Hanley, Michael Rosenblum Johns Hopkins University Correspondence: Jon Steingrimsson Trials 2017, 18(Suppl 1):P326 p Jon Steingrimsson, Daniel F. Hanley, Michael Rosenblum Johns Hopkins University Correspondence: Jon Steingrimsson Trials 2017, 18(Suppl 1):P326 This abstract is not included here as it has already been published. P328 P328 A response-adaptive trial to determine the optimal IL-2 dose-frequency to achieve multiple target increases on regulatory T cells in type 1 diabetes: dilfrequency study James Howlett1, Adrian Mander1, Simon Bond2, Frank Waldron-Lynch3 1MRC Biostatistics Unit Hub for Trials Methodology Research; 2Cambridge Clinical Trials Unit, Cambridge University Hospitals NHS Foundation Trust; 3Division of Experimental Medicine & Immunotherapeutics, Department of Medicine, University of Cambridge Correspondence: James Howlett Trials 2017, 18(Suppl 1):P328 P326 3 6 Improving precision by adjusting for prognostic baseline variables in randomized trials with binary outcomes, without regression model assumptions Conclusions Conclusions When randomisation has been stratified on a categorised continuous variable, there is no difference between adjusting for the underlying continuous variable and adjusting for the categorisation. When the continuous variable has a large effect on the outcome and the vari- able has not been stratified on, power is lost if the analysis is ad- justed for the categorisation as opposed to the continuous variable. It is safer to always adjust for the underlying continuous variable. Three approaches to this problem are apparent: i) analyse any partici- pants that have currently completed the study accepting that the success rate is temporarily reduced; ii) analyse a full cohort of pa- tients that have completed the study using a pre-defined cut-off period, e.g. Only those randomised greater than nine months previ- ously; and iii) switch analytical methods during the interim period to utilise survival analysis methodology (e.g. Kaplan-Meier, Cox Propor- tional Hazards), censoring participants at the point of last know follow-up if not yet completed the study. Aim The aim of this work is to use formal causal inference techniques to help trial researchers understand when the treatment effect estimate will be biased due to recruitment issues, by using Directed Acyclic Graphs (DAGs) and d-separation, and to give a measure of the size of these biases. Methods: We considered several situations that could lead to recruitment bias in a CRT, namely where individual recruitment is Discussion In studies of this type, Trial Statisticians should consider planning the use of survival analysis methodology during the interim period regard- less of planned final analysis methods to enable provision of inform- ative estimates to independent Data Monitoring Committees. Results When the randomisation was stratified, the nominal power and sig- nificance levels were maintained unconditionally. When the random- isation was unstratified, the nominal power and significance levels were maintained except when both (i) the analysis was adjusted for the categorisation; and (ii) the effect of the underlying continuous variable on the outcome was large. Understanding, quantifying and reducing recruitment bias in cluster randomized trials Karla Diazordaz LSHTM Trials 2017, 18(Suppl 1):P331 Understanding, quantifying and reducing recruitment bias cluster randomized trials Karla Diazordaz LSHTM Trials 2017, 18(Suppl 1):P331 Understanding, quantifying cluster randomized trials Karla Diazordaz LSHTM Trials 2017, 18(Suppl 1):P331 Trials 2017, 18(Suppl 1):P331 Discussion p Results p y p y Results ll h h ll d f We will show that: option i) will not provide appropriate estimates of current success rates and could potentially bias interim efficacy esti- mates if there is a difference between groups in early failures; option ii) will provide appropriate estimates of current live birth rate but will limit the amount of data available for analysis and therefore poten- tially hamper decision-making; and option iii) will utilise all available data for analysis of efficacy and also provide appropriate interim esti- mates of the live birth-rate. Adjusting trial analyses for continuous stratification variables Tom Morris, Shaun Barber, Cassey Brookes Leicester Clinical Trials Unit Trials 2017, 18(Suppl 1):P330 Adjusting trial analyses for continuous stratification variables Tom Morris, Shaun Barber, Cassey Brookes Leicester Clinical Trials Unit Trials 2017, 18(Suppl 1):P330 p Results p g We discuss the existing research on how the value of correlation coefficient r influences the sample size, using change score from baseline vs. Post-intervention score alone. We found at each pre-planned interim analyses, that the optimal dose- frequency was estimated with increasing accuracy, thereby allowing more participants to be allocated dose-frequencies close to the optimal than would be possible in a non-adaptive design. The results from the final interim analysis suggest that the optimal aldesleukin dose to We perform a sensitivity analysis on different values of r to show the effect of the strength of the correlation on the sample size by Page 125 of 235 Page 125 of 235 Trials 2017, 18(Suppl 1):200 maintain steady state increases in Tregs and CD25 expression is be- tween 0.20 x106 IU/m2 and 0.32x106 IU/m2 at a frequency of every 3 days. Results from the final analysis are ongoing and will be pre- sented when available. guarantees that similar numbers of patients are allocated to each arm, within limits determined by the block size(s). This type of ran- domisation can be stratified, so that balance is achieved within the strata, rather than overall. The analysis of data from such trials should be adjusted for the stratification variables [1]. Stratification variables must be categorical, and therefore, if randomisation is to be stratified on a continuous variable, the variable must first be split into categor- ies (e.g. BMI is often categorised as underweight/normal, overweight, or obese). It is well known that a continuous covariate should not be categorised in an analysis (or anywhere else) without good reason. The question this investigation aims to answer is: should the analysis of a trial in which randomisation has been stratified on a categorised continuous variable be adjusted for the categorisation, or the under- lying continuous variable? Methods Reference [1] Improper analysis of trials randomised using stratified blocks or minimisation, Brennan Kahan, Tim Morris, Statist. Med. 2012, 31 328–340. Statistical issues in data monitoring of clinical trials that incorporate assessment of pregnancy loss Statistical issues in data monitoring of clinical trials that incorporate assessment of pregnancy loss Lee Middleton, Konstantinos Tryposkiadis, Versha Cheed University of Birmingham Correspondence: Lee Middleton Trials 2017, 18(Suppl 1):P329 p p g y Lee Middleton, Konstantinos Tryposkiadis, Versha Cheed University of Birmingham Correspondence: Lee Middleton Trials 2017, 18(Suppl 1):P329 Background Independent data monitoring in clinical trials of pregnant women where successful pregnancy is one of the outcomes is hugely important given the sensitive nature of these studies. Recommendations regarding early stopping or protocol modification can only be made with appropriate data available. Our experience in the NIHR-funded TABLET, PRISM and C- STICH studies suggests certain elements of data monitoring - namely oversight of interim estimates of the overall event rate and efficacy esti- mates - require careful consideration and forward planning. Methods Simulations were performed to assess the effect on the significance level and power of analyses in which the (continuous) outcome de- pends on a continuous covariate, and the randomisation has been stratified on a categorisation of this continuous variable. Three differ- ent relationships between the variable and the outcome were tested: linear, non-linear, and none, where the non-linear relationship meant that different slopes were used for each level of the (binary) categor- isation. The slopes in the linear and non-linear cases were varied in magnitude. For each simulation, 10,000 data sets were generated, and each was analysed in two models, one adjusting for the continu- ous variable and the other for the categorisation. The simulations were also conducted with unstratified randomisation. Primary outcomes for the aforementioned studies are variations of suc- cessful pregnancy (either live birth > 34 weeks or pregnancy loss up to the first week of life) and are planned to be analysed at the end of the trial as dichotomous outcomes (success/fail) through the generation of relative risks and associated confidence intervals using standard meth- odology. However, interim assessment during the recruitment period requires further thought as the failure rate is temporarily inflated due to the fact that treatment failures (e.g. Miscarriage or still births) are ?A3B2 show $132#?>accumulated sooner than successful outcomes (live births). In these circumstances, the Trial Statistician and Data Moni- toring Committee need to consider their approach in how to monitor both the sample size assumptions and interim estimates of efficacy. Background Cluster randomized trials (CRTs) are becoming increasingly common in primary care and public health. Often clusters are recruited and randomised before suitable individuals are identified and recruited to participate or consent to data collection, leading to possible bias if the recruited/consented individuals differ systematically from those who do not. It is often thought that this biases may be particularly important when recruitment rates vary across clusters and interven- tion arms. Adjusting trial analyses for continuous stratification variables Tom Morris, Shaun Barber, Cassey Brookes Leicester Clinical Trials Unit Trials 2017, 18(Suppl 1):P330 Background g A competing risk is an event that prevents an event of interest, such as a primary trial outcome, from occurring. The most common com- peting risk is death. Ignoring a competing risk in the analysis of a trial results in invalid estimates of the cumulative incidence (absolute risk) of the event of interest. Ignoring competing risks can also result in invalid comparisons between treatment and control groups, for example by biasing the estimate of the hazard ratio. Methods We reviewed currently methods for dealing with competing risks. The two most commonly used methods were the Fine and Gray model and cause-specific hazards models. We aimed to illustrate the effect of competing risks on estimates of cumulative incidence or es- timates or hazard ratios. We aimed to characterise the scenarios where bias is most likely to occur and most likely to be large. We reviewed currently methods for dealing with competing risks. The two most commonly used methods were the Fine and Gray model and cause-specific hazards models. We aimed to illustrate the effect of competing risks on estimates of cumulative incidence or es- timates or hazard ratios. We aimed to characterise the scenarios where bias is most likely to occur and most likely to be large. We used data from 3 large Phase III randomised clinical trials in car- diovascular disease: EMPHASIS-HF, EPHESUS and RALES. We chose heart failure hospitalisation as the event of interest and cardiovascu- lar death as the competing event. Missing data can be a serious problem in longitudinal studies be- cause of the increased chance of drop-out and other non-response across the multiple time points, and can be particularly challenging when there are different causes of the missing values. For instance, the reasons that patients completely drop-out of the study (mono- tone missingness) may be very different from those for failing to at- tend a particular follow-up appointment (intermittent missingness). Also, for some types of missingness, it is often plausible to assume that data may be “missing not at random” (MNAR), i.e. after condi- tioning on the observed data, the probability of missing data may depend on the underlying unobserved values. For example, in critical care trials the collection of hourly/daily biomedical data may take place at the local physician’s discretion and lead to intermittent miss- ingness that is related to the severity of the patient’s illness. Conclusions Recruitment bias in CRTs happens when recruitment/consent is dif- ferential by treatment allocation and associated to a variable which is also associated with the outcome. If this variable is observed, we can control for it in the models, and our treatment effect will be un- biased. However, if the variable is unobserved, the treatment effect will be biased. This bias is small when recruitment rates are high. The possibility that recruitment is associated with treatment assignment can be minimised by identifying/recruiting individuals prior to cluster randomisation, or by blinding recruiters and potential participants as much as possible). In addition, if researchers know which individual characteristic is likely to be associated with the systematic differences in recruitment/consent, measuring this and adjusting for it, can miti- gate the recruitment bias. Results Cause-specific hazards over-estimate cumulative incidence, whereas the Fine and Gray method correctly adjusts estimates of cumulative incidence to take into account competing risk. Both cause-specific hazards models and Fine and Gray models give biased estimates of the hazard ratio for treatment effect. When using the cause-specific hazards model, the likely size of the bias was small or moderate in the examples we studied, but the bias was larger when using the Fine and Gray model. Competing risks caused larger biases when the event occurred in larger numbers of patients; oc- curred earlier during follow up; or occurred more frequently in either the treatment or control group. Results We have formal results showing when there is bias present, for ex- ample when the probability of recruitment and the outcome both depend on a common individual-level covariate, and these associa- tions are differential by treatment allocated. In the simulations, we found that where this bias is present, it can be over half a standard deviation of the true causal treatment effect. Background For PIVOT, our interest is in the health- related quality of life outcome, which was collected every 12 weeks over a 3-year period, but suffered from substantial intermittent (35%) and monotone (20%) missingness in both arms. For each outcome, we compare the results from alternative assumptions about the longitudinal missing data mechanisms with the pub- lished trial results and assess the implications for decision uncer- tainty. As an example, provisional results from the sensitivity analysis for VASST find that the average cardiac index over time was 9% higher for patients treated with vasopressin compared with those treated with norepinephrine (95% credible interval: 1%-17%), whereas the original analysis reported no difference. We conclude that this approach to sensitivity analysis provides a flexible frame- work to assess the implications of the missing data for the trial conclusions. associated with treatment allocation and/or another variable (either measured or unmeasured), and use d-separation to show when the treatment effect is biased due to the associations induced by condi- tioning the analyses to those individuals in the population who are recruited (or consented). We also conducted a simulation study in which we varied the magnitude of association among the individual- level covariate, treatment allocation at the cluster-level, the probability of recruitment, and the outcome variable. We considered two different individual recruitment rates: 50% and 75%. l associated with treatment allocation and/or another variable (either measured or unmeasured), and use d-separation to show when the treatment effect is biased due to the associations induced by condi- tioning the analyses to those individuals in the population who are recruited (or consented). We also conducted a simulation study in which we varied the magnitude of association among the individual- level covariate, treatment allocation at the cluster-level, the probability of recruitment, and the outcome variable. We considered two different individual recruitment rates: 50% and 75%. l Background Faced with MNAR data, missing data guidelines recommend sensitivity ana- lysis to allow for alternative assumptions about the missing data. A useful approach is to use selection models, which specify a marginal distribution for the outcomes (analysis model) and a conditional distribution for the missing value indicators given the outcomes (missingness model). Selection models are particularly attractive in longitudinal studies, because they can recognise that the missing data mechanism may be distinct across the different types of miss- ingness. This research proposes flexible Bayesian selection models for assessing the robustness of trial results to alternative realistic as- sumptions about the different forms of missingness. In particular, we consider i) the implications of different model choices to allow for y y g We used data from 3 large Phase III randomised clinical trials in car- diovascular disease: EMPHASIS-HF, EPHESUS and RALES. We chose heart failure hospitalisation as the event of interest and cardiovascu- lar death as the competing event. Background Randomisation in clinical trials is often performed using permuted blocks, in which the randomisation of a patient is dependent on the randomisation of the other patients in the same block. This method Page 126 of 235 Page 126 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 126 of 235 complex longitudinal data structures and ii) the incorporation of clinical expert knowledge about the reasons for the missing values through informative priors in the missingness model. We illustrate the methods using two examples: the Vasopressin and Septic Shock Trial (VASST) and the Protease Inhibitor Monotherapy Versus Ongoing Triple Therapy Trial (PIVOT). For VASST, we reanalyse the cardiac index data, collected at baseline and 9 subsequent time- points over the following 96 hours. Monitoring started after base- line for a third of the patients and was discontinued as a result of both death and recovery. For PIVOT, our interest is in the health- related quality of life outcome, which was collected every 12 weeks over a 3-year period, but suffered from substantial intermittent (35%) and monotone (20%) missingness in both arms. For each outcome, we compare the results from alternative assumptions about the longitudinal missing data mechanisms with the pub- lished trial results and assess the implications for decision uncer- tainty. As an example, provisional results from the sensitivity analysis for VASST find that the average cardiac index over time was 9% higher for patients treated with vasopressin compared with those treated with norepinephrine (95% credible interval: 1%-17%), whereas the original analysis reported no difference. We conclude that this approach to sensitivity analysis provides a flexible frame- work to assess the implications of the missing data for the trial conclusions. complex longitudinal data structures and ii) the incorporation of clinical expert knowledge about the reasons for the missing values through informative priors in the missingness model. We illustrate the methods using two examples: the Vasopressin and Septic Shock Trial (VASST) and the Protease Inhibitor Monotherapy Versus Ongoing Triple Therapy Trial (PIVOT). For VASST, we reanalyse the cardiac index data, collected at baseline and 9 subsequent time- points over the following 96 hours. Monitoring started after base- line for a third of the patients and was discontinued as a result of both death and recovery. P334 How and when do competing risks influence results from clinical trials? John Gregson London Schoole of Hygiene and Tropical Medicine Trials 2017, 18(Suppl 1):P334 John Gregson London Schoole of Hygiene and Tropical Medicine Trials 2017, 18(Suppl 1):P334 P333 A bayesian framework to address missing not at random data in longitudinal studies with multiple types of missingness Alexina Mason1, Richard Grieve1, Anthony C. Gordon2, James A. Russell3, Simon Walker4, Nick Paton5, James Carpenter1, Manuel Gomes1 1London School of Hygiene and Tropical Medicine; 2Imperial College London; 3University of British Columbia; 4University of York; 5National University of Singapore Correspondence: Alexina Mason Trials 2017, 18(Suppl 1):P333 Methods We explored a series of ways of modifying the KM graph with two ob- jectives: (1) clearly and accurately representing the numbers censored, experiencing events, and still ‘at risk’, and (2) displaying uncertainty. We included combinations of often-used basics, such as censoring marks and simple risk tables, to more sophisticated risk tables, companion risk-and-event graphs, area shading graphs which represent at-risk pop- ulations, and re-construction of the KM lines themselves with sampling. We used trial data to illustrate the strengths and weaknesses of each possible approach. An international survey is in development which will seek responses during winter 2016 17 from people with a wide range of relevant perspectives, including statisticians, clinicians, journal editors and regulators. A fun, supplemental interactive vote would be under- taken on-site during the conference. 1Imperial College London; 2Imperial College London, School of Public Health, Imperial Clinical Trials Unit; 3Cardiff University, College of Biomedical & Life Sciences, Centre for Trials Research; 4Imperial College London, Department of Surgery & Cancer, Division of Cancer Potential Impact There is potential to improve the presentation of KM graphs and, furthermore, to convey more information about the results of clinical trials. However, implementation in manuscripts would likely depend on the willingness of editors to make the necessary space. Discussion Conclusions The cumulative incidence of a primary outcome can be accurately es- timated using the Fine and Gray method. However, when estimating the hazard ratio for treatment effect of an event of interest, current methods do not adequately deal with competing risks. Trials 2017, 18(Suppl 1):200 Page 127 of 235 the selection rules are not properly taken into account by the estima- tion strategy, then intuitively one might expect overly optimistic esti- mates of the treatment effect of the selected candidate, given that it had to perform ‘well’ in the first stage in order to proceed to the sec- ond stage. Improving Kaplan – Meier graphs: better presentation of numbers-at-risk, cumulative events and measures of uncertainty Matthew Sydes1, Christopher C. Jarvis2, Babak Choodari-Oskooei1, Patrick P. J. Phillips1, Tim P. Morris1 1MRC Clinical Trials Unit at UCL, Institute of Clinical Trials Methodology, UCL, London, UK; MRC London Hub for Trials Methodology Research, London, UK; 2London School of Hygiene and Tropical Medicine, London, UK; MRC London Hub for Trials Methodology Research, London, UK Correspondence: Matthew Sydes Trials 2017, 18(Suppl 1):P335 To efficiently and completely correct for selection bias in adaptive two-stage clinical trials, uniformly minimum variance conditionally unbiased estimators (UMVCUEs) have been derived for a variety of trial designs with normally distributed data. However, a common as- sumption is that the variances are known exactly, which is unlikely to be the case in clinical practice. In this paper, we extend the work of Cohen and Sackrowitz (Statistics & Probability Letters 1989), who proposed an UMVCUE for the best performing candidate in the normal setting with a common un- known variance, but only when the first stage sample sizes are all equal and the second stage sample size is equal to one. y Methods Variable selection methods were performed on SCOTROC4 trial data collected from Scottish Gynecologic Cancer Trials Group. The original cohort included 964 patients, of which 155 patients had both available protein expression data in tumour samples assessed by in- dependent scorers, and evaluable CA125 data which monitored pa- tients’ therapeutic response. Following clinical consultation, response was defined as >50% reduction in CA125 baseline post-treatment. A pre-selection method to improve variable selection efficiency re- duced 26 candidate proteins to 6: cycline E, SENP2, p53, folr2, larp1 and Ki-67. Backwards selection (BS), Akaike Information Criterion (AIC) and LASSO methods were then applied to create predictive models. Accuracy (sensitivity and specificity) of models was assessed through receiver operating characteristic (ROC) curve and area under the curve (AUC). Discrimination ability was assessed through box-and- whisker plots of predicted probability of responding/non-responding groups (127 and 28 patients respectively). 10-fold stratified cross- validation was applied to BS and AIC to control for over-fitting. Performance ability from the full model, BS, AIC, LASSO, cross- validated BS and cross-validated AIC were compared. To assess clinical usefulness, Positive predictive value (PPV) and negative predictive value (NPV) were calculated by extracting accuracy values from the most accurate model and prevalence of thera- peutic response from the original cohort. If there is agreement on a new standard which is not yet routinely available in the major statistical packages, work will be required to make these routinely and simply available. Conditionally unbiased estimation in two-stage adaptive trials with unknown variances Results Several ways to improve depictions of survival data will be presented on the poster. Results of the survey will be presented at the meeting. We will summarise the strengths and weaknesses. Background Clinical trials are valuable resources for biomarker exploration as prospectively collected data and experimental designs minimise bias. Models using multiple biomarkers to predict therapeutic re- sponse are of particular interest; however, the high-dimensional, small datasets come with challenges. This study aimed to high- light over-fitting issues with producing predictive models using commonly-used methods for Stratified Medicine in typical clinical trial datasets, using an ovarian cancer trial derived dataset as a case study. Background Kaplan – Meier (KM) graphs are the standard approach for depicting outcomes and risks over time for time-to-event outcome measures, in- cluding, for example, survival-based outcome measures which are widely used in many disease areas. In the context of clinical trials using these outcome measures, a KM graph is ubiquitous, and is intended to provide a visual representation of any difference between groups or lack thereof and is therefore critical to the interpretation and impact of the trial results. We believe, however, that the standard version of KM graphs can sometimes mislead. One challenge is that the number of patients contributing information decreases as time increases, but the eye is naturally drawn to the right-hand side of the graph where there are fewer data. Another challenge is the uncertainty in the data under- pinning the lines. There is no widely agreed way to depict this informa- tion, and is insufficiently clearly presented in most journals’ graphs. M th d Our extension allows for arbitrary first and second stage sample sizes for the different treatment arms, and can also be used to estimate the outcome measure of the j-th best candidate out of k. We show through a simulation study that the UMVCUE that assumes a known variance and estimates it from the trial data is no longer unbiased, and will have a higher mean squared error than our new estimator if the variance is overestimated. Issues with over-fitting in predictive models produced for stratified medicine: a case study on an ovarian cancer trial Meredith Martyn1, Xinxue Liu2, Charlotte Wilhelm-Benartzi3, Robert Brown4, Deborah Ashby2 1Imperial College London; 2Imperial College London, School of Public Health, Imperial Clinical Trials Unit; 3Cardiff University, College of Biomedical & Life Sciences, Centre for Trials Research; 4Imperial College London, Department of Surgery & Cancer, Division of Cancer Correspondence: Meredith Martyn Trials 2017, 18(Suppl 1):P337 p Conclusion Evidence of over-fitting issues were present in all variable selection methods, including LASSO which supposedly controls for over-fitting within its algorithm. LASSO proved advantageous with its enhanced pre- cision in dichotomising patients, however, NPV and PPV values suggested that a clinically useful model is unlikely to be found unless a dataset is large, or odds ratios of biomarkers added in models are extreme. Conditionally unbiased estimation in two-stage adaptive trials with unknown variances 1 2 David Robertson1, Ekkehard Glimm2 1MRC Biostatistics Unit; 2Novartis Pharma AG Correspondence: David Robertson Trials 2017, 18(Suppl 1):P336 Two-stage adaptive trial designs offer an efficient way of selecting and validating multiple candidate treatments within a single trial. A common strategy is to select the best performing treatment (accord- ing to some ranking criteria) after an interim analysis, and to then validate its properties in an independent sample in the second stage. However, selecting and ranking candidates in this way can induce bias into the naive estimates that combine data from both stages. If Trials 2017, 18(Suppl 1):200 Page 128 of 235 A simple relationship between power and expected confidence interval width 1 2 Most trials had missing data, most used a complete case analysis, about half accounted for missing data in the sample size calculation and very few carried out a sensitivity analysis. About one-fifth ana- lyzed both the intention-to-treat and per-protocol sets. Andrew Forbes1, Richard Hooper2 1Monash University; 2Queen Mary University of London Correspondence: Andrew Forbes Trials 2017, 18(Suppl 1):P340 There is room for improvement in handling missing data in noninferi- ority trials. There is also a need to carry out research in sensitivity analyses for noninferiorityi trials with respect to missing data. Results The futility boundaries in Simon’s minimax design are 0/13 and 3/27 with a sample size of 27 patients. In Bayesian PP design, the futility boundaries are 0/14, 1/24, 2/26 and 3/27 with the same sample size as Simon’s minimax design. The exact type I errors in Simon’s design and PP design are both 0.042, while the powers are 0.80 and 0.81, re- spectively. Although the probability of early stopping under null hy- pothesis is significantly higher in PP design than that in Simon’s design (87% vs 51%), the expected sample size under null hypothesis for the two designs are the same (E(N|H0) = 19.8). y Melanie Bell, Brooke Rabe University of Arizona Correspondence: Melanie Bell Trials 2017, 18(Suppl 1):P338 p Method The phase II cancer was original designed as a Simon’s two-stage Minimax with the primary outcome of clinical benefit, defined as complete response, partial response or stable disease for 6 months. The trial tested H0: p0 < =0.05 versus H1: p1 > =0.20 with type I error of 0.05 and type II error of 0.20.The Bayesian PP design monitors the trial continuously so that the Bayesian posterior probability is up- dated after the outcome from each participant becomes available. The predictive probability of concluding a positive result by the end of the trial is calculated based on the updated posterior probability. In this study, we used p0 = 0.05 and p1 = 0.20 to design the trial. If the probability that the clinical benefit rate of p is larger than p0 ex- ceeds a threshold of Theta-T at the end of the trial, the drug will be concluded as effective. During the monitoring, the trial will stop for futility if the PP is less than a threshold of Theta-L, and the trial will not stop for efficacy (Theta-U = 1). To compare with the Simon’s minimax design, the minimum sample size is selected among the sample sizes satisfying the constraints of above type I and type II error. The corresponding Theta-L and Theta-T are 0.001 and [0.86, 0.95], respectively. Bayesian predictive probability design in single-arm cancer phase II trials: is it superior to frequentist design? Xinxue Liu, Victoria R. Cornelius Imperial Clinical Trials Unit, Imperial College London Correspondence: Xinxue Liu Trials 2017, 18(Suppl 1):P339 p p g Correspondence: Xinxue Liu Trials 2017, 18(Suppl 1):P339 Result Most phase II cancer trials apply a single-arm design with a binary out- come, and multi-stage designs are commonly used to stop for futility in these settings. The most common design for single-arm phase II cancer trial is Simon’s two-stage design. However, Bayesian design with con- tinuous monitoring is getting popular in recently years as it is flexible and efficient given its intensive statistical input. In this study, we compared the operating characteristics of Simon’s two stage design and Bayesian predictive probability (PP) design using a real life cancer phase II trial. esu t The full model, BS, AIC and LASSO produced similar performance levels of accuracy (AUC = 0.80, 0.78, 0.80, 0.80). Discriminative ability was also similar, as 75% of distributions between responding and non-responding patients in box-and-whisker plots were distinctly different from each other. LASSO demonstrated advantageous pre- cision in discrimination ability. High correlation between the full model, BS, AIC and LASSO models predictive probability (r ranged from 0.8-1) suggested over-fitting in models produced by these variable selection methods. This was supported by the substantial drop in accuracy once BS and AIC models were cross-validated (AUC = 0.57, 0.54 respectively). Cross-validated models showed lim- ited ability to distinguish between responding/non-responding pa- tients. PPV and NPV calculations implied that 10% of patients in this dataset predicted as responders would not respond to therapy, and 55% patients who would be predicted as non-responders would respond to therapy using the most optimal sensitivity and specificity values from the full model (70%, 75%) assuming the prevalence of response is 77.4%. Methods We carried out a systematic review to investigate how researchers are handling missing data in noninferiority trials: the amount of miss- ing data; the analyses used and the missing data assumptions; whether missing data were considered in the sample size calculation; and whether any sensitivity analyses were carried out. Background There have been intermittent calls in the health sciences for sample size planning for randomised trials to be based on, or include, the ex- pected width of the 95% confidence interval (CI) for the parameter of interest. The relationship between power of a test at a 5% signifi- cance level and the expected 95% CI width has appeared in the lit- erature for trials planned with 80% or 90% power, most notably by Goodman and Berlin over 20 years ago. However, this relationship does not appear to be well known, has not been extended to treat- ment effect parameters other than differences between randomised arms, and it does not seem to have been realised that an even sim- pler approximate relationship also exists. Background Missing data pose a serious threat to the validity and interpretation of noninferiority trials and may result in the rejection of a promising new noninferior agent or the acceptance of what is, in fact, an infer- ior treatment. While there are recommendations for principled ap- proaches to handling missing data in superiority trials, there are none for NI trials, and missing data can affect them differently. M th d In this cancer phase II trial, where the clinical benefit rate of standard treatment (p0) was relatively low, the Simon’s two stage design had similar operating characteristics compared to Bayesian PP design. In practice, this suggests that if a phase II trial has a stop boundary of 0 in the interim analysis of Simon’s design, the Bayesian PP design is unlikely to be superior. P339 P339 Bayesian predictive probability design in single-arm cancer phase II trials: is it superior to frequentist design? Xinxue Liu, Victoria R. Cornelius Imperial Clinical Trials Unit, Imperial College London Correspondence: Xinxue Liu Trials 2017, 18(Suppl 1):P339 P341 Funnel plots for statistical quality control in a large, multi-site registry 1 1 2 2 g y Claire Boyle1, Nicole C. Foster1, Kenneth Scheer2, Henry Anhalt2, Avni Shah3, Joyce Lee4, Sarah Corathers5 1Jaeb Center for Health Research; 2T1D Exchange; 3Stanford University; 4University of Michigan; 5Cincinnati Children's Hospital Medical Center Correspondence: Claire Boyle Trials 2017, 18(Suppl 1):P341 y Claire Boyle1, Nicole C. Foster1, Kenneth Scheer2, Henry Anhalt2, Avni Shah3, Joyce Lee4, Sarah Corathers5 Claire Boyle1, Nicole C. Foster1, Kenneth Scheer2, Henry Anhalt2, Avni Shah3, Joyce Lee4, Sarah Corathers5 y 1Jaeb Center for Health Research; 2T1D Exchange; 3Stanford University; 4University of Michigan; 5Cincinnati Children's Hospital Medical Center Correspondence: Claire Boyle Trials 2017, 18(Suppl 1):P341 y 1Jaeb Center for Health Research; 2T1D Exchange; 3Stanford University; 4University of Michigan; 5Cincinnati Children's Hospital Medical Center Correspondence: Claire Boyle Trials 2017, 18(Suppl 1):P341 The T1D Exchange Clinic Network consists of 81 endocrinology practices throughout the United States. Eighteen of the centers primarily care for adult patients, 38 for pediatric patients, and 25 care for both. Among the more than 100,000 patients with type 1 diabetes (T1D) who receive care at these centers, more than 30,000 have been enrolled in the T1D Exchange clinic registry. The diverse size, resources, and practices among registry clinics may have an impact on the diabetes management and diabetes-related out- comes of participants. Understanding the variation in these re- sources and practices is an important step in determining how to improve diabetes management and diabetes outcomes. Statistical quality control is a method for monitoring quality of conformance and eliminating distinct causes of variability in a process through the use of graphical displays. One such graphical display is a funnel plot, which plots effect estimates from individuals against a measure of size or precision. Funnel plots also include lines for expected value of the effect and lower and upper control limits. These plots can be useful in assessing the variation in mean, median, or proportion of diabetes management factors and outcomes across clinic size. For example, a funnel plot of the proportion of diabetes patients achieving target glycemic control as measured by hba1c is an effective visual display of glycemic variation across clinical centers. The funnel plot enables iden- tification of high performing centers that may provide insights to in- form practice improvements for other participants in the network. g Existing Methods We outline two conceptually different methods for optimising the expected outcome of a pharmaceutical portfolio from the literature and provide a discussion and comparison of these methods. The first method is based on real options analysis and draws upon the way in which the sequential nature of the investments made in a drug development programme corresponds to a series of call op- tions. The resulting model formulation is a mixed integer linear programme which maximises the real options value of the portfolio. The second method is similar to the stochastic version of the re- source constrained project scheduling problem. In this setting, the development programmes for each of the drugs within the portfo- lio will be treated as projects which are made up of stochastic and deterministic tasks. The resulting model formulation is a multi-stage stochastic programme and has a particular focus on the technical uncertainty involved in the process. Conclusions One can determine the expected 95% CI width given a certain level of power, or vice versa, using an extremely simple relationship which makes it easy to conceptualise the consequences of one for the other. The relationship can be a useful rule of thumb to consider when planning trials. P341 Funnel plots for statistical quality control in a large, multi-site registry 1 1 2 2 Knowledge of variation in glycemic control, current use of advanced diabetes technologies, and occurrence of acute diabetes-related out- comes across varying clinic size is useful for learning and improving practices and resources in delivering diabetes care. Background Preterm newborns are a very vulnerable population in which clinical trials are extremely difficult and therefore rarely conducted. A phase I/II trial aiming at finding the recommended dose of Levetiracetam for treating neonate’s seizures was planned with a maximum sample size of 50. In the trial, 4 dose levels (consisting in a loading dose and up to 8 maintenance doses) are considered with 3 primary outcomes: efficacy, short term toxicity (Ts) and long term toxicity (Tl). Tl occurs at the same time as short term toxicity but can only be measured at Emily Graham1, Thomas Jaki1, Nelson Kinnersley2, Chris Harbron2 1Lancaster University; 2Roche Products Ltd Correspondence: Emily Graham Trials 2017, 18(Suppl 1):P342 y Our Contribution The existing methods for portfolio decision making do not allow in- formation about combination therapies specifically to be incorpo- rated into the decision making process itself. Therefore, we provide a comparison and discussion of these methods in the context of a portfolio containing combination therapies before providing our own extension. Our extension builds on network meta-analytic techniques and allows information to be shared between studies for similar combination therapies. Learning across trials of similar combinations will allow us to improve the accuracy of our treatment effect esti- mates which in turn will lead to better informed decision making and hence better outcomes for the portfolio. A Bayesian weighted quasi-likelihood design for phase I/II clinical trial with repeated dose administration in preterm newborns Moreno Ursino1, Ying Yuan2, Corinne Alberti3, Emmanuelle Comets4, Tim Friede5, Frederike Lents6, Nigel Stallard7, Sarah Zohar1 1INSERM, UMRS1138 - team22; 2The University of Texas MD Anderson Cancer Center; 3INSERM, UMR 1123; 4INSERM, CIC 1414; INSERM IAME, UMR 1137; 5University Medical Center Göttingen; 6Federal Institute for Drugs and Medical Devices; 7Warwick Medical School, The University of Warwick Correspondence: Moreno Ursino Trials 2017, 18(Suppl 1):P343 Correspondence: Moreno Ursino Trials 2017, 18(Suppl 1):P343 p pp Methods Phase II trials play a vital role in cancer drug development as they de- termine whether a new drug should continue for further investigation. We derive the basic relationship between power and expected CI width, state its underlying assumptions, and illustrate its use in a Page 129 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 129 of 235 series of examples for difference and ratio treatment effect measures used in randomised trials. We demonstrate that a linear approxima- tion simplifies this relationship further. Results series of examples for difference and ratio treatment effect measures used in randomised trials. We demonstrate that a linear approxima- tion simplifies this relationship further. Results therapies. There has been a recent rise in popularity of combination therapies, particularly those containing new molecular entities. Our particular area of interest is oncology, due to the recent develop- ment of cancer immunotherapy treatments. While this development is an exciting one, it poses new challenges for pharmaceutical companies. One of these challenges is how to decide which combi- nations, from the large set of possible combinations, is the most promising and hence which therapies should be added to a com- pany’s portfolio. In order to make the best decisions for the portfolio, emerging information should be included alongside the available his- torical data. However, in the context of combination therapies we have a different source of information: the information from studies involving similar combinations. We believe that incorporating infor- mation from similar studies will lead to improved portfolio level deci- sion making. The expected 95% CI width calculated from the relationship with power compares very favourably with asymptotic analytical formulae. The simpler linear approximation is appropriate for any level of power between 50% and 95%. The use of unequal allocation ratios in the design of randomised phase II trials Richard Jackson, Paul Silcocks, Trevor Cox University of Liverpool Correspondence: Richard Jackson Trials 2017, 18(Suppl 1):P344 Objectives Equal allocation of patients to one of two treatments is accepted al- most universally in the design of randomised clinical trials and it is often assumed that this approach provides the most efficient use of available resources. Background Background W i We are interested in the problem of portfolio level decision making in the context of a pharmaceutical portfolio containing combination Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 130 of 235 allocation will ensure a smaller standard error about the odds ratio for studies where there is a positive response. a later time. In the absence of efficacy, physicians could add a sec- ond agent as rescue medication, which could differ from centre to centre. allocation will ensure a smaller standard error about the odds ratio for studies where there is a positive response. Background g Interstitial lung disease (ILD) frequently complicates systemic auto- immune disorders resulting in considerable morbidity and mortality. The connective tissue diseases (CTDs) most frequently resulting in ILD include; systemic sclerosis, idiopathic inflammatory myositis (including dermato- myositis, polymyositis and anti-synthetase syndrome) and mixed con- nective tissue disease. Despite the development, over the last two decades, of a range of biologic therapies which have resulted in signifi- cant improvements in the treatment of the systemic manifestations of CTD, the management of CTD-associated ILD has changed little. At present there are no approved therapies for CTD-ILD. Following trials in scleroderma-ILD, cyclophosphamide is the accepted standard of care for individuals with severe or progressive CTD-related ILD. Observational studies have suggested that the anti-CD20 monoclonal antibody, rituxi- mab, is an effective rescue therapy in treatment refractory CTD-ILD. How- ever, before now, there have been no randomised controlled trials assessing the efficacy of rituximab in this treatment population. Methods On average, the proposed design leads to recommendation of the correct dose at about 60% for a sample size of 30, increasing up to more than 80% in many scenarios for a sample size of 50. This model maintains an acceptable number of neonates with toxicities when compared to the same design without quasi-likelihood part and without relevance weights. Materials and methods A Bayesian design was developed for this trial. The 3 primary outcomes were modelled via a logistic model for efficacy, a time-to-event quasi- likelihood for Ts and a quasi-likelihood with Ts as covariate for Tl, as Ts is predictive for Tl. The quasi-likelihood method allows us to take into account the fact that toxicity may be due to Levetiracetam or to the added second agent or to both, in case the Levetiracetam shows no efficacy and a second agent is added. Relevance weights were added to the model to avoid stickiness (that is, to be stuck for several patients at the same suboptimal dose level) due to early toxicities along with small target probability. Finally, this model al- lows sequential analyses on accumulating data. Dose escalation rules were based on adaptive thresholds for posterior probabilities, in the start-up phase considering only Ts while later considering both Ts and Tl. A simulation study was conducted to assess the design under several scenarios for sample size of 30, 40 and 50, respectively. The same design without quasi likelihood part, that is considering all toxicities due to Levetiracetam, and without rele- vance weight was used for comparison. R lt P345 Rituximab versus cyclophosphamide for the treatment of connective tissue disease associated interstitial lung disease (RECITAL): a randomised controlled trial Vicky Tsipouri1, Peter Saunders1, Greg J. Keir2, Deborah Ashby3, Sophie V. Fletcher4, Michael Gibbons5, Matyas Szigeti3, Helen Parfrey6, Elizabeth A. Renzoni1, Chris P. Denton7 1Royal Brompton Hospital; 2Princess Alexandra Hospital; 3Imperial College London; 4Southampton General Hospital; 5Royal Devon and Exeter Hospital; 6Papworth Hospital; 7Royal Free Hospital Correspondence: Vicky Tsipouri Trials 2017, 18(Suppl 1):P345 Discussion Design of phase II studies with a binary endpoint is often carried out in a two-stages following the principles of Simon and A’Hern and ex- tended into randomised trials by Jung and Sargent. Assessments of efficacy are often made via the odds ratio, the precision of which is only optimal under equal allocation when there is no difference in the response rates between the two treatment arms. M th d This is the first randomised control trial to study the efficacy of rituxi- mab as first line treatment in CTD-associated ILD. To date we have recruited 34 patients from 3 UK sites. Our recruitment accruals repre- sent one of the largest cohorts worldwide in these rare diseases. Herewith, we are presenting baseline characteristics of this unique cohort. The results anticipated at the conclusion of the trial should provide important information on the treatment of a life-threatening complication affecting a rare group of CTDs. For trials where the response rates are p_x and p_y in the experi- mental and control arm respectively, we propose to allocate 0 1 pa- tients to the experimental arm such that = (1 + A)^(−1) Where Calculating expected survival from high-dimensional cox models with treatment-by-biomarker interactions in randomized clinical trials A = (p_x (1-p_x))/(p_y (1-p_y)) Acknowledgments This work was conducted as part of the inspire (Innovative methodology for small populations re- search) project funded by the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement number FP HEALTH 2013–602144, but does not necessarily represent the view of all inspire partners. assessing t Methods RECITAL is a multicentre, randomized, double-blind, double-dummy, controlled trial funded by the Efficacy and Mechanism Evaluation Programme of the Medical Research Council and National Institute for Health Research. The trial, which has to date recruited ~30% of its target recruitment, will compare rituximab 1 g given intravenously, twice at an interval of two weeks, with intravenous cyclophospha- mide given monthly at a dose of 600 mg/m2 body surface area in in- dividuals with ILD due to systemic sclerosis, idiopathic inflammatory myositis (including anti-synthetase syndrome) or mixed connective tissue disease. A total of 116 individuals will be randomised 1:1 to each of the two treatment arms, with stratification based on under- lying CTD, and will be followed for a total of 48 weeks from first dose. The primary endpoint for the study is change in forced vital capacity (FVC) at 24 weeks. Key secondary endpoints include; safety, change in FVC at 48 weeks as well as survival, change in oxygen re- quirements, total 48 week corticosteroid exposure and utilisation of healthcare resources. Results Sample size calculations based on the exact methodology of Jung and Sargent show that estimates are smaller than those where equal allocation is used, with the discrepancy being greater as the re- sponse rate tends towards 0 or 1. In studies where standard sample size calculations are used, for a fixed sample size using unequal Nils Ternès, Federico Rotolo, Stefan Michiels Gustave Roussy Correspondence: Nils Ternès Trials 2017, 18(Suppl 1):P346 Nils Ternès, Federico Rotolo, Stefan Michiels Gustave Roussy Correspondence: Nils Ternès Trials 2017, 18(Suppl 1):P346 Page 131 of 235 Trials 2017, 18(Suppl 1):200 Page 131 of 235 cance of Results Out of 29´119 articles 378 RCT were included in the analysis investi- gating a total of 62´522 patients of which 15´025 patients were blinded (24.0%). Regarding performance bias 88 of 378 RCT (23.3%) were at high risk of performance bias and 290 of 378 RCT (76.7%) were not. Hereby, 50 of 88 high risk RCT (56.8%) showed significant trial results compared to 134 of 290 non-high risk RCT (46.2%) result- ing in non-significant association (OR 1.53; 95%-CI: 0.95 to 2.48; p = 0.08) of performance bias and trial results. Further, 59 of 378 RCT (15.6%) were at high risk of detection bias and 319 of 378 RCT (84.4%) were not. Hereby, 28 of 59 high risk RCT (47.5%) showed significant trial results compared to 156 of 319 non-high risk RCT (48.9%) resulting in non-significant association (OR 0.94; 95%-CI: 0.52 to 1.65; p = 0.84) of detection bias and trial results. Discussion Considerations in designing equity-relevant clinical trials Lawrence Mbuagbaw1, Beverley Shea2, Theresa Aves1, Vivian Welch2, Monica Taljaard3, George Wells3, Peter Tugwell2 1McMaster University; 2Bruyère Research Institute; 3Ottawa Hospital Research Institute Considerations in designing equity-relevant clinical trials Lawrence Mbuagbaw1, Beverley Shea2, Theresa Aves1, Vivian Welch2, Monica Taljaard3, George Wells3, Peter Tugwell2 1McMaster University; 2Bruyère Research Institute; 3Ottawa Hospital Research Institute Correspondence: Lawrence Mbuagbaw Trials 2017, 18(Suppl 1):P347 Correspondence: Lawrence Mbuagbaw Trials 2017, 18(Suppl 1):P347 Correspondence: Lawrence Mbuagbaw Trials 2017, 18(Suppl 1):P347 Background equity-relevant trial, the Consolidated Standards for Reporting (CONSORT) equity advisory group came together to address these issues. Thanks to the advances in genomics and targeted treatments, an in- creasing interest is being devoted to develop prediction models with biomarkers or gene signatures to predict how likely patients will benefit from particular treatments. Despite the methodological framework for the development and validation of gene signatures in a high-dimensional setting is quite well established, no clear guid- ance exists yet on how to estimate expected survival probabilities. We propose a unified framework for developing and validating a high-dimensional Cox model integrating clinical and genomic vari- ables in a randomized clinical trial to estimate the expected absolute treatment effect according to signature values, and to estimate ex- pected survival probabilities for patients with associated confidence intervals. Content This work is part of a broader project that includes the development of a framework for defining equity-relevant trials and a CONSORT ex- tension for equity-relevant trials. This work discusses approaches to integrating equity considerations in equity-relevant randomized trials by building upon the PROGRESS-Plus framework (Place of residence, Race, Occupation, Gender, Religion, Education, Socioeconomic status, Social capital and other context-specific factors) and covers research questions formulation, two scenarios of equity relevant trials and how the PROGRESS-Plus factors may influence trial design, conduct, and analyses. Background g Blinding is a measure in randomized controlled trials (RCT) to reduce performance and detection bias. There is evidence that lack of blinding leads to overestimated treatment effects. Since, surgical trials use inter- ventions with a physical component blinding is often complicated to apply. The aim of this study was to analyse the actual impact of blind- ing on outcomes in general and abdominal surgery RCT. Methods A systematic literature search in CENTRAL, MEDLINE and Web of science was conducted to locate RCT between 1996 and 2015 with a surgical intervention. General study characteristics and information on blinding methods were extracted. The risk of performance and detection bias was rated as low, unclear or high according to the Cochrane Collaboration's tool for assessing risk of bias. The main out- come was the association of a high risk of performance or detection bias with significant trial results and was tested at a level of signifi- cance of 5%. y g Conclusion We propose a unified framework for developing and validating a gene signature in a high-dimensional survival setting in order to calculate expected survival probabilities at a given horizon for future patients, and to visualize the survival predictions. Based on our simulations, the adaptive lasso penalty can be useful to identify a signature and then, to accurately estimate the expected survival probability of future patients. y Correspondence: Pascal Probst Trials 2017, 18(Suppl 1):P349 Correspondence: Pascal Probst Trials 2017, 18(Suppl 1):P349 Correspondence: Pascal Probst Trials 2017, 18(Suppl 1):P349 Simulation results suggest that a penalized regression model estimated using adaptive lasso estimates the survival probability of new patients with low bias and standard error, and that bootstrapped confidence in- tervals have empirical coverage probability close to the nominal level across very different scenarios. The double cross-validation allows mim- icking internally the prediction performance in absence of external val- idation data. We also propose a visual representation of the expected survival probabilities using splines. In the breast cancer trial, we identi- fied a prediction model with 4 clinical covariates, the main effect of 98 biomarkers and 24 biomarker-by-treatment interactions. This illustration also highlights the high variability of the expected survival probabilities, with very large confidence intervals. P349 Blinding in randomized controlled trials in general and abdominal surgery: a systematic review and empirical study g y y y Pascal Probst, Steffen Zaschke, Patrick Heger, Phillip Knebel, Alexis Ulrich, Markus W. Büchler, Markus K. Diener y Conclusion Based on a parsimonious selection model in a penalized (lasso or adaptive lasso) high-dimensional Cox model, we investigated several strategies to: estimate the individual survival probabilities at a given timepoint (using single or double cross-validation); construct confi- dence intervals thereof (analytical or bootstrap); and visualize them graphically (pointwise or spline). We compared these strategies through a simulation study covering null and alternative scenarios and we eval- uated them by prediction criteria. We applied the strategies to a large randomized controlled phase III trial in 1574 early breast patients that evaluated the effect of adding trastuzumab to chemotherapy and for which the expression of 462 genes were measured. With an a-priori focus on certain equity items, trials can be designed to optimize their ability to provide actionable and credible evidence on equity, by careful consideration of design, conduct and analytical issues that play a role in equity. p Results In M1 disease, we found a clear benefit of docetaxel on survival. FAME gave us confidence that the primary question was answered definitively, without needing to wait for results of the remaining 2 tri- als, or collecting IPD. Collaborating with trialists through FAME gave us access to pre-publication trial results, and facilitated contemporan- eous publication of the systematic review and the largest trial. In M1 disease, we found a clear benefit of docetaxel on survival. FAME gave us confidence that the primary question was answered definitively, without needing to wait for results of the remaining 2 tri- als, or collecting IPD. Collaborating with trialists through FAME gave us access to pre-publication trial results, and facilitated contemporan- eous publication of the systematic review and the largest trial. In M0 disease, there was a clear effect of docetaxel on failure-free survival, but overall survival results were inconclusive. Therefore, FAME provided an early signal of potential benefit, and highlighted the value of a future update that includes longer-term follow-up of included trials and results of currently unreported trials. Ongoing collaboration with trialists will provide up-to-date information, en- abling better prediction of the timing and feasibility of a definitive p y g In M0 disease, there was a clear effect of docetaxel on failure-free survival, but overall survival results were inconclusive. Therefore, FAME provided an early signal of potential benefit, and highlighted the value of a future update that includes longer-term follow-up of included trials and results of currently unreported trials. Ongoing collaboration with trialists will provide up-to-date information, en- abling better prediction of the timing and feasibility of a definitive Post-trial follow-up methodology in large randomized controlled trials: a systematic review Timely and reliable evaluation of the effects of interventions: a framework for adaptive meta-analysis (FAME) Jayne Tierney1, Claire L. Vale2, Sarah Burdett2, David Fisher2, Larysa Rydzewska2, Mahesh K. B. Parmar2 1University College London; 2MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL; MRC London Hub for Trials Methodology Research Correspondence: Jayne Tierney Trials 2017, 18(Suppl 1):P351 Rebecca Llewellyn-Bennett1, Danielle Edwards2, Richard Bulbulia2, Louise Bowman2 1University of Oxford; 2Clinical Trial Service Unit, Nuffield Department of Population Health, University of Oxford Correspondence: Rebecca Llewellyn-Bennett Trials 2017, 18(Suppl 1):P350 1University of Oxford; 2Clinical Trial Service Unit, Nuffield Department of Population Health, University of Oxford Correspondence: Rebecca Llewellyn-Bennett 1University of Oxford; 2Clinical Trial Service Unit, Nuffield Department of Population Health, University of Oxford Correspondence: Rebecca Llewellyn-Bennett Trials 2017, 18(Suppl 1):P350 Background Large randomised trials tend to have a relatively short “in-trial” Follow- up period and hence may underestimate any long-term benefits of the assessed intervention or fail to detect delayed hazards. Post-trial follow- up (PTFU), which we define as extended follow-up either after the scheduled trial period or publication of the primary results, allows detection of both persistent or enhanced beneficial effects following cessation of study treatment (ie, a legacy effect) and the emergence of possible adverse effects (eg, cancer). Despite these advantages, PTFU is not routinely undertaken and, when implemented, methods vary widely. This review describes methods of PTFU used in recent large ran- domised trials, and will compare retention rates and study costs where such information is available, and may help promote the use of effect- ive PTFU for ongoing and future large trials. Methods A systematic search of electronic databases and clinical trial registries was conducted using a pre-defined search strategy with the following inclusion criteria: i) randomized trials with 1000 or more participants, ii) published between March 2006–2016; iii) evaluation of medical, surgi- cal or psychological interventions; iv) implementation of post-trial follow-up reported. Two reviewers screened and extracted data from eligible papers with the aim of 95% concordance and any discrepancies were resolved by a third reviewer. Retention rates, costs and other de- scriptive differences of PTFU were reviewed. The systematic review was conducted following PRISMA guidelines. Key principles of FAME are: 1) Start the systematic review process early, before all trials have completed 2) Comprehensively search for published, unpublished and ongoing eligible trials; 3) Develop a de- tailed picture of these trials, particularly how information and results are likely to accumulate; 3) Predict the feasibility and timing of a reli- able meta-analysis; 4) Interpret meta-analysis results accounting for trials that have not yet completed/reported; 5) Determine if an up- date based on AD or IPD is needed. In 2014, using FAME, we initiated two systematic reviews to evaluate the effects of adding docetaxel to standard care in men with HSPC. We predicted that, by mid-2015, results of 3 of 5 eligible trials in M1 disease would become available, each with median follow-up of around 4 years. They would represent around 90% of all men rando- mised, giving 70 to >99% power to detect a 5% -10% absolute differ- ence in 4-year survival. This provided a clear trigger for a robust meta-analysis. Background Most systematic reviews are retrospective and use aggregate data (AD) from publications, meaning they can be unreliable, lag behind therapeutic developments and fail to influence ongoing or new trials. Commonly, the potential influence of unpublished or ongoing trials is overlooked when interpreting results, or determining the value of updating the meta-analysis or need to collect individual participant data (IPD). Therefore, we developed a Framework for Adaptive Meta- analysis (FAME) to determine prospectively the earliest opportunity for reliable AD meta-analysis. We illustrate FAME using two system- atic reviews in men with metastatic (M1) and non-metastatic (M0) hormone-sensitive prostate cancer (HSPC). Results The search strategy incorporated relevant papers from Cochrane Central Register, Embase, Medline and clinical trial registries yielding 50,153 papers from databases (49,915) and trial registries (218). After excluding duplicates (22,168), studies of children and animals (1649) and papers published before 2006 (9289). 17,027 abstracts were screened by 2 reviewers using a concordance strategy. Reviewers were 73% concordant for the first 10% of abstracts screened, but after discussion concordance rose to 99%. Following abstract screening, 239 papers and 218 protocols were eligible for full review and preliminary results suggest that around half will represent unique studies with relevant data to extract in the review. The length of PTFU ranged from 1–20 years and PTFU methods varied, including direct patient contact via clinic appointments, postal questionnaires, telephone interviews and indirect follow-up via national registries. Some trials used incen- tives for participant retention, including free healthcare relevant to the intervention. Several PTFUs were prompted by the Data Monitoring Committee because of concerns about potential delayed treatment hazards. Occasionally trials investigated an outcome different to the in- trial primary endpoint. Where industry supported the in-trial period, such funding for PTFU was infrequent. Final results of the review are pending and will be presented. y Also, for M0 disease, we anticipated the availability of results from 4 of 11 eligible trials, again with median follow-up around 4 years. Power would be reasonable (60 to >99%) to detect similar absolute effects, but only 60% of randomised men would be represented. Al- though a meta-analysis would not be definitive, it could provide use- ful context for the M1 results and for ascertaining when a robust update of the meta-analysis might be feasible. Background Disparities in health and health outcomes are a common feature in health research. When these disparities are unfair and avoidable they may be referred to as inequities. Due consideration of inequities is important to inform the design and conduct of trials so that they do not aggravate inequities, but instead capture the role of inequities in a credible and informative way. In light of the lack of evidence on equity and the absence of guidance on how to design a purposefully Surprisingly, performance and detection bias do not distort treatment effects in general and abdominal surgery RCT. Therefore, surgical re- searcher can rely on this evidence and leave out complicated ways of blinding methods. However, easily applicable blinding measures should Page 132 of 235 Trials 2017, 18(Suppl 1):200 Page 132 of 235 be taken for the theoretical advantage. During critical appraisal of a surgical RCT the threat to validity of trial results by performance and detection bias should not be overestimated. follow-up used in a range of recent randomized trials. We anticipate that PTFU using routinely collected health records will be more com- prehensive and cost-effective than studies involving direct patient contact. On synthesis evidence from explanatory and pragmatic trials: a comparison of meta-analytic methods Randomized controlled trials of treatments and interventions are typically described as either explanatory or pragmatic. Meta-analysis of RCT studies typically pools evidence of treatment effects from included studies, regardless of their classification as ‘pragmatic’ or ‘explanatory trials. Given that treatment effects in explanatory trials may be greater than those obtained in pragmatic trials, conventional meta-analytic approaches may not accurately account for the hetero- geneity among the studies and may result in biased estimates of treatment effects. Stratified meta-analysis of systematically review studies in which treatment effects from explanatory trials are meta- analyzed and reported separately from pragmatic trials is increasingly being adopted in meta-analysis studies. But this approach might not necessarily inform decision-making especially when stratum-specific pooled treatment effects are in opposite directions. In this study we investigate a variety of meta-analytic approaches for synthesizing evidence from pragmatic and explanatory trials, including mixture random-effects meta regression, robust random-effects meta- regression, and hierarchical Bayesian meta-analysis techniques for synthesizing evidence from pragmatic and explanatory trials. Data from a systematic review of 55 published obesity prevention trials, which investigated the effectiveness of public health intervention on reduction of obesity, was used to demonstrate and compare these methods. Discussions about the key statistical and design consider- ations when pooling evidence from both types of trial designs are provided. Review of treatment allocation schemes reported in published clinical trial results Review of treatment allocation schemes reported in publi clinical trial results Jody Ciolino, Hannah L. Palac, Amy Yang, Mireya Vaca Northwestern University Correspondence: Jody Ciolino Trials 2017, 18(Suppl 1):P354 Jody Ciolino, Hannah L. Palac, Amy Yang, Mireya Vaca Northwestern University Correspondence: Jody Ciolino Trials 2017, 18(Suppl 1):P354 Jody Ciolino, Hannah L. Palac, Amy Yang, Mireya Vaca Northwestern University Correspondence: Jody Ciolino Trials 2017, 18(Suppl 1):P354 Clinical trial units of medical scientific societies to close evidence gaps g p Gabriele Dreier University Medical Center Freiburg Trials 2017, 18(Suppl 1):P353 University Medical Center Freiburg Trials 2017, 18(Suppl 1):P353 Background Properly designed and implemented randomized controlled trials (RCTs) serve as the ideal form of evidence-based research to estab- lish efficacy of new therapies; however, substantial debate regarding most appropriate trial designs persists today. Areas of confusion in- clude: appropriate treatment allocation techniques to ensure com- parable baseline arms, best reporting practices, and controlling for influential variables at the analysis phase. While randomization litera- ture promotes covariate adaptive methods (e.g., minimization, devel- oped 1974) to protect against baseline imbalance and provide more efficient analyses, many investigators prefer simpler methods (e.g., stratified blocking schemes) for their understandability and ease of implementation. This manuscript reviews recently published rcts to illustrate current practice. p g Conclusions Post-trial follow-up of large RCTs may allow more reliable estimation of the long-term benefits of the study treatment and the detection of any delayed adverse effects which might not emerge during the relative “in-trial” period. This review will describe the methods of post-trial Page 133 of 235 Page 133 of 235 Page 133 of 235 Trials 2017, 18(Suppl 1):200 meta-analysis, and whether AD or IPD will be required. It will also facilitate a co-ordinated dissemination strategy. Conclusions committees of DGHNOKHC and BVHNO have together founded the German Clinical Trials Unit for Ear, Nose and Throat medicine, Head and Neck Surgery (DSZ-HNO) to assist their members in the identification of evidence gaps and the planning and conduct of systematic reviews and clinical trials. An interdisciplinary team of statisticians, physicians, project managers, study nurses, data managers and monitors provides the required expertise. The first projects have been started, including a BMBF (German Ministry for Education and Research)-funded clinical trial for the treatment of sudden hearing loss. A survey among all members of both as- sociations to detect evidence gaps was conducted. The results led to a prioritization process and planning of trials, registries, sys- tematic reviews and other projects with industry and academia alike. A presentation at the Guideline Commission of the Working Group of German Medical Scientific Societies led to further Soci- eties wanting to copy the ENT example, thus a Clinical Trial Unit as presented here can be a suitable model for closing evidence gaps and fostering clinical trials. In piloting FAME, we have shown that meta-analysis can be done in a timely and transparent manner without compromising reliability P352 On synthesis evidence from explanatory and pragmatic trials: a comparison of meta-analytic methods Tolulope Sajobi1, Oluwagbohunmi Awosoga2, Meng Wang1, Anita Brobbey1, Guowei Li3, Bijoy K. Menon1, Michael D. Hill1, Lehana Thabane3 1University of Calgary; 2University of Lethbridge; 3McMaster University Correspondence: Tolulope Sajobi Trials 2017, 18(Suppl 1):P352 Methods We searched pubmed for articles indexed ‘randomized controlled trial’, published in the New England Journal of Medicine, Journal of the American Medical Association, British Medical Journal, or Lancet for two time periods: 2009 and 2014 (before and after establishment of up- dated Consolidated Standards of Reporting Trials [CONSORT] guide- lines). Upon completion of screening, articles underwent full review to collect data related to trial characteristics, the type of randomization scheme used, and clarity of reporting. Background Background In the last 25 years, ebm has increasingly found its way into clinical practice and research. Existing evidence primarily serves doctors to sup- port their decision-making, but is also the basis for providing scientific proof for a health care intervention`s benefit to patients and ultimately payers/health insurances. The closure of existing evidence gaps re- quires substantial human and financial resources, and can only succeed with the involvement of clinical and methodological expertise. Objectives Our search returned 343 articles, 298 of which we included in full re- view. The majority reported on superiority (86%), multicenter (92%), two-armed (79%) trials. With respect to CONSORT adherence, 68% of trials indicated a ‘randomizedr Trial in the title, and the randomization scheme could not be determined in 10% of studies. Consistent with our hypothesis, the majority of articles reported a stratified block method (69%) of allocation, but 81% of trials involved covariates in the treatment allocation procedure. The majority (84%) of trials reported adjusted analyses, with 91% of these adjustments in analyses pre- specified. Trials published in the later time period (2014 vs. 2009) were more likely to have clearer report of randomization scheme (84% vs. 66%, p = 0.0003), report adjusted analyses (87% vs. 79%, p = 0.0100), and pre-specify adjustment in analyses (95% vs. 85%, p = 0.0045). Study start year significantly predicted whether design involved a covariate adaptive method of allocation, but in the opposite hypothesized direc- tion: odds of adaptive method use decreased for every one-year in- crease in study start (OR = 0.89 [0.82, 0.96], p = 0.0045). However, odds of pre-specified adjusted analyses tended to increase over time (OR = 1.13 [1.02, 1.24], p = 0.0145). Scientific Societies have a natural interest in detecting and closing evidence gaps. Here we report a project of the German Society of Otolaryngology, Head and Neck Surgery (DGHNOKHC) and the Ger- man professional association of otolaryngologists (BVHNO) which can serve as a master example for similar projects. Methods The two institutions have a vested interest in supporting their members in the generation and dissemination of evidence, and to foster the transfer of knowledge into practice. This includes the areas of diagnosis, treatment, prognosis and prevention, comprising the application of me- dicinal products, medical devices or surgical procedures. The executive Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 134 of 235 Methods To capture as many anal cancer patients as possible, we developed an umbrella protocol that would capture patients across the spectrum of disease. PLATO (personalising Anal cancer radiotherapy dose) (ISRCTN88455282) is an integrated protocol, comprising 3 sep- arate trials (ACT3, ACT4 and ACT5) in which the most relevant clinical research questions are asked across three distinct risk strata. Each trial asks separate questions and has separate eligibility criteria and sample sizes. Background p g Daniel Sabanes Bove, Jiawen Zhu, Ulrich Beyer Hoffmann-La Roche Ltd Correspondence: Daniel Sabanes Bove Trials 2017, 18(Suppl 1):P355 Anal cancer is a rare disease, but its incidence is rising rapidly. Approxi- mately 1000 cases in the UK and 5,000 in the USA are diagnosed each year. Standard treatment for anal cancer includes concurrent Mitomy- cin C, 5-Fluorouracil (or more recently capecitabine) and radiotherapy. Due to advances in radiotherapy technology, a new generation of clin- ical trials is now required that optimises radiotherapy dose based on a stratified risk assessment of the disease. This abstract is not included here as it has already been published. Training the clinical investigators of the future - A clinical trials clerkship Natalie Ives, Jon Deeks University of Birmingham Correspondence: Natalie Ives Trials 2017, 18(Suppl 1):P356 Background f The ACT3 trial (n = 90) is a non randomised phase II study for low- risk disease that will evaluate a strategy of local excision only versus local excision plus radiotherapy, depending on the size of tumour margin post local excision (>1 mm versus < =1 mm, respectively). The ACT4 trial (n = 162) is a randomised phase II trial (2:1) for intermediate-risk disease comparing standard dose chemoradiother- apy with reduced-dose chemoradiotherapy. The ACT5 trial (n = 640) is a seamless pilot (n = 60)/phase II (n = 140)/phase III trial (n = 672 total) for patients with high risk disease that will compare standard dose chemoradiotherapy with two increased doses. Only one of the dose escalated experimental arms will be evaluated for the phase III component. The primary end point for each trial is 3 year locoregio- nal failure. PLATO is funded by Cancer Research UK and is due to open to recruitment in the UK and Ireland in Q4 2016. Discussion To support future clinical trial research it is important that those planning a career in clinical trials are supported and trained to lead on these trials. Training clinical investigators of the future in the design, management and analysis of clinical trials is key [Sackett]. Clinical Trials Units (CTUs) have extensive experience in the design and delivery of clinical trials, and provide an excellent training envir- onment in which to embed researchers of the future. Ctus provide a unique opportunity for researchers to learn about clinical trials in a highly active research environment, alongside staff who work on trials every day. The pros and cons of an ‘umbrella’ trial design for a rare disease from a trial management and data management perspective Sue Bell, Jo Copeland, Alexandra Smith Correspondence: Sue Bell Trials 2017, 18(Suppl 1):P357 Correspondence: Sue Bell Trials 2017, 18(Suppl 1):P357 Rolling dose escalation with overdose control: an efficient and safe phase 1 design P357 P357 The pros and cons of an ‘umbrella’ trial design for a rare disease from a trial management and data management perspective Sue Bell, Jo Copeland, Alexandra Smith University of Leeds Correspondence: Sue Bell Trials 2017, 18(Suppl 1):P357 Reference David L. Sackett. Clinician-trialist rounds: 20. Shouldn’t “Trialists-in-training” Rotate through RCT-clerkships; Clinical Trials 2013;0:1–4. Our findings suggest that while optimal reporting procedures and pre-specification of adjustment in analyses for RCTs tend to be pro- gressively more prevalent over time, we have evidence of the op- posite effect on use of sophisticated covariate adaptive methods in clinical trial practice. Many authors suggest covariate adaptive methods as ideal in designing clinical trials, but there is a discon- nect between theory and practice. Moreover, our results suggest a widening of this gap as time moves on. y Methods To develop a clinical trials clerkship for clinical investigators that combines a programme of training with hands-on experience, men- torship and access to experts working in clinical trials. R l P358 Partnership of cancer center core facilities with community-based research networks in the coordination and management of multi-center clinical trials Rani Jayswal, Stacey Slone, Mark Stevens, Rushi Goswami, Lara Sutherland, Kris Damron, Emily Dressler, Brent Shelton, Eric Durbin, Heidi L. Weiss Markey Cancer Center, University of Kentucky Correspondence: Rani Jayswal Trials 2017, 18(Suppl 1):P358 p Discussion Time, money and resources could potentially be saved by incorporat- ing more than one trial under the umbrella of one protocol. The PLATO trial concept allows different research questions across the locoregional disease spectrum to be addressed efficiently using a sin- gle protocol and clinical trial funding application. This type of trial design is increasingly important in the era of personalised medicine and the need for clinical studies to address different research ques- tions within the same disease. Sharing the details of this concept should assist other investigators to develop similar future studies. De- tails of our experience of implementing an integrated protocol along with the pros and cons of this approach from a trial and data man- agement perspective will be presented in more detail. At the Birmingham Clinical Trials Unit (BCTU) at the University of Bir- mingham, we have developed a clinical trials clerkship where fellows spend on average 15 days (including a 3 day Research Methods Course) in BCTU over a 12 month period. The fellows will have the opportunity to learn about various trial processes from study set-up, protocol and case report form development, regulatory require- ments, trial management, database development, statistical aspects of clinical studies, recruitment strategies, data management and monitoring, trial steering committees, interim and final data analysis and submitting for publication. Each fellow is assigned a senior trial- ist who acts as their mentor, who is responsible for working with the fellow to tailor the training and learning experience. The fellow is ex- pected to maintain a reflective log of their taught and experiential training through completion of a workbook. For those planning to run a trial as part of their fellowship, these can be embedded within BCTU, with mentorship on delivering the project provided by a se- nior trial manager, trial co-ordinator, database programmer and stat- istician. Appropriate CTU costs to help the fellow deliver the study can be included in NIHR fellowship applications. C l i Conclusions Within BCTU, we are currently running our first cohort of fellows following the above programme, and initial feedback is that it is an enjoyable and highly valuable learning experience. Markey Cancer Center, University of Kentucky Correspondence: Rani Jayswal Trials 2017, 18(Suppl 1):P358 Trials 2017, 18(Suppl 1):200 Page 135 of 235 Page 135 of 235 The importance of translation of clinical trials into catchment popula- tions of Cancer Centers coupled with the advent of molecularly tar- geted agents and emphasis in precision medicine resulting in smaller patient pool within a single institution entail the need to engage multiple sites for the design and implementation of clinical trials. The conduct of multi-center studies is necessarily complex, requiring in- formatics tools and data management processes that need a coordi- nated effort necessitating an infrastructure akin to Data Coordinating Centers. We present a model whereby biostatistics and informatics core facilities partner with community based research networks to manage multi-center clinical trials. More specifically, we focus on three critical areas in informatics and data management namely i) development of an integrated set of standard operating procedures (SOPs) between the community based network and MRU pertaining to all aspects of data management; ii) improving utilization of a clin- ical trial management system (CTMS), a biospecimen management system and customized database applications to accommodate multi-center studies and iii) adopting and expanding automated statistical programs to monitor protocol-specific triggers including subject accrual, safety, and efficacy endpoints into a multiple site setting. The community research network focuses on administrative coordination and site communication and management to serve as a clinical coordinating center. We demonstrate this model for the con- duct of a therapeutic intervention trial and non-intervention study; provide the specific informatics, data management and statistical tools we have implemented to manage multi-center studies; and dis- cuss challenges and areas of improvement in this partnership infra- structure for provision of an integrated clinical and data science coordination for multi-center clinical studies. Database through an iterative process of regression analysis and re- assessment. P360 P360 Consolidated trial management: an example of a purpose built clinical trial management system for an academic research organization Rebecca Mister1, Seshu Atluri2, Burcu Vachan3, Wendy Hague4 1Head of Site Management; 2Analyst Programmer; 3Oncology Program Manager; 4Clinical Trials Program Director Correspondence: Rebecca Mister Trials 2017, 18(Suppl 1):P360 Funding h g The ARRISA-UK study is funded by the National Institute for Health Research's Health Technology Assessment Programme (13.34.70). The views and opinions expressed are those of the authors and do not necessarily reflect those of the HTA, NIHR, NHS or the Department of Health. Concepts important to the design of an innovative risk register in general practice databases? Developing methodology from ARRISA-UK Stanley Musgrave1, Erika Sims1, David Price2, Annie Burden2, Allan Clark1, Susan Stirling1, Mohammad Al Sallakh3, Gwyneth Davies3, Estelle Payerne1, Ann Swart1 1Norwich Medical School; 2Research in Real Life, Ltd.; 3Swansea University Medical School Correspondence: Stanley Musgrave Trials 2017, 18(Suppl 1):P359 Background Many clinical conditions require the identification and stratification of risk to ensure that interventions can be targeted appropriately. Challenges to identification of ‘at-risk’ patients using data from elec- tronic health records include identification of relevant characteristics, how data availability informs decision making, coding and storage of data, and how data can be searched for, accessed and managed. Each week in the UK, 22 patients die and 1400 are hospitalised due to asthma (Asthma UK). Sixty per cent of patients with at-risk asthma defined according to British Thoracic Society (BTS) guidelines have an exacerbation requiring prednisolone per year compared to 10% of the total asthma population and BTS guidelines suggest at-risk regis- ters may be useful for asthma. The At-Risk Registers Integrated into primary care to Stop Asthma crises - UK (ARRISA-UK) study group are evaluating the effectiveness and cost effectiveness of generating and implementing an at-risk asthma register. Background g The NHMRC Clinical Trials Centre (CTC) based in Sydney, Australia and affiliated with the University of Sydney is an Academic Research Organisation (ARO) which develops and co-ordinates multi-centre clinical trials in Australia, New Zealand and internationally. Working across number of different fields including cardiology, oncology, neo- natology and endocrinology the group collaborates with a number of institutions including study sites, other international co-ordinating centres, cros, cmos and central laboratories. As central coordinating centre for a number of clinical trials, the CTC frequently works with the same study sites (and personnel) across a number of different trials. A need was identified to collate trial operations information centrally to reduce time for individual trials collecting this informa- tion in their own bespoke systems. There was also a need to be able to collate core information (timelines, approvals) across trials and report these centrally in order to generate metrics to review performance. After consideration and review of the cost and func- tionality of existing commercial software packages it was decided to develop a custom system in-house, tailored to CTC specific trial co-ordination requirements. Consolidated trial management: an example of a purpose built clinical trial management system for an academic research organization Stanley Musgrave1, Erika Sims1, David Price2, Annie Burden2, Allan Clark1, Susan Stirling1, Mohammad Al Sallakh3, Gwyneth Davies3, Estelle Payerne1, Ann Swart1 1 2 3 Rebecca Mister1, Seshu Atluri2, Burcu Vachan3, Wendy Hague4 1Head of Site Management; 2Analyst Programmer; 3Oncology Program Manager; 4Clinical Trials Program Director Correspondence: Rebecca Mister Trials 2017, 18(Suppl 1):P360 Rebecca Mister1, Seshu Atluri2, Burcu Vachan3, Wendy Hague4 1Head of Site Management; 2Analyst Programmer; 3Oncology Program Manager; 4Clinical Trials Program Director Correspondence: Rebecca Mister Trials 2017, 18(Suppl 1):P360 y 1Norwich Medical School; 2Research in Real Life, Ltd.; 3Swansea University Medical School Correspondence: Stanley Musgrave Trials 2017, 18(Suppl 1):P359 1Norwich Medical School; 2Research in Real Life, Ltd.; 3Swansea University Medical School Beyond ARRISA-UK These experiences will be used to develop strategies using a multi- disciplinary approach for identification and recruitment of at-risk individuals in other disease areas. This will permit development of methodology for efficient trial design, delivery and planning in primary care. Developing a risk profile for an at-risk register: importance of a multi-disciplinary team Developing a risk profile for an at-risk register: importance of a multi-disciplinary team Candidate characteristic values to be included in the risk profile were identified based on expert opinion, prior work and literature review. This list was reviewed by a working group within the ARRISA-UK team to identify additional characteristics based on clinical experience of managing asthma, consider limitations/restrictions of GP Practices. Clinical data systems in relation to the characteristics identified, and evaluate reliability and variability of the characteristics in terms of real world coding of clinical information. The characteristics contributing to the identification of patients with a statistically significant risk of hospi- talisation were determined in the Optimum Patient Care Research Conclusions The coefficients of the characteristics (including age, smok- ing status, comorbidities (rhinitis, diabetes, ischaemic heart disease, anxiety and/or depression, and anaphylaxis), BTS treatment step, para- cetamol treatment, lower respiratory tract infection, oral corticosteroid therapy or hospitalisation in the previous year, body mass index and blood eosinophil count) then are used in an algorithm to calculate a risk score which was validated in a second database, the Secure Anon- ymised Information Linkage databank. Implementing an At-Risk Register Using this algorithm, the ARRISA-UK search tool identifies at-risk individuals in general practices. Search re- ports from the GP clinical database system for the characteristics above are analysed, and the risk assessment is flagged in relevant patient re- cords via specific Read or SNOMED codes. These inform the compu- terised decision support system in the form of popup information boxes prompting clinical action. They can also facilitate care manage- ment tasks, data collection and further clinical coding. Case study h ' h y Who's who - PW is a Clinical Trials Fellow and occupational therapist with previous experience of feasibility RCTs in his NIHR Doctoral Research Fellowship. A core aim of the fellowship was to develop skills to become a future CI of a multicentre study. The Nottingham Clinical Trials Unit (NCTU) is a UK Clinical Research Collaboration registered unit, based at the University of Nottingham. The unit cur- rently hosts a number of Fellowships and research training awards. Application process - Collaborative meetings with NCTU helped bal- ance the learning objectives of the trainee with the learning opportun- ities available at NCTU and identify suitable trials. Surgical procedures can be difficult to standardise due to the num- ber of factors involved, including the surgeon’s skill and experience, and decisions taken regarding the surgical procedure based on pa- tient characteristics or fitness, variations in anatomy, etc. Each surgi- cal procedure will, therefore, be open to variability which the QA programme within inpact aims to minimise. Discussions among inter- national surgical collaborators have led to agreement on precise sur- gical details to be included in the trial protocol and supplementary surgical trial guidance notes. Each surgeon will be accredited before participation in the trial. Accreditation will involve independent re- view of a number of surgical procedures by the inpact surgical QA committee comprising US and UK surgical leads for the trial. During the trial, photographs and operative notes will be reviewed and feed- back will be given to individual surgeons at participating sites. y p g Application process - Collaborative meetings with NCTU helped bal- ance the learning objectives of the trainee with the learning opportun- ities available at NCTU and identify suitable trials. Training programme - NCTU developed an extensive ‘menu’ Of activ- ities from which a tailored programme was produced covering: trial oversight, quality management and sops, pharmacy, trial set-up, site set-up, recruitment, data management, follow-up, write up and dis- semination. KS, as CTU mentor, led in the development and oversight of PW’s training programme. The randomisation schema within inpact requires knowledge of lymph node involvement. Correct interpretation of protocol criteria is crucial. Initially, prospective central review of all patient scans (to assess lymph node involvement) prior to randomisation was envisaged, but during protocol development it became evident that the logistics of this Integration into the unit - PW worked within three trials teams to maximise experience and learning. Quality assurance (QA) challenges in the development of international trials in rare diseases 1 2 Quality assurance (QA) challenges in the development of international trials in rare diseases 1 2 Quality assurance (QA) challenges in the development of international trials in rare diseases Clare Cruickshank1, Steve Nicholson, Curtis Pettaway2, Nick Watkin3, Jelle Teertstra4, James Gimpel5, Elizabeth Miles6, Cathy Corbishley3, Pheroze Tamboli2, Stephanie Burnett1 1The Institute of Cancer Research; 2M.D. Anderson Cancer Center; 3St. George's University Hospital NHS Foundation Trust; 4The Netherlands Cancer Institute; 5American College of Radiology, Center for Research and Innovation; 6National Radiotherapy Trials QA (RTTQA) Group Correspondence: Clare Cruickshank Trials 2017, 18(Suppl 1):P362 NIHR clinical trials fellowship: reflections from a fellow and a mentor Phillip Whitehead, Kirsty Sprange, Alan Montgomery University of Nottingham Correspondence: Phillip Whitehead Trials 2017, 18(Suppl 1):P361 Phillip Whitehead, Kirsty Sprange, Alan Montgomery University of Nottingham Correspondence: Phillip Whitehead Trials 2017, 18(Suppl 1):P361 Aim To develop and implement a user-friendly Clinical Trial Management System (CTMS) to support the clinical trials team at the CTC that was cost effective to develop and maintain. Project specification: The first step was to develop a requirements document by seeking input from relevant parties. The following key content domains were identified: projects, organisations, people and documents and their relation- ships specified. Key user requirements were ease of data entry and reporting. Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 136 of 235 Project development Supplementary training - The “hands on” Experience in NCTU was supplemented with formal training opportunities involving method- ology and statistical courses, and presenting at national conferences. Reflections System specifications were then prepared collaboration with a data systems developer. A relational database design and (Oracle) written in Java was used for development. After an initial prototype was developed the system was released for user testing by trials staff from the discrete functions within the trials teams. Once the system was qualified, but prior to rollout, existing study tracking data was imported into the new CTMS system. Reports (both within and across projects) were developed prior to release to enable staff to access key information. Benefits and Challenges Benefits included experiential learning by involvement and integration into the unit and the various trials teams, involvement in multidisciplinary working, observing multiple chief investigators across multiple studies at various stages of the trials, knowledge of processes and procedures and making contacts’ internal and external. Background The National Institute for Health Research (NIHR) introduced Clinical Trials Fellowships in 2012 with the aim of further developing existing NIHR trainees’ skills and experience in clinical trials. Fellowships are hosted within Clinical Trial Units (CTU) that are in receipt of NIHR CTU Support Funding as these offer the best environment in which to: expose trainees to all aspects/stages of clinical trials; cover mul- tiple studies; understand how proposals are developed from initial concept through to funding application by interdisciplinary, collab- orative working; and tailor training to individuals’ needs. We reflect on our experience of the fellowship from the perspective of the trainee (PW) and the CTU mentor (KS). inpact (CRUK/13/005, EA8134) is an international trial in penis cancer developed under the auspices of the International Rare Cancers Initiative (IRCI). It evaluates the combination and sequence of four common treatments for penis cancer: Inguinal Lymph Node Dissec- tion (ILND), chemotherapy, chemoradiotherapy, and Pelvic Lymph Node Dissection (PLND). The interventions used within the trial present a number of QA challenges to ensure that any differences in trial outcomes are related to the randomisation schedules and not deviations from the trial protocol. The rarity of the disease means that, whilst networks of specialists have developed, experience at an individual clinical team level can be limited. A number of inter- national specialist subgroups were therefore set up during protocol development to discuss areas of QA need and to agree on QA pro- cesses for the trial. Project deployment Challenges included capacity and availability of key activities across trials and the unit, time-lag between the application (summer 2014) and the commencement of the fellowship (2016) making it difficult to plan specific activities in the unit. Some flexibility was required due to the uncertain nature of clinical trials particularly in the set-up phase when timelines can be fluid. This resulted in some adaptations to the training package. Training sessions were conducted on the use of the new system. Staff were also invited to specify what reports would be helpful to their teams. Project Evaluation: After implementation, a process of continual user feedback and enhancement was undertaken to im- prove system usability and acceptability. Conclusion: Development of the initial system took approximately 12 months from the decision to develop, through specification and user testing, import of existing data to release. Since this time additional functionality and reporting has been developed and released periodically. The system has now been in use for 3 years and feedback from users demonstrates in- creasing acceptance of the system. However, there were key learn- ings from the experience of implementing a new software system e.g., unforeseen costs related to the lack of staff dedicated solely to this project (and impact on timelines), resistance to change, and the expansion of the original scope of the project with requests for fur- ther functionality. Conclusions CT Fellowships offer a unique opportunity for trialists of the future to get hands on experience at an early career stage and also to enable CTUs to develop researchers leading to high quality multi-centre tri- als. Both fellow & NCTU found the experience highly beneficial and strongly support continuation of this NIHR training programme. Background g In 2005, the Panamerican network for the harmonisation of pharmaceutical regulation (Red PARF in Spanish), based on the ICH guidelines, introduced the Document of the Americas for the Good Clinical Practices (DA-GCP) with the aim of harmonising clinical re- search practices in the region. The DA-GCP was not mandatory to all regulatory authorities. Therefore, each country had the inde- pendence to develop their guidelines and regulations to allow clin- ical trials. Colombia and Brazil in 2008 presented their resolution to implement clinical research in the country adopting the ICH-GCP guidelines, and in 2012 Mexico did the same. However, only Colombia and Brazil stated in their regulation the adoption of DA- GCP. Therefore, the question that emerges is how does the differ- ence on the normativity between these countries have influenced the sponsor strategies to coordinate, manage and implement multi- national clinical trials in Latin America? M th d Methods To answer this question, in first place three multi-site clinical trials to evaluate vaccines were studied in Mexico, Colombia, and Brazil to assess the influence of the national regulation on multinational pro- jects. Also in each country, members of clinical research associations were interviewed to understand better the local dynamics and the rela- tionship between local normativity and the pharmaceutical industry. Sixty-six semi-structured interviews were conducted with members of the research site, sponsors, clinical monitors, ethic committees, regula- tory agencies, and members of clinical research associations. Objective Substantial variability in adverse event (AE) reporting practices may exist between sites, particularly in multicenter clinical trials involving patient populations for whom AEs are prevalent. Variability is likely to be multifactorial, involving differences in training, culture, docu- mentation, and other parameters, but also, perhaps, upon the quality of trial performance. We hypothesize that sites with very low or very high numbers of AEs reported are more likely to also have excessive data corrections identified during source document review by site monitors. Methods In a recently completed randomized clinical trial of acute treatment of patients with traumatic brain injury (protect NCT00822900), we retrospectively determined the coincidence of enrollment sites being outliers on both AE reporting and data corrections found by site monitoring. Outlier sites were those outside 95% boundaries on fun- nel plots of AE reporting and of data corrections. Variability in AE reporting was assessed by examining the average number of AEs reported at each site (the total number of AEs reported at a site di- vided by the number of subjects enrolled at that site). Data correc- tion at each site was assessed as the average number of data clarification requests (DCRs) written by a site monitor during source document verification visits that resulted in the site correcting erro- neous data in the case report form (CRF). Analysis of coincidence was descriptive in this exploratory study. Sensitivity analyses using 90% boundaries and looking at only serious AEs (SAEs) were also visualized. This qualitative study reveals that despite Red PARF efforts to har- monise GCP in the American continent, this objective has not been achieved in practice. Between Colombia, Brazil, and Mexico, it does not exist a harmonisation which is reflected in four aspects. 1) The di- vergence on requirements and procedures to approve the trial. 2) The number of institutions involved in protocols evaluation. 3) The restriction of Colombia regulation to hire certain professional profiles to be part of research teams 4) The research capabilities requested by each regulatory agency to implement the trial. These differences made that each sponsor had to develop management strategies to implement the vaccine trial in Colombia, Brazil, and Mexico which demanded: 1) coordinate times among different countries to start their trials. 2) Invest in the creation of research capabilities to imple- ment its protocol. 3) Hire smos to coordinate trials at local levels and manage research sites, and 4) design new training strategies to create a knowledge-base among all clinical teams according to the local requirements. In conclusion, despite Red PARF’s efforts, har- monisation of clinical trial regulation in Latin America has not been achieved. The difference between regulatory frameworks induced the creation of unique strategies by sponsor to coordinate and management the evaluation and implementation of multinational clinical trials in the region. Objective The primary objective of this paper is to explain the differences be- tween the Mexican, Colombian, and Brazilian clinical research regula- tions and how these influenced the evaluation and implementation of multi-national vaccine trials in these three countries. P363 Variability in adverse event reporting rates per subject by enrollment site in a multicenter acute care clinical trial Erin Bengelink1, Valerie Stevenson1, Jordan Elm2, Sharon Yeatts2, Robert Silbergleit1 1University of Michigan; 2Medical University of South Carolina Correspondence: Erin Bengelink Trials 2017, 18(Suppl 1):P363 P364 Regulation in Latin America and its impact on the execution of multinational clinical trials to evaluate vaccines Sara Valencia phd Student Science and technology studies Trials 2017, 18(Suppl 1):P364 P364 Regulation in Latin America and its impact on the execution of multinational clinical trials to evaluate vaccines Sara Valencia phd Student Science and technology studies Trials 2017, 18(Suppl 1):P364 Case study h ' h During the course of the fellow- ship, regular meetings were held between the fellow and mentor to reflect on personal development and for NCTU to offer feedback and guidance. Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 137 of 235 high and 2 were low AE reporting outliers. Findings were similar using 90% boundaries and rates of SAE reporting. Conclusions would be prohibitive. The ECOG-ACRIN Cancer Research Group and The Netherlands Cancer Institute shared anonymised images to enable development of a web-based teaching and testing solution using the ACR Radiology Curriculum Management System. Radiologists respon- sible for assessing patient lymph node involvement at each of the par- ticipating sites will be assessed through this training tool and accredited prior to the trial opening at that site. Extensive variability in both AE reporting and data collection quality exceed that expected by chance alone in this example trial. AE reporting rates may be useful as a metric to incorporate into risk- based site monitoring plans if similar patterns are found with larger numbers of sites across additional clinical trials. Other areas identified as requiring international QA consensus were pathology and radiotherapy, the latter being led by the UK’s NCRI Radiotherapy Trials QA Team and in the US, the National Clinical Trials Network’s QARC. The organisation of separate QA subgroups in addition to the standard trial set-up processes and protocol develop- ment has been challenging, but the QA programme ultimately underpins the quality of trial treatment in this rare cancer. Regular international communication and the sharing of knowledge and experi- ence with existing national QA processes and infrastructure have ensured consensus on trial protocol and associated QA. Internationally- harmonised QA programmes should optimise deliverability of this trial across multiple countries. Background Good sites are vital to ensure that multicentre randomised con- trolled trials (RCTs) are delivered on time, within budget and to a high standard. For example, over optimistic recruitment targets often mean the trial goes over budget and fails to complete on time. To help improve trial efficiency, site selection questionnaires (SSQs) to gather relevant information about potential sites are con- sidered ‘best practice’ for selecting new sites in multicentre trials. However, there is limited evidence about their effectiveness in im- proving trial conduct. Correspondence: Diane Whitham Trials 2017, 18(Suppl 1):P365 p Methods For each of the five trials, SSQs were developed using questions that were both generic and protocol specific. The SSQ was emailed to the Principal Investigators (PIs) for potential sites, requesting its completion and return. The Chief Investigators (CIs) had access to these responses, and it was at their discretion whether they used the information to select sites. P366 P365 A study aimed at improving the conduct and efficiency of trials by developing a standardised set of site performance metrics and a systematic approach to reporting Diane Whitham1, Julie Turzanski1, Alan Montgomery1, Lelia Duley1, Shaun Treweek2, Paula Williamson3, Lucy Culliford4, Mike Clarke5, Julia Brown6, Louise Lambert7 1Nottingham University; 2Aberdeen University; 3Liverpool University; 4Bristol University; 5Queen's University; 6Leeds University; 7CRN National Coordinating Centre (CRNCC) | NIHR Clinical Research Network (CRN), Leeds Correspondence: Diane Whitham Trials 2017, 18(Suppl 1):P365 Can site recruitment be predicted? Results of a retrospective, blinded evaluation of a site selection questionnaire in five multicentre trials Diane Whitham, Dawn Coleby, Wei Tan, Julie Turzanski, Lelia Duley Nottingham University Correspondence: Diane Whitham Trials 2017, 18(Suppl 1):P366 y Conclusion We will present the outcomes of the focus groups and literature search and discuss the design and development of our Delphi survey questionnaire. For CI selected sites, where the SSQ was reviewed by a blinded assessor, those that were selected based on the blinded assess- ment seemed to perform significantly better in terms of recruit- ment than those rejected following blinded assessment. This suggests that SSQs have potential as a tool to improve the se- lection of sites for clinical trial. They merit further development and evaluation as to whether they can improve efficiency of trial conduct. Research Plan ll f Key Stakeholders: Trial Managers, Clinical Trials Units, NETSCC, NIHR Clinical Research Network, Chief Investigators, Statisticians. Results Results An SSQ had been returned for all sites selected by the CI. There were 105 sites across the five trials. Three trials had completed recruit- ment, and overall sites had been recruiting for between 0.7 months and 47 months. Finally we will develop a simple tool (probably within Excel) for the presentation of key metrics to Trial Managers, Trial Management Groups and Trial Steering Committees in a standardised format. The median monthly recruitment (actual/target) was higher in the 54 sites selected by blinded assessment of the SSQs (median re- cruitment =78.5) compared with the 51 sites not selected by the SSQ assessment (median recruitment =50.0) (p = 0.0019, Mann- Whitney U test). p g Key Stakeholders: Trial Managers, Clinical Trials Units, NETSCC, NIHR Clinical Research Network, Chief Investigators, Statisticians. Results Can site recruitment be predicted? Results of a retrospective, blinded evaluation of a site selection questionnaire in five multicentre trials A study aimed at improving the conduct and efficiency of trials by developing a standardised set of site performance metrics and a systematic approach to reporting 1 1 1 1 Can site recruitment be predicted? Results of a retrospective, blinded evaluation of a site selection questionnaire in five multicentre trials Diane Whitham, Dawn Coleby, Wei Tan, Julie Turzanski, Lelia Duley Nottingham University Correspondence: Diane Whitham Trials 2017, 18(Suppl 1):P366 Diane Whitham1, Julie Turzanski1, Alan Montgomery1, Lelia Duley1, Shaun Treweek2, Paula Williamson3, Lucy Culliford4, Mike Clarke5, Julia Brown6, Louise Lambert7 1Nottingham University; 2Aberdeen University; 3Liverpool University; 4Bristol University; 5Queen's University; 6Leeds University; 7CRN National Coordinating Centre (CRNCC) | NIHR Clinical Research Network (CRN), Leeds 1Nottingham University; 2Aberdeen University; 3Liverpool University; 4Bristol University; 5Queen's University; 6Leeds University; 7CRN National Coordinating Centre (CRNCC) | NIHR Clinical Research Network (CRN) Leeds Nottingham University; Aberdeen 4Bristol University; 5Queen's Univers National Coordinating Centre (CRNC Network (CRN), Leeds Correspondence: Diane Whitham Trials 2017, 18(Suppl 1):P365 Background Standardising the collection, reporting and monitoring of data rele- vant to site performance could improve the effective and efficient oversight of clinical trial delivery. Our surveys of UK Trial Manager Network (UK TMN) members and NIHR chief investigators revealed wide variations in how trial data are used to assess performance. However, without consensus on optimal ways of utilizing perform- ance metrics, trialists may focus on too many or uninformative indicators, causing inefficiency in trial conduct and difficulty in com- paring between studies. Ideally, This study aimed to evaluate the performance of an SSQ developed by the Nottingham Clinical trials Unit (NCTU), using data on key metrics collected from five randomised controlled trials. Previously presented preliminary data comparing mean number of days to re- cruit the first participant found that sites selected by both the Chief Investigator (CI) and by blinded assessment of SSQs were 68% more likely to have recruited their first participant than those where the CI and the blinded assessment disagreed (Trials 2015, 16(suppl 2):P176.). This project aims to improve trial conduct and efficiency by: Reach- ing consensus on important metrics that should be monitored rou- tinely in multicentre trials. Establishing initial baseline benchmark indicators for each performance metric for trending and predicting potential issues, so minimizing their impact and improving trial per- formance and efficiency. Developing a standardised systematic method for reporting and presenting these metrics to trial mangers, tmgs and tscs. We now update our study and present data assessing how well the SSQ predicted site recruitment. Research Plan ll f Small focus groups of stakeholders will establish an initial list of per- formance metrics and parameters that could be measured routinely in trials. We will then design a Delphi survey using data from litera- ture searches and the focus groups to develop a comprehensive list of performance metrics and parameters for inclusion in the Delphi survey. For sites selected by the CI, each completed SSQ was assessed by an assessor who was ‘blind’ to the site and to the PI. This assess- ment used seven pre-defined criteria: SSQ not returned, potential pool of participants, available staff resources, clinical trials experi- ence of PI, competing trials for target population, number of trials competing for resources, and equipoise for the trial interventions. If any one of the first three criteria was not satisfied, the site was excluded. The Delphi survey will be sent to Trial Managers and CTU directors as they play key roles in ensuring the efficient delivery of multicentre trials. Three Delphi rounds will be used to steer the groups towards consensus, on a list of important performance metrics. We will docu- ment the reasons for their decision-making with regard to selection of metrics. For CI selected sites, the monthly recruitment rates (actual/target) of sites that were and were not selected by blinded assessment of the SSQs from these sites were compared. Data from the Delphi survey will be presented to stakeholders in a priority-setting workshop with a wide range of trial stakeholders, pro- viding participants with the opportunity to express their views, hear different perspectives and think about monitoring of site perform- ance. We will seek agreement on the top key performance metrics (expected to be around 8–12 in number) and benchmark indicators for each metric to trigger action to improve site performance. Data from the Delphi survey will be presented to stakeholders in a priority-setting workshop with a wide range of trial stakeholders, pro- viding participants with the opportunity to express their views, hear different perspectives and think about monitoring of site perform- ance. We will seek agreement on the top key performance metrics (expected to be around 8–12 in number) and benchmark indicators for each metric to trigger action to improve site performance. Finally we will develop a simple tool (probably within Excel) for the presentation of key metrics to Trial Managers, Trial Management Groups and Trial Steering Committees in a standardised format. Results 882 subjects were enrolled at 49 sites between 2010 and 2013. 11 sites that did not enroll any subjects in the study were excluded, leaving 38 sites for inclusion. Site enrollment ranged widely from 1 to 85 subjects with a median of 18. The average number of reported AEs by site ranged from 0.5 to 12 (median 3.14). The average number of DCRs resulting in data correction by site ranged from 0.75 to 15 (median 3.56). On funnel plots, 14/38 (37%) sites were outliers with regard to AE reporting outliers (6 low, 8 high), and 7/38 (34%) were outliers with regard to high data correction rate. Coincidence was suggestive but not significant given the small numbers; 4/14 (29%) of the AE reporting outliers were also high data correction outliers, as compared to only 3/24 (13%) of the sites that were not AE report- ing outliers. Unexpectedly, among the 4 coincident outliers, 2 were Trials 2017, 18(Suppl 1):200 Page 138 of 235 P369 g y Of the total 1075 registrations, 496 (46%) have submitted the form noting that a patient will not register to a sub-study. Of note, pa- tients without a matching biomarker who previously received im- munotherapy are not currently eligible for any sub-study. Conduct of a precision medicine trial: screening, tissue adequacy, study registration, and reasons for not participating on lung-map (lung cancer master protocol) Conduct of a precision medicine trial: screening, tissue adequacy, study registration, and reasons for not participating on lung-map (lung cancer master protocol) Katie Griffin, Shannon McDonough, Jieling Miao, James Moo Mary W. Redman SWOG Statistics and Data Management Center at FHCRC Correspondence: Katie Griffin Trials 2017, 18(Suppl 1):P367 Correspondence: Guy Peryer Trials 2017, 18(Suppl 1):P369 Correspondence: Guy Peryer Trials 2017, 18(Suppl 1):P369 Methods Lung-MAP has two steps, a screening step followed by sub-study regis- tration step. In the screening step, tissue is submitted to determine pa- tient eligibility for biomarker-selected or non-match sub-studies. For patients with tissue that is determined to not be adequate for bio- marker testing, either additional tissue or tissue from a fresh biopsy can be submitted for retesting. Patients can either be screened at progres- sion on therapy or pre-screened while receiving therapy for stage IV or recurrent disease. The trial did not open with the pre-screening option; this option was added at the end of 2015. The protocol-specified tar- gets are that patients screened at progression receive their sub-study assignment within 16 days from tissue submission and pre-screened patients receive their sub-study assignment within one day of notifying the study they have progressed on the prior treatment. If at any point in time it is determined that a patient will not enroll on a sub-study, the site submits a form noting the reasons for not registering. R lt Exploration of possible measurement methods to best collect opin- ions on trade-off levels was undertaken. One option explored was a simple cross-tabulation of hypothesised levels of DFS and cardiotoxi- city. Another avenue explored was the use of gaming chips placed on separate continuums of perceived “costs” and “benefits” of the two treatment arms. To assist in interpretation of the trade-off be- tween perceived advantages and disadvantages, graphical aids were also considered. One option investigated used pictures of old fash- ioned weighing scales with two pans, one representing one treat- ment arm with a hypothesised DFS and cardiotoxicity level, and the other just a hypothesised cardiotoxicity level. Responders were asked to choose the level of DFS required to make the scales balance. As of November 4, 2016, 1075 patients have registered to be screened (714 (66%) screened at PD, 361 (34%) pre-screened). Upon initial sub- mission, about 12% of submitted tissue was inadequate, with the most common reason being an insufficient amount of tissue (N = 58). Pa- tients resubmit tissue samples about 37% of the time and 79% of those were analyzable; the tissue inadequacy rate overall is 8.8%. Eliciting clinician’s opinions on acceptable trade-offs within one trial endpoint for various levels of detriment in another endpoint is a complex one. Surveys were sent out to all 152 hospitals involved in the PERSEPHONE trial. Background Another avenue explored was the use of gaming chips placed on separate continuums of perceived “costs” and “benefits” of the two treatment arms. To assist in interpretation of the trade-off be- tween perceived advantages and disadvantages, graphical aids were also considered. One option investigated used pictures of old fash- ioned weighing scales with two pans, one representing one treat- ment arm with a hypothesised DFS and cardiotoxicity level, and the other just a hypothesised cardiotoxicity level. Responders were asked to choose the level of DFS required to make the scales balance. Eliciting clinician’s opinions on acceptable trade-offs within one trial endpoint for various levels of detriment in another endpoint is a l ll h l l d q Discussion Discussion The project will result in a reporting tool showing a standardised set of clear, meaningful and easily accessible performance metrics. The metrics will assist researchers to indicate change over time and iden- tify potential problem areas early, allow better utilisation of resources and timely action to be taken. Trials 2017, 18(Suppl 1):200 Page 139 of 235 Page 139 of 235 PERSEPHONE is a phase III non-inferiority RCT comparing six months of trastuzumab to the standard twelve months in patients with HER2 positive early breast cancer. The primary endpoint is disease-free survival (DFS), with cardiac function as a secondary endpoint. It was assumed that the standard 12 months trastuzumab results in 80% DFS at 4 years. With 5% 1-sided significance and 85% power, 4000 patients gives the ability to prove non-inferiority of the experimen- tal arm, defining non-inferiority as no worse than 3% below the es- timated 4-year DFS of the standard arm. The trial reached its 4000 patient target in July 2015, making this UK trial the largest of its kind in the world. P367 Conduct of a precision medicine trial: screening, tissue adequacy, study registration, and reasons for not participating on lung-map (lung cancer master protocol) Katie Griffin, Shannon McDonough, Jieling Miao, James Moon, Mary W. Redman SWOG Statistics and Data Management Center at FHCRC Correspondence: Katie Griffin Trials 2017, 18(Suppl 1):P367 P367 Conduct of a precision medicine trial: screening, tissue adequacy, study registration, and reasons for not participating on lung-map (lung cancer master protocol) Katie Griffin, Shannon McDonough, Jieling Miao, James Moon, Mary W. Redman SWOG Statistics and Data Management Center at FHCRC Correspondence: Katie Griffin Trials 2017, 18(Suppl 1):P367 Standard operating procedures for managing adverse events in trials that do not involve an investigational medicinal product: a protocol for a Delphi consensus study 1 2 1 3 Guy Peryer1, Catherine Minns Lowe2, Yoon Loke1, Catherine Sackley3 1University of East Anglia; 2University of Hertfordshire; 3King's College, London Background Whilst waiting for the follow-up data to mature, we embarked on de- signing a survey to canvass current clinician’s opinions on trastuzu- mab duration that would provide insight into not only the potential practice-changing impact of PERSEPHONE’S results, but also the most appropriate non-inferiority limits to define for the future meta- analysis of the “twelve month trastuzumab versus less” trials for fur- ther investigation into pre-specified sub-groups of patients. The Lung-MAP trial (Lung Cancer Master Protocol), launched in 2014, is an umbrella protocol to evaluate targeted therapies in biomarker se- lected patients for previously-treated stage IV or recurrent squamous non-small cell lung cancer. The trial infrastructure also includes a “non- match” study or set of studies for patients without any of the bio- markers under study. Lung-MAP, conducted by SWOG, and involving the National Clinical Trials Network of the National Cancer Institute (NCI), is the first precision medicine trial launched with the support of the NCI in the United States. The survey aimed to record opinions on what clinicians considered the effectiveness of each of PERSEPHONE’S two randomised treatment du- rations, followed by what difference between them they would require the results to prove in order to change their current practice. Opinions on the two randomised arms’ rates of cardiotoxicity were also collected. The next section of the survey depicted various hypothetical scenarios of cardiotoxicity differences between treatment arms, with responders asked what trade-off they would require in terms of the primary end- point of DFS to change their current practice within those scenarios. The survey aimed to record opinions on what clinicians considered the effectiveness of each of PERSEPHONE’S two randomised treatment du- rations, followed by what difference between them they would require the results to prove in order to change their current practice. Opinions on the two randomised arms’ rates of cardiotoxicity were also collected. The next section of the survey depicted various hypothetical scenarios of cardiotoxicity differences between treatment arms, with responders asked what trade-off they would require in terms of the primary end- point of DFS to change their current practice within those scenarios. Exploration of possible measurement methods to best collect opin- ions on trade-off levels was undertaken. One option explored was a simple cross-tabulation of hypothesised levels of DFS and cardiotoxi- city. p Discussion Conduct of a complex trial platform including biomarker testing and evaluation of multiple investigational therapies may continue to be a valued approach for evaluating biomarker/investigational therapy combinations. Lessons learned and views into their conduct are im- portant to help inform future endeavors. Background Medical research methods, technologies and tools evolve rapidly. It is essential guidance prioritising the safety of human volunteers is reviewed at timely intervals. This study aims to provide clarity and consistency to the assessment and reporting of adverse events in clinical trials that do not involve an investigational medicinal product (non-CTIMP). Non-CTIMP governance covers a broad spectrum of non-pharmacological disciplines (e.g. Surgery, nutrition, psychological and physical therapies etc.). Currently, this is a neglected area of clin- ical trial research. The lack of consistent identification, categorization, and reporting of harms prevent researchers from conducting reliable meta-analyses and comprehensive systematic reviews on the bene- fits and risks of non-drug interventions that help to guide clinical practice. Non-systematic methods of assessing harms increase the potential for reduced effect sizes, resulting in a bias towards the null Measurement methods for eliciting opinions on treatment benefits, toxicities and acceptable trade-offs of the two, within the PERSEPHONE trial 1 1 2 1 Methods Results will be presented of the success of the methodology adopted to undertake this task. Once a patient’s tissue has been successfully tested, the patient is assigned and can then register to a Lung-MAP sub-study. To date, 785 patients have been notified of their sub-study assignment and 387 patients have registered to a sub-study. p p Methods A variety of strategies were utilised for the success of the trial. These included; Inclusion of a medical ethicist on the investigator team; A longer than normal set-up phase of the study for educating sites about placebo surgery; A pre-trial survey where surgeons in- terested in participating in the CSAW outlined their practices, followed by in-depth interviews between the surgeons and the study’s clinical leads; Use of a Prospective Patient Assessment (PPA) at the main site. This involved presenting a hypothetical placebo surgery trial to patients to gain feedback and ask whether they would consider participation. A Qualitative Recruitment Investiga- tion (QRI) was also undertaken in the early phases of the trial to ob- serve transparency of information given to patients and to assess the level of surgeon equipoise. Standard evaluation of the fre- quency of study procedures was also undertaken. Results In addition to the protocol design the poster will present preliminary survey data collected with 70 chief investigators of non-CTIMPs. The survey questions and results are attached. Questions covered a series of themes evaluating the range of inconsistency in defining, categor- izing and reporting serious adverse events, and evaluated prefer- ences for increased harmonisation in this area. The strategies resulted in successful recruitment to the study. Feed- back showed the benefit of involving the participating surgeons in defining the placebo procedure arm. Regular monitoring of the study showed surgeons were fully compliant with the restrictions of the placebo operation (the arthroscopy only). The placebo elem- ent was not an issue in relation to recruitment nor in implementa- tion of the study arms. The PPA completed showed 90% of patients would be interested in participating in a placebo surgery trial. Feed- back on the hypothetical study also informed the research ethics application. The QRI generated a “top tips for recruitment” list and enabled training on the best approach to patients. CSAW success- fully reached their recruitment target of over 300 patients, recruited from 25 NHS sites. Measurement methods for eliciting opinions on treatment benefits, toxicities and acceptable trade-offs of the two, within the PERSEPHONE trial 1 1 2 1 Louise Hiller1, Shrushma Loi1, Anne-Laure Vallier2, Donna Howe1, Peter Bell1, John Carey1, Uzma Manazar1, David Cameron3, David Miles4, Andrew Wardley5 1 2 Trials 2017, 18(Suppl 1):200 Page 140 of 235 (Type II error). Critically, a lack of evidence of harm does not equate to evidence of safety. (Type II error). Critically, a lack of evidence of harm does not equate to evidence of safety. Placebo surgery trials in the NHS are possible Naomi Merritt1, David Beard2, Andrew Carr2, Cushla Cooper2 1University of Oxford; 2NDORMS, University of Oxford Correspondence: Naomi Merritt Trials 2017, 18(Suppl 1):P371 The study will address variability in practice, defined in Standard Operating Procedures, that UK Clinical Trials Units (CTU) have in place for: i) defining, ii) classifying, and iii) reporting adverse events in non-ctimps. Compared to drug trials, adverse events in non-ctimps are not managed well. There is considerable inconsistency in report- ing styles between trials of similar design and intervention type. Placebo Surgery Trials in the NHS are Possible Introduction Placebo sur- gery trials are controversial and are not routinely conducted in the NHS. Evidence related to the management of such studies is limited and teams planning a placebo surgery trial need to carefully consider how to manage such a trial. To promote increased consistency, we will conduct a consensus exer- cise among non-CTIMP experts using a Delphi technique followed by a face-to-face meeting. This method adheres to the recommended sequence outlined by the international network for Enhancing the Quality and Transparency of Health Research (EQUATOR) for develop- ing health research guidelines. Background CSAW is a multicentre randomised placebo controlled blinded surgi- cal trial assessing the effectiveness of arthroscopic sub-acromial de- compression surgery versus an arthroscopy alone (the placebo or sham procedure) versus a period of active monitoring with specialist reassessment. Previously, a placebo surgery trial has been deemed difficult to run in the NHS with additional challenges for the study management team. These include (and supported by the literature) increased concerns regarding risk, ethics, perceived patient decep- tion, ability to recruit and the surgical community acceptance of the placebo procedure. A non-CTIMP expert is defined as: a CTU representative, a Chief Investigator or trial manager of non-ctimps with >3 trials experience in this role, or a senior member of the Health Research Authority’s Operations team or Ethics Committee. As such, the participants in the consensus exercises will also be the direct beneficiaries from the project maximising its pathway to impact. Following the face-to-face meeting, guidance and explanatory state- ments will be drafted. The guidance statement will focus on: How ad- verse events should be defined in relation to the non-pharmacological intervention, How CTU standard operating procedures should be de- signed to reflect the results of the Delphi exercise, How adverse events should be classified following a judicious causal assessment, and Recommended reporting methods that will promote more effective meta-analyses of non-pharmacological interventions that provide a balanced benefit-harm evaluation. Following study completion, we will work with a selection of UK CTUs to evaluate the implementation of any agreed modifications to current practice. Improving the quality of NIH funded clinical trials Carmen Rosa National Institute on Drug Abuse Trials 2017, 18(Suppl 1):P370 The National Institutes of Health (NIH), as the largest public funder of clinical trials in the United States, recognizes the importance of clin- ical trials and well as recognizes the major challenges in the design, efficiency and reporting of clinical trials. Over the years, NIH has funded trials that are too complex, have small sample sizes, rely on surrogate endpoints, have unrealistic enrollment goals, inadequate budgets, etc. Many times these trials are not published nor data sub- mitted to a public site. On September 16, 2016, the NIH announced a series of efforts directed towards the improvement of clinical trials efficiency, accountability and transparency. This presentation will briefly discuss these activities, which are aimed to address the clinical trials process from the time new ideas are generated to sharing data to the public. The initiatives covers NIH review and selection of trials to fund, clinical trials management and oversight, and data sharing. More specifically, this presentation will discuss the variety of new NIH policies, including Good Clinical Practice (GCP) training require- ments for investigator and NIH staff, using clinical trials specific Fund- ing Opportunity Announcements (FOAs), including appropriate expertise to review sessions, using a protocol template (required for FDA studies), using a single Institution Review Board (SIRB), and util- izing clinicaltrials.Gov to register and upload results. P372 Randomization balance in multicenter clinical trials with short drug life and rapid allocation Jeff Szychowski, Alan T. N. Tita, Gary R. Cutter University of Alabama at Birmingham Correspondence: Jeff Szychowski Trials 2017, 18(Suppl 1):P372 Sample size calculations using bayesian optimisation 1 2 1 Sample size calculations using bayesian optimisation Duncan Wilson1, Richard Hooper2, Rebecca Walwyn1, Amanda Farrin1 1University of Leeds; 2Queen Mary, University of London Correspondence: Duncan Wilson Trials 2017, 18(Suppl 1):P374 Conclusion With effective strategies in management and monitoring a placebo surgery trial is possible in the NHS. No major challenges were faced in the conduct of the study. CSAW has now successfully completed and results will be published early 2017. Jeff Szychowski, Alan T. N. Tita, Gary R. Cutter University of Alabama at Birmingham Correspondence: Jeff Szychowski Trials 2017, 18(Suppl 1):P372 Page 141 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 141 of 235 Background 1The University of Warwick; 2Oxford University Hospitals NHS Trust; 3Royal Devon & Exeter NHS Foundation Trust; 4University Hospitals of Coventry and Warwickshire; 5Guys and St Thomas’ NHS Foundation Trust; 6The University of Oxford; 7Glasgow Caledonian University Correspondence: Susanne Finnegan Finding the optimal sample size for a trial is an important step in its design. In many trials of complex interventions (such as psychother- apies and behavioural interventions) this task is complicated by two factors. Firstly, sample size can be defined by several design parame- ters rather than a single n. For example, a trial which compares a psychotherapy intervention with treatment as usual may be partially nested, with patients nested within therapists in the intervention arm but not in the control arm. The design parameters of such a trial are the number of therapists in the intervention arm, the number of patients seen by each therapist, and the number of patients in the control arm. The second complication is that analytical formulae for p p Development of the PREFIT MFFP At each site the investigational pharmacists prepared a pre-specified number of study bags to be used or discarded within 7 days. Given pharmacy costs and constraints, bags were typically prepared once per week. The number of prepared bags was estimated to be the number of patients enrolled over the course of the next 7 days. In successful recruiting weeks, all prepared study bags were used. In lower enrollment weeks, bags were discarded. Because of these con- straints, the rates of study drug preparation and randomization were continually monitored and modified as needed. Intervention: Multifactorial interventions are defined as that where in- dividuals receive ‘an assessment of known risk factors for falling and an intervention matched to their risk profile’. To determine which risk factors to include in PREFIT, we considered evidence from systematic reviews, including a large Cochrane review of 34 RCTs of multifactor- ial interventions to prevent falls in older people and evidence exam- ining the effectiveness of MFFP delivered in primary care. We referred to clinical guidelines from the American Geriatrics Society (AGS), British Geriatrics Society (BGS) and National Institute for Health and Care Excellence (NICE) to further inform selection of risk factors. In addition, we elicited a range of views from clinical and practice ex- perts within the field of falls and bone health. The final PREFIT MFFP intervention comprised of seven risk factors which were assessed on every participant referred for treatment. The model was based upon individual assessment and onward referral to specialist services where indicated. Results Pilot study We undertook a pilot study in 12 general practices (n = 1801) in Devon to determine the acceptability and feasibility of delivering an exercise programme and a complex MFFP intervention to older adults recruited from primary care. The interventions were then rolled out to other regions within the main trial. Methods Prefit is a three-arm, cluster RCT, conducted within primary care across England. We aimed to recruit 9000 participants, aged 70 and above, from 63 general practices. Practices were randomised to deliver one of three falls prevention interventions: (1) advice only; (2) advice with ex- ercise; (3) advice with multifactorial falls prevention (MFFP). The Age UK Staying Steady booklet was sent to all trial participants. The process of developing the complex ‘active’ interventions is described below. Development of the PREFIT Exercise Intervention: We undertook a review of systematic reviews of exercise interventions to prevent falls in community-dwelling older people. Based upon evidence from Cochrane systematic reviews and UK clinical guidelines, we shortlisted three standardised programmes for possible inclusion. To reflect the pragmatic nature of the trial, we also reviewed surveys of NHS falls ser- vices and conducted consensus work with clinicians working in falls prevention. We selected the Otago Exercise Programme which targets balance and strength, using ankle weights. The intervention is a six- month programme, which is individualised and supported by trained therapists who prescribe exercises and progress over time. y Methods and Results Methods and Results We simulate the effects of underutilization of prepared study drug on randomization balance and total drug waste. We consider differ- ent combinations of randomization schemes (fixed blocks of 2, 4, and 6, and variable block designs), total number randomized by site, and ratio of prepared-to-used study drug. Randomization bal- ance is lost, and waste increases, as the preparation rate exceeds the randomization rate. Development of complex interventions to prevent falls and fractures in older people living in the community: the prevention of fall injury trial (PREFIT) 1 1 1 7 Susanne Finnegan1, Julie Bruce1, Emma Withers1, Dawn Skelton7, Ranjit Lall1, Shvaita Ralhan2,, Ray Sheridan3, Katherine Westacott4, Finbarr Martin5, Sarah Lamb6 1 2 Susanne Finnegan1, Julie Bruce1, Emma Withers1, Dawn Skelton7, Ranjit Lall1, Shvaita Ralhan2,, Ray Sheridan3, Katherine Westacott4, Finbarr Martin5, Sarah Lamb6 1 2 Conclusions It is extremely important that we understand the characteristics of potentially suboptimal randomization procedures, as they may lead to increased waste, increased costs, and randomization imbalance. The complications introduced by short drug shelf life and by the need for rapid randomization are important in multiple contexts in- cluding labor & delivery and emergency medicine. We discuss strat- egies to optimize resources and minimize waste. Background d Our study is inspired by the Cesarean Section Optimal Antibiotic Prophylaxis (C/SOAP) trial, a double-blind, pragmatic, randomized clinical trial conducted at 14 hospitals in the United States. Women with a singleton pregnancy, at least 24 weeks gestation, and under- going nonelective cesarean delivery were randomized to receive ei- ther azithromycin (500 mg in 250 ml saline) or an identical-appearing saline placebo prior to incision. All women were to receive standard prophylaxis (cefazolin) prior to incision. The 250 ml bags were prepared in advance by investigational pharmacists according to site-stratified randomization schemes, kept in a secure refrigerator, and had a 7 day shelf life. Only the investigational pharmacists who prepared the study drug had access to the randomization scheme through a dedicated password-protected website. Randomized women received the next sequentially numbered study bag in the refrigerator. Expired study bags were discarded. Introduction to investigate the efficacy of falls preventions strategies on rate and risk of falls, there is a lack of strong, robust evidence for multifactorial or exercise interventions in preventing fractures. We developed two complex falls prevention interventions for evaluation within the framework of a large multicentre, pragmatic, randomised controlled trial (RCT) (ISCTRN 71002650). Methods The logistics of allocating an assigned drug treatment in a multicen- ter randomized controlled clinical trial may be complicated by a short drug shelf life and by the need for rapid allocation after randomization. We conduct a simulation study to examine the effects on randomization balance under these conditions, where the ratio of rates of drug preparation and recruitment at study centers vary from low to high. We further explore practical strategies to address these logistical needs. Conclusions These complex interventions are currently being evaluated within the largest multicentre falls prevention clinical trial conducted in the UK. P373 Development of complex interventions to prevent falls and fractures in older people living in the community: the prevention of fall injury trial (PREFIT) Susanne Finnegan1, Julie Bruce1, Emma Withers1, Dawn Skelton7, Ranjit Lall1, Shvaita Ralhan2,, Ray Sheridan3, Katherine Westacott4, Finbarr Martin5, Sarah Lamb6 1The University of Warwick; 2Oxford University Hospitals NHS Trust; 3Royal Devon & Exeter NHS Foundation Trust; 4University Hospitals of Coventry and Warwickshire; 5Guys and St Thomas’ NHS Foundation Trust; 6The University of Oxford; 7Glasgow Caledonian University Correspondence: Susanne Finnegan Trials 2017, 18(Suppl 1):P373 P373 Development of complex interventions to prevent falls and fractures in older people living in the community: the prevention of fall injury trial (PREFIT) Susanne Finnegan1, Julie Bruce1, Emma Withers1, Dawn Skelton7, Ranjit Lall1, Shvaita Ralhan2,, Ray Sheridan3, Katherine Westacott4, Finbarr Martin5, Sarah Lamb6 1The University of Warwick; 2Oxford University Hospitals NHS Trust; 3Royal Devon & Exeter NHS Foundation Trust; 4University Hospitals of Coventry and Warwickshire; 5Guys and St Thomas’ NHS Foundation Trust; 6The University of Oxford; 7Glasgow Caledonian University Correspondence: Susanne Finnegan Trials 2017, 18(Suppl 1):P373 Background Falls are the leading cause of accident-related mortality in older adults. Injurious falls, including fractures, are associated with func- tional decline, loss of independence, disability, and significant health and social care costs. Although numerous trials have been conducted Page 142 of 235 Trials 2017, 18(Suppl 1):200 Page 142 of 235 Developing a framework to aid purposive sampling in process evaluation of a critical care trial Bayesian optimisation can be an effective technique for performing sample size calculations when power must be estimated using simu- lation, particularly when sample size is characterised by several de- sign parameters. By improving the efficiency of these calculations, increasingly complex sample size problems can be solved without the need for unrealistic simplifying assumptions. Correspondence: Lydia Emerson Trials 2017, 18(Suppl 1):P376 p Methods We propose using Bayesian optimisation to solve the sample size problem. This method allows optimal or near-optimal choices for sample size to be found with minimal computational effort. The gen- eral approach involves the careful choice of the design parameter values where power should be estimated using simulation. Conduct- ing the simulations at these points, a statistical model is then fitted to the output to describe the general relationship between the design parameters and the trial power. This model is then used to find the smallest design parameter values which will give power of at least the nominal level. The method is flexible, can be used for al- most any problem for which power can be estimated using Monte Carlo simulation, and can be implemented using existing statistical software packages. Could adaptive research designs be useful in designing an effective ehealth intervention? A methodological analysis R B j i J i C Lee Kong Chian School of Medicine, Nanyang Technological University Correspondence: Ram Bajpai Trials 2017, 18(Suppl 1):P375 Conclusions The adaptive designs showed potential to address various ehealth specific challenges. Such designs could lead to simplified operational complexities involved and make these interventions more efficient and cost-effective. There is a need to encourage researchers to use adaptive designs and set regulatory guidelines to handle practical challenges. Background The use of ehealth or digital health interventions has increased due to rapid growth in information and communication technologies (ICTs). This results quick appearance and change in digital interven- tions that challenge the robust designing of such interventions. The traditional intervention designs are somehow incapable to tackle specific challenges of digital interventions. It is therefore important to explore innovative research designs to handle the unique chal- lenges of ehealth interventions. g Objective h h This methodological research aims to analyse how different adaptive research designs could be used in evaluation of digital behaviour change interventions (DBCIs) without altering the nature of random- ized designs. Results calculating power are not always available. As a result power must instead be estimated through Monte Carlo simulation methods, which may be computationally demanding. For example, such a simulation of the TIGA-CUB study would involve the generation of a large number of hypothetical trial data sets and fitting a multilevel model to each one. In combination, these factors make finding an optimal sample size a difficult and time consuming problem. We ex- plore how modern optimisation algorithms can be used to solve these problems in an effective, timely manner. We did not find any evidence so far of using adaptive designs either in ongoing or published digital intervention trials literature. There- fore, we discuss different adaptive design choices conceptually such as adaptive randomisation design, group sequential design, sample size re-estimation design, hypothesis-adaptive design and seamless phase II/III design etc. Literature suggested that sequential, seamless phase II/III and multi-arm multi-stage designs could improve effi- ciency and maintain ethical considerations; changing- sample size and hypothesis designs could handle the uncertainty and be flexible to define end points; and enrichment designs could handle hetero- geneity among responses and ease the data monitoring process. On the other hand, multiple adaptive designs in a single trial require more control in execution and should be handled with care. How- ever, other adaptive designs such as treatment-switching and dose- finding are yet to explore their usability in relation to ehealth intervention. There are some statistical challenges that need address- ing when designing such trials. For example, any adoption to the design may increase the Type I error rate, difficulties in the analysis of trial data including Bayesian approach (especially in deciding prior distribution) and interpretation of results. Conclusions p Evaluation p Evaluation To illustrate the approach we apply it to a partially nested psychother- apy trial in an illustrative case study. We use the proposed method to identify a set of candidate sample size options, each of which will give power of at least the nominal rate. From this set of options, that which is considered best in terms of its balance between number of therapists and number of patients can be chosen. We compare this with an alter- native approach using simpler heuristics, in terms of both the computa- tion time required and the quality of the resulting solutions. Conclusions P376 Developing a framework to aid purposive sampling in process evaluation of a critical care trial Lydia Emerson1, Danny McAuley1, Mike Clarke2, Thomas P. Hellyer3, A. John Simpson3, Bronagh Blackwood1 1Centre for Experimental Medicine, Queen's University Belfast; 2Centre for Public Health, Queen's University Belfast; 3Institute of Cellular Medicine, Newcastle University Correspondence: Lydia Emerson Trials 2017, 18(Suppl 1):P376 Background h P375 Could adaptive research designs be useful in designing an effective ehealth intervention? A methodological analysis Ram Bajpai, Josip Car Lee Kong Chian School of Medicine, Nanyang Technological University Correspondence: Ram Bajpai Trials 2017, 18(Suppl 1):P375 To answer the question ‘does a complex intervention work?’ in a way that distinguishes between failure of the intervention and failure of its implementation, an evaluation of the process of intervention delivery is required. Process evaluation data are collected from a sample of the practitioners involved in implementation and interven- tion delivery. However, the mechanism of selecting a sample is rarely described in the literature. The aim of this project was to define a framework to purposively sample clinicians in a trial conducted in 22 intensive care units (ICUs) which used a new invasive test for detect- ing ventilator associated pneumonia in critically ill patients (the vaprapid-2 trial, ISRCTN65937227). Methods Data analysis of context and usual practice collected at the beginning of the trial, alongside qualitative data collected from doctors, nurses, and laboratory staff during the trial provided information on adoption and delivery of the intervention in the ICUs. From this information, we constructed themes describing what worked well, for whom and in what contexts in terms of intervention delivery. These themes were explored with clinicians at the end of the trial in order to identify factors and the mechanisms of their interaction that were likely to impact on trial outcomes. We purposively sampled 40% of ICUs for end of trial inter- views, ensuring that we obtained maximum variation in barriers and facilitators to the trial. Results An adaptive design allows modifications during the trial based on the reports from interim analysis. We reviewed Medline available literature related to adaptive designs and hand searched relevant medical and statistical journals. We also assessed the published and on- going (registered at www.clinicaltrials.gov/) ehealth interventions from Jan 2011 to Oct 2016 to search any evidence of adaptive designs in ehealth or digital health interventions. In the analysis of data collected before and during the trial, we identi- fied five themes. To enable easier sampling, we grouped the themes into two broad categories to form a framework: (1) ICU situation which reflected (positive or negative) issues with laboratories, workload, staff availability, and fitting the trial into the ICU; and (2) perceived risk (classed as high or low risk). This categorisation enabled sites to be Trials 2017, 18(Suppl 1):200 Page 143 of 235 ‘mapped’ onto the framework. In addition to these themes, we also examined recruitment data to assess the reach of the intervention i.e. The percentage of eligible patients who were actually recruited into the trial. We subsequently sampled ICUs from each of the four cells that also captured the variation in reach. Background Blinding, the process of withholding knowledge of treatment allo- cation from participants and trial personnel, is critical in the design of RCTs. It may reduce differences between trials groups in the as- sessment of outcomes (detection bias), in the way interventions and co-interventions are delivered (performance bias) and in with- drawals from the trial (attrition bias). In addition, it may minimise bias in the interpretation and reporting of analyses if data analysts are successfully blinded. Indeed if there is inadequate blinding this can exaggerate estimates of treatment effects by up to 25%. Blind- ing, however, can be hard to achieve and maintain in trials asses- sing non-pharmacological interventions such as surgery. Challenges specific to surgical trials include, but are not limited to, difficulties in delivering a control intervention indistinguishable from the active intervention, and blinding personal who deliver the interven- tion. This systematic review will describe the current methods used to blind participants, intervention providers, care givers, outcome assessors and data analysts in trials of invasive surgical interven- tions across all surgical specialties. In addition, we will present examples of trials where blinding was not attempted but may have been possible, outlining how novel methods of blinding may be utilised. Presently we outline results to date with a complete report available in March 2017. 1. SAS Formats cannot be applied while creating excel files using proc export Solution: SAS macro program is written to handle the format issue. 2. The Source data path changes of Pivot reports while moving the reports Solution: There is a wide discussion can be seen in various online forums on how to solve the pivot source data path changes while moving the files with pivot tables. None of the solutions dis- cussed online are simple and reliable. The simplest solution is open the file and using SAVE AS option save it in the desired location in- stead of the coping the master dashboard. How simple it is. Conclusion Little information is available on the methods that might be used for purposive sampling of practitioners implementing new interventions in critical care research. We suggest a novel and practical method of categorising data to produce a framework to guide maximum vari- ation sampling. P378 Cost effective interactive live clinical data monitoring dashboards with drill-down Amarnath Vijayarangan Emmes Services Pvt Ltd. Trials 2017, 18(Suppl 1):P378 P378 Cost effective interactive live clinical data monitoring dashboards with drill-down Cost effective interactive live clinical data monitoring dashboards with drill-down Amarnath Vijayarangan Emmes Services Pvt Ltd. Trials 2017, 18(Suppl 1):P378 with drill-down Amarnath Vijayarangan Emmes Services Pvt Ltd. Trials 2017, 18(Suppl 1):P378 P377 Minimising bias in surgical RCTs through blinding: a systematic review Sian Cousins, Katy Chalmers, Kerry Avery, Natalie Blencowe, Sara Brookes, Jane Blazeby, M. Kobetic, T. Munder University of Bristol Correspondence: Sian Cousins Trials 2017, 18(Suppl 1):P377 Minimising bias in surgical RCTs through blinding: a systematic review Periodical data review is very important and highly recommended for all the ongoing clinical studies to ensure the data integrity and quality. Each clinical study requires experts from various functional groups like SAS programming, Biostatistics, Data Management and so on. Each one of them have various data review requirements and also one cannot expect everyone to familiar with SAS program- ming as clinical datasets are often available as SAS datasets. Statisti- cians prefer summary level data whereas others might need to look at the summary level as well as granular level data. These reports are static and hence end users do not have any choice to customize or drill down the reports on their own. Currently it is al- ways directed to a SAS programmer to update the reports which is an overall time consuming process. Every clinical study is con- strained with budget and it might be expensive for them for a so- phisticated tool. These dashboards are created only once for each study using SAS, Excel VBA & Excel Pivot Table and they are –Multi user access at a time –Live Interactive Summary Reports and Graphs –No Programming is required for the end user –100% Menu Driven –Auto Refresh –Custom Filters –Drill Down to Raw Data The master dashboard excel file can be copied by each user to their local machine and each one can play around with the locally saved reports without impacting the master dashboard & source files. The following are the challenges faced during the developmental stage along with their solutions. Sian Cousins, Katy Chalmers, Kerry Avery, Natalie Blencowe, Sara Brookes, Jane Blazeby, M. Kobetic, T. Munder University of Bristol Correspondence: Sian Cousins Trials 2017, 18(Suppl 1):P377 Sian Cousins, Katy Chalmers, Kerry Avery, Natalie Blencowe, Sara Brookes, Jane Blazeby, M. Kobetic, T. Munder University of Bristol Correspondence: Sian Cousins Trials 2017, 18(Suppl 1):P377 Sian Cousins, Katy Chalmers, Kerry Avery, Natalie Blencowe, Sara Brookes, Jane Blazeby, M. Kobetic, T. Munder Correspondence: Sian Cousins Trials 2017, 18(Suppl 1):P377 Methods A A systematic search was carried out in Medline (ovidsp), Embase and CENTRAL databases for articles published between January 2006 and June 2016 in the top 10 surgical and general medical journals accord- ing to impact factor. Articles eligible for inclusion were RCTs of invasive interventions, in which blinding of any participants or trial personnel had been attempted. We define an invasive procedure to be where a cut is made or access to the body is gained via cutting, instrumentation via a natural orifice or percutaneous skin puncture where instruments are used in addition to the puncture needle. Trials in which a medicinal product is delivered via an invasive procedure and where there is ad- ministration to targeted anatomical districts or where an action is per- formed internally to administer the product, will be included. General study characteristics will be extracted, in addition to data regarding blinding specifically. Blinding status of participants and trial personnel, method of blinding, instances where blinding may have been possible but was not attempted and details of any reported tests of success of blinding will be extracted. Quality of included trials will also be assessed using the Cochrane Risk of Bias tool. 3. Pivot based reports are static with the number of rows in excel. But for an ongoing study, this will be increasing and every time user cannot change their reports for the rows. It becomes tedious to manually do the same on several reports and it is error prone. Solu- tion: While creating the pivot reports, 50000 rows are selected and Whenever any SAS dataset exceeds 50000 rows, email notification is sent. 4. Date based filtering criteria is one of the frequently used criteria for the data monitoring. For example, number of subjects enrolled during last 1 week or 1 month. Excel pivot does not provide an op- tion to filter data using specified date ranges. Solution: Fortunately, this is solved using the concept of SLICER available in excel 2010 onwards. This paper proposes an easy and cost effective approach to develop an Interactive Live Clinical Data Monitoring Dashboards with Drill-Down Using SAS & Microsoft Excel. Discussion We outline and summarise the methods of blinding used in high quality surgical RCTs assessing invasive interventions. We highlight good practice and will make recommendations for future research in this field to minimise bias in RCTs in surgery. Conclusion Putting in place small changes and guidelines can improve staff productivity and save duplication of their workload. Having a sys- tem which all staff can easily implement and use helps everyone keep track of what has been done to deal with data queries. The in- vestigations that come from these queries improves data quality and efficiency of trial conduct. A similar approach is used in our BHF RIGHT-2 trial. Subsequently, there was a need identified within ECTU for a forma- lised procedure for data management activity, in particular where there is no allocated Trial Manager. By first establishing a desig- nated Data Manager role, ECTU have undertaken a schedule of evaluation and improvement procedures to redefine data manage- ment activity within the unit. The entire lifecycle of the evaluation of the existing data management activity and formalisation of new procedures will be detailed, including the development of an ECTU data management plan and its standardised content. Through on- going monitoring and feedback from Trial Managers and external clients, future plans will also be identified, including suggestions for future discussion with the wider data management community. P381 Changing platforms without stopping the train : a data management perspective on the operational aspects of adaptive platform trials Dominic Hague1, Stephen Townsend1, Lindsey Masters1, Mary Rauchenberger1, Matthew R. Sydes1 1MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL, London, UK; 2MRC London Hub for Trials Methodology Research, London, UK Correspondence: Dominic Hague Trials 2017, 18(Suppl 1):P381 Methods We critically reviewed data management challenges in STAMPEDE and FOCUS4. These included implementation of case report forms (CRFs), Clinical Data Management Systems (CDMS), randomisation systems, report development, documentation, and other operational challenges. We also sought specific challenges arising from electronic (FOCUS4) or paper (STAMPEDE) CRFs. P380 Data validation traffic light system: data from the tranexamic acid for intracerebral haemorrhage (TICH-2) trial Data validation traffic light system: data from the tranexamic aci for intracerebral haemorrhage (TICH-2) trial Katie Flaherty, Lelia Duley, Zhe Law, Philip M. Bath, Nikola Sprigg University of Nottingham Correspondence: Katie Flaherty Trials 2017, 18(Suppl 1):P380 for intracerebral haemorrhage (TICH-2) trial Katie Flaherty, Lelia Duley, Zhe Law, Philip M. Bath, Nikola Sprigg University of Nottingham Correspondence: Katie Flaherty Trials 2017, 18(Suppl 1):P380 Discussion We found similar adaptive platform trial-specific challenges in both trials. Adding and removing comparisons to open trials provides extra layers of complexity to CRF and CDMS development. At the start of an adaptive platform trial, CRFs and CDMS must be designed to be scalable in order to cope with the continuous changes in this trial design, ensuring future data requirements are considered where possible. When adding or stopping a comparison, the challenge is to incorporate new data requirements while ensuring data collection within ongoing comparisons is unaffected. Some changes may apply to all comparisons; others may be comparison-specific or only applic- able to patients recruited during a specific time period. We will dis- cuss the advantages and disadvantages of the different approaches to CRF and CDMS design we implemented in these trials, particularly g Results Th Twenty seven queries were raised from a sample of 14 centres; 6 (22%) of these were coded green and were data corrected, the remaining 21 (78%) were red and removed from the checks. data checks were run recently for TICH-2, so all data are not yet avail- able. Twenty seven queries were raised from a sample of 14 centres; 6 (22%) of these were coded green and were data corrected, the remaining 21 (78%) were red and removed from the checks. This system also led to changes in ECRFs; it was noticed that partici- pant weight was often missing on the baseline form, so this was moved to the day 2 form to give hospital staff more time to get the information. Since the change, the number of completed entries has increased from 69% to 75% (p = 0.03). Background To date, STAMPEDE has added four new research comparisons, closed two research comparisons following pre-planned interim analysis (lack-of-benefit) and completed recruitment to six research comparisons. FOCUS4 has closed one research comparison follow- ing pre-planned interim analysis (lack-of-benefit) and added one new research comparison, with a number of further comparisons in the pipeline. We share our experiences from the operational as- pects of running these adaptive platform trials, focusing on data management. Data validation in clinical trials is an important procedure. Real time validation checks may not pick up all errors and these need correct- ing. A flagging system would help trial staff be more time effective by preventing re-checking of problems that cannot be resolved. A traffic light system was created and implemented at the Nottingham Stroke Trials Unit. g Results Th The search retrieved 3946 articles. 1873 duplicates were removed and 613 were removed on basis of journal. 1460 titles and abstracts were screened for eligibility using standardised screening forms. 1129 articles were excluded after abstract review and 331 articles were included for full text review. Full results will be available by March 2017 and reported at the meeting. Data management lifecycle evaluation Michelle Steven Edinburgh Clinical Trials Unit Trials 2017, 18(Suppl 1):P379 Data management lifecycle evaluation Michelle Steven Edinburgh Clinical Trials Unit Trials 2017, 18(Suppl 1):P379 Michelle Steven Edinburgh Clinical Trials Unit Trials 2017, 18(Suppl 1):P379 Trials 2017, 18(Suppl 1):200 Page 144 of 235 Page 144 of 235 data checks were run recently for TICH-2, so all data are not yet avail- able. Twenty seven queries were raised from a sample of 14 centres; 6 (22%) of these were coded green and were data corrected, the remaining 21 (78%) were red and removed from the checks. This system also led to changes in ECRFs; it was noticed that partici- pant weight was often missing on the baseline form, so this was moved to the day 2 form to give hospital staff more time to get the information. Since the change, the number of completed entries has increased from 69% to 75% (p = 0.03). Data management processes are required to ensure clinical trial data is high-quality and captured according to protocol and regula- tory requirements; a critical phase within clinical research. The Edinburgh Clinical Trials Unit (ECTU) Data and IT Systems Team are small yet well-established with a wide ranging remit from CRF ?A3B2 show $132#?>design, data entry, and data cleaning through to the design and hosting of complex bespoke electronic data cap- ture (EDC) systems. Historically, the majority of the EDC systems de- veloped were used in studies fully supported by ECTU, with an identified project manager assigned to the study who completed many of the data management tasks together with the IT Systems Team. However, there has been a recent demand for support only from data management, with ECTU developing EDC systems for tri- als with external trial coordination. In addition, with the continued growth of the Trial Management teams, there was significant vari- ation in CRF design and data management procedures between the individual trial managers. data checks were run recently for TICH-2, so all data are not yet avail- able. Background There is limited research and literature on the data management challenges encountered in adaptive platform trials. This trial design allows both (i) seamless addition of new research comparisons, and (ii) early stopping of accrual to individual comparisons that do not show sufficient activity without affecting other active comparisons. FOCUS4 (colorectal cancer) and STAMPEDE (prostate cancer), run from the MRC CTU at UCL, are two leading UK examples of clinical trials implementing adaptive platform designs. Methods The NIHR HTA Tranexamic acid for intracerebral Haemorrhage (TICH-2) trial is a randomised, controlled, international, multicentre trial aiming to recruit 2,000 patients. Data volume in such a trial is large, and having numerous centres makes solving data queries time consuming. The database in TICH-2 has built in logic checks allowing real time validation and this identifies most but not all data inconsistencies. A program was created that looks at the data and checks it for values which lie outside the normal ranges or are inconsistent between different data fields, this picks up queries that the real time validation missed to ensure data is as complete as possible. The checks are run for one centre at a time and sent to trial coordinators, within the trial coordinating centre, to go through them and resolve with sites. Coordinators highlight each of the checks in either green, orange or red. Green shows the query has been resolved, usually due to incorrect data that has been checked with the recruiting site and updated. Orange means it has not been resolved because the site has yet to respond; such queries may be resolved at a later date. Red indicates that data cannot be resolved or is correct as is and the statistician should remove the value from further data checks. [Note: For lessons learnt from a central trial management perspec- tive, see our companion abstract] Method The Collaborative Studies Coordinating Center (CSCC) in the Depart- ment of Biostatistics at the University of North Carolina serves as the data coordinating center for the trial. A dynamically allocated biased- coin minimization algorithm (Pocock & Simon, Biometrics 1975) was designed and programmed at the CSCC as an application called from the randomization electronic case report form (ecrf) from the CSCC- developed, web-based data management system, Carolina Data Acquisition and Reporting Tool (CDART). Each time a patient is to be enrolled, the site enters the enrollment data, including all of the stratification factors into the ecrf. The randomization application within CDART calculates an imbalance score for each purported treat- ment assignment that is based on stratification co-factor levels of previously enrolled, randomized patients. The lowest imbalance score has some influence when the treatment is assigned. g ( Methods Randomization seeks to balance background factors across treatment groups through random assignment, and stratified randomization im- poses structure to help ensure balance of the stratification factors. Yet randomization schemes that are over-stratified may not result in treat- ment group balances in factors related to disease outcome, sometimes making study interpretations difficult. Using dynamic randomization al- gorithms in trials with a small sample size relative to the number of stratification groups may improve the co-factor balance, allowing for a clearer study signal. Periodontal Treatment to Eliminate Minority Inequality and Rural Disparities in Stroke (PREMIERS) (NIH Minority Health and Health Disparities Research) is a 2-center phase III randomized, controlled trial that will enroll 400 patients to test whether intensive periodontal treatment reduces the risk of recur- rent vascular events among ischemic stroke and TIA survivors. Re- searchers wanted to ensure balance of 4 different factors: race, stroke severity, socio-economic status, and stroke risk. The SIP team applied the PRECIS-2 tool to assess the pragmatism of the SIP feasibility study design, using training materials and the soft- ware on the PRECIS-2 website www.PRECIS-2.org to create their own sip study wheel. The individual PRECIS-2 scores of the SIP trial team were aggregated into a single PRECIS-2 wheel to indicate the median and range of scores for each of the nine PRECIS-2 domains. The trial team then used aggregated scores to discuss the design of the feasi- bility study and reach consensus. Discussion focussed on domains with the greatest range of scores (i.e. Widest discrepancy between individuals). Members of the team were asked to complete an evalu- ation form to determine the utility of using the tool with a view to designing a future randomised trial. Operationalizing and testing a minimization randomization algorithm for use in clinical trials g Sheila Burgard1, James Bartow1, Hope Bryan1, Sonia Davis1, Steven Offenbacher1, Souvik Sen2 1University of North Carolina; 2University of South Carolina Correspondence: Sheila Burgard Trials 2017, 18(Suppl 1):P382 1University of North Carolina; 2University of South Carolina Correspondence: Sheila Burgard Trials 2017, 18(Suppl 1):P382 Conclusions Adaptive platform trials offer an efficient model to run randomised controlled trials but setting-up and conducting the data manage- ment activities in these trials can be operationally challenging. Trialists and Funders must plan for scalability in data collection, and the resource required to cope with additional competing data man- agement tasks. Correspondence: Kirsty Loudon Trials 2017, 18(Suppl 1):P383 Impact case study: using PRECIS-2 to discuss the design of a pilot and feasibility study - summary of swallowing intervention package (SIP) 1 2 2 Impact case study: using PRECIS-2 to discuss the design of a pilot and feasibility study - summary of swallowing intervention package (SIP) p g Kirsty Loudon1, Mary Wells2, Emma King2 1University of Stilring; 2Nursing, Midwives and Allied Health Professions Research Unit Kirsty Loudon1, Mary Wells2, Emma King2 1University of Stilring; 2Nursing, Midwives and Allied Health Professions Research Unit Background The PRECIS-2 tool was developed to help trialists match their design decisions to the information needs of those they hope will use the trial results. PRECIS-2 has a highly visual wheel format with nine design do- mains including Eligibility, Recruitment, Setting, Organisation and Pri- mary outcome which are scored on a Likert scale from “1” Very explanatory - Ideal world, to “5” Very pragmatic - Just like usual care. The tool was developed to design Randomised Controlled Trials and is being used internationally by health care professionals in a variety of clinical areas. The aim of our study was to evaluate the usefulness of PRECIS-2 for discussing the design of a feasibility study of an interven- tion developed by a trial team including speech and language thera- pists, oncology nurses, and a psychologist: a Swallowing intervention Package (SIP). M h d Operationalizing and testing a minimization randomization algorithm for use in clinical trials Sheila Burgard1, James Bartow1, Hope Bryan1, Sonia Davis1, Steven Offenbacher1, Souvik Sen2 1University of North Carolina; 2University of South Carolina Correspondence: Sheila Burgard Trials 2017, 18(Suppl 1):P382 g Results Ten out of the 14 members of the sip trial team used the PRECIS-2 tool to assess the pragmatism of the SIP feasibility study. Prior to the meeting, the discrepancy between domain scores was up to 3 points on a scale of 5, suggesting differing views of how explanatory or pragmatic the study design was. The PRECIS-2 domains with greatest consensus were Eligibility - To what extent are the participants in the trial similar to those who would receive this intervention if it was part of usual care, and Setting - How different are the settings of the trial from the usual care setting. Some of the trial group believed that certain aspects of the feasibility study for the sip trial were quite ex- planatory, with four out of nine of the domains scoring "2": Flexibility (Adherence), Follow up, Primary Outcome and Primary Analysis. Fol- lowing discussion, the team reached consensus on scoring 7 out of 9 domains, assessing the overall design of the sip feasibility study as more pragmatic than explanatory. With all but one of the meeting participants independently scoring the sip trial using PRECIS-2, this enabled meaningful discussion of the key elements of a future trial design. Results Results This traffic light system has proven easy to follow and understand and has been effective at getting queries organised. It ensures coor- dinators are not re-checking queries that cannot be resolved. The Page 145 of 235 Trials 2017, 18(Suppl 1):200 Page 145 of 235 Page 145 of 235 in relation to use and maintenance of generic versus comparison- specific CRFs and CDMS. of implementing the algorithm, the additional steps to safeguard against changes in stratification data after a patient has been ran- domized, and the double-programmed validation job confirming that the treatment assignments are being correctly assigned. in relation to use and maintenance of generic versus comparison- specific CRFs and CDMS. The work required to add or remove a comparison, including the development and testing of changes, updating of documentation, and training of sites, must be undertaken alongside data manage- ment of ongoing comparisons. Adequate resource is required for these competing data management tasks, especially in trials with long follow-up. A plan is needed for regular and pre-analysis data cleaning for comparisons that could recruit at different rates and times. We will discuss the ways that these data cleaning activities can be split and prioritised. P383 Impact case study: using PRECIS-2 to discuss the design of a pilot and feasibility study - summary of swallowing intervention package (SIP) Kirsty Loudon1, Mary Wells2, Emma King2 1University of Stilring; 2Nursing, Midwives and Allied Health Professions Research Unit Correspondence: Kirsty Loudon Background d 34 sites have opened to recruitment across the UK, with enthusiasm, commitment and support evident from the site research teams. Pa- tients have responded favourably to the trial and the opportunity to contribute to ground breaking research. Patients found the Patient Information DVD informative and welcomed receiving information early on in the patient pathway. Corticosteroids (CS) are key to achieving rapid disease control in chil- dren and young people (CYP) presenting with new or flaring juvenile idiopathic arthritis (JIA). Efficacy, duration of action and side effect pro- files vary with the route of administration. Current routes of CS adminis- tration are based on physician and patient preference, rather than scientific evidence. A randomised controlled trial is needed to ascertain the most effective routes and doses of CS. This paper will report on the feasibility of a potential CS induction regimen randomised controlled trial (RCT) in JIA from the perspective of CYP and their families. Methods Reasons patients gave for participating in the trial include “the trial offered the best treatment options” and that “others would benefit from the results of the trial”. Patients have also commented that they welcome the opportunity for independent pathology review of their biopsies; a key quality assurance element of the trial. Feedback from sites highlights the difficulty in conveying to patients the rationale of replacing surgery with active monitoring, with 30% of patients stat- ing a preference for surgery. To address this, regular communication workshops are offered, phone-in sessions allow sites to share best practice and frequent newsletters offer hints and tips to aid recruit- ment. Media channels are being explored to raise public awareness of the overtreatment of DCIS. Dissemination of this information will help to engage future LORIS patients. Semi-structured in-depth interviews were conducted with a purpos- ive sample of CYP with JIA and their families, recruited via rheuma- tology clinics across the UK. All CYP and their families had recent (<12 months) experience of CS treatment. The Framework Method was used to support thematic analysis of the data. Results Findings will be reported on CYP’S and parents’ experiences of four different CS delivery routes, treatment preferences and willingness to participate in a CS induction regimen RCT, randomising patients be- tween different steroid delivery routes and dose regimes. CYP’s and parents’ questions about the planned RCT and their recommenda- tions regarding its design will also be reported. Overtreatment of DCIS – 4 years on from the marmot review - progress with the LORIS trial 1 2 3 Overtreatment of DCIS – 4 years on from the marmot review - progress with the LORIS trial 1 2 3 Overtreatment of DCIS – 4 years on from the marmot review - progress with the LORIS trial 1 2 3 Jennie Young1, Adele Francis2, Jeremy Thomas 3, Matthew Wallis4, Andrew Hanby5, Lesley Fallowfield6, Valerie Jenkins6, Lucy Matthews6, Patricia Fairbrother7, Maggie Wilcox7, John Bartlett8, Lucinda Billingham9, Sarah Bowden9, Claire Gaunt9, Daniel Rea9, Tracy Roberts9, Sarah Pinder10 1University of Birmingham; 2University Hospitals Birmingham; 3Western General Hospital; 4Addenbrooke's Hospital; 5University of Leeds; 6SHORE-C, Brighton and Sussex Medical School; 7Independent Cancer Patients' Voice; 8Ontario Institute for Cancer Research; 9University of Birmingham, Cancer Research UK Clinical Trials Unit; 10King's College London g Discussion Collaborative efforts and feedback from site staff, patients and over- sight committees during the LORIS feasibility phase has allowed us to establish LORIS as a world leading trial addressing the overtreat- ment of DCIS. The findings from this qualitative study will inform judgements about the feasibility and design of a future RCT of CS induction regimens for JIA. Corticosteroids are a very effective treatment for flaring JIA but carry significant and challenging side-effects. The views and ex- periences of CYP with JIA are an important outcome of this study and are key to informing the feasibility and acceptability of a future RCT from the patient perspective. Methodological issues about the validity of pre-trial feasibility studies for informing decisions about potential future trials will also be considered. The LORIS Trial is funded by the National Institute for Health Research Health Technology Assessment programme. The LORIS Trial is funded by the National Institute for Health Research Health Technology Assessment programme. Correspondence: Jennie Young Trials 2017, 18(Suppl 1):P385 The Marmot independent review of breast screening report pub- lished in 2012 concluded that the breast screening programme saves lives but significant overtreatment exists [1]. The review called for randomised clinical trials to address overtreatment of screen-detected DCIS. Correspondence: Cliona McDowell Trials 2017, 18(Suppl 1):P386 Correspondence: Cliona McDowell Trials 2017, 18(Suppl 1):P386 Family perspectives on the feasibility of a corticosteroid induction regimen randomised controlled trial in juvenile idiopathic arthritis: results of a qualitative study 2 2 Family perspectives on the feasibility of a corticosteroid induction regimen randomised controlled trial in juvenile idiopathic arthritis: results of a qualitative study 1 1 2 2 LORIS is a multi-centre, randomised controlled phase III trial of sur- gery versus active monitoring in patients with low risk DCIS designed to address the problem of overtreatment. An independent patient advocate group (Independent Cancer Patients' Voice) have been involved in the study since conception and provided a direct patient perspective throughout the LORIS Trial. Key eligibility criteria are; women aged 46 years and over with screen- detected or incidental microcalcification with no previous invasive breast cancer, no comedo necrosis and low risk disease confirmed by Central Digital Histopathology Review. The primary outcome is ipsilat- eral invasive breast cancer free survival up to 5 years. y Correspondence: Frances Sherratt Trials 2017, 18(Suppl 1):P384 g Conclusions CDART has the functionality for complex calculations and custom re- ports, yet operationalizing the dynamic allocation application was new to CDART. A separate application developed in SAS was used to validate the randomization algorithm within the data management system is working as expected. The presentation will show the details This exercise was useful for assessing the design of both the SIP feasibility study and a potential future trial. PRECIS-2 is a relevant framework for reaching consensus on design aspects of pilot and feasibility studies as well as full trials. Trials 2017, 18(Suppl 1):200 Page 146 of 235 Page 146 of 235 cancer specific survival between patients who had surgery and those who did not in the low grade DCIS group. cancer specific survival between patients who had surgery and those who did not in the low grade DCIS group. Family perspectives on the feasibility of a corticosteroid induction regimen randomised controlled trial in juvenile idiopathic arthritis: results of a qualitative study Frances Sherratt1, Louise Roper1, Eileen Baildam2, Matthew Peak2, Flora McErlane3, Simon Stones4, Bridget Young1 1University of Liverpool; 2Alder Hey Children's NHS Foundation Trust; 3Great North Children's Hospital, Newcastle Hospitals NHS Foundation Trust; 4University of Leeds Correspondence: Frances Sherratt Trials 2017, 18(Suppl 1):P384 Background d Following on from the successful launch of LORIS in the UK, the trial is now leading the way internationally. LORIS is collaborating with COMET (USA) and LORD (Netherlands). Statistical analysis plans for internal pilots in randomised controlled trials 1 2 3 Cliona McDowell1, Evie Gardner2, David Harrison3, James McNamee4, Daniel F. McAuley5 1 2 p ; p ; y ; 6SHORE-C, Brighton and Sussex Medical School; 7Independent Cancer Patients' Voice; 8Ontario Institute for Cancer Research; 9University of Birmingham, Cancer Research UK Clinical Trials Unit; 10King's College London 1Northern Ireland Clinical Trials Unit; 2Northern Ireland Clinical Trials Unit, Belfast; 3Intensive Care National Audit & Research Centre, London; 4Regional Intensive Care Unit, Royal Victoria Hospital, BHSCT and Wellcome-Wolfson Institute for Experimental Medicine, Belfast; 5Northern Ireland Clinical Trials Unit, Regional Intensive Care Unit, Royal Victoria Hospital, BHSCT and Wellcome-Wolfson Institute for Experimental Medicine, Belfast Correspondence: Jennie Young Trials 2017, 18(Suppl 1):P385 References [1] Marmot MG, Altman DG, Cameron DA, Dewar JA, Thompson SG, Wilcox M. The benefits and harms of breast cancer screening: an independent review. Br J Cancer. 2013;108(11):2205–40. [2] Sagara Y, Mallory MA, Wong S, Aydogan F, desantis S, Barry WT, et al. Survival Benefit of Breast Surgery for Low-Grade Ductal Carcinoma In Situ: A Population-Based Cohort Study. JAMA surgery, 2015. Background It is common practice for clinical effectiveness trials to include an in- ternal pilot but guidance on how they should be analysed is lacking. The REST study is a pragmatic randomised controlled trial to deter- mine whether Veno-Venous Extracorporeal Carbon Dioxide Removal More recently, Sagara et al. [2] conducted a retrospective longitu- dinal cohort study in the USA investigating the survival benefit of breast surgery for DCIS. They found no significant difference in breast Trials 2017, 18(Suppl 1):200 Page 147 of 235 (VV-ECCO2R) in mechanically ventilated patients with hypoxaemic re- spiratory failure improves 90 day mortality. An internal 6-month pilot study in 10 sites to confirm both recruitment and adherence assump- tions that have contributed to study design will precede the main trial. p Discussion A formal SAP for an internal pilot is not standard clinical trial meth- odology and there are pros and cons to having one which are open for discussion. In general, the advantages are as for the SAP for the final analysis: to pre-specify the analysis and reporting in order to avoid intentional or unintentional bias caused by multiple unplanned analyses and selective reporting of data. Without a SAP, investigators who are convinced of the usefulness of an intervention may manipu- late pilot results to make a convincing case for progression to a full trial ultimately leading to a trial showing no difference and a waste of research funding. On the other hand, one may argue that the risks of multiple analyses and “data dredging” leading to spurious findings are primarily associated with large sample sizes that will not be present in a pilot study. Furthermore, relying too strictly on pre- specified criteria for progression to a full trial could result in stopping a trial of a promising treatment in the presence of practical issues with delivery that may be easy to overcome. One specific pro to having a SAP for the internal pilot that is separate from the full SAP is the ability to have the pilot analyses documented without the need to have the full SAP signed off at an early stage of the trial. The SPIRIT guidelines should give consideration to internal pilot studies and the need to document planned analyses and progression criteria. 1University of Leeds; 2University of Warwick Correspondence: Nada Elbeltagi Trials 2017, 18(Suppl 1):P388 Methods We will select all studies that were defined as pilot or feasibility studies in the study protocol, as well as any study that agrees with the definition of pilot or feasibility studies under the guidelines. We will follow the guidelines and report all of the elements proposed (e.g. Design, sample size calculation, objectives, type of outcomes, analyses, and whether they were used to estimate parameters that supported sample size calculations for a full trial). We will also re- port whether the study was an internal pilot or not, and whether the results of the pilot/feasibility study were published separately from the main trial. Results The literature review identified a number of parameters to be investi- gated, including phase II sample size, distribution of the underlying true treatment effect and phase II design. Additional parameters we will investigate are the strength of relationship between endpoints at phase II and III, and the population size. From the literature, a num- ber of measures were found to quantify the impact of these parame- ters on the success of phase II trials, including the number of patients required in both phases II and III before the first successful phase III.This measure takes into account the expected probability of success in phase II, E(P_1), and the expected probability of success in phases II and III, E(P_1 P_2). Since the phase III trial will only com- mence if the phase II trial is successful, these probabilities can be used to calculate the conditional probability of success at phase III, E(P_1 P_2)/(E(P_1)).Using this measure, we analytically evaluate the impact of each of the design parameters on the ability to successfully screen treatments at phase II. To start with, the phase II and phase III trials are assumed randomised, two-arm trials, with continuous nor- mally distributed endpoints in both phases II and III, and with an underlying true treatment effect that follows a normal distribution. Conclusions 1Population Health Research Institute, Mcmaster University Correspondence: Amparo Casanova Trials 2017, 18(Suppl 1):P387 P388 Investigating the impact of phase II trial design parameters on the ability to successfully screen new treatments for phase III trials Nada Elbeltagi1, Sarah Brown1, Nigel Stallard2, Julia Brown1, Walter Gregory1, Fiona Collinson1 1 2 Investigating the impact of phase II trial design parameters on the ability to successfully screen new treatments for phase III trials Nada Elbeltagi1, Sarah Brown1, Nigel Stallard2, Julia Brown1, Walter Gregory1, Fiona Collinson1 1 2 Method A statistical analysis plan (SAP) has been written for the internal pilot for the REST trial which will be signed off separately to the full trial SAP but will form an appendix to it once it’s written. It covers the analyses required for the internal pilot to assist with making the decision to progress to the full trial. For the REST internal pilot the following analyses will be completed: The overall recruitment rate will be compared to target recruitment of 7 per month. Analysis of separation in terms of tidal volume (ml/kg predicted bodyweight) between the two arms will be undertaken on an intention to treat and a per protocol basis including the patients in receipt of VV-ECCO2R on day 2 and 3. Completeness of datasets with respect to the primary outcome measure of 90 day mortality will be assessed for patients recruited in the first 3 months. Like a full trial SAP, the SAP for the internal pilot was reviewed by the Data Monitoring and Ethics Committee and the Trial Management Group. Background The purpose of phase II trials is to assess the potential activity of new therapies, which will then be investigated in larger, more time and resource consuming phase III trials. Currently, in oncology, the success rate of phase III trials is considerably low, suggesting that the phase II trials are allowing the progression of too many inefficacious therapies. Thus, early rejection of inactive treatments and moving therapies that are most likely to be efficacious to phase III testing would benefit patients and the drug development industry. The aim of this research is to investigate the impact of various design param- eters on the phase II trials’ ability to successfully screen new treat- ments in phase III. M h d p Methods A literature review was conducted in order to determine which phase II design parameters have previously been considered for impact on phase III outcomes, and to identify any additional parameters that may be considered. It also aimed to identify an appropriate measure of success which allows comparisons of phase II trial efficiency to be made. The impact of the varying design parameters on the identified measure of success will then be evaluated. P387 Pilot and feasibility studies: a systematic review of studies conducted at the population health research institute Amparo Casanova, Shun Fu Lee, Chinthanie Ramasundarahettige, Salim Yusuf 1Population Health Research Institute, Mcmaster University Correspondence: Amparo Casanova Trials 2017, 18(Suppl 1):P387 Conclusion A systematic review of pilot and feasibility studies under the recently published CONSORT guidelines may improve understating their ap- plication in a practical setting, and easy the implementation amongst researchers. p Results Utilty extrapolation is feasible. The more complex 5-parameter model appears to be the most useful (lowest AIC value of 92.4) in terms of predictive ability beyond 12 months. Two parameters were statisti- cally significant (p < 0.001). The Lorentz, Rational and 5-parameter models generated the most accurate estimates of mean PP utilities and QALYs: 0.474 vs. 0.508, 0.509 and 0.487 respectively for utilities; and 3.176 vs. 3.37, 3.37 and 3.26 for QALYs. Conclusion Modelling post progression utilities as well as extrapolation of util- ities beyond the study follow up appears feasible and is an alterna- tive to mapping or using published utility estimates. Age, recent cardiovascular events and contemporaneous CKD status were the most important determinants of adverse events. Predicted event rates from the model corresponded mostly well to those in the external cohorts. The model was superior to the PCREs in predicting cardiovascular risk, with predictions by the PCREs not adequately capturing variation in risk across CKD stages especially in CKD stage 3B (observed 5-year probability of a major vascular event in SHARP 8.6 [95% confidence interval 6.2-11.0] vs 9.4 predicted by the SHARP model vs 15.3 predicted by the PCREs) and dialysis (19.3 [16.2-22.2] observed in SHARP vs 17.9 predicted by the SHARP model vs 12.4 predicted by the PCREs). In predicting end-stage renal disease, the model performance was comparable to that of the Tangri model ?A3B2 show $132#?>(observed 5-year probability of end-stage renal disease in SHARP 36.5 [35.2-37.8] vs 34.2 predicted by the SHARP model vs 34.3 predicted by the Tangri model). P392 Embedding trials into established service improvement initiatives: challenges and lessons from an implementation laboratory Suzanne Hartley1, Robert Cicero1, Amanda Farrin1, Jill Francis2, Liz Glidewell1, Natalie Gould2, John Grant-Casey3, Fabiana Lorencatto2, Lauren Moreau1, Simon Stanworth3 1University of Leeds; 2City, University of London; 3NHS Blood & Transplant Correspondence: Suzanne Hartley Trials 2017, 18(Suppl 1):P392 1University of Leeds; 2City, University of London; 3NHS Blood & Transplant Correspondence: Suzanne Hartley Trials 2017, 18(Suppl 1):P392 There are opportunities to embed randomised trials of interventions to promote the uptake of evidence-based practice within established, large-scale improvement initiatives. Such “implementation laboratories” offer an efficient means of producing robust evidence generalizable to service settings. However, there are practical and methodological chal- lenges in delivering such programmes of work. Iryna Schlackow1, Borislava Mihaylova2, on behalf of SHARP Collaborative Group2 p 1University of Oxford; 2Nuffield Department of Population Health, University of Oxford Correspondence: Iryna Schlackow Trials 2017, 18(Suppl 1):P389 p 1University of Oxford; 2Nuffield Department of Population Health, University of Oxford 1University of Oxford; 2Nuffield Departme University of Oxford Correspondence: Iryna Schlackow Trials 2017, 18(Suppl 1):P389 Correspondence: Iryna Schlackow Trials 2017, 18(Suppl 1):P389 Methods The model is based on the individual patient-level data from the Study of Heart and Renal Protection (SHARP), which included 9,270 participants with moderate-to-severe CKD but no major coronary disease. Detailed sociodemographic and clinical characteristics at study entry and serious adverse events during the 5 years’ follow- up were recorded. A two-stage lifetime Markov model was devel- oped, comprising of a CKD submodel simulating progression of chronic kidney disease, and a CVD component simulating annual experience of fatal and nonfatal cardiovascular events and non- vascular death. Risk equations included baseline characteristics as well as important time-updated covariates (age, cardiovascular event and CKD status). Three external CKD cohorts (an observa- tional study, CRIB, and two randomised controlled trials, 4D and AURORA) were used to validate the model. Additionally, the model performance was compared against an external CVD risk score (the Pooled Cohort Risk Equations, PCREs) and an external risk score of progression to end-stage renal disease (the model by Tangri et al.). Results Background Background Chronic kidney disease (CKD) increases the risk of cardiovascular dis- ease (CVD) but, conversely, CVD may accelerate kidney disease progression. y In this research, we demonstrate the feasibility of extrapolation of utilities beyond disease progression and standard trial follow up for the purposes of estimating quality adjusted life years (QALYs) over a life time horizon through non-linear mixed effects modelling. Methods To develop, validate and make widely accessible a CKD disease model that takes into account interdependence of CKD and CVD and can be used to simulate disease outcomes and (quality-adjusted) life expect- ancy and incorporate effects of interventions that modify cardiovascu- lar risk in CKD patients. Data from an observational study in 100 lung cancer patients followed up for at least 12 months was used to extrapolate EQ-5D-3 L utilities after patients have progressed. Several non-linear models were postu- lated including a Lorentz, Rational, 5-Parameter, Pareto, Exponential and Linear models. Extrapolation of survival times were generated using a Royston-Parmar (3 Knott) flexible parametric survival model in order to estimate the QALY. Models were compared in terms of AIC and impact on QALY estimates. Background In many cancer trials, health related quality of life (HRQOL) are often collected up to disease progression. For health economic evaluation, a lifetime perspective of both costs and utilities is required to assess cost-effectiveness of cancer drugs. Therefore, ideally, utility data is re- quired beyond disease progression. In some cases assumptions are made about the behaviour of utility data (e.g. Such as constant, linear decline, or decaying). Other options include the possibility to determine utilities from historical data and rarely is any attempt made to extrapolate utility data beyond trial follow up, although this is commonly used for survival data. Iryna Schlackow1, Borislava Mihaylova2, on behalf of SHARP Collaborative Group2 Background Weaknesses in the design, conduct and reporting of pilot/feasibility studies have Weaknesses in the design, conduct and reporting of pilot/feasibility studies have Been shown by systematic reviews. Moreover, lack of agreement on the definition of a pilot or a feasibility study has made difficult to propose guidelines on the analyses and reporting of these studies, which often ended up not being published. The working group on developing reporting guidelines on pilot and feasibility studies, as an extension to the Consolidated Standards of Reporting Trials (CONSORT) Statement, created a conceptual framework to propose definitions for pilot and feasibility studies (Eldridge et al. 2016), and has recently published their guidelines (Thabane et al. 2016). We propose to systematically review the pilot and feasibility studies conducted at the Population Health Research Institute, McMaster University (our group) in the last 10 years, and assess the applica- tion of the new guidelines. The impact of the strength of relationship between the endpoints, and sample size, on the conditional probability of success of phase III trials will be presented. Understanding the impact of each of the Trials 2017, 18(Suppl 1):200 Page 148 of 235 Page 148 of 235 Trials 2017, 18(Suppl 1):200 design parameters on phase III trials will allow us to design efficient phase II trials which can more quickly move effective treatments to phase III testing. design parameters on phase III trials will allow us to design efficient phase II trials which can more quickly move effective treatments to phase III testing. Extrapolation of utilities between disease progression and death in cancer trials for economic evaluation Iftekhar Khan University of Warwick Trials 2017, 18(Suppl 1):P391 Extrapolation of utilities between disease progression and death in cancer trials for economic evaluation Iftekhar Khan University of Warwick Trials 2017, 18(Suppl 1):P391 Extrapolation of utilities between disease progression and death in cancer trials for economic evaluation Iftekhar Khan University of Warwick Trials 2017, 18(Suppl 1):P391 p Results P394 Developing a web-based central laboratory shipment scheduler and information system Kayla Daniels, Michael Frasketi, Dikla Shmueli-Blumberg, Peter Dawson The EMMES Corporation Correspondence: Kayla Daniels Trials 2017, 18(Suppl 1):P394 A number of challenges related to, or increased by, embedding re- search within an existing national audit programme have been highlighted to date in the AFFINITIE programme. These include: Communicating a message about equipoise to clinicians developing, delivering and receiving different feedback interventions who might otherwise feel advantaged or disadvantaged; Identifying and mitigat- ing threats of contamination between the enhanced and standard feedback arms of the trial; Preserving fidelity of intended enhanced feedback interventions on the pathway to their delivery; Ensuring that data quality and governance processes sufficiently meet the needs of both a national audit programme and a rigorous evaluation; Aligning research timelines with those of a rolling and evolving na- tional audit programme. The use of central laboratories in multi-site clinical trials is common to ensure consistency in reporting and analysis of assay results. The additional time between sample collection and analysis is typically not a burden for frozen or otherwise properly stored specimens. When assay results are time sensitive due either to the sample type (e.g. Fresh cells) or need for immediate results, local laboratories are often utilized. If an assay is novel or proprietary, however, the local laboratory may not be able to perform the assay and a specific cen- tral or research laboratory must be utilized. In several recent multi- center studies, an assay required shipment of whole blood samples and immediate labor intensive processing by a specific central la- boratory. Despite extensive piloting to reduce time from collection to analysis and obtain quality assay results, an unexpected predicament developed during the trial in which sites were shipping more speci- mens than the lab could successfully test in a timely fashion due to limited central laboratory resources; resulting in failed assays. The co- ordinating center for these studies had to find an immediate solution that was convenient and accessible to both central laboratory and site staff. In response, coordinating center staff developed and de- ployed an access controlled shipment scheduler web site to allow sites and lab staff to more efficiently coordinate. Clinical site users entered shipment details and a validation routine capped each site at a certain number and type of clinical sample shipments. p Results To facilitate the use of the model, a practical and flexible web inter- face was developed, which allows the user to execute analyses on individuals or patient cohorts and provides estimates, including the uncertainty, of the disease outcomes, life expectancy and cost- effectiveness of interventions. We will illustrate the model, and its interface, with examples of potential applications. g g p g Audit and Feedback (A&F) - defined as any summary of clinical per- formance of health care over a specified period of time, to provide healthcare professionals with data on performance - is widely used to improve the quality of healthcare. However, its effects are often unreli- able, indicating the need for coordinated research including more head-to-head trials comparing different ways of delivering feedback. Blood transfusions are a commonly used intervention in hospital care. Repeated national audits in the United Kingdom suggest that up to a fifth may be unnecessary when judged against recommen- dations for good clinical practice. The AFFINITIE programme The SHARP CKD-CVD lifetime risk model is a novel resource for simu- lating lifetime health outcomes, and effects of interventions to re- duce cardiovascular risk and kidney disease progression, in people with moderate-to-severe CKD. The freely available web-based inter- face allows for a wide range of policy relevant analyses. Page 149 of 235 Trials 2017, 18(Suppl 1):200 Page 149 of 235 Page 149 of 235 Many of these challenges, and their resolution, will be illustrated using a randomised controlled trial of ex-vivo normothermic perfu- sion, compared to standard cold storage in kidney transplantation. (Development & Evaluation of Audit and Feedback interventions to Increase evidence-based Transfusion practice) aims to test different ways of delivering feedback within the existing UK National Com- parative Audit of Blood Transfusion (NCABT). It includes two linked 2x2 factorial, cluster-randomised trials evaluating two theoretically- enhanced A&F interventions to reduce unnecessary blood transfu- sions in hospitals. Trial outcomes were derived from data collected for the national audit. Many of these challenges, and their resolution, will be illustrated using a randomised controlled trial of ex-vivo normothermic perfu- sion, compared to standard cold storage in kidney transplantation. P393 Clinical trials in organ donation and transplantation in the UK - benefits and challenges g Laura Pankhurst, Dave Collett NHS Blood and Transplant Correspondence: Laura Pankhurst Trials 2017, 18(Suppl 1):P393 Clinical trials in organ donation and transplantation are increasing in the UK but each presents their own set of challenges. The centrally maintained UK Transplant Registry, held by NHS Blood and Transplant, prospectively collects data on all organ donors, transplants and trans- plant recipients, including periodic follow up data, and is a particularly valuable resource for the conduct of clinical trials in this field. In particu- lar it facilitates power calculations, and data collection, especially for longer term outcomes. Alongside this, NHS Blood Transplant has a na- tional network of Specialist Nurses for Organ Donation, who seek con- sent for organ donation and the use of such organs in a clinical trial. There is very little time from when the organ is deemed suitable for transplantation, the potential recipient is notified and the organ is transplanted. Consent from the recipient to participate, and randomised treatment allocation, usually takes place in this short window. Streamlining and simplifying of these processes is essen- tial. Other challenges arise in trials in kidney transplantation, where deceased donors typically donate both kidneys, which have differ- ent anatomy, and are allocated in such a way a centre may receive one or both kidneys from a donor. Randomisation therefore re- quires careful consideration to take account of features such as this. Typically in transplantation, the key outcome of interest is graft or patient survival years. However, survival rates are high and differ- ences to be detected are generally small, and hence such an out- come can rarely be used as the primary outcome. Composite outcomes, or biomarker data have then to be used. P395 Ensuring a GCP compliant audit trail for EDC/EPROM Jonathan Gibb, Sharon Kean Robertson Centre for Biostatistics Correspondence: Jonathan Gibb Trials 2017, 18(Suppl 1):P395 P393 P393 Clinical trials in organ donation and transplantation in the UK - benefits and challenges Laura Pankhurst, Dave Collett NHS Blood and Transplant Correspondence: Laura Pankhurst Trials 2017, 18(Suppl 1):P393 p Results Central la- boratory users could then monitor when specimens would be shipped to them and indicate lab closures or other dates shipments could not be accepted, thereby allowing better allocation of lab staff resources. Addition of the shipment scheduler to the laboratory man- agement process resolved the difficulties involving laboratory cap- acity and the studies were able to continue obtaining novel, high quality assay results. This presentation will highlight some of the in- frastructure and functionality of the system that may be relevant and applicable for studies with existing laboratory management systems or those interested in creating such a process. Furthermore, we will discuss the importance of flexibility among the clinical trial manage- ment team and the value of adapting processes to correspond with constantly changing requirements of a research trial. From a methodological perspective these challenges suggest a ten- sion between internal and external validity (that is, bias and general- isability), characteristic of pragmatic trials, where greater importance is placed on generalisability. Embedding research within major improvement initiatives is, how- ever, feasible. We will present our recommendations in response to each challenge we have identified. These may assist researchers in optimising the conditions for sustainable implementation laborator- ies in the context of existing A&F programmes. These include: Embedding research within major improvement initiatives is, how- ever, feasible. We will present our recommendations in response to each challenge we have identified. These may assist researchers in optimising the conditions for sustainable implementation laborator- ies in the context of existing A&F programmes. These include: Negotiating shared expectations and ground rules for collaboration; Establishing joint processes for assuring the quality of data for audit and research; Aligning audit standards and trial endpoints wherever possible. Negotiating shared expectations and ground rules for collaboration; Establishing joint processes for assuring the quality of data for audit and research; Aligning audit standards and trial endpoints wherever possible. Our recommendations will be discussed in the context of a wider literature on designing pragmatic trials. y Methods The authors have been involved in the delivery of eight separate mo- bile device EDC systems, supporting online and/or offline modes, four different languages, web based and native application and have gained quite a lot of experience in this area in a short time due to the nature of the studies involved. Background Sales in mobile devices are out stripping traditional PC sales and a number of analysts expect this trend to continue and the gap to widen. In line with this trend, the number of trials that wish to in- clude mobile device based EDC systems has risen significantly in the last five years. Jonathan Gibb, Sharon Kean, William Aitchison 1Robertson Centre for Biostatistics Correspondence: Jonathan Gibb Trials 2017, 18(Suppl 1):P396 Jonathan Gibb, Sharon Kean, William Aitchison 1Robertson Centre for Biostatistics Correspondence: Jonathan Gibb Trials 2017, 18(Suppl 1):P396 Jonathan Gibb, Sharon Kean, William Aitchison 1Robertson Centre for Biostatistics Correspondence: Jonathan Gibb Trials 2017, 18(Suppl 1):P396 Jonathan Gibb, Sharon Kean, William Aitchison 1Robertson Centre for Biostatistics Correspondence: Jonathan Gibb Developing tablet based EDC apps Jonathan Gibb, Dionne Russell, William Aitchison, Sharon Kean Glasgow Clinical Trials Unit Developing tablet based EDC apps Jonathan Gibb, Dionne Russell, William Aitchison, Sharon Kean Glasgow Clinical Trials Unit g Correspondence: Jonathan Gibb Trials 2017, 18(Suppl 1):P397 Background An eval() mechanism is one which evaluates and executes code at run- time, it exists in different forms in almost every programming ?A3B2 show $132#?>language and platform. In computing it has long been known that the use of eval() mechanisms pose a security threat, the code being executed likely originated with the user of a system and therefore should be treated with caution. A large proportion of Clinical Trial Management Systems (CTMS) and Electronic Data Capture (EDC) systems allow users who design forms to type in snippets of code and then run these during system use by using an eval() mechanism. Eval() is employed despite the security concerns posed because it is an easy way to incorporate user defined rules into a system e.g. Form skip fill rules. The ability to pass the rule content to the underlying platform for execution without the requirement to understand what is being passed, drastically simplifies the development task when building the system. Apart from the security concerns, not understanding the user defined rules significantly limits the EDC’a abilities and function includ- ing but not limited to: All user entered code blocks are subject to GCP validation requirements however the EDC is limited in what tooling and support it can provide when the user defined code block is not understood e.g. Automated creation of test scenarios. Code blocks that are not “understood” cannot be translated; therefore one is limited to the language/platform they were written for initially. To run the code on any new platform, one has to essentially emulate the old platform in the new platform. Background g In September 2016 the UK competent authority in relation to clinical trial regulations, the Medicines and Healthcare products Regulatory Agency (MHRA) Ran an annual good clinical practise (GCP) sympo- sium. One of the main topics discussed was data integrity and numerous common problems found during site inspections. The MHRA highlighted numerous deficiencies found in electronic systems, the majority of these were well known and it could be con- sidered surprising that such deficiencies still exist. There was how- ever one particular notice from the MHRA that has wide reaching implications for EDC systems as it is believed almost no systems are currently compliant. The MHRA highlighted the case that when a electronic record is saved, all of the variables on the page are saved Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 150 of 235 with the identical timestamp. The timestamp represents the point of saving, not when the individual fields were actually entered. Further- more, the MHRA highlighted that the audit mechanism in electronic systems usually operated in such a way that the electronic system could allow unaudited changes to data which would be captured on paper when following GCP guidelines. On paper, an edit to most pieces of data require that the piece of data be scored through once, and then a replacement value written down, dated and initialled. In most electronic systems, only the state of data at the time of saving is recorded; any changes made prior to saving are completely lost. One could argue that keyboard usage is more prone to error, there- fore typographical errors should not be recorded. However, how could the system distinguish between typographical errors and change of answers? There is a very real potential cost to this require- ment in terms of data storage. A naïve solution would be to capture the answer at every stage of entry; however this leads to huge over- heads in data storage. If a surname is 10 characters long then typic- ally that requires 20 bytes of storage, if however you storage each state of the surname as it is entered, assuming there are no actual edits or corrections then the storage requirement balloons to 100 bytes. those who are fluent in the specific programming language – to ver- ify that the code meets the requirements. with the identical timestamp. P398 The cocoa supplement and multivitamin outcomes study in the mind (cosmos-mind): design and rationale g Sarah Gaussoin, Mark A. Espeland, Sally A. Shumaker, Stephen R. Rapp, Darrin Harris, Debbie Pleasants, Julia Robertson, Laura D. Baker Wake Forest School of Medicine Correspondence: Sarah Gaussoin Trials 2017, 18(Suppl 1):P398 Identifying a safe, affordable, and well-tolerated intervention that prevents or delays cognitive decline in older adults is of critical importance. There is growing evidence from basic science and small randomized trials that cocoa flavanols may provide protec- tion against this decline. COSMOS-Mind is an NIA-funded ancillary study to the Cocoa Supplement and Multivitamin Outcomes Study, Conclusion The authors will present the best practises learned from delivering these applications in their various modes and explain why as an or- ganisation we have decided to develop future native applications in their operating system specific development environment, despite the duplication of work, rather than rely on an intermediary platform like Xamarin. Background The timestamp represents the point of saving, not when the individual fields were actually entered. Further- more, the MHRA highlighted that the audit mechanism in electronic systems usually operated in such a way that the electronic system could allow unaudited changes to data which would be captured on paper when following GCP guidelines. On paper, an edit to most pieces of data require that the piece of data be scored through once, and then a replacement value written down, dated and initialled. In most electronic systems, only the state of data at the time of saving is recorded; any changes made prior to saving are completely lost. One could argue that keyboard usage is more prone to error, there- fore typographical errors should not be recorded. However, how could the system distinguish between typographical errors and change of answers? There is a very real potential cost to this require- ment in terms of data storage. A naïve solution would be to capture the answer at every stage of entry; however this leads to huge over- heads in data storage. If a surname is 10 characters long then typic- ally that requires 20 bytes of storage, if however you storage each state of the surname as it is entered, assuming there are no actual edits or corrections then the storage requirement balloons to 100 bytes. those who are fluent in the specific programming language – to ver- ify that the code meets the requirements. Conclusion The authors will present an overview of different mechanisms to break- down code blocks into understandable units from classic compiler design to more adhoc methods and the various things that can be achieved including: test data generation, automatic test execution, documentation production and programming language translation. P397 Developing tablet based EDC apps Jonathan Gibb, Dionne Russell, William Aitchison, Sharon Kean Glasgow Clinical Trials Unit Correspondence: Jonathan Gibb Trials 2017, 18(Suppl 1):P397 y Methods Various problems in audit trail mechanisms highlighted by the MHRA will be discussed along with common solutions from production sys- tems. A detailed investigation at the problem of between save auditing will take place including discussion of various solution approaches and their merit on a number of different electronic platforms. y There are two main types of EDC: Native application based or web based. While the web based approach does support offline function- ality, there are serious concerns over the security of data in offline web based systems with the current technology. Therefore, if you wish your mobile device EDC to function offline you are limited to native applications. The evil of eval() in CTMS – how to get more from user defined code The authors utilised Xamarin, a system that advertises multiplatform support to allow applications to be delivered for multiple operating systems. Conclusion All staff involved with the creation, maintenance or usage of EDC sys- tems should be aware of the GCP audit requirements highlighted by the MHRA in the UK. Even to those who deal with other competent authorities, as this is a direct GCP requirement, it is likely the issue will arise across all GCP bound component authorities. There are three main mobile device operating systems: android, ios and windows. Therefore having decided to produce a native applica- tion one must consider which operating system(s) you will build applications for. Obviously building the same application for multiple operating systems seems counter intuitive. To this end, a number of products exist on the market offering a middle ground that allows you to build one application that will run natively on all three operating systems. Methods If the code blocks are “Understood” then they can be further trans- lated to a non-programming language to allow all job roles – not just Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 151 of 235 a 2x2 factorial randomized controlled trial testing the effects of cocoa flavanols (600 mg/d) and a multivitamin with matching placebo on cardiovascular disease and cancer endpoints. The primary aim of COSMOS-MIND is to assess the effects of COSMOS supplements on cognitive trajectory over 3 years of follow-up using a composite meas- ure of cognitive function. An important secondary outcome is incident Mild Cognitive Impairment or Alzheimer’s disease and other dementias. A validated telephone-based protocol conducted at baseline and annually thereafter assesses attention, memory, executive function, lan- guage, and global cognitive functioning in 2,000 women and men aged 65 years and older without insulin-dependent diabetes. Add- itional measures administered by telephone assess subjective memory concerns, depression, and insomnia. For participants who score below a pre-specified threshold on a test of global cognition, a study partner will be interviewed to obtain additional information regarding cognitive and functional status. A web-based telephone call tracking system is used to prioritize COSMOS-Mind enrollment to ensure diverse represen- tation. Real-time reports monitoring study calls provide information to detect issues that may impact recruitment goals, data collection, personnel resources, and costs. At the end of follow-up, cognitive status will be adjudicated by an expert panel to identify Mild Cognitive Im- pairment, and Alzheimer’s and related dementias. Enrollment will be completed in the summer of 2017. COSMOS-Mind will establish whether daily use of a cocoa flavanol supplement, with or without a multivitamin, can protect cognitive function and reduce incidence of cognitive impairment, including Alzheimer’s dementia. Conclusion The purpose, activities and outputs of the ISOG in collaboration with other UKCRC operational groups will be presented. Increasing the availability of statistical tools through mobile development 2 p Chris Cook1, Taylor Phillips1, Michael LeBlanc2 1 2 Chris Cook1, Taylor Phillips1, Michael LeBlanc2 1Cancer Research And Biostatistics; 2Fred Hutchinson Cancer Chris Cook1, Taylor Phillips1, Michael LeBlanc2 1Cancer Research And Biostatistics; 2Fred Hutchinson Cancer Research Center Correspondence: Chris Cook Trials 2017, 18(Suppl 1):P401 P399 Development of a safe haven analytical platform Sharon Kean1, Alan Stevenson1, Allen Tervit1, Marion Flood2 1University of Glasgow; 2Greater Glasgow & Clyde Health Board Correspondence: Sharon Kean Trials 2017, 18(Suppl 1):P399 p Trials 2017, 18(Suppl 1):P401 SWOG has a free set of online statistical tools that are used widely across the world at stattools.crab.org. We were interested in further expanding the reach of these tools by making them ‘mobile friendly’. We wanted users to have a similar user experience on smartphones and tablets as they would on a normal desktop computer. This would give users an opportunity to use statistical tools wherever they went. SWOG wanted to also give training sessions where we can demonstrate statistical tools and then have attendees follow along on their mobile device to provide an active learning experience. SWOG has a free set of online statistical tools that are used widely across the world at stattools.crab.org. We were interested in further expanding the reach of these tools by making them ‘mobile friendly’. We wanted users to have a similar user experience on smartphones and tablets as they would on a normal desktop computer. This would give users an opportunity to use statistical tools wherever they went. SWOG wanted to also give training sessions where we can demonstrate statistical tools and then have attendees follow along on their mobile device to provide an active learning experience. Method The Glasgow Safe Haven is a collaboration between University of Glasgow (UOG) and Greater Glasgow & Clyde Health Board. Approval is sought from a local privacy advisory committee for access to non- consented data on a per research project basis. Once approved, the data are extracted, anonymised and placed on an analytical platform hosted by UOG where researchers can then perform their analysis in a strictly monitored environment. In order to become a “safe haven” analytical platform, certain requirements need to be adhered to. Se- curity is paramount and for very sensitive datasets the creation and use of a “safe room” is required. y To ensure a successful implementation of the site, SWOG added website analytics to obtain more metrics on what devices are using the site and how they’re using it. There were several questions we wanted to answer: What devices are most commonly used on the website and were they able to use the site successfully? Did allowing mobile access give opportunities for new users? Are mobile devices utilized differently by users in different countries? We plan to gather this data to help answer these questions over the coming months and use them to improve the site. Background Using routinely collected data for clinical trials is on the increase and funders are recognising that this is an excellent method to get answers to many research questions. In order to utilise non-consented electronic records such as hospitalisation admission and discharge records, deaths and prescribing data requires appropriate governance and a secure IT environment which fulfils the requirements of the data providers. p g p In July 2016, SWOG upgraded its statistical tools site to be ‘mobile friendly’ or responsive. The site is now able to dynamically change its interface to accommodate the different devices, screen sizes, and browsers of the different users that access it. The current site sup- ports all mobile platforms including iphones, ipads, Android, black- berry, and Microsoft devices. This presented a technical challenge due to the sheer variety of devices and interfaces on the market. Conclusion The technical infrastructure to comply with data providers require- ments will be presented. Governance procedures and oversight will be discussed. Exemplar project to demonstrate the value of using this method for research will be demonstrated. p Support: NIH/NCI/NCTN grants CA180819, CA180888. Methods The Information Systems Operational Group has a mandate to foster collaboration and improve quality through self-support (within the group) to help ensure robust secure and regulatory compliant provision of IS systems to registered CTUs. The objectives and sup- port themes undertaken by the group evolve over time to take into account changes in the regulatory environment and technological methodology are directed by the UK group (consisting of representa- tives from all registered CTUs) as a whole following national meet- ings or feedback from working groups. The activities of the ISOG are overseen and directed by a steering group consisting of members proposed by the directors of the registered CTU’s and mandated by the UKCRC executive. Conclusion Conclusion Background The UK Clinical Research Collaboration (UKCRC) partner’s goal is to establish the UK as a world leader in clinical research. The UKCRC provides a forum that enables all partners to work together to trans- form the clinical research environment within the UK. M th d P400 P402 Architecture design of an automated study drug distribution coordination module integrated in a web-based clinical trial management system The UKCRC information systems operational group: what is it we do? Sharon Kean1, Will Crocombe2, Ian Kennedy3, Danny Kirby4, Duncan Appelbe5, Carolyn McNamara6, Tim Chater7 1Glasgow Clinical Trials Unit; 2Leeds CTU, University of Leeds; 3Diabetes Trials Unit Oxford, University of Oxford; 4Leicester CTU, University of Leicester; 5Liverpool CTU, University of Liverpool; 6Institute of Cancer Research Clinical trials and Statistics unit; 7Sheffield CTRU, University of Sheffield g y Wenle Zhao Medical University of South Carolina Trials 2017, 18(Suppl 1):P402 High quality study drug distribution management is essential to the operation efficiency and quality of large multicenter clinical trials. First, each site must have a sufficient drug inventory, i.e. At least one Correspondence: Sharon Kean Trials 2017, 18(Suppl 1):P400 Trials 2017, 18(Suppl 1):200 Page 152 of 235 Page 152 of 235 drug kit for each treatment arm, in order to perform subject randomization. When response adaptive randomization is applied, the treatment allocation ratio may change multiple times during the study period, and corresponding site drug inventory adjustment may be needed. Second, the high cost and short shelf life of study drug and the slow subject recruitment speed may demand to minimize the site drug inventory. Finally, study drug distribution management must take into account the protection of treatment allocation con- cealment and treatment blinding, the time and financial cost of study drug shipping. To accomplish these critical tasks, traditional manual drug distribution coordination, often managed using spread sheets, is not reliable. As the national data management center for several NIH-funded clinical trial networks, we have developed a gen- eric study drug tracking module integrated in the web-based clinical trial management system. It provides automated coordination of study drug distribution for multicenter clinical trials. Study drug re- quests are automatically generated when the site drug inventory is less than the pre-specified threshold and are triggered by a new subject randomization, confirmation of drug removal from site inventory for any reason. The entire study drug shipping process, from the central pharmacy to the clinical sites, including optional multiple regional drug depots, is tracked and information is shared among collaborators in real time. Central and local pharmacy staff are notified by automated emails when study drug distribution actions are requested. This module has been implemented in 8 multicenter trials, and received very positive feedback from investigators. p Objective d f Objective To identify studies which have involved patients and members of the public (as research partners, co-investigators, advisors or research team members), to develop COS or proms and describe the ways in which PPI has been conceptualised and reported in these studies, the methods of PPI used, and the contexts in which PPI has been employed. Challenges CTC-STOP blood samples are received and processed by the central laboratory. CTC counts are recorded in the central laboratory’s information management system (LIMS). The LIMS cannot be programmed to calculate or alert staff to CTC progressions and the ICR-CTSU cannot access LIMS. CTC counts are required by the ICR-CTSU trial team for CTC progression calculations within the Electronic Data Capture (EDC) system, so treatment switch recommendations can be issued promptly. Manual data re-entry of the CTC counts into an EDC database would be time consuming and could result in transcription errors; therefore a system was designed to automatically import the CTC counts from the LIMS into the EDC system. Solution tinuation of t Conclusion The application provides a robust, time critical, automatic solution to accurately import LIMS data into the EDC system, reducing workload for both the ICR-CTSU team and laboratory staff and removing error rates associated with manual data entry. The application also provides the ICR-CTSU trial team and site staff, including treating clinicians, with real-time access to the CTC counts. 0 Collating the evidence base to facilitate patient and public involvement in core outcome set development - A qualitative meta-synthesis Lucy Brading1, Kerry Woolfall2, Paula Williamson3, Bridget Young2 1University of Liverpool; 2University of Liverpool, Institute of Psychology Health and Society/North West Hub for Trials Methodology Research; 3University of Liverpool, Department of Biostatistics/North West Hub for Trials Methodology Research Correspondence: Lucy Brading Trials 2017, 18(Suppl 1):P404 P403 Automated solution for importing lab test results from a laboratory information management system into an electronic data capture system Elizabeth Hill, Nuria Porta, Miguel Miranda, Marie Hyslop, Penelope Flohr, Rebecca Lewis, Edward Heath, Claire Snowdon, Johann de Bono, Emma Hall 1The Institute of Cancer Research Correspondence: Elizabeth Hill Trials 2017, 18(Suppl 1):P403 Background p y Elizabeth Hill, Nuria Porta, Miguel Miranda, Marie Hyslop, Penelope Flohr, Rebecca Lewis, Edward Heath, Claire Snowdon, Johann de Bono, Emma Hall 1The Institute of Cancer Research Correspondence: Elizabeth Hill Trials 2017, 18(Suppl 1):P403 Patient and public involvement (PPI) in health research has grown rap- idly and is considered a key component of good research. Patient and public input into the development of core outcome sets (COS), which specify which outcomes should be measured and reported as a mini- mum in trials within specific health areas, is also increasing. It is vital to the credibility of a core outcome set that the chosen outcomes are rele- vant to both health professionals and patients and it is believed that this can be assisted through PPI in the development process. Patient and public involvement (PPI) in health research has grown rap- idly and is considered a key component of good research. Patient and public input into the development of core outcome sets (COS), which specify which outcomes should be measured and reported as a mini- mum in trials within specific health areas, is also increasing. It is vital to the credibility of a core outcome set that the chosen outcomes are rele- vant to both health professionals and patients and it is believed that this can be assisted through PPI in the development process. Whilst research has investigated PPI in clinical trials and service im- provement, PPI in the development of COS is yet to be explored; des- pite some distinct challenges and the need for guidance on how best to involve patients in COS development. As part of a wider project to inform methods to facilitate PPI in COS development, we are con- ducted a review and qualitative meta-synthesis of published COS stud- ies that have reported on PPI. Meta-synthesis involves the integration of themes from numerous qualitative studies; the technique is inter- pretive and yields findings that are ‘greater than the sum of the parts’. As few published COS studies have reported on PPI, the scope of the review was widened to also include PPI in the development of patient-reported outcome measures (proms). It was anticipated that the challenges encountered in the development of both COS and proms would be analogous. P400 This presentation will discuss the database design and implementation of the automated study drug distribution coordination module in the clinical trial management system. the number of data rows successfully imported into the EDC system. When the application has completed processing the log file is emailed to the ICR-CTSU trial team for review. Details of each export file processed are also written to a database history table. the number of data rows successfully imported into the EDC system. When the application has completed processing the log file is emailed to the ICR-CTSU trial team for review. Details of each export file processed are also written to a database history table. The CTC counts imported into the EDC system are used to perform computations to ascertain whether a treatment switch is required by calculating whether each subject has progressed or responded. The EDC system’s event management tool automatically alerts the ICR- CTSU trial team if the imported results reveal an initial progression event requiring a second confirmatory CTC count, a confirmation of progression requiring a treatment switch recommendation or a con- tinuation of therapy confirmation for any participant. Conclusion Background CTC-STOP is a multicentre phase III trial for castration resistant prostate cancer patients with bone metastases (MCRPC). The trial is designed to determine if the use of serial circulating tumour cells (CTC) counts can direct early discontinuation of docetaxel chemotherapy in mcrpc patients without adversely impacting overall survival, when compared with stand- ard approaches to guide treatment switch decisions. Treatment switch recommendations based on CTC progression are centrally managed by the co-ordinating clinical trials unit (ICR-CTSU) and require real time trans- fer of CTC counts from the central laboratory to the ICR-CTSU. From molecule to medicine: a case study in how a statistician can provide strategic input to drug development P407 Clinical trials in neonatology: design and analyses issues Abhik Das1, Jon Tyson2, Claudia Pedroza2, Barbara Schmidt3, Marie Gantz1, Dennis Wallace1, William Truog4, Rosemary Higgins5 1RTI International; 2University of Texas Health Sciences Center; 3University of Pennsylvania; 4Children’s Mercy Hospital; 5NICHD, NIH Correspondence: Abhik Das Trials 2017, 18(Suppl 1):P407 Challenges in teaching clinical trials: the experience of teaching biostatistics in online post-graduate academic courses, target to industrial biometrician Laura Cavaliere1, Egle Perissinotto1, Ileana Baldi1, Beatrice Barbetta2, Dario Gregori1 1 2 Impressive advances in neonatology have occurred over the 30 years of life of The Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network (NRN). However, substantial room for improvement remains in investigating and further developing the evidence base for improving outcomes among the extremely premature. We discuss some of the specific methodological challenges in the statistical design and analysis of randomized trials in this population. Challenges faced by the NRN, applicable to all neonatal trials, include designing trials for unusual or rare outcomes, accounting for and explaining center variations, identifying other subgroup differences, and balancing safety and effi- cacy concerns between short-term hospital outcomes and longer term neurodevelopmental outcomes. The constellation of unique pa- tient characteristics in neonates calls for broad understanding and careful consideration of the issues identified in this presentation for conducting rigorous randomized trials in this population. g 1University of Padova; 2Rottapharm Biotech Correspondence: Laura Cavaliere Trials 2017, 18(Suppl 1):P406 Impressive advances in neonatology have occurred over the 30 years of life of The Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network (NRN). However, substantial room for improvement remains in investigating and further developing the evidence base for improving outcomes among the extremely premature. We discuss some of the specific methodological challenges in the statistical design and analysis of randomized trials in this population. Challenges faced by the NRN, applicable to all neonatal trials, include designing trials for unusual or rare outcomes, accounting for and explaining center variations, identifying other subgroup differences, and balancing safety and effi- cacy concerns between short-term hospital outcomes and longer term neurodevelopmental outcomes. The constellation of unique pa- tient characteristics in neonates calls for broad understanding and careful consideration of the issues identified in this presentation for conducting rigorous randomized trials in this population. From molecule to medicine: a case study in how a statistician can provide strategic input to drug development From molecule to medicine: a case study in how a statistician can provide strategic input to drug development Nelson Kinnersley Roche Products Ltd Trials 2017, 18(Suppl 1):P405 provide strategic input to d Nelson Kinnersley Roche Products Ltd Trials 2017, 18(Suppl 1):P405 Twelve students in the basic course and thirteen in the advanced course were able to access to Moodle platform. Most of the students were workers. Median number of individual accesses to Moodle plat- form was 458 for the basic course and 931 for the advanced one. They mostly appreciated: self-administration of hour, number and time of ac- cess and stimulating discussion board online. They reported as worst limits: inadequate preliminary knowledge to understand new biostatis- tics concepts (median score 6.3 for the basic course, 6.6 for the ad- vanced one), too elevated study load in the advanced course (median score 6.7), time-spending searching concepts in videos during study and reviewing topics due to streaming modality. C l i Trials 2017, 18(Suppl 1):P405 When evolving the design of a clinical trial, statisticians may spend a considerable amount of time to optimise certain characteristics of the proposed trial and there is considerable literature to support such work. However, the literature is more sparse on the strategic factors that a statistician should consider when involved with designing an entire drug development programme. The aim of this work is to use a suitably anonymised case study to inform the practising statistician about the considerations for strategic drug development. Through liberal use of scenarios covering topics such as Target Product Profile (TPP), Clinical Development Plans (CDP), gating criteria and probability of success we will offer suggestions for how a statistician can contribute to strategic drug development. Many concepts are applicable across a variety of therapeutic areas even if the technical implementations may differ. It is hoped that with a wider understanding of strategic drug development, more statisticians can be better equipped to contribute to the cross- functional teams who perform this type of work when the plans are be- ing developed for how to turn a molecule into a medicine. Our experience supports feasibility and efficacy of online distance learning in teaching biostatistics. The experience suggests elaborating the following tools: videos length shorter than 20 min, lists of main concept and definition indicating the position (minute) in videos, wide- spread operative examples, timely matching of concepts and examples. Background At today, new technologies and widespread web access are moving the traditional face-to-face teaching towards online and open digital teaching processes. New technologies provide widespread flexible and convenient learning tools overcoming any temporal and geo- graphical constrains. The instances of innovation gradually enhanced higher education institutions towards new models of e-learning. The challenge is furtherly hard for teaching Biostatistics to health profes- sionals and medical students, who are in general very little motivated to learn statistics in the traditional academic courses. Method The review is currently underway; we are searching the COMET Initia- tive’s database of all completed work in selecting COS to identify studies with PPI. We developed a search strategy to identify PROM development projects with PPI, via searches of MEDLINE, psycinfo, hapi and the Cochrane Methodology Register. Conclusion An in-house C#.net application was developed by ICR-CTSU to verify and import CTC counts provided by the central laboratory LIMS. The application locates and verifies the LIMS export file before validating the subject identifiers and trial visit timepoint for each CTC count. Once validated, the data is imported into the EDC system using its Application Programming Interface. The application runs automatic- ally every day via Windows Task Scheduler and generates a log file for each export file to detail any errors found during processing and Previous research has highlighted the need for guidance on how best to seek the input of patients and the public in developing COS. As part Page 153 of 235 Trials 2017, 18(Suppl 1):200 Page 153 of 235 Trials 2017, 18(Suppl 1):200 of a wider project investigating PPI in COS development, the findings from this review will inform the development of guidelines for the COS development community on methods for involving patients as research partners, co-investigators, advisors or research team members. These guidelines will facilitate improved engagement with patients - one of the key stakeholders in COS development. work tools was planned, the quality and accessibility of platform was conveniently tested and the students’ Access to the platform was monitored. The computerized portal for education was based on MOODLE platform. To realize the fully online courses, different tools were built: streaming videos (10–40 minutes) with in person teachers’ explanation, slides highlighting central concepts, self-tests with multiple-choice questions and simulation-based tests with unlimited access, and homework with supervisor. Moodle platform allowed stu- dents for documents download and upload. At the end of each mod- ule a questionnaire regarding the assessment of teaching has been reported, the questions were proposed in a Likert scale from 1 to 10. Results of a wider project investigating PPI in COS development, the findings from this review will inform the development of guidelines for the COS development community on methods for involving patients as research partners, co-investigators, advisors or research team members. These guidelines will facilitate improved engagement with patients - one of the key stakeholders in COS development. Material and methods In the academic year 2015/2016, first in the teaching experience at the University of Padova, two fully online post-graduate courses of Biostatistics were established: the first one taught basic biostatistics and research methodology; the second one, advanced topics in biostatistics. The courses were organized into two phases: modules dealing with structured topics (25 weeks, each unit from 2 to 5 weeks) and a project-work (20 weeks). To perform statistical ana- lysis, the R software was adopted. The student-centered model was used instead of the traditional teacher-centered model. Each student independently manages her/his access to the web pages contents, without limits in number of accesses, within the timetable. She/he attends the didactic activities individually, preforming self-test and participating in the discussion online. From the teaching point of view, the face-to-face lessons components of contents, interaction and assessment were translated into digital and online contents and tools; from the logistic point of view, the publication time of the Method work tools was planned, the quality and accessibility of platform was conveniently tested and the students’ Access to the platform was monitored. The computerized portal for education was based on MOODLE platform. To realize the fully online courses, different tools were built: streaming videos (10–40 minutes) with in person teachers’ explanation, slides highlighting central concepts, self-tests with multiple-choice questions and simulation-based tests with unlimited access, and homework with supervisor. Moodle platform allowed stu- dents for documents download and upload. At the end of each mod- ule a questionnaire regarding the assessment of teaching has been reported, the questions were proposed in a Likert scale from 1 to 10. Results P405 From molecule to medicine: a case study in how a statistician can provide strategic input to drug development y Results Trialists spend much more time on secondary outcomes than primar- ies. This is not surprising; there are more secondary outcomes. What is more surprising is how much more: some trials spend more than 20 times as much time collecting secondary outcome data as primary outcome data. As an example, one trial spent 66 hours collecting primary outcome data and 1466 hours on secondaries. Using UK costing data, this is approximately £2908 on primary data collection and £63990 on secondaries. Trials that spend less than 10% of data collection effort on primary outcomes seem common. The median ra- tio of time to obtain primary to secondary outcomes is 1:8. Independent adjudication of neonatal cranial ultrasound scans in a pilot randomised trial 1 2 1 1 Lucy Bradshaw1, Jon Dorling2, Lelia Duley1, Lindsay Armstrong-Buisseret1, Joe Fawke3, Bernard Schoonakker4, Eleanor Mitchell1, Rob Dineen5 1Nottingham Clinical Trials Unit, University of Nottingham; 2Early Life Research Group, University of Nottingham; 3Leicester Neonatal Service, University Hosptials Leicester NHS Trust; 4Nottingham Neonatal Service, Nottingham University Hospitals NHS Trust; 5Division of Clinical Neuroscience, University of Nottingham Trials 2017, 18(Suppl 1):P408 This abstract is not included here as it has already been published. Trials 2017, 18(Suppl 1):200 Page 154 of 235 P409 Time well spent? A comparison of the work associated with collecting primary and secondary outcomes David Pickles1, Shaun Treweek2 1Leeds Teaching Hospitals NHS Trust; 2University of Aberdeen, Health Services Research Unit Correspondence: David Pickles Trials 2017, 18(Suppl 1):P409 P409 Time well spent? A comparison of the work associated with collecting primary and secondary outcomes David Pickles1, Shaun Treweek2 1Leeds Teaching Hospitals NHS Trust; 2University of Aberdeen, Health Services Research Unit Correspondence: David Pickles Trials 2017, 18(Suppl 1):P409 Background I i l Increasingly, researchers are being asked to demonstrate the impact of their research to their sponsors, funders and fellow academics. However, the most appropriate way of measuring the impact of healthcare research is subject to debate. We aimed to identify the existing frameworks used to measure healthcare research impact and to summarise the common themes and metrics in an impact matrix. Methods P409 Time well spent? A comparison of the work associated with collecting primary and secondary outcomes David Pickles1, Shaun Treweek2 Twenty-four unique frameworks were identified, addressing five broad categories of impact: (1) ‘Primary research-related impact’, (2) ‘Influence on policy-making’, (3) ‘Health and health systems impact’, (4) ‘Health-related and societal impact’, and (5) ‘Broader economic im- pact’. These categories were subdivided into 16 common impact subgroups. Authors of the included publications proposed 80 different metrics aimed at measuring impact in these areas. Conclusions 1Leeds Teaching Hospitals NHS Trust; 2University of Aberdeen, Health Services Research Unit Correspondence: David Pickles Trials 2017, 18(Suppl 1):P409 Methods The COMET database currently has 146 ongoing registered studies, of which 124 aim to include patient and public representatives. We will identify the COS developer leads for these studies and invite them to participate in a short online survey. The survey will establish the capacity in which patients are being included (involvement vs. Participation), developers’ methods for enabling patient inclusion, and their rationale for choosing a particular method(s). This survey will be conducted in English. Correspondence: Samantha Cruz Rivera Trials 2017, 18(Suppl 1):P410 Correspondence: Samantha Cruz Rivera Trials 2017, 18(Suppl 1):P410 Background The measurement of research impact is an essential exercise to help direct the allocation of limited research resources, to maximise bene- fit and help minimise research waste. This review provides a collect- ive summary of existing impact frameworks, which funders may use to inform the measurement of research impact and researchers may use to inform study design decisions aimed at maximising the short, medium and long-term impact of their research. Trials are essential but often inefficient. Some of this inefficiency is due to designs that burden both trial participants and trial teams with measurements that are not essential to answer the trial’s main research questions. Trialists are generally good at selecting their pri- mary outcomes - the outcomes they consider most important. Trial- ists have less focus when it comes to secondary outcomes. Maximising research impact: a systematic review of research impact frameworks Maximising research impact: a systematic review of research impact frameworks Samantha Cruz Rivera, Derek G. Kyte, Olalekan Aiyegbusi, Thomas J. Keeley, Melanie Calvert Centre for Patient Reported Outcomes Research, University of Birmingham Correspondence: Samantha Cruz Rivera Trials 2017, 18(Suppl 1):P410 To investigate which methods are being used by COS developers to facilitate patient participation in ongoing studies and the rationale behind using that method. Samantha Cruz Rivera, Derek G. Kyte, Olalekan Aiyegbusi, Thomas J. Keeley, Melanie Calvert y Centre for Patient Reported Outcomes Research, University of Birmingham Method Keywords: medical research impact, impact metrics, research impact framework A random selection of 115 protocols for publicly funded, randomised trials published 2010–2014 were selected (roughly 24 per year) for analysis. To date, twenty trials have been examined. The primary and secondary outcomes were extracted from protocols. Data on time to complete each outcome were sought from protocols; where timing was not available, these data were requested from the corresponding author, or from trial managers familiar with the outcome. To date, twenty trials have been examined. Methods for including patients in core outcome set deve Alice Biggane1, Lucy Brading1, Bridget Young1, Philippe Ravau Paula R. Williamson1 1University of Liverpool; 2Universite Paris Descartes Correspondence: Alice Biggane Trials 2017, 18(Suppl 1):P411 Methods for including patients in core outcome set development Alice Biggane1, Lucy Brading1, Bridget Young1, Philippe Ravaud2, Paula R. Williamson1 1 2 1University of Liverpool; 2Universite Paris Descartes Correspondence: Alice Biggane Trials 2017, 18(Suppl 1):P411 Background The usefulness and importance of a core outcome set (COS) is well recognised, as is the need for patient participation in its develop- ment. A COS needs patient input to ensure it is credible and that future studies using the COS can provide patients and clinicians with relevant knowledge regarding interventions, consequently re- ducing the amount of wasteful research. Researchers are increas- ingly aware of this and are progressively including patients and the public alongside other stakeholders in identifying what outcomes to measure in clinical trials. Whilst only 22% of 300 published COS reported that there was input from patients in their development, nearly 90% of 146 ongoing studies report including patients in some capacity. However, nobody knows the best methods for facili- tating the participation of patients in COS development. There are numerous challenges in enabling patient Trialists routinely spend a far greater proportion of their time obtaining outcomes that they themselves deem of lesser import- ance than they do on primary outcomes. Given the significant ex- pense of collecting data and the widely reported fact that much trial data goes unreported, we suggest that trialists should have an increased awareness of the burden and cost associated with each outcome when making their selections. Participation in a COS study and these will depend on the pa- tient group and the methods chosen. Therefore, this project aims to investigate and develop methods for including patients as par- ticipants in COS development in a meaningful and productive manner. This work is part of the Trial Forge initiative to improve trial efficiency. P413 P413 A comparison of stroke diagnosis at trial entry by local clinicians versus independent adjudicators: secondary analysis and simulation Peter Godolphin1, Trish Hepburn1, Liz Walker 1, Nikola Sprigg1, Joanna M. Wardlaw2, Philip M. Bath1, Alan A. Montgomery1 1University of Nottingham; 2University of Edinburgh Correspondence: Peter Godolphin Trials 2017, 18(Suppl 1):P413 Designing trials that aim to evaluate therapies that target brain metastases in cancer patients: challenges and recommendations Sujata Patil Memorial Sloan Kettering Cancer Center Trials 2017, 18(Suppl 1):P412 A comparison of stroke diagnosis at trial entry by local clinicians versus independent adjudicators: secondary analysis and simulation Peter Godolphin1, Trish Hepburn1, Liz Walker 1, Nikola Sprigg1, Joanna M. Wardlaw2, Philip M. Bath1, Alan A. Montgomery1 1University of Nottingham; 2University of Edinburgh Peter Godolphin1, Trish Hepburn1, Liz Walker 1, Nikola Sprigg1, Joanna M. Wardlaw2, Philip M. Bath1, Alan A. Montgomery1 1University of Nottingham; 2University of Edinburgh Objective Correspondence: Peter Godolphin Trials 2017, 18(Suppl 1):P413 To ascertain the impact of the BERC review on the reported inci- dence of LOII and NEC, compared to those derived from the original data collection forms (DCFs). Methods The aim of this study was to investigate the benefit of adjudication of stroke type at trial entry in a large stroke trial. The three objectives were to: (1) compare stroke diagnoses made by site clinicians and in- dependent adjudicators; (2) assess the impact of adjudication on the primary analysis and a subgroup analysis by stroke type; (3) using simulation, explore the effects of increasing levels of misclassification on analyses. Pairs of BERC reviewers independently reviewed Gut Signs and Infec- tion dcfs, feeding log data and any additional data requested (e.g. Discharge summaries) and completed a diagnostic classification form. These were cross-validated for discrepancies and referred to a third BERC reviewer if agreement could not be reached. The incidence of LOII and NEC were calculated for each arm before and after BERC review; firstly, using data obtained from the Gut Signs and Infection dcfs, applying an algorithm detailed in the statistical analysis plan; and secondly, using the diagnostic classifications deter- mined by the BERC. Results To provide a comprehensive review of the methodological challenges in designing trials where progression in the brain is the primary end- point and to provide concrete clinical design recommendations. Background Of 4011 participants randomised, 3857 (96%) had baseline scans that were assessed by adjudicators. There was excellent agreement between Hospital and Trial diagnoses (crude agreement 98%, unweighted kappa, k = 0.92). Adjudication of stroke type had no impact on the primary outcome (p = 0.95) or subgroup analysis by stroke type. These findings were robust to all except the most extreme simulated non-differential misclassification of stroke diag- nosis and subgroup effect. The presence of brain metastases in cancer patients often indicates poor prognosis. Additionally, the presence of brain metastases can directly impact a patient’s quality of life. Controlling brain disease is im- portant and has been one current focus of clinical trials and retrospect- ive reviews [Preusser et al., Eur J Cancer 2012; Lin, ecancer 2013]. However, there are challenges in conducting such studies and interpre- tations of results are not uniform. For instance, patients may progress extracrainially before progression in the brain can be assessed, thereby creating a competing risks analytic setting. Assessing true brain recur- rence versus radionecrosis and the use of consistent criteria to assess brain recurrence have also been methodological issues. Methods Simulations modifying the following factors 1) sample size, 2) censor- ing, 3) effect size, 4) correlation between competing events, 5) degree of endpoint misclassification (pseudo-progression), and 6) method used for analysis (Kaplan-Meier, Cox regression, cumulative incidence, subdistribution regression) are conducted. The effect of these factors on power and type I error in Phase II clinical trials are reported. P414 The impact of blinded endpoint review on the incidence of primary short-term outcomes in the SIFT trial Christopher Partlett1, Louise Linsell1, Oliver Hewer1, Ed Juszczak1, Jon Dorling2 1NPEU, University of Oxford; 2Division of Child Health, Obstetrics and Gynaecology, University of Nottingham Trials 2017, 18(Suppl 1):P414 p Results Simulations on the randomized phase II design show that per arm sam- ple sizes of 75 to 100 have sufficient power to detect hazard ratios in the range of 1.7 and 2.0 where the endpoint is brain-specific. Higher correlation between competing risks (e.g. Brain vs systemic progres- sion) and the method used for analysis (e.g. Cause-specific hazard or cumulative incidence subdistribution) have effects on sample size. Misclassification of the endpoint (eg pseudo- progression) also has a demonstrable effect on inference. These simulation findings will be de- scribed in detail. Results from ongoing simulations under other Phase II designs will also be described along with design recommendations. Conclusion This study found that clinicians at ENOS trial sites largely were correct in their diagnosis of stroke, and adjudication did not impact on the trial results. Diagnostic adjudication may be important if diagnosis is com- plex and a treatment-diagnosis interaction is expected. Researchers should consider the value adjudication may bring to their study by using pilot or feasibility studies to estimate misclassification and poten- tially avoid substantial resource implications. Expected results The results will provide insights into the COS developers’ Plans and rationale for facilitating patient participants in their studies. Details about methods used for recruitment (social media, NHS services etc.) And methods used for eliciting the outcomes will be obtained (qualitative interviews, Delphi survey etc.). We will also establish the reasoning for using these methods. We will then use this information to purposively sample COS developers and pa- tients to participate in a subsequent qualitative interview phase of our study. Two independent investigators systematically searched, MEDLINE, EMBASE, CINAHL+, the Health Management Information Consortium and the Journal of Research Evaluation from inception until May 2016 for publications that presented an impact framework. We then summarised the common concepts and themes across frameworks and identified the metrics used to evaluate differing forms of impact. Trials 2017, 18(Suppl 1):200 Page 155 of 235 Page 155 of 235 diagnosis was the diagnosis used in all ENOS analyses. Agreement between Hospital and Trial diagnoses was determined using un- weighted kappa. The trial primary analysis and subgroup analysis by stroke type were re-analysed using Hospital diagnosis as base- line covariate and interaction factor respectively. Statistical simulations were created to: (1) increase misclassification of Hospital compared with Trial diagnosis; (2) introduce an interaction (subgroup effect) be- tween ENOS treatment arm and stroke type. Designing trials that aim to evaluate therapies that target brain metastases in cancer patients: challenges and recommendations Sujata Patil Background l d d d Blinded endpoint review committees (BERCs) comprise of a panel of clinical experts blinded to trial allocation. They are responsible for reviewing trial outcome data reported by participating centres, to ?A3B2 show $132#?>ensure they meet the protocol-specified criteria. This can be particularly useful for outcomes which are complex to assess, include subjective components, or when the original data collection could not be blinded. SIFT (ISRCTN: 76463425) is an open-label multicentre randomised controlled trial of a feeding intervention in very preterm or very low birthweight infants in neonatal units in the United Kingdom and Ireland. Infants were randomised to receive either a faster rate of feeding (30 ml/kg/day) or a slower rate of feeding (18 ml/kg/day). BERCs were set up to assess the incidence of two primary short-term outcomes; late onset invasive infection (LOII) and necrotising entero- colitis (NEC). Conclusion The two methods were highly concordant, however, there was marginal evidence that unblinded local investigators were more likely to assign a diagnosis of NEC in the fast feed arm, in infants deemed not to have NEC by the BERC. This may suggest a poten- tial bias, reflecting concerns about rapid advancement of feeds and its possible effect on the gut. Thus, while the addition of BERC reviews did not alter the conclusions of the trial, this investi- gation highlights their importance in reinforcing confidence in the outcome results. Splintered adverse event reporting in multicenter clinical trials Joy Black, Valerie LW. Stevenson, Robert Silbergleit University of Michigan Correspondence: Joy Black Trials 2017, 18(Suppl 1):P416 How are surrogate outcomes defined in critical care trials? Preliminary results of a systematic review 1 2 3 Preliminary results of a systematic review Rejina Verghis1, Bronagh Blackwood2, Cliona McDowell3, Daniel Hadfield4, Philip Toner5, Marianne. Fitzgerald6, Daniel F. Mcauley6, Mike Clarke6 1Queens University Belfast QUB; 2Centre for Experimental Medicine, Queen’s University Belfast; 3Northern Ireland Clinical Trials Unit; 4Kings College and Hospital; 5Belfast Health and Social Care Trust; 6Centre for Public Health, Queen’s University Belfast We reviewed all AE reported in two clinical trials performed in our clinical trials network, protect (NCT00822900) and ATACH (NCT01176565) both of which were reported in the New England Journal of Medicine. For each trial, a splintered and collapsed list of PT were compared. Protect published the collapsed list and ATACH the splintered list. Splintered lists were generated primar- ily by autocoding, with manual coding by a data manager during the conduct of the trial when autocoding failed. Collapsed lists were generated from the splintered lists using clinical judgement by the trial investigators in protect at the end of the trial. For ATACH, the collapsed list was generated in part by investigators at the end of the trial and in part by the authors of this abstract. All splintered and collapsed lists used only meddra PT. Descrip- tive statistics were used to characterize and compare splintered and collapsed AE lists. Correspondence: Rejina Verghis Trials 2017, 18(Suppl 1):P415 Background h h The choice of outcome measure is a critical decision in the design of any clinical trial, but many phase III clinical trials in critical care fail to detect a difference between the interventions being compared. This may be because the surrogate outcomes used to show beneficial ef- fects in early phase trials (which informed the design of the subse- quent phase III trials) are not valid guides to the differences between the interventions for the main outcomes of the phase III trials. We did this review to determine the variability in reported surrogate out- comes in early phase, critical care trials. Results and reported in a variety of ways. The definition of specific measure- ment (mechanical ventilation), participant level analysis metric (dur- ation of mechanical ventilation or time to extubation), method of aggregation (mean & SD or median & IQR) and time points vary across trials. There was little change in the risk of LOII for either arm, however there was a slight reduction in the risk ratio for NEC after BERC re- view; (RR 0.89, 95% CI 0.65 to 1.23) compared to (RR 1.00, 95% CI 0.73 to 1.36). y Methods The Efficacy of Nitric Oxide in Stroke (ENOS) trial examined the safety and efficacy of glyceryl trinitrate (GTN) versus no GTN in patients with acute ischaemic or haemorrhagic stroke. Independent expert as- sessors, referred to as adjudicators, who were masked to treatment allocation, centrally assessed cranial scans to inform diagnosis of stroke type. For this study, diagnoses made by local site clinicians are referred to as Hospital diagnosis, whilst diagnoses with input from in- dependent adjudicators are referred to as Trial diagnosis. The Trial For both outcomes we compared the risk ratio (fast/slow) and 95% confidence interval derived from the DCF and BERC classification. For each arm we also investigated the concordance between the classification of infants before and after BERC review, using the kappa statistic and mcnemar’s test. Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 156 of 235 Page 156 of 235 Methods d We undertook a systematic review to generate a list of outcome measures used in early phase critical care trials. We searched for trials published in the six top-ranked critical care journals between 2010 and 2015. The review was conducted according to the protocol published on the PROSPERO website (http://www.crd.york.ac.uk/ PROSPERO/display_record.asp?ID=CRD42015017607). We searched MEDLINE and EMBASE using key words such as intensive care unit, crit- ical care and randomised controlled trials. Two independent reviewers were involved in the search and article screening. All articles meeting inclusion criteria and published in 2010 were selected for data extrac- tion and data saturation was achieved during this process. Therefore, we included only an additional 10% of the articles from 2014 and 2015 to boost the sample with some more recent papers. We extracted de- scriptive data including trial registration details, outcome measures re- ported in the methods, definition, and time-points. We classified outcomes into body organ systems, severity of disease and quality of life with sub-categories based on clinical judgement, and tabulated them to understand underlying patterns and variations. R lt Results Substantial splintering was found in both trials. 3032 AE occurring in 810 patients in protect were coded under 399 unique PT in the splintered list, and under 235 unique PT in the collapsed list. Simi- larly, in 1000 subjects enrolled in ATACH, 3140 AE were coded under 344 unique PT in the splintered list and 193 unique PT in the collapsed list. There were 235 and 193 collapsed PT terms in pro- tect and ATACH respectively and collapsed terms included a mean of 3.00 splintered terms with a range of 2 to 9 PT. Illustrative exam- ples of splintered and collapsed terms in these two trials include: bronchopneumonia, lung infection, pneumonia aspiration, and 7 other PT that collapsed under the PT ‘pneumonia’; and embolic stroke, cerebral artery embolism, cerebral infarction and 5 other PT clinically equivalent to the collapsed PT ‘ischemic stroke’. An ex- ample of the potential effect of splintering was found in ATACH where splintered terms related to renal injury were similar between the two treatment groups as individually reported, but demon- strated a potentially significant difference when collapsed. Conclusions P415 How are surrogate outcomes defined in critical care trials? Preliminary results of a systematic review Rejina Verghis1, Bronagh Blackwood2, Cliona McDowell3, Daniel Hadfield4, Philip Toner5, Marianne. Fitzgerald6, Daniel F. Mcauley6, Mike Clarke6 1Queens University Belfast QUB; 2Centre for Experimental Medicine, Queen’s University Belfast; 3Northern Ireland Clinical Trials Unit; 4Kings College and Hospital; 5Belfast Health and Social Care Trust; 6Centre for Public Health, Queen’s University Belfast Correspondence: Rejina Verghis Trials 2017, 18(Suppl 1):P415 How are surrogate outcomes defined in critical care trials? Preliminary results of a systematic review Rejina Verghis1, Bronagh Blackwood2, Cliona McDowell3, Daniel Hadfield4, Philip Toner5, Marianne. Fitzgerald6, Daniel F. Mcauley6, Mike Clarke6 1Queens University Belfast QUB; 2Centre for Experimental Medicine, Queen’s University Belfast; 3Northern Ireland Clinical Trials Unit; 4Kings College and Hospital; 5Belfast Health and Social Care Trust; 6Centre for Public Health, Queen’s University Belfast Correspondence: Rejina Verghis Trials 2017, 18(Suppl 1):P415 Results A total of 5448 references were screened. The total number of in- cluded articles was 48, and based on the preliminary analysis, these mentioned over 300 outcomes in their methods sections. Focusing specifically on outcomes reported in the respiratory category, there were ten sub-categories and the number of different outcomes in the subcategories. The reported outcome measures were analysed Conclusions There was strong concordance between the classification of infants before and after BERC review, with over 95% agreement for both outcomes in both arms. Among the discordant cases the original DCFs were more likely to classify an infant as a case than the BERC, however this discordance was only marginally statistically significant for NEC in the fast feeding arm (p = 0.04). There is large variability in outcome reporting in early phase, critical care trials. This creates difficulties for synthesizing data in systematic reviews and planning definitive trials. This review highlights an ur- gent need for standardization and validation of surrogate outcomes reported in critical care trials. Future work will validate and develop a core outcome set for surrogate outcomes in critical care trials. Objective The use of controlled vocabularies like meddra are essential to prac- tical useful adverse event (AE) reporting, but have limitations. The use of autocoding in meddra allows objective mapping of verbatim terms (VT) to preferred terms (PT) but can result in the listing of clin- ically identical events into a variety of effectively synonymous PT, an effect we call splintering. A potential solution involves a clinician grouping these splintered PT into a single collapsed PT relevant to the medical context of the trial. Both splintering and collapsing AE have the potential to obscure safety signals. g Results Twenty-six themes were identified that related to six topics; i.e. 1) overall views of the intervention; 2) experience of implementing the intervention; 3) changes made/suggested to the intervention; 4) views on participants’ experience of the intervention; 5) perception of the intervention’s feasibility for future delivery; and 6) views and experiences of training, and were mapped to the feasibility criteria: acceptability, demand, implementation, practicality, adaptation and integration. Case study methodology is advocated for exploring real life phenom- ena within a contemporary context. The nature of the design lends it- self to addressing questions that aim to understand, in detail, how or why events occur. The two-tailed design, offers a comparative focus; in the case of clinical trials the tails can be the control and intervention arms enabling comparison of relevant features of the control and inter- vention. OPAL uses a two-tailed longitudinal case study, where women are interviewed at four time points (baseline, post-treatment, 12 months and 24 months post-randomisation); mirroring the trial data collection. The nature of the analysis encourages a move beyond description to explanation; for example, identifying factors that may interact to influ- ence participant outcomes, and the mechanisms of action. PTs were positive about their experience of training and implementing the SOLAS intervention and its support materials to a mixed group of participants, reporting it acceptable and feasible to deliver. Key de- mands in delivering the intervention that impacted on implementation included the high volume of education content during class one, which required shortening the exercise session, and the challenge of using the needs supportive communication skills during goal setting and the group exercise component, highlighting the need for additional train- ing. However, PTs felt that overall they implemented the intervention content and structure according to the protocol. Some PTs reported adapting the education component to include additional information based on their clinical expertise and participant needs. Practical consid- erations for future integration of the intervention into health services included attaining a minimum of six participants to run a successful group, the accessibility of the intervention in some primary care set- tings, and the need to address health literacy for some participants. Conclusions In the OPAL qualitative longitudinal study the aim is to understand the links between context, delivery, and outcomes in each arm for women with UI. Conclusions We have demonstrated that autocoding AE VT to PT in meddra is objective but results in significant splintering as compared to clinically relevant collapsed terms, obscuring medically important safety effects. Use of clinical judgement to combine effectively synonymous PT is subjective, but is a practical solution. Trials 2017, 18(Suppl 1):200 Page 157 of 235 Page 157 of 235 y Methods Individual semi-structured telephone interviews were conducted by an independent researcher with 10 physiotherapists (PTs) within one week of their completion of delivery of the SOLAS intervention in the feasibility trial. The interviews were audio-recorded, tran- scribed verbatim and analysed using inductive thematic analysis, based on Braun and Clarke’s method. Coding frames were developed, re-examined and refined. The reliability of the identified themes was established by a second researcher who independently coded a ran- dom sample of 25% of the data using the coding frame, with 70% agreement taken as the minimum cut-off rate of agreement. R l y OPAL (optimising pelvic floor exercises to achieve long-term benefits) has three elements: a large multi-centre trial, a mixed methods process evalu- ation, and a longitudinal qualitative case study. The trial investigates the effectiveness of biofeedback intensified pelvic floor muscle training (PFMT) versus PFMT alone in improving UI symptoms for women with stress or mixed UI. The case study is a two-tailed design, one tail is the control (PFMT) the other the intervention (intensified PFMT), exploring the views and experiences of trial women about UI and the interventions to identify barriers and facilitators to intervention delivery and adherence and to inform potential roll-out of the intervention. P417 What does qualitative case study methodology have to offer the interpretation of findings from trials of complex interventions? Reflections from a large complex intervention study Caarol Bugge1, Aileen Grant1, Sarah Dean2, Jean Hay Smith3, Doreen McClurg4, Suzanne Hagen4 1University of Stilring; 2University of Exeter; 3University of Otago; 4NMAHP RU, Glasgow Caledonian University Correspondence: Caarol Bugge Trials 2017, 18(Suppl 1):P417 Self-management (SM) is endorsed by clinical guidelines for osteo- arthritis (OA) and chronic low back pain (CLBP), but there is a current lack of multi-joint interventions to target both conditions in group settings. The 6 week group-based self-determination theory (SDT) driven education and exercise SOLAS intervention was developed in consultation with primary care physiotherapists through the interven- tion mapping process. Following Medical Research Council (MRC) guidelines for complex interventions, the SOLAS cluster randomised controlled feasibility trial aims to assess the (1) acceptability and feasibility of the SOLAS intervention to patients and physiotherapists compared to usual individual physiotherapy, (2) feasibility of trial procedures and sample size for a definitive trial and (3) effect on secondary outcomes. The aim of the present study was to explore physiotherapists' views of the SOLAS intervention’s acceptability and feasibility. The contribution qualitative research can make to improving interven- tion and trial design, evaluation and implementation is well recognised (O’Cathain et al., 2014; Moore et al. 2011). Qualitative methods are often used alongside quantitative methods within a process evaluation to ex- plore trial processes, intervention components and mechanisms in rela- tion to context (Grant et al. 2013). This paper describes the use, and presents the advantages, of a two-tailed qualitative case study meth- odological design linked to a trial of a complex intervention for women with urinary incontinence (UI) (OPAL ISRCTN 57746448). g Results In a complex intervention such as that evaluated in OPAL, many factors could impact on the final outcome for a woman; only some of these factors may relate to intervention delivery. For example, women are asked to exercise at home in both trial arms and in the intensified arm women are asked to use biofeedback to support PFMT at home. There may be many psychological, social, or practical variables that influence a woman’s ability to use biofeed- back, or do this in the home environment. The case study design aims to support the identification of these factors and, importantly, how their influence may differ on the trial primary outcome (UI at two years) between the control and intervention arms. The SOLAS intervention content and support materials were considered acceptable and feasible to deliver within the trial and in future health- care services provided sufficient numbers of clients could be enrolled and retained. Further training in the intervention SDT-based needs sup- portive communication skills is being developed through E-learning. In this paper we will explore: 1. The nature of case study methodology. 2. Why case study methodology might be useful for qualitative studies linked to complex intervention trials. 3. Lessons for researchers from our use of case study methodology within OPAL. Image modification journaling for reproducibility and fraud prevention Image modification journaling for reproducibility and fraud prevention l h Physiotherapists’ views of the acceptability and feasibility of the self-management of osteoarthritis and low back pain through activity and skills (SOLAS) complex intervention within a cluster randomised controlled feasibility trial [ISRCTN 49875385] Deirdre Hurley, David Hayes, Danielle McArdle, James Matthews, Suzanne Guerin p Paul Thompson Sanford Research Trials 2017, 18(Suppl 1):P419 Paul Thompson Sanford Research Trials 2017, 18(Suppl 1):P419 Background P417 What does qualitative case study methodology have to offer the interpretation of findings from trials of complex interventions? Reflections from a large complex intervention study Caarol Bugge1, Aileen Grant1, Sarah Dean2, Jean Hay Smith3, Doreen McClurg4, Suzanne Hagen4 1University of Stilring; 2University of Exeter; 3University of Otago; 4NMAHP RU, Glasgow Caledonian University Correspondence: Caarol Bugge Trials 2017, 18(Suppl 1):P417 Background Background In scientific discussions, images are analyzed to make scientific points. In basic biology, in clinical trials, in clinical research, images are used to present information, show differences between conditions, and define University College Dublin Correspondence: Deirdre Hurley Trials 2017, 18(Suppl 1):P418 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 158 of 235 Page 158 of 235 was mainly UK based but does have some representation from further afield. Interviews are scheduled for fall 2016. We have approached 27 individuals in roles such as Research Nurse, Head of Patient Engagement, Clinical Trial Educator, Senior Research Man- ager and Professor of Health Services. Everyone we approached agreed to take part, giving our sample a split of 14 “recruiters” And 13 “designers”, 23 of which are UK-based, 1 from Holland, 2 from South Africa and 1 from Italy. A Framework analysis approach will be used to analyse the data from one-to-one interviews. Antici- pated and emergent themes will be identified, defined and linked through continual comparison of data both within and across stakeholder groups. C l i was mainly UK based but does have some representation from further afield. Interviews are scheduled for fall 2016. We have approached 27 individuals in roles such as Research Nurse, Head of Patient Engagement, Clinical Trial Educator, Senior Research Man- ager and Professor of Health Services. Everyone we approached agreed to take part, giving our sample a split of 14 “recruiters” And 13 “designers”, 23 of which are UK-based, 1 from Holland, 2 from South Africa and 1 from Italy. A Framework analysis approach will be used to analyse the data from one-to-one interviews. Antici- pated and emergent themes will be identified, defined and linked through continual comparison of data both within and across stakeholder groups. phenomena. The presentation and manipulation of images is governed by rules which have been defined by editors of scientific publications. The images can be cropped (to select parts of a larger image). The con- trast of the image can be increased so long as the changes are made over the entire image. The colors of the image can be adjusted over the entire image. Images can be places together, so long as it is clear what is done to the final image and that the component portions are not part of the original image (this can be done by lines of a black color). P420 Perceptions and experiences of participant recruitment: a qualitative interview study with trial stakeholders Heidi Gardner, Katie Gillies, Shaun Treweek University of Aberdeen Correspondence: Heidi Gardner Trials 2017, 18(Suppl 1):P420 Heidi Gardner, Katie Gillies, Shaun Treweek University of Aberdeen Correspondence: Heidi Gardner Trials 2017, 18(Suppl 1):P420 Background In recent years, the amount of fraudulent manipulation in im- ages is becoming alarming. Fraud takes many forms, including re-using the same image for different purposes, adding new components to an existing image without clear markings and so forth. Image fraud is fre- quently discussed on the Retraction Watch blog. Background Recruitment to randomised controlled trials can be extremely difficult, and poor recruitment can lead to extensions to both time and budget, potentially resulting in an underpowered study which does not satisfac- torily answer the original research question. In the worst cases, a trial may be abandoned, causing huge waste. Consequently, recruitment is considered the number one problem in trial methods research. To understand how the process of participant recruitment impacts the day-to-day lives of those charged with the task, we conducted a qualitative semi-structured interview study with a wide range of trial stakeholders. This study will help trial methodologists to understand the challenges that trial recruitment generates on the ground, which will enable them to better design future research work so that its re- sults are more relevant and applicable to the challenges faced by those tasked with recruiting individuals to trials. Presentation Several examples of image manipulation will be presented to demon- strate this new tool and capability. Other image manipulation programs will be discussed to determine if the capability can be extended to them. Over the last several years, oncology drug development has fo- cused on molecularly targeted agents necessitating development of early phase clinical studies driven by a need to better under- stand underlying molecular mechanisms, provide delivery of tar- geted interventions in enriched patient subgroups and evaluate biological outcomes. Given the relatively safe profile of these tar- geted therapies, dose-finding clinical trials aim to evaluate both toxicity and biological effectiveness. An isotonic regression model was utilized in the design and conduct of a dose escalation trial to determine an admissible set of drug doses based on toxicity out- comes and selects the lowest dose with the highest biological re- sponse rate within the admissible set of doses. Simulations under different scenarios of dose toxicity and biological effectiveness rates demonstrate optimal operating characteristics of this design based on high selection probabilities of the correct safe and bio- logic effective dose, increased number of patients allocated to the right dose and low toxicities on the correct dose. Implementation of the isotonic regression is underway to guide dose escalation de- cisions. We present the performance of the model based on ob- served toxicity and pharmacodynamic (PD) biological response at each dose level as well as varying scenarios of toxicity and PD rates. As expected, dose escalation was guided by doses closest to the target biologic response rate within doses with acceptable toxicity rates. We also present comparison of performance and dose escal- ation decisions of this model compared to algorithm-based method for assessing biological or tumor response rate for dose escalation recommendations. The application of this model has provided a flexible and efficient use of limited patient data to determine not only safety but incorporation of proof of concept of biological re- sponse in the very early phases drug development. q y Results The results of this study will give a clear description of current recruit- ment practice. This is turn will make it easier for trial methodologists and others to design and present evidence-based recruitment strat- egies and highlight what sort of evidence future research should provide. Several new approaches are presented here for partial solutions to these problems. When images are manipulated in an interactive tool, a record of the actions can be kept. This is called a “journal”. This is a common part of a number of statistical analysis programs (JMP keeps a journal of analysis steps, which can then be used to analyze the data). GIMP has been modified to journal the process of manipulat- ing the image to allow the process to be repeated and to allow in- spection of the process. A shiny app (in R) has also been created to perform analysis and journal that analysis using imagemagick code. By making the process of image analysis transparent, fraud in the image manipulation will be reduced. This work is part of the Trial Forge initiative to improve trial efficiency. P421 P421 Implementation into practice of isotonic regression for simultaneous assessment of toxicity and pharmacodynamic endpoints in a dose escalation trial Rani Jayswal, Stacey Slone, Peng Wang, Vivek Rangnekar, Heidi L. Weiss Markey Cancer Center, University of Kentucky Correspondence: Rani Jayswal Trials 2017, 18(Suppl 1):P421 Methods A search of clinical trials registers (globally) was conducted and deci- sions were made as to the suitability of the registry. Included registries were searched using the term ‘qualitative’ and returns logged and analysed by; 1. Year Registered 2. Country of Sponsor 3. Type of Trial (Drug, medical device, surgical or other). Trials were only included if the researcher confirmed that they included qualitative methodology (i.e. Using qualitative data collection methods and analysis techniques). Trials reporting qualitative testing and statistical analysis were excluded (i.e. ‘qualitative’ was tagged to quality of life measures, reports about medical tests were included such as ‘qualitative urine test’ or statistical tests, such as ‘qualitative Fishers Extract test’ were in evidence). Initially, all registers were searched from first record available up to 31st October 2013. The activity was repeated to update records up to November 2016. Interviewees recognised that fostering positive relationships with participants was a key strategy for enhancing participant retention. Interpersonal connections were forged by social relational actions such as making cups of tea during trial appointments and offering flexible appointments to suit the participant’s needs. However, these activities required additional time which the trial researchers felt was not always acknowledged by funders or valued by the wider trial team. Interviewees were not aware of how their own ‘moral compass’ influ- enced retention of participants. However such unrecognised or un- conscious strategies were present in their accounts. They expressed how they often utilised their own beliefs and values regarding how to interact with participants, reflecting for example on how they would want their own parents to be treated, or projecting their own feelings onto a situation which may conflict with obtaining data. Three registries were included and searched; clinicaltrials.gov, Inter- national Standard Randomised Controlled Trial Number Register (ISRCTN) and World Health Organisation (WHO) International Clinical Trials Registry Platform (ICTRP). The initial search in 2013 found an extremely small proportion of the 382,944 trials being carried out worldwide were utilising qualitative methods. The overall percentage of registered trials confirmed as including qualitative methods was less than 0.2%. This number appears to have increased over time, but is limited to ‘developed countries’ such as the U.K. and U.S.A. Most trials using qualitative methods appeared to be non-clinical, and were mostly testing behavioural interventions (87%). Methods Methods A mix of purposive and convenience sampling generated trial stake- holders that represented views of those that work within the Na- tional Health Service, academia and industry. Individuals categorised themselves as “designers”; those directly responsible for the design or recruitment methods, or “recruiters”; those who implement recruit- ment strategies to recruit participants to trials. Currently we have de- veloped an interview topic guide that will allow us to investigate and explore the views of trial designers and recruiters. We will also ex- plore how best to present research evidence about recruitment methods so that evidence-based recruitment strategies are effect- ively disseminated and implemented. Enquiry into how qualitative methods influence trials and their yields (equity): exploring registered trials that include a reported qualitative component 1 1 1 2 q p Clare Clement1, Suzanne Edwards1, Hayley Hutchings1, Frances Rapport2 1Swansea University; 2Macquarie University Correspondence: Clare Clement Trials 2017, 18(Suppl 1):P422 Role type and recruitment experiences were varied, spanned various therapeutic indications, intervention types and trials units. Our sample Trials 2017, 18(Suppl 1):200 Page 159 of 235 Page 159 of 235 Conclusions This study has highlighted the increasing use of qualitative methods over time and the use of these methods worldwide. However, the use of qualitative methods is restricted to ‘developed countries’ and non-clinical trials. More needs to be done to increase the use and benefits of qualitative methods in ‘under-developed’, or ‘developing’ countries, and the reasons for their lack of inclusion in clinical trials needs further investigation and development. Strategies deployed by trial researchers to enhance retention include a combination of recognised and unrecognised influences. These are underpinned by relational factors as well as researchers beliefs about their responsibilities and professional values. However, the pursuit of retention is constrained by a systemic emphasis on recruitment. "To have and to hold, from this day forward": understanding current practice regarding retention of trial participants Clare Clement1, Anne Daykin1, Carol Gamble2, Anna Kearney2, Jane Blazeby1, Mike Clarke3, Athene Lane1, Ali Heawood1 1University of Bristol; 2Universityof Liverpool; 3Queen's University Correspondence: Clare Clement Trials 2017, 18(Suppl 1):P423 1Royal Brompton Hospital; 2Imperial College London; 3Southmead Hospital; 4University Hospital Aintree Correspondence: Vicky Tsipouri Trials 2017, 18(Suppl 1):P424 Methods Of the small percentage of those trials which appeared clinical in nature, drug trials appeared to utilise qualitative methods more than either medical device or surgical trials (7%, 4%, 2% respectively). This was consistent across the three trial registries. Early indications from the repeated 2016 activity show a continuation of the initial pattern of less than 0.2% of a total 428,175 trials recorded using qualitative methods. Full findings will be reported at the conference. Conclusions The influence of the level of experience of team members on reten- tion practices also appeared unrecognised. Researchers lacking experience reported having less confidence to pursue participants for outcome measure data, especially when participants wished to withdraw from the trial, worrying about coercion. More experienced researchers were happy to negotiate with participants in order to at least collect primary outcome data. Novice researchers presumed the participants wanted to withdraw from all aspects of the trial and made no further contact with them. Researchers recognised that incentives influenced retention but seemed unaware that incentives may affect their own behaviour. Trial staff felt more confident and comfortable maintaining contact with participants over a period of time and more motivated to pur- sue acquisition of data from participants. y Results Nineteen semi-structured interviews were conducted with trial team members along with three supplementary interviews with experi- enced senior trial managers. Participants recognised the context of the wider focus on recruitment to the detriment of retention by limiting motivation and resources to maximise retention. In trial re- searchers’ accounts, their retention practices were shaped by factors which were recognised and conscious, and unrecognised and unconscious. Background trial teams within ongoing trials and factors which may influence the adoption of such strategies. trial teams within ongoing trials and factors which may influence the adoption of such strategies. Support for the use of qualitative methods within trials is widely recognised; however, reports indicate their full potential is not being realised, and issues ensue with the visibility, recognition and report- ing of the qualitative approach in trials. It is important to understand the global view of the historical and contemporary make-up of quali- tative research linked to trials if we are to identify potential areas of improvement. As part of a larger project to explore patterns in, and status of, the use of qualitative methods in trials, a review of trial registries was conducted to determine the extent of qualitative methods' use in trials. p Methods p g Methods A purposive sample of five trials was selected from the NIHR HTA portfolio of ongoing trials. Semi-structured interviews explored strat- egies utilised by trial teams when collecting outcome data and in retaining participants. With the aid of nvivo, the interview data were analysed thematically using techniques of constant comparison. Results A case study method to support and promote recruitment at a multi-centre RCT comparing surgical versus non-surgical treatments 1 1 1 1 Alba Realpe1, Edward Dickenson1, Rachel Hobson1, Damian Griffin1, Marcus Jepson2, Jenny L. Donovan2 1University of Warwick; 2University of Bristol Correspondence: Alba Realpe Trials 2017, 18(Suppl 1):P427 Implementing training for recruiters based on a new simple six-step model to promote information sharing and recruitment to RCTs: challenges and opportunities 1 1 1 1 g Alba Realpe1, Edward Dickenson1, Rachel Hobson1, Damian Griffin1, Marcus Jepson2, Jenny L. Donovan2 1University of Warwick; 2University of Bristol Correspondence: Alba Realpe Trials 2017, 18(Suppl 1):P426 Results Recruitment to trial was successful, with 60% of patients approached across 20 centres agreeing to take part in the RCT. Recruiters used the six-step model to structure their consultations. However there were differences in the way recruiters addressed patient questions and concerns in participants’ versus decliners’ consultations. Differ- ences were also observed in patients’ view of the trial, those who de- clined to take part expressed more concerns and preferences and asked fewer questions than participants in the trial. C l i Results We have completed recruitment of 87 patients to the study which rep- resents one of the largest such cohorts world-wide. We are presenting here the trial design and baseline characteristics. Disc ssion P426 P426 Implementing training for recruiters based on a new simple six-step model to promote information sharing and recruitment to RCTs: challenges and opportunities Alba Realpe1, Edward Dickenson1, Rachel Hobson1, Damian Griffin1, Marcus Jepson2, Jenny L. Donovan2 1University of Warwick; 2University of Bristol Correspondence: Alba Realpe Trials 2017, 18(Suppl 1):P426 Objective This qualitative study added to the growing evidence of how aspects of team functioning are important for recruitment to complex RCTs. Trial Management Groups can help research teams identify and ad- dress issues, and therefore contributing to a sense of ownership by the research team. Empowering research teams to find solutions at local level is essential to conduct multi-centre RCTs successfully. The way trial information is presented is a key determinant of re- cruitment to randomised controlled trials (RCTs), which can be modified in order to encourage patients to participate. Recruiters in a full-scale surgical RCT comparing a surgical procedure with physiotherapy were trained based on a simple six-step model to support recruitment (Realpe et al. 2016). This paper shows how the model was implemented. We compared communication practices in consultations where patients decided to take part versus those consultations in which patients declined participation in the trial in order to validate and expand the six-step model. P428 When is enough, enough? Replication of behaviour change interventions to minimise attrition of follow up questionnaires Anne Duncan1, Beatriz Goulao1, Patrick Fee2, Fiona McLaren-Neil2, Ruth Floate2, Fiona Ord2, Hazel Braid2, Debbie Bonetti2, Jan Clarkson2, Craig Ramsay1 Objective Multi-centre RCT designs provide robust evidence of therapeutic ef- fect of health interventions. However participating centres often dif- fer in how well they conduct the trial and the number of patients successfully recruited. This paper describes barriers different research teams encountered when conducting a complex RCT comparing a surgical procedure with physiotherapy, and the actions taken by the trial management group to overcome obstacles that were hindering recruitment. Methods We conducted 22 interviews with principal investigators and research associates at 14 sites involved in the delivery of a surgical RCT that compared hip arthroscopy and physiotherapy for hip pain. Interview transcripts were analysed thematically and case study approaches were utilised to present results to the trial management group. Results This is the first RCT of the effects of ambulatory oxygen during daily life on health status and breathlessness in fibrotic lung disease. The results generated should provide the basis for setting up ILD specific guidelines for the use of ambulatory oxygen. Research teams reported difficulties related to logistics (e.g. Room space); motivation (e.g. PI reluctant to approach patients); and skill (e.g. Lack of knowledge about the treatment arms). Similar Issues were shared by sites that recruited to target and those that did not, however there were differences in the team’s response to challenges. Whilst on-target sites found local solutions to issues or support through their research infrastructure or the trial TMG, off-target sites usually did not show proactivity. Site profiles were created and action plans designed based on aspects that were particular to the individual sites. These plans were implemented in collaboration with site teams. Background A frequent problem in clinical trials is the failure to attain complete outcome data for all randomised participants. Loss to follow-up (attri- tion) is problematic as it can introduce bias and reduce the power of a trial. However, until recently the main focus has been on enhancing recruitment rather than retention. As part of a multi-method study, this qualitative study, sought to explore retention strategies used by Fibrotic Interstitial Lung Diseases (ILD) are rare, chronic and often progressive conditions resulting in substantial morbidity and mor- tality. Shortness of breath, a symptom often linked to oxygen desat- uration on exertion, is tightly linked to worsening quality of life in these patients. Although ambulatory oxygen is used empirically in Page 160 of 235 Trials 2017, 18(Suppl 1):200 Page 160 of 235 these patients, there are no ILD specific guidelines on its use. To our knowledge, no studies are available on the effects of ambula- tory oxygen on day to day life in patients with ILD. Methods to maintain patient equipoise is required when addressing patient questions and preferences. Patient views and their particular circum- stances are important factors when deciding whether or not to par- ticipate in a surgical RCT with a less intensive comparison arm. Ambulatory oxygen in fibrotic lung disease (AMBOX) is a multicentre, randomized, cross-over controlled trial (RCT) funded by the Research for Patient Benefit Programme of the National Institute for Health Research. The RCT will evaluate the effects on health status (measured by the King’s Brief ILD questionnaire: K-BILD) of ambulatory oxygen used at home, at an optimal flow rate determined by titration at screening visit, and administered for a two-week period, compared to two weeks off oxygen. Key secondary outcomes will include breathless- ness on activity scores, as measured by the University of California San Diego Shortness of Breath questionnaire, global patient assessment of change scores, as well as quality of life scores (St George’s Respiratory Questionnaire), anxiety and depression scores (Hospital Anxiety and Depression Scale), activity markers measured by sensewear Armbands, pulse oximetry measurements, patient reported daily activities, patient and oxygen company reported oxygen cylinder use. The study also in- cludes a qualitative component and will explore in interviews patients' experiences of the use of a portable oxygen supply and trial participa- tion in a subgroup of 20 patients. Methods A sample of recruitment consultations with participants (n = 32) and decliners (n = 28) were recorded during a full-scale RCT. Recordings were analysed using techniques of thematic analysis and focused conversation analysis pioneered in previous studies. 1University of Aberdeen; 2University of Dundee Correspondence: Anne Duncan Trials 2017, 18(Suppl 1):P428 1University of Aberdeen; 2University of Dundee Correspondence: Anne Duncan Trials 2017, 18(Suppl 1):P428 Objective To describe the development of the trial’s screening tool to recruit KR patients at greater risk of poor outcome and who therefore might benefit more from the intervention. Methods The screening tool was developed based on the principles of prognos- tic model development, using data from the KAT randomized clinical trial [1] which contains pre-operative and 12 months outcome data on more than 2,192 KR patients. As a proxy for poor outcome, since the KAT trial did not include the LLFDI score, poor outcome was defined as a score 26 in the Oxford Knee Score (OKS). Pre-operative characteristics considered as candidate predictors in the development of the screen- ing tool included age, sex, height, body mass index (BMI), mobility, ASA grade, SF-12 (questions 6 and 11) and OKS (question 1) components. Multivariate imputation by chained equations (MICE) was used to han- dle missing data in the KAT dataset. Ten complete datasets were pro- duced by MICE. One of these datasets was selected at random and multivariable logistic regression models were fitted to identify the sta- tistically significant predictors of poor outcome after KR. Predictors were selected by using backwards elimination (stepwise) procedure. The final model was aimed to be simple and easy to implement in the clinical setting, considering both the clinical and statistical relevance of the predictors. Model simplification was done by rounding up the logis- tic regression coefficients (odds ratio) of the predictors in the final model to the nearest integer. Model performance was evaluated in terms of discrimination and calibration. Discrimination was quantified by the c-index (area under the receiver operating characteristics curve). Calibration was assessed by grouping individuals into tenths of pre- dicted risk and graphically comparing the agreement between the mean predicted risk and the observed events in each tenth. The cut- offs to classify individuals at increased risk of poor outcome was deter- mined with the aim of achieving a balance between model’s specificity and recruitment feasibility. Background Low response rates to participant follow-up questionnaires in trials place the validity and generalisability of results in jeopardy. Evidence provided by the iquad Trial demonstrated that using the Theoretical Domains Framework (TDF) to identify theoretical targets for behaviour change interventions and incorporating these into a theory-based cover letter randomly issued to 1192 participants with their postal questionnaire at year 1 and year 2 of annual follow-up had a beneficial impact on response rates [1]. Lack of replication of research findings The six-step model provided a useful framework for recruitment to RCTs that was easy to implement. However further skill development Trials 2017, 18(Suppl 1):200 Page 161 of 235 has been highlighted as a key limitation across health and related disci- plines. To address this limitation, the strategy was replicated in the INTERVAL Dental Recalls trial to investigate if the intervention would improve participant questionnaire response rates in a similar patient population (primary dental care), with a similar level of non-response. Background It’s important when designing clinical trials to select an appropriate method of recruitment. Traditionally research nurses recruit participants from GP practices. They are often familiar to the patients which could mean those patients are more likely to enter and complete clinical trials. A randomised controlled trial to test the effectiveness of a brief intervention for weight management in primary care compared practices using research nurses (N = 17) with practices using research assistants (RA) (N = 44) to opportunistically recruit participants. Aims Results 93.8% in the RA group and 96.6% in the nurse group (RR 0.97 95% CI 0.94, 0.99) were enrolled of those that were eligible. 58.1% in the nurse group and 81.1% in the RA group (RR 0.71 95% CI 0.65, 0.79) were followed up at 3 months. g Results The participants in both the iquad and INTERVAL trials had similar baseline characteristics; the mean age of INTERVAL participants was 48.4 (14.9) years, 60% female and iquad participants 47.8 (15.7) years, 64% fe- male. The response rate in INTERVAL was 67% for the intervention cohort and 66% in the control group. There was a +1% difference (95% CI −3 to 5%) between groups favouring the intervention. In iquad the response rate was 72% in the intervention cohort and 65% in the control group. There was a +7% difference (95% CI +2 to +12%) between groups favouring the intervention. On meta-analysis of results there is a risk dif- ference of 3% (95% CI 0 to +7%) in favour of the intervention. C l i Development of the CORKA trial screening tool for identifying patients at increased risk of poor outcome following knee replacement Michael Schlussel, Gary Collins, Susan Dutton, Karen Barker University of Oxford Correspondence: Michael Schlussel Trials 2017, 18(Suppl 1):P430 Method 1867 INTERVAL participants were sent annual questionnaires at year 2 and 3 of follow-up and randomly allocated to receive either the standard covering letter (control group) or theory-based cover letter (intervention cohort). The response rates between the groups to both the iquad and INTERVAL replicated SWAT were estimated with 95% confidence intervals. A fixed effect meta-analysis was calculated using the Mantel-Haenszel method. Research nurses were slightly more effective at successfully enrolling eligible participants into the trial than research assistants however the difference between the groups is barely significant. Once en- rolled, participants were more likely to return for follow up in the RA group. This significant result suggests that using research assistants for recruitment is more likely to result in better follow up rates. Conclusions To our knowledge, this is the first true replication of a behaviour change intervention for improving response rates in a similar popula- tion. These results indicate that inclusion of a theory-based cover let- ter with postal questionnaires provides a cheap and effective method for improving participant response rates by 3% compared with a standard letter in a dental primary care population. We be- lieve this study provides strong evidence of the effectiveness of the intervention in this population. However, the study has raised inter- esting methodological challenges around when should replication stop and the role of context (settings and population). As such fur- ther replication of this strategy is planned in different trial settings and populations through the Trial Forge initiative (http://trialfor- ge.org) and through the Medical Research Council (MRC) Hubs for Trial Methodology Research (https://www.qub.ac.uk/sites/thenorthern irelandnetworkfortrialsmethodologyresearch/swatswarinformation/) to add to the evidence base. Background Community based Rehabilitation after Knee Arthroplasty (CORKA) is a multicentre two-arm individually randomised controlled trial with blinded outcome assessment at 12 months. It aims to determine if a multi-component rehabilitation programme, provided to patients who had knee replacement (KR) and are deemed at risk of poor outcome, as measured by the Late Life Function and Disability Instrument (LLFDI) score, is better than usual care. Reference 1. Duncan A, Bonetti D, Clarkson J, Ramsay C. Improving trial questionnaire response rates using behaviour change theory. Trials 2015, 16(Suppl 2):P92 1. Duncan A, Bonetti D, Clarkson J, Ramsay C. Improving trial questionnaire response rates using behaviour change theory. Trials 2015, 16(Suppl 2):P92 Results Compare two different methods of recruitment, specifically the effect on participant enrolment and follow up. Subjects in the KAT dataset were aged 71(SD = 7.1) years on average, with a mean ASA grade of 2(SD = 0.6). From a total set of nine candi- date predictors, four were selected for the screening tool: BMI, ASA grade, OKS question 1 and SF-12 question 6. Model discrimination, as measured by the c-index was 0.67. The screening tool score range is 0–10 and patients scoring 5 or more (29% of the KAT sample) are considered at increased risk of poor outcome following KR. Data was analysed as proportions. We reported the number of those enrolled and those being followed up in each group, divided by the total number eligible and the total number enrolled in each group respectively i.e. the risk ratio with 95% confidence intervals. Trials 2017, 18(Suppl 1):200 Page 162 of 235 Page 162 of 235 Trials 2017, 18(Suppl 1):200 Page 162 of 235 Search strategy h The ORRCA (Online Resource for Recruitment Research in Clinical Trials; www.orrca.org.uk) database was utilised in this systematic re- view. ORRCA includes studies of all designs, systematically extracted from the literature, reporting on recruitment into RCTs and non- randomised clinical studies. In this review, ORRCA was searched for primary research reports of studies that reported on recruitment to RCTs in adult patients receiving UHC. Informed consent and proxy decision making for research involving adults lacking capacity: a systematic review (framework synthesis) Cardiff University Correspondence: Victoria Shepherd Trials 2017, 18(Suppl 1):P431 Conclusions met. The review and the qualitative study will then be used to deter- mine the factors that must be included in a decision support inter- vention for research participation by proxy decision makers for adults lacking capacity. We developed a simple and objective screening tool to identify pa- tients at increased risk of poor outcome for inclusion in to the CORKA randomized clinical trial, with a moderate discriminatory ability. Background Decisions about the participation of adults lacking mental capacity in medical research are complex, and raise considerable legal and eth- ical issues. There are differences between decisions relating to the medical treatment of adults lacking capacity, and those concerning their participation in medical research. Carers and relatives of adults lacking capacity are regularly called upon to make such decisions on their behalf, however little is known about the ethical basis on which these proxy decisions are made and there is a dearth of information or support available. The coming decades are expected to see a sig- nificant rise in health challenges resulting from ageing populations, with a proportionate rise in conditions characterised by cognitive dis- orders. Ambitious UK research agendas have been set out in order to address these challenges, however these will require considerable numbers of research participants. Research into optimising recruitment to RCTs is commonly undertaken, however there is no agreed method for organising and reporting studies. Adequately describing and classifying recruitment study types may enable researchers to evaluate and compare studies more reliably. Aim This study developed and applied a categorisation system for differ- ent recruitment studies, encountered during a systematic review of recruitment to RCTs in unplanned hospital care (UHC), to inform fu- ture recruitment research. Results Category A - Randomised controlled trials of interventions to optimise re- cruitment within one or more host RCTs Category B - Non-randomised studies of interventions to optimise recruitment within one or more host RCTs Category C - Non-randomised studies without interventions evaluat- ing recruitment to one or more host RCTs Category D - Randomised studies to consider recruitment within proposed hypothetical RCTs (community consultations) Category E - Non-randomised studies to consider recruitment within proposed hypothetical RCTs (community consultations). The findings from the systematic review will be presented, which will include an examination of the ethical issues encountered, what fac- tors are involved when proxy decisions are made, and factors that affect the quality of informed consent and proxy decision making in practice. The review will provide an overarching synthesis of proxy decision-making for research participation, and the development of a conceptual framework. Reference [1] J Bone Joint Surg Am. 2009;91:134–41. [1] J Bone Joint Surg Am. 2009;91:134–41. New methods for categorising recruitment research: a case study Ceri Rowlands1, Leila Rooshenhas1, Jonathan Rees2, Jane M. Blazeby3 1MRC conduct-II Hub for Trials Methodology Research, School of Social & Community Medicine, University of Bristol; 2Division of Surgery, Head & Neck, University Hospitals Bristol NHS Foundation Trust; 3MRC conduct-II Hub for Trials Methodology Research, School of Social & Community Medicine, University of Bristol and Division of Surgery, Head & Neck, University Hospitals Bristol NHS Foundation Trust Background There are specific legal provisions in England and Wales governing proxy decision making by another individual, such as a family mem- ber of friend, for those unable to provide consent for themselves to participate in research. Data regarding the ethical and regulatory fac- tors influencing these decisions, and interventions to inform and sup- port those involved, are urgently required in order to maximise the participation of adults lacking capacity in research. Research partici- pants, their families and carers, clinicians and researchers require a clear, evidence-based ethical framework for research enrolment of adults lacking capacity. This systematic review forms part of an NIHR Doctoral Research Fellowship to investigate informed consent and proxy decision making in research involving adults lacking capacity, and the development of an intervention to support informed proxy decision making, set within ethical and legal frameworks. M h d Development of study categories Reading the articles led to initial categorisation of the recruitment studies into those with a rando- mised or non-randomised recruitment designs. Iterative refinement of the study structured categories through discussion between study authors (CR, JMB, LR, JR). It was noted that papers reporting surveys in the community (commu- nity consultations) had been undertaken to establish the likelihood of recruitment success or acceptability of a trial. In recognition of this, a clear differentiation was made between studies that focused on recruit- ment to an actual clinical RCT (a ‘host RCT’) versus potential recruitment to a RCT that did not yet exist (a ‘hypothetical RCT’). Latterly a further categorisation was introduced to classify whether the recruitment study evaluated an intervention to modify recruit- ment, or simply reported on recruitment experiences. The final classi- fication for papers was formulated based on whether i) randomised or non-randomised study design was employed during the recruit- ment study ii) an intervention to optimise recruitment was evaluated, and iii) a host or hypothetical RCT was used. A mixed methods systematic review will be conducted to determine the ethical and legal issues encountered in proxy decision-making for research participation by adults lacking capacity, using a frame- work synthesis approach. The aim is to synthesise empirical evidence from qualitative, quantitative or observational studies which examine the relevant ethical issues. The review will be registered with PROS- PERO database of systematic reviews. Background g Many clinical trials experience recruitment difficulties, leading to underpowered studies, costly extensions or early closure. Trials in paediatric critical care encounter additional practical and ethical difficulties as there is no time to seek prior informed consent in an emergency situation. Eclipse is an unblinded pragmatic randomised controlled trial that explores the treatment (levetiracetam versus phenytoin) of status epilepticus in children. Challenges to the suc- cess of eclipse include: a vulnerable target population (children aged 6 months to <18 years); the need to administer the interven- tion without prior informed consent (deferred consent); and use of levetiracetam, an anti-epileptic medication which is not traditionally usually used in this clinical setting. We evaluated the effectiveness of site training on healthcare practitioners’ (HCP) confidence in the recruitment of patients to eclipse. The interactive site training in- cluded: protocol presentations from the trial team; screening and randomisation simulation (video and real-time); a deferred consent scenario video informed by pre-trial feasibility work with parents, and a question and answer session. h d Results This systematic review will examine a range of factors encountered in research involving adults lacking capacity, and what influence these and other factors have on informed consent and proxy deci- sion making in practice. The findings will be used to develop a con- ceptual framework of proxy decision making which will form the basis of a subsequent qualitative study to explore how proxy deci- sions are made, and whether legal and ethical obligations are being 3114 papers were available in ORRCA and 39 met the inclusion cri- teria. The new categorisation was able to be applied to all papers with 1, 11, 16, 0 and 11 within categories A to E respectively. Conclusions This case study illustrates new methods for categorising recruitment studies. It has potential utility to researchers by encompassing the Page 163 of 235 Trials 2017, 18(Suppl 1):200 Page 163 of 235 Trials 2017, 18(Suppl 1):200 different aspects of the recruitment study design and the use of real/ hypothetical RCTs. This categorisation requires further evaluation in other recruitment settings to establish its validity and role. q Methods Mixed method study including a 14 item questionnaire administered before and immediately after the site training as well as telephone in- terviews with eclipse HCPs in the first 12 months after site opening. Results In total, 156 HCPs from 25 UK hospital completed a before and after site training questionnaire. We interviewed eight HCPs involved in patient-recruitment and deferred consent in eclipse. Prior to site training, HCPs were concerned about recruitment because of a lack of knowledge about the trial protocol and apprehensions about par- ents’ response to deferred consent. We found that site training im- proved HCP confidence in the trial, including being better able to discuss the study with parents (p < 0.001), explain randomisation (p < 0.001) explain deferred consent (p < 0.001) and address parents’ objections to their child being randomised (p < 0.001). HCPs valued the clarity and content of site training and described how videos helped them to visualise recruitment and consent processes. HCPs offered suggestions about how the trial management team could provide ongoing recruitment and consent support through study up- dates, recruitment-training tips and advice from study team mem- bers as and when required. However, many challenges abound in conducting sporadic remote visits by an external examination service technician. A clear and precise protocol is essential to ensure fidelity and consistency in data collection and equipment. Prioritizing data collection for non- clinic visits will help simplify the visit flow for external technicians to balance the capture of essential outcomes and participant bur- den. Training and communication are critical to facilitate interac- tions among the external examination service central office, the technician completing the visit, the clinical coordinator, the coord- inating center staff, and the participant. In this presentation, we will describe the process of working closely with an external examin- ation service for a long-term multi-center clinical trial with an aging cohort. We will present our experiences, both the successes and failures, over the first year of remote visit implementation within the framework of a national multi-center clinical trial. If long-term studies can overcome these obstacles, the use of external examin- ation services to conduct remote visits may provide a cost-effective solution to boost participant retention and support study validity in otherwise hard to reach populations. q Conclusions P433 Utilizing remote participant visits to boost retention in a long-term clinical trial Ashley Hogan, Hanna Sherif, Nicole Butler, Tsedenia Bezabeh, Adrienne Gottlieb, Ella Temprosa George Washington University Correspondence: Ashley Hogan Trials 2017, 18(Suppl 1):P433 The success of long-term studies rely heavily on the ability to retain participants for the entire study duration which may span much of the participant’s adult life. Researchers must accommodate partici- pants’ life changes including moving to locations that are no longer near a clinical center, personal circumstances that prevent in-person clinic visits, and most importantly aging. Prolonged illness and de- creased mobility of aging participants create barriers to clinic access for data collection. In such cases, performing collection at convenient locations for participants including their home, work or nursing home may boost retention. In studies with event driven analysis approach, the data for every participant is valued. Their individual contribution may be small but their retention is essential to the suc- cess of the study; as a result, a critical concern is how we can expand our reach and continue to maximize data collection on all partici- pants. Questionnaire data collected over the phone may not be enough and phenotypic data can offer a more complete picture. Thus to improve retention and minimize participant burden, cost- effective approaches to conduct remote visits can be implemented to collect anthropometric measurements and biospecimens with the use of external examination services. Conclusions Interactive site training can assist important HCP ‘buy in’ for challen- ging clinical trials. Our findings highlight how pre-trial feasibility work with parents can improve HCP confidence in recruitment and de- ferred consent seeking in a paediatric critical care trial. P435 P435 Seeing the light at the end of the carpal tunnel: the challenges of recruiting sites to a clinical trial of an investigational medicinal product in primary care Helen Myers1, Claire Burton2, Michelle Robinson1, Graham Davenport2, Krysia Dziedzic2, Danielle van der Windt2, Linda Chesterton2, Edward Roddy2, Elaine Hay2 1 2 f Seeing the light at the end of the carpal tunnel: the challenges of recruiting sites to a clinical trial of an investigational medicinal product in primary care 1 2 1 2 p p y Helen Myers1, Claire Burton2, Michelle Robinson1, Graham Davenport2, Krysia Dziedzic2, Danielle van der Windt2, Linda Chesterton2, Edward Roddy2, Elaine Hay2 1 2 1Keele Clinical Trials Unit; 2Institute for Primary Care and Health Sciences Correspondence: Helen Myers Trials 2017, 18(Suppl 1):P435 1Keele Clinical Trials Unit; 2Institute for Primary Care and Health Sciences Correspondence: Helen Myers Trials 2017, 18(Suppl 1):P435 Site training to improve healthcare practitioners? Confidence in recruiting to a challenging critical care trial Kerry Woolfall1, Louise Roper2, Amy Humphreys3, Mark D. Lyttle4, Shrouk Messahel5, Elizabeth Lee5, Joanne Noblet5, Anand Iyer6, Carrol Gamble7, Helen Hickey7 1The University of Liverpool; 2Department of Psychological Sciences, The University of Liverpool; 3Clinical Trials Research Centre (CTRC); 4Emergency Department, Bristol Royal Hospital for Children; Faculty of Health and Applied Sciences, University of the West of England; 5Emergency Department, Alder Hey Children’s NHS Foundation Trust; 6Neurology Department Alder Hey Children’s NHS Foundation Trust; 7Clinical Trials Research Centre (CTRC), North West Hub for Trials Methodology Research Correspondence: Kerry Woolfall Trials 2017, 18(Suppl 1):P434 P434 Site training to improve healthcare practitioners? Confidence in recruiting to a challenging critical care trial Kerry Woolfall1, Louise Roper2, Amy Humphreys3, Mark D. Lyttle4, Shrouk Messahel5, Elizabeth Lee5, Joanne Noblet5, Anand Iyer6, Carrol Gamble7, Helen Hickey7 1The University of Liverpool; 2Department of Psychological Sciences, The University of Liverpool; 3Clinical Trials Research Centre (CTRC); 4Emergency Department, Bristol Royal Hospital for Children; Faculty of Health and Applied Sciences, University of the West of England; 5Emergency Department, Alder Hey Children’s NHS Foundation Trust; 6Neurology Department Alder Hey Children’s NHS Foundation Trust; 7Clinical Trials Research Centre (CTRC), North West Hub for Trials Methodology Research Correspondence: Kerry Woolfall Trials 2017, 18(Suppl 1):P434 Background Sub-optimal participant recruitment rates are common in trials. A multi- tude of factors have been suggested to explain this [1], including the task of opening sites to recruitment. Clinical trials of investigational me- dicinal products (CTIMPs) may pose additional challenges in primary care compared to other healthcare settings due to the need to engage busy general practitioners (GPs) in research processes. A recent report [2] highlighted the increasing pressures on GPs to meet the clinical needs of patients during routine consultations. Given this backdrop, fa- cilitating GPs to recruit participants can prove challenging. This abstract presents the experiences of opening sites to recruitment for a nation- wide clinical trial based in primary care: Injection versus Splinting in Carpal Tunnel Syndrome (instincts) [3]. ( Methodology Research Correspondence: Kerry Woolfall Trials 2017, 18(Suppl 1):P434 Page 164 of 235 Trials 2017, 18(Suppl 1):200 Page 164 of 235 p Methods Trial managers based in a registered CTU involved in setting-up re- cruitment sites during 2014/15 predicted the recruitment success of each site they opened: whether the site would recruit to target (ie re- cruit the number of participants documented in the site agreement) or not, and reasons for this. Predictions were placed in sealed enve- lopes and opened after a minimum of 8 months recruitment at each site. A focus group was held with the trial managers where predic- tions were revealed and compared with the actual recruitment achieved by the site; reasons for and accuracy of the prediction were discussed. Background It is estimated that around 50% of trials fail to recruit to target. Po- tential implications of this is that trials require extensions to their re- cruitment period, their sample size is revised, or the trial is closed, leaving the clinical question unanswered. In multi-centre trials, sub- stantial time and effort is spent setting up clinical recruitment sites, and this has inherent cost. Experience suggests that some sites will recruit to target and others will not. The more sites that fail to recruit to target, the less likely the trial will meet recruitment targets. Aims Retention in randomised trials: what matters to trial managers? Katie Gillies, Shaun Treweek University of Aberdeen Correspondence: Katie Gillies Trials 2017, 18(Suppl 1):P436 Retention in randomised trials: what matters to trial managers? Katie Gillies, Shaun Treweek University of Aberdeen Correspondence: Katie Gillies Trials 2017, 18(Suppl 1):P436 Retention in randomised trials: what m Katie Gillies, Shaun Treweek University of Aberdeen Correspondence: Katie Gillies Trials 2017, 18(Suppl 1):P436 p Conclusions Targeting GPs using a ‘hub-and-spoke’ model and Interface Clinics was generally most efficient for site opening and most productive for subsequent participant recruitment. Web-based initial training would have allowed sites to register interest after seeing what the trial involved, thereby saving time and resources. Ensuring sites were ‘research ready’ and had completed site set-up paperwork be- fore training would have reduced the delay in site opening. GPs were required to offer each participant several appointment slots to screen, consent, randomise and treat, which may have dissuaded them from engaging: providing support with these activities may have encouraged more sites to participate. Can trial managers predict whether sites will recruit to target or not - Results from the estimating site performance (ESP) study Seonaidh Cotton, Kirsty Shearer, Anne Duncan, Hanne Bruhn, Shaun Treweek University of Aberdeen Correspondence: Seonaidh Cotton Trials 2017, 18(Suppl 1):P437 Background We explored whether trial managers (involved in recruitment site set-up) were able to predict whether a site would recruit to target or not before the site was opened to recruitment, the reasons for these predictions, and their subsequent reflections. It is common for many trial participants (sometimes more than 20%) to drop out before the trial finishes. Drop out seriously affects the credibility of trial results and significantly affects a trials potential to influence clinical practice. Recent estimates have shown that in up to 53% of trials the results could have been overturned if the outcomes from those who had dropped out were known. Trial Managers are often faced with identifying ways to improve retention but the evi- dence base on effective methods in this area is lacking. Exploring and identifying Trial Managers experience and tacit knowledge re- garding what they perceive as the biggest barriers and facilitators for retention in RCTs can contribute to this evidence base and identify areas for further research. pp Discussion This survey identifies some of the main barriers and facilitators to trial retention as perceived by Trial Managers. Specifically, the results focus on trial design and participant perspectives, how poor proto- cols make retention harder, and identify potential priorities for future evaluation. Methods are employed on a publicly funded trial and registered with the UK TMN). In addition to questions about demographics, there were 4 broad open-ended questions that asked Trial Managers about: experi- ence of retention and what they perceive as main issues; thoughts on ways to improve retention; how trial design could be changed to im- prove retention; and to identify one thing in relation to retention that would make their lives easier. Demographic data was analysed using descriptive statistics and the free text responses were coded using a thematic analysis approach. are employed on a publicly funded trial and registered with the UK TMN). In addition to questions about demographics, there were 4 broad open-ended questions that asked Trial Managers about: experi- ence of retention and what they perceive as main issues; thoughts on ways to improve retention; how trial design could be changed to im- prove retention; and to identify one thing in relation to retention that would make their lives easier. Demographic data was analysed using descriptive statistics and the free text responses were coded using a thematic analysis approach. Recruiting sites were GP practices, GPs using a ‘hub-and-spoke’ model, and primary-secondary care Interface Clinics. Practices were identified via the Primary Care Rheumatology Society and through the relevant Clinical Research Networks (CRN). Training was undertaken at regional workshops and individual sites. Some sites had access to Research Nurses, or support from the CRN for set-up. Support with completing site set-up paperwork was offered to sites by the Clinical Trials Unit (telephone or visit). p Results The email invite was sent to 501 list SERV members and we received 48 responses (9.6% response rate). The duration of trial management experience of respondents ranged from 0.8 to 34 years (median 5 (Q1:3; Q3: 12.25)). The types of trial managed by respondents showed wide variation and covered all phases of trial (Phase I-Phase IV) includ- ing pilot trials; trials set within primary, secondary and tertiary care; with children and adults; in a range of clinical areas; and interventions under investigation also varied (included CTIMPS, devices, surgery, educa- tional, service level). The findings could be grouped in two main themes: considerations relating to trial design; and considerations relat- ing to participant perspectives. The overwhelming issue raised in the 48 responses from across the UK was that many aspects of retention are context dependent: at the level of the clinical condition; its associ- ated population; and the individual trial (i.e. Type of follow-up, differ- ences across sites with relation to how retention is discussed and the rapport between recruiter and participant). Discussion Of the 59 sites expressing interest, 27 opened, 12 were trained but did not open, and 20 were not trained and did not proceed further. Of the sites which opened to recruitment, 13 were GP practices, seven were GPs using a ‘hub-and-spoke’ model, and seven were Interface Clinics. Time from training to site opening ranged from two to 24 months. Delays between training and site opening were experienced for a variety of reasons related to overall trial set-up and site-specific set-up. The change from Primary Care Trusts to Clinical Commissioning Groups delayed sites engaging with the trial. Site- specific issues included delays completing site set-up paperwork and meeting the additional requirements for conducting CTIMPs (e.g. Good Clinical Practice training). Delays to site opening meant some sites needing re-training and others never opening. Reasons for trained sites never opening included changes in clinician availability and service re-structure. As a consequence, there was a need to recruit sites for longer than anticipated. Support from a Research Nurse or CRN had a positive effect on site set-up. GPs using a ‘hub-and-spoke’ model and Interface Clinics were eventually targeted as priority for site opening, as initial patterns of recruitment showed these sites gave a better return in terms of recruitment for the time invested in site set-up. Methods A web-based survey was conducted as a scoping exercise to explore what Trial Managers perceive as the biggest barriers and facilitators for retention in randomised trials. Trial Managers were sent an invita- tion to complete the survey through the UK Trial Managers Network (UK TMN) listserv to all listed members (i.e. Those trial managers who Results 10 trial managers working across 7 randomised trials and one non- randomised diagnostic study participated; 56 predictions were made. Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 165 of 235 Of 39 predictions about sites involved in randomised trials, 31 (79%) were that the site would recruit to target; 17 of these (55%) were correct and the site met target. Of 17 predictions made about sites in the diagnostic study, 12 (71%) were that the site would recruit to target; all 12 did so. Reasons for positive predictions were similar across the types of studies and included engaged/enthusiastic PI/ team, experienced research nurses, consideration of trial logistics prior to site initiation. Eight sites involved in randomised trials were predicted not to recruit to target; 6 of these predictions were correct. Five sites involved in the diagnostic study were predicted not to recruit to target; only 1 site did not recruit to target. Reasons given for these predictions included lack of interest/engagement, slow responses, involvement in multiple studies. Results from the focus group will be presented. We will also explore whether there is any difference between accuracy of prediction in sites where training was on-site or carried out remotely. Methods ARIC’s key to successful retention include information that was gath- ered at visit 1, and consistent contact with the participant over the years. At visit 1, information on the participants’ address, physicians, contacts, and other items were well documented. Spousal participation in the study was also encouraged in hopes of keeping both spouses in- volved. Annual and semi-annual phone interviews that include health related questions also verify contact information. This includes current participant physical and mailing address; multiple contact names, ad- dresses and phone numbers; physician name, address and phone num- ber; as well as a designated proxy who can represent the participant given any future health or cognitive problems (which is especially im- portant with an aging cohort). Phone interviews are done at optimal times such as evenings or weekends. At the time of the call, inter- viewers have at their fingertips all the relevant information on the par- ticipant via reports produced on a web-based data management system called Carolina Data Acquisition and Reporting Tool, or CDART (developed by the ARIC coordinating center at the University of North Carolina at Chapel Hill). This report provides participant information to facilitate the call such as living arrangements, potential hearing loss, study history and other administrative notes. Long-term and dedicated interviewing staff provide familiarity for the participant, and go through annual interviewing recertification to ensure quality. Periodic clinic visits are done (currently conducting visit 6) where various interviews and medical tests are completed. The participants are offered a small sti- pend for the visit, as well as comprehensive test results that they can share with their physicians. If the participant is unable to come to the clinic, in-home visits are offered. To further engage the participant, peri- odic newsletters and birthday/greeting cards are mailed, and a partici- pant focused public website (in both English and Spanish) is available. In the iquad trial the theory-based letter was associated with a 6% increase in questionnaire return rate when compared with the standard letter [5]. This is promising but needs replication to inves- tigate if this effect can be repeated in other trials with different pa- tient populations that may have different barriers to “behaviour change”. P439 P439 Can behaviour change theory increase questionnaire response rates within trials? Kirsteen Goodman1, Suzanne Hagen1, Doreen McClurg, Nicole Sergenson1, Susan Stratton1, Shaun Treweek2 1Nursing, Midwifery and Allied Health Professional Research Unit; 2Health Services Research Unit, University of Aberdeen Correspondence: Kirsteen Goodman Trials 2017, 18(Suppl 1):P439 Conclusions Trial managers were generally optimistic in their predictions. For the randomised studies, predictions were more likely to be correct when trial managers predicted that a site would not meet recruitment tar- gets. We propose that the trial manager’s tacit knowledge at site set up may help to target time and resources more effectively during site set-up: the implication of this is that time and resource may be directed away from sites predicted to not meet recruitment targets. Results Sixty-four letters (group 1:32, group 2: 32) will be sent in the AMBER trial (41 sent to date). 384 letters (group 1: 205, group 2: 179) will be sent in the OPAL trial (174 sent to date). These data will be pooled with the iquad data to create a cumulative meta-analysis, which will also form part of future versions of the Cochrane review of interven- tions to improve retention. g Objectives To ascertain whether a theory-based cover letter accompanying pa- tient follow-up questionnaires improves response rates in two trials with differing patient groups; the AMBER (neurogenic bowel) and the OPAL trial (urogynaecology) [6,7]. Keys to successful participant response rates in a 30 year longitudinal study Keys to successful participant response rates in a 30 year longitudinal study Lisa Reeves University of North Carolina Trials 2017, 18(Suppl 1):P438 g y Lisa Reeves University of North Carolina Trials 2017, 18(Suppl 1):P438 Results Currently, the ARIC study follow-up has over 80% response rate of those participants still alive, providing over 30 years of robust data sets for investigators worldwide, and contributed to over 1600 peer- reviewed articles. ARIC continues to follow these participants and has plans for its seventh clinic visit in 2017. Background ARIC (Atherosclerosis Risk in Communities) is a prospective study funded by the National Heart, Lung and Blood Institute (NHLBI) that began over 30 years ago in 4 communities in the United States: Forsyth County, North Carolina; Jackson, Mississippi; Minneapolis Minnesota; and Washington County Maryland. At that time, 15,792 participants be- tween the ages of 45 and 64 were enrolled and have been successfully followed over the years with an impressive response rate. The iquad trial team [3] has developed a template for a theory- based cover letter (the intervention) using the Theoretical Domains Framework (TDF) [4] to identify theoretical targets for behaviour change interventions. This letter is issued to participants with follow-up questionnaires. Methods Methods AMBER and OPAL trial participants are randomly assigned to receive a theory-based cover letter (group 1) or a standard letter (group 2) with follow-up postal questionnaires; 24 weeks in AMBER; 12 and 24 months in OPAL. Questionnaire response rates across time-points in the AMBER and OPAL trials will be presented. Results Background Maximising recruitment in trials is an important goal for trial teams; however a trial that has met recruitment targets can be underpow- ered with inconclusive results if retention rates are poor. Trialists an- ticipate around 10% of participants will withdraw from a trial or are “lost to follow up”, however, there are few interventions with high quality evidence of benefit for increasing retention in trials [1–2]. The need for more rigorous evaluations of interventions to increase retention in trials has resulted in the Trial Forge Initiative (http:// trialforge.org) coordinating SWAT studies (“study within a trial”): evaluations of trial methods innovations embedded within a host trial Research teams can use the same SWAT protocol, thus the re- sults can be combined in a meta-analysis which may provide high- quality evidence to inform their trial design. The SWAT 24 protocol focuses on increasing the return of outcome data collected through participant-completed questionnaires. The completion and return of the questionnaires is a “behaviour” Which could be influenced by an intervention targeting participants’ willingness “to do” this behaviour. Conclusions We will provide evidence as to whether a theory-informed question- naire cover letter can improve trial questionnaire response rates, and whether some patient populations are more receptive to these letters than others. Trials 2017, 18(Suppl 1):200 Page 166 of 235 Page 166 of 235 ; p Results Results will be presented reporting the bias, power, precision and ICC estimation using the different analysis models. The effect of the choice of imposed clustering on the intracluster correlation estimate will be presented and the most appropriate method for imposing clustering in the unclustered control arm. p g Methods Simulation studies will be used to explore varying scenarios of cluster size, number of clusters, intra-cluster correlation, and differential variance between the two trial arms and their impact on bias, power, precision and ICC estimation. In theory the mixed effect models for partially nested trials do not model clustering in the control arm, how- ever, when fitting these models in statistical software it is necessary to impose clustering in the unclustered control arm. We will explore the various methods for imposing clustering in the control arm: a unique singleton cluster of size one for every individual; one large single clus- ter; or pseudo random clusters. The two-stage treatment selection (TSTS) design: a novel approach to treatment selection in clinical trials 1 1 2 3 Matthew Parkes1, Mark Lunt1, Philip S. Pallmann2, David T. Felson3 1University of Manchester; 2Lancaster University; 3Boston University Correspondence: Matthew Parkes Trials 2017, 18(Suppl 1):P440 References Correspondence: Jane Candlish Trials 2017, 18(Suppl 1):P441 7. The OPAL Study is a UK collaborative study funded by the National Institute for Health Research, Health Technology Assessment Programme (Project:11/71/03) and sponsored by Glasgow Caledonian University (CI: Professor Suzanne Hagen). Ethical approval was granted by the West of Scotland Research Ethics Committee. 7. The OPAL Study is a UK collaborative study funded by the National Institute for Health Research, Health Technology Assessment Programme (Project:11/71/03) and sponsored by Glasgow Caledonian University (CI: Professor Suzanne Hagen). Ethical approval was granted by the West of Scotland Research Ethics Committee. Background Individually randomised trials often have the added complication of a comparison of interventions administered in different ways, where groups of outcomes are correlated in one trial arm and not the other, termed a partially nested design. The correlation of outcomes is defined by the nature of the intervention itself, for example, a comparison of group therapy intervention and drug therapy control. Small clusters, small intracluster correlations, and differential variance between the control and intervention arms are often present in partially nested trials. If not accounted for in the design or analysis this may result in biased effect size estimates with spurious precision. The two-stage treatment selection (TSTS) design: a novel approach to treatment selection in clinical trials Matthew Parkes1, Mark Lunt1, Philip S. Pallmann2, David T. Felson3 1University of Manchester; 2Lancaster University; 3Boston University Correspondence: Matthew Parkes Trials 2017, 18(Suppl 1):P440 Objective To evaluate statistical methods to analyse partially nested trials and provide practical advice on the analysis of partially nested trials using a simulation study, with focus on the most appropriate method for imposing clustering in the unclustered control arm. M th d Clinical trial designs which focus resources on treatments that show promise in early accrued data are efficient and desirable, particularly when resources are constrained. To help stop trials, or trial arms, which show a low likelihood of confirming a treatment effect at final analysis, typically one could include an interim analysis in a parallel-groups ?A3B2 show $132#?>confirmatory clinical trial, or adopt an adaptive multi-arm design, for example a seamless phase II/III design. These strategies all feature one or more interim analyses during trial recruit- ment to assess whether the trial is to continue to completion. In a typ- ical interim analysis, the hypotheses tested at interim/transition are principally concerned with whether the trial is likely to observe a treat- ment effect which meets the chosen limit for statistical significance, at the final analysis. This analysis only indirectly tests whether the treat- ment is likely to produce a useful clinical effect at final analysis. Treat- ment selection designs may be better served by confidence intervals and estimation methods, than more traditional hypothesis testing ap- proaches. Using confidence intervals at interim gives the researcher a better idea of an interim estimate’s precision, and therefore provides more information about a treatment’s potential efficacy than a p-value alone allows. A researcher interested in establishing the efficacy of an intervention may wish to continue a trial showing a large variance (imprecise estimate) at interim, in the hope that the later analyses (either interim or final), which feature more participants, may reveal a more precise estimate of the true treatment effect of the intervention. Interim analyses using p-values as the stopping criteria do not address this issue.Instead, imprecision in the estimate increases the likelihood of the trial stopping. To resolve this problem, the stopping criterion of a typical interim analysis for futility could be modified to instead stop a treatment arm when the interim treatment effect confidence interval is contained entirely within a region deemed clinically unimportant – an indication that the treatment is likely to not be of benefit. Interim estimates which are imprecise (have wide confidence intervals) are pro- tected from stopping using this rule, rather than with a hypothesis References test using a p-value as the criterion. Confidence intervals produced using interim data have limitations – most obviously the fact that they will be wider (less precise) than those expected at the final analysis of the trial, given the lower sample size, which, using the proposed stopping criteria. To avoid these intervals being so inad- equately wide at a low sample size that all trials continue to com- pletion, the use of normal-based confidence intervals at a lower nominal confidence level (e.g. 90%), ‘predicted intervals’, which re- place elements of the confidence interval calculation with assumed values, Bayesian estimation, or novel, bootstrapping-based methods, could be used instead, all of which have varying implica- tions on the analysis. A comparison of this method is discussed, using simulated data. 1. Treweek S, Mitchell E, Pitkethly M, Cook J, Kjeldstrøm M, Johansen M, Taskila TK, Sullivan F, Wilson S, Jackson C, Jones R, Lockhart P. Strategies to improve recruitment to randomised controlled trials. Cochrane Database of Systematic Reviews 2010, Issue 4. Art. No.: MR000013. doi:10.1002/14651858.MR000013.pub5. 2. Brueton VC, Tierney J, Stenning S, Harding S, Meredith S, Nazareth I, Rait G. Strategies to improve retention in randomised trials. Cochrane Database of Systematic Reviews 2013, Issue 3. Jan E Clarkson et al. IQuaD dental trial; improving the quality of dentistry: a multicentre randomised controlled trial comparing oral hygiene advice and periodontal instrumentation for the prevention and management of periodontal disease in dentate adults attending dental primary care.BMC Oral Health 10/2013; 13(1):58 4. Michie et al. Making psychological theory useful for implementing evidence based practice. Qual & Safety in Health Care 2005; 14:26–33 5. Duncan et al. Improving trial questionnaire response rates using behaviour change theory. Trials 2015, 16(Suppl 2):P92 P441 Methods to analyse partially nested randomised controlled trials Jane Candlish, Dawn Teare, Judith Cohen, Munyaradzi Dimairo, Laura Flight, Laura Mandefield, Stephen Walters University of Sheffield Correspondence: Jane Candlish Trials 2017, 18(Suppl 1):P441 P441 Methods to analyse partially nested randomised controlled trials Jane Candlish, Dawn Teare, Judith Cohen, Munyaradzi Dimairo, Laura Flight, Laura Mandefield, Stephen Walters University of Sheffield C d J C dli h 6. The AMBER Study is A UK collaborative study funded by the National Institute for Health Research, Health Technology Assessment Programme (Project: 12/127/12) and sponsored by Glasgow Caledonian University (CI: Professor Doreen McClurg). Ethical approval was granted by the West of Scotland Research Ethics Committee. Overview of statistical methods to monitor harms during the conduct of a randomised controlled trial 1 2 The target difference, or ‘effect size’, is an important component of a sample size calculation as the calculations are extremely sensitive to assumptions as to what the effect size will be. A review of 117 NIHR Health Technology Assessment funded randomised controlled trials indicated that over 50% of randomised controlled trials report that they have used a previous study or previous research to estimate their target effect size. Conclusions Since they are implemented after each observed event they are best suited to the evaluation of serious adverse events where immediate evaluation is ne- cessary in order to determine whether the trial should continue The review identified 11 methods for use for harm monitoring and analysis in clinical trials. We have categorised these as: sequential methods, group sequential methods and surveillance methods. The four sequential methods have been designed to be implemented after each observed harm event. They have been developed for use in a single treatment arm setting and require a pre-specified harm of interest and a pre-specified hypotheses to be tested. Since they are implemented after each observed event they are best suited to the evaluation of serious adverse events where immediate evaluation is ne- cessary in order to determine whether the trial should continue. Group sequential methods primarily proposed for use in monitoring efficacy outcomes have been extended by several authors for the purpose of harm monitoring. Analogous to the methods for efficacy each require a pre-specified harm of interest with a pre-specified hy- pothesis to be tested. A binary endpoint (success/failure) was modelled using a multi-level lo- gistic model, treating operating surgeon as the unit of clustering. The number of patients assigned to each surgeon was simulated using a gamma distribution, which allowed the small and/or unbalanced clus- ter sizes described above to be simulated. Four surveillance methods have been developed for multi-arm stud- ies with the purpose of monitoring emerging harm events i.e. The harm is not pre-specified. The applications of these methods to date have been applied at body system level rather than reported adverse event level. A ‘surgeon effect’ was included in the simulation that would increase the probability of success based on the experience, skill etc. of the sur- geon. Another ‘surgeon treatment effect’ was included that allowed the surgeon effect to be different depending on the treatment being performed. In a review we undertook to examine the methods and reporting of harms in rcts we found none of the 189 included trials used any of these methods. Patients were allocated to one of two treatments using a minimisa- tion algorithm, stratifying for the operating surgeon. Background b Data obtained from randomised controlled trials (RCTs) contribute important information to the harm profile of a drug as they provide unbiased estimates of harm effects and provide a controlled com- parison allowing causality to be evaluated. The objective of this presentation is to examine the issues that can arise when designing one trial based on the results of a previous trial, or previous research using a simulation study. When basing one study on the results of another, there is a bias which is introduced called regression to the mean. This bias means that there would be an over-estimation of the effect size, and the effect size observed in the second trial is likely to be considerably less than that which the study was powered on. The most common approach for harm monitoring and analysis dur- ing a RCT is to tabulate event rates by treatment arm and sometimes the difference in event rates is estimated and p-values from hypoth- esis tests are presented. Data are examined by an independent data monitoring committee (DMCs) who will make a recommendation to proceed or halt a trial based on these presentations. More formal as- sessments and integration of existing or emerging knowledge for drug harm into the DMC report is rare and as a result there is an in- efficiency present when monitoring and analysing harms in trials. In Conclusions Statistical methods have been proposed for use in a clinical trials set- ting to flag signals for adverse drug reactions for both pre-specified harm events and for emerging harm events. However the clinical trials community are not currently implementing these harm moni- toring methods and tabulations of adverse events remains the most popular choice to evaluate disproportionalities between treatment arms. The reasons for this are unclear but could be due to: their rela- tive infancy; sophisticated methodology; the computational intensity and increased resource level needed; and no formal requirement from regulatory bodies and the wider clinical community for more robust methods. The patients were assigned a probability of success based on their treatment. The surgeon effects were incorporated to calculate a different probability for each surgeon and treatment combination. The outcome was then generated from a binomial distribution using the calculated probability as the probability of success. Both random intercept and random slope models were investigated. The effects of changing the number of surgeons, changing the variance of the ?A3B2 show $132#?>surgeon effect and changing the variance of the surgeon treatment effect on model bias and convergence were in- vestigated, as well as their effects on the power of the trial. Early results suggest that unbalanced and small cluster sizes do not appear to effect the convergence of the model or cause bias in the fixed effects of the model. The effects of different cluster size distri- butions on the power of the study will be investigated. The conse- quences of changing the variance of the ‘surgeon effect’ and the ‘surgeon treatment effect’ will also be investigated as these may vary depending on the difficulty of the operation and the difference in the skill required for each operation within a trial. P444 Quantifying effect sizes in clinical trials Joanne Rothwell, Steven A Julious, Cindy Cooper University of Sheffield Correspondence: Joanne Rothwell Trials 2017, 18(Suppl 1):P444 Background Overview of statistical methods to monitor harms durin conduct of a randomised controlled trial Rachel Phillips1, Victoria Cornelius2 1King's College London; 2Imperial College London Correspondence: Rachel Phillips Trials 2017, 18(Suppl 1):P443 Conclusions Partially nested trials are commonly used in complex intervention re- search. The design and analysis of these trials can take account of the hierarchical data structure and needs to consider the choice of imposing clustering in the unclustered control arm. Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 167 of 235 Page 167 of 235 The effect of small or unbalanced clusters of patients on logistic re- gression models in surgical trials In surgical trials it is necessary to adjust for the clustering effect of the operating surgeon, as outcomes will be more similar for patients with the same operating surgeon than for those with a different surgeon. Due to the incremental na- ture of surgeon recruitment into a large surgical trial, it is likely that within a trial there will be a large number of surgeons that operate on only a few patients each, causing small cluster sizes. It is also likely that there will be a few surgeons’ recruited to the trial earlier - that operate on a lot more patients than the rest of the surgeons in the trial, creating unbalanced cluster sizes. Practically, the potential effects of these small or unbalanced cluster sizes on the bias and convergence of a multi-level model can be a concern when a trial is forced to recruit many more centres or surgeons than originally planned, for example to bolster recruitment. A simulation approach was used to explore and quantify the potential risk of recruiting many additional surgeons (while keeping the sample size fixed). the last 15 years new methods for improving the monitoring and ana- lysis of harms in trials have been proposed. We review these methods, outline when they are appropriate to implement, examine their use in published studies and discuss challenges of their implementation. Results The review identified 11 methods for use for harm monitoring and analysis in clinical trials. We have categorised these as: sequential methods, group sequential methods and surveillance methods. The four sequential methods have been designed to be implemented after each observed harm event. They have been developed for use in a single treatment arm setting and require a pre-specified harm of interest and a pre-specified hypotheses to be tested. udy a Methods Simulations were performed to quantify the impact of using previ- ously observed responses to design future studies. The inputs used in the simulations were based on the findings of the review. The sim- ulations were completed under the context of having one trial based Trials 2017, 18(Suppl 1):200 Page 168 of 235 Page 168 of 235 on the results of another. Various end-points were used to build from the simplest case to cases where biomarkers or surrogate end-points were used. The simulation results will be used to inform a mathemat- ical solution using a truncated Normal distribution. This mathematical solution will provide an adjustment which can be used to better estimate sample sizes when using previous results. The results will be extended to different powers and significance levels. Results on the results of another. Various end-points were used to build from the simplest case to cases where biomarkers or surrogate end-points were used. The simulation results will be used to inform a mathemat- ical solution using a truncated Normal distribution. This mathematical solution will provide an adjustment which can be used to better estimate sample sizes when using previous results. The results will be extended to different powers and significance levels. Results Current trends in data monitoring committees Kent Koprowicz, David R. Kerr Axio Research Correspondence: Kent Koprowicz Trials 2017, 18(Suppl 1):P446 Current trends in data monitoring committees Kent Koprowicz, David R. Kerr Axio Research Correspondence: Kent Koprowicz Trials 2017, 18(Suppl 1):P446 Axio Research has served as an independent statistical group serving Data Monitoring Committees (DMCs) for industry and government clinical trials in pharmaceuticals and devices for over two decades. Accepted, best, and most common practices have changed greatly over this period. The practice of DMCs continues to evolve and emer- ging trends will be discussed. Current trends include: program-wide DMCs, teleconference and web meetings, reduced sponsor and DMC interaction, focused DMC recommendation delivery, electronic reporting, DMCs for more studies (early phase, single-arm, open- label), expertise of DMC members. Pros and cons, implementation, and what to expect in the near future will be covered from the per- spective of an independent statistician with guidance for both spon- sors and DMC members. Using effect sizes previously observed to design a new study can lead to an over estimation of the treatment effect. Background g Randomised controlled trials (RCTs) are widely considered the gold standard study design for quantifying the effect of an intervention, due to the minimal risk of bias from confounding. Some RCTs are de- signed whereby subjects are randomised to different strategies, for example differing criteria for red blood cell (RBC) transfusions to be given, rather than specifically to an intervention or control treatment. In such studies the groups should differ substantially overall in terms of the intervention received (e.g. The average number of RBC units transfused), but within each of the randomised groups there will be heterogeneity in the intervention received. Such situations give an opportunity to estimate across the RCT as a whole the effect of dif- fering amounts of intervention (i.e. An observational analysis within the RCT). The latter can be estimated using instrumental variable (IV) techniques with randomised allocation as an instrument, avoiding the problem of confounding (measured or unmeasured) that is often a concern in observational analyses. Methods We have used this approach in three RCTs. In the titre2 trial a liberal RBC transfusion strategy after cardiac surgery was compared with a more restrictive strategy, creating two groups with different risks of transfusion and distributions of numbers of RBC units transfused. The Thermic trials compared paediatric cardiac surgery performed at warmer (normothermic) vs colder (hypothermic) temperatures, gen- erating groups with different average surgery temperatures. Finally, the RAPIDO trial compared a rapid diagnostic pathway with the con- ventional method for patients with blood stream infections, with the resultant groups differing substantially in terms of the time until microbiological information is returned from the laboratory. In addition to the primary intention-to-treat (ITT) analyses an IV analysis was performed for each trial, with randomised allocation as an instru- ment. Such models estimate different effects to the ITT analyses, namely: each RBC unit transfused on severe post-operative complica- tion (titre2); each degree Celsius on intubation duration (Thermic); each We have used this approach in three RCTs. In the titre2 trial a liberal RBC transfusion strategy after cardiac surgery was compared with a more restrictive strategy, creating two groups with different risks of transfusion and distributions of numbers of RBC units transfused. The Thermic trials compared paediatric cardiac surgery performed at warmer (normothermic) vs colder (hypothermic) temperatures, gen- erating groups with different average surgery temperatures. Finally, the RAPIDO trial compared a rapid diagnostic pathway with the con- ventional method for patients with blood stream infections, with the resultant groups differing substantially in terms of the time until microbiological information is returned from the laboratory. Background The intra-cluster correlation coefficient (ICC) is a statistic that is used to describe the variation between and within clusters. A trial ICC can be calculated from a pilot study but when calculated has a large confident interval. Alternatively we can select an ICC for a trial from previous trials ICC’s that have similar cluster type and outcome. Aim We aim to collect data available on ICC’s from previous trials to create a prior distribution of ICC’s and then combine these with the data from pilot studies in a Bayesian analysis. Bayesian prediction of the intra-cluster correlation for sample size calculation of cluster randomised trials Bayesian prediction of the intra-cluster correlation for sample size calculation of cluster randomised trials Chris Newby, Sandra Eldridge Chris Newby, Sandra Eldridge Quenn Mary, University of London Correspondence: Chris Newby Trials 2017, 18(Suppl 1):P445 y y Correspondence: Chris Newby Trials 2017, 18(Suppl 1):P445 p Methods As an example we use simulated data of 10 clusters with 200 patients in each cluster with an ICC of 0.05 with four priors for the ICC. A non- informative prior and three informative priors, one based on 56 studies for continuous outcomes of GP surgeries from University of Aberdeen, one based on QMUL’s cluster randomised course containing 150 ICC’s and a prior based on 10 studies specific to asthma questionnaire data from GP surgeries. Results The ICC was calculated from Bayesian software winbugs and returned to R. The mean and credible interval for ICC were calculated from the posterior distribution. The different methods of ICC calculation along with their means and confidence/credible intervals are summarised and compared. udy a Methods This over estima- tion could be as much as 15% which, if not allowed for, could lead to studies with sample sizes that are too small and therefore are underpowered. Designing a trial dependent on the results of a first trial impacts on the distribution of plausible responses for this initial trial and leads to bias in effect size estimation. Methods will be pre- sented that allow for this over estimation. The level of adjustment will depend on factors such as the statistical power of the first study or the p-value of a meta-analysis to combine previous studies. C l i p Conclusions When designing a clinical trial which is dependent on the results of a first trial, the effect size used will be overestimated and so as a result the sample size will be too small. The effect size should be adjusted to account for the sequential nature of the trials being investigated. Use of instrumental variables within randomised controlled trials Katie Pike1, Chris A. Rogers1, Gavin J. Murphy2, Massimo Caputo3, Alasdair MacGowan4, Barnaby C. Reeves1 1Clinical Trials and Evaluation Unit, University of Bristol; 2University of Leicester; 3Bristol Royal Hospital for Children; 4Southmead Hospital Correspondence: Katie Pike Trials 2017, 18(Suppl 1):P447 Bayesian prediction of the intra-cluster correlation for sample size calculation of cluster randomised trials Chris Newby, Sandra Eldridge Quenn Mary, University of London Correspondence: Chris Newby Trials 2017, 18(Suppl 1):P445 Discussion Although the ITT and IV models are estimating different effects we anticipated that the direction of effects would be consistent, which was the case in the examples we considered. The use of IV tech- niques to address secondary objectives in RCTs can be a useful tool in certain settings, although such models are generally low powered. Methods Cox’s proportional hazards regression modelling is a common method for analysing time-to-event data in clinical trials, and pro- vides an estimate of the hazard ratio (HR) as a measure of the overall treatment effect. This semi-parametric model relies on the assump- tion that the hazard ratio remains constant over time, such that the hazards between the treatment groups are proportional. If this assumption is violated, the Cox’s proportional hazards model can lead to a reduction in power for the corresponding tests of signifi- cance and more crucially, imprecise and misleading estimates of the treatment effect. We are using the simulation model to generate biomarker data of the S and E type. Four different analysis methods are being used to analyse the simulated biomarker data and estimate the relative precision of each model: t-test of the maximum change from baseline; area under the curve; multiple comparisons with Bonfer- roni correction; and a multilevel model. We are boot-strapping data on creatinine (type S biomarker) and troponin (type E bio- marker) collected in a randomised controlled trial to mirror the simulated scenarios with real data. We are also investigating the impact on relative precision of removing varying amounts of data (5%, 10% and 20%) at random, since repeated biomarker mea- sures are often incomplete. ADMIRE is a two-arm Phase II randomised-controlled trial, in 215 pa- tients with Chronic Lymphocytic Leukaemia (CLL). Progression-free sur- vival (PFS) was one of the key secondary endpoints, the intention for analysis was via a multivariable Cox regression model and presentation of the Kaplan-Meier survival estimates. On analysis of PFS, there was strong evidence that the proportional hazards (PH) assumption did not hold, as indicated by the crossing of the survival curves, putting into question the reliability of the estimate of the HR in the Cox regression model. Use of longitudinal data in the analysis of biomarkers: lessons from simulation and reality Use of longitudinal data in the analysis of biomarkers: lessons from simulation and reality Francesca Fiorentino1, Chris A. Rogers2, Gianni Davide Angelini2, Shahrul Mt-Isa1, Barnaby C. Reeves2 1Imperial College London; 2Bristol University Correspondence: Francesca Fiorentino Trials 2017, 18(Suppl 1):P449 Use of longitudinal data in the analysis of biomarkers: lessons from simulation and reality y Francesca Fiorentino1, Chris A. Rogers2, Gianni Davide Angelini2, Shahrul Mt-Isa1, Barnaby C. Reeves2 1Imperial College London; 2Bristol University Correspondence: Francesca Fiorentino Trials 2017, 18(Suppl 1):P449 y Francesca Fiorentino1, Chris A. Rogers2, Gianni Davide Angelini2, Shahrul Mt-Isa1, Barnaby C. Reeves2 1Imperial College London; 2Bristol University Correspondence: Francesca Fiorentino Trials 2017, 18(Suppl 1):P449 g Results g Results For titre2 the ITT estimate of the odds ratio for allocation (liberal vs restrictive) on post-operative severe complications was 0.87, 95% confidence interval (CI) (0.72-1.05). The IV estimate of the relative risk of each unit transfused on outcome was 0.89, 95% CI (0.75-1.06). In the Thermic trials the geometric mean ratio (GMR) from the ITT ana- lysis of the effect of allocation (normothermic vs hypothermic) on in- tubation duration was 0.77, 95% CI (0.57-1.04). The IV estimate of the GMR of each degree Celsius was 0.95 (0.89, 1.02). Results from the RAPIDO trial are forthcoming. Background Repeated post-randomization longitudinal measurements are often not used to maximum efficiency at the analysis stage, with baseline data being disregarded or used simply to derive a single ‘change from baseline’ measurement. We have previously presented a simula- tion model comparing different statistical methods of dealing with repeated biomarker measurements over time. This abstract extends that work to consider how the relative precision of the different methods are affected in different biomarker scenarios, both by simu- lation and using real data. Biomarkers can represent a physiological state (S) at any time (e.g. Reflecting a comorbidity such as chronic kidney dysfunction) or only reach measurable levels after an event (E; e.g. Organ-specific response to injury). Our previous work only considered the former scenario, using a simulation model. Here, we use data for creatinine and myocardial troponin from a trial to illus- trate the two scenarios. Using the simulation model, or boot- strapped estimates for the trial data, we quantify how the relative precision of different methods for analysing repeated longitudinal measurements is affected, when: (a) adjusting for a baseline meas- urement or not, and (b) when varying amounts of data are missing- at-random. h d Methods for testing for and handling non-proportional hazards in a phase II rct in chronic lymphocytic leukaemia p y p y Lucy McParland, Dena R. Howard University of Leeds Correspondence: Lucy McParland Trials 2017, 18(Suppl 1):P448 Conclusions I will present common methods for testing for non-proportional hazards in the analysis of survival data. I will then present an alter- native method for estimating the treatment effect when the pro- portional hazards assumption is violated known as restricted mean survival time (RMST). Awareness of the greater precision afforded by modern statistical methods of analysis is limited, leading to inefficiencies in translat- ing discovery science into clinical settings. This research will high- light to researchers and funders the extent of the inefficiency and how practical constraints in doing the research, such as complete- ness of data, modify the penalty of using old-fashioned methods of analysis. RMST provides a way of estimating the treatment effect when the PH assumption is in doubt or has clearly been violated as recom- mended by Royston and Parmar (2011). It is a measure of average survival from time 0 up to a restricted pre-specified time t, and can be estimated as the area under the survival curve using a pseudova- lue approach. The difference in RMST between treatment groups can be calculated using standard regression methods and provide an ap- propriate estimate of the treatment effect, when non-proportional hazards exist. Results This work is ongoing. We know from our previous work that the multilevel model has the best precision compared to the other methods. What is unknown is whether the relative precision of the methods varies, and if so by how much, in these different scenarios. Results will be presented at the conference. Discussion and Conclusion Bayesian methods of calculating the ICC are similar to frequentist methods when a non-informative prior is used. If a more informative prior is used based on existing trials we can reduce the credible interval for the ICC in order to better inform sample size calculations and sensitivity analysis of sample size calculations. More disease spe- cific trial ICC’s need to be found to create more prior distributions for specific disease outcomes. In addition to the primary intention-to-treat (ITT) analyses an IV analysis was performed for each trial, with randomised allocation as an instru- ment. Such models estimate different effects to the ITT analyses, namely: each RBC unit transfused on severe post-operative complica- tion (titre2); each degree Celsius on intubation duration (Thermic); each Page 169 of 235 Trials 2017, 18(Suppl 1):200 hour in the time to provision of microbiological information on mortal- ity (RAPIDO). Models were fitted in Stata. For titre2 and RAPIDO IV pois- son models for binary outcomes were used. For Thermic an IV linear regression model was used. Conclusions Provided that the overall treatment effect is maintained, deviations from proportional odds marginally reduce power. However, devia- tions from proportional odds can modify the effect of misclassifica- tion, the number of categories, and the distribution of the placebo group on power. In general, adjacent pairs of categories with many patients should be kept separate to help ensure that power is maintained at the pre-specified level. Introduction h We suggest that use of more appropriate statistical methods will im- prove interpretability and inferences for early phase II trials that use continuous fractional outcomes. Typically such trials are designed and analysed based on transformed data (e.g. Log transformation), with sample size calculations based on standardised effect sizes and results summarised using less familiar measures such as geometric means. We illustrate these issues in terms of Ki67, a common meas- ure of tumour response in early breast cancer studies. gy Southwestern Medical Center, Department of Internal Medicine; 7CRG, Infection and Population Health, UCL and MRC CTU at UCL, University College London Correspondence: Ross Peterson Trials 2017, 18(Suppl 1):P452 Correspondence: Ross Peterson Trials 2017, 18(Suppl 1):P452 Background A single best endpoint for evaluating treatments of severe influenza requiring hospitalization has not been identified. A novel 6-category ordinal endpoint of patient status is being used in a randomized con- trolled trial (FLU-IVIG) of intravenous immunoglobulin (IVIG). We sys- tematically examine four factors regarding the use of this ordinal endpoint that may affect power from fitting a proportional odds model: 1) deviations from the proportional odds assumption which result in the same overall treatment effect as specified in the FLU-IVIG trial protocol and which result in a diminished overall treatment effect; 2) deviations from the distribution of the placebo group that researchers assumed in the FLU-IVIG trial protocol; 3) the effect of patient misclassi- fication among the 6 categories; and 4) the number of categories of the ordinal endpoint. We also consider interacting the treatment effect (i.e., Factor 1) with each other factor. Guidelines on assessment of Ki67 scores in breast cancer (Dowsett et al., 2011) give advice on the role of Ki67 in clinical management and methodological issues for its measurement, but neglect methods for statistical analysis. Ki67 scores are expressed as a percentage and hence restricted to the range 0–100. Despite the natural bounds of the data, recommendations propose Ki67 be analysed assuming a log-normal distribution. Methods We conducted a Monte Carlo simulation study to assess the effect of each factor. To study factor 1, we developed an algorithm for deriving distributions of the IVIG group that deviated from proportional odds while maintaining the same overall treatment effect in the form of an average log odds ratio. To construct the algorithm, we know that for large samples the average log odds ratio of a misspecified model is the value for which the expected score function equals zero. Given infor- mation about the trial, our algorithm constrains the distribution of the IVIG group to maintain the average log odds ratio across deviations from proportional odds. Our algorithm can handle ordinal endpoints with any number of levels. For factor 2, we considered placebo group distributions which were more or less skewed than the one specified in the FLU-IVIG trial protocol by adding or subtracting a constant from the cumulative log odds ratios. To assess factor 3, we added misclassifica- tion between adjacent pairs of categories that depend on subjective We show how beta regression and fractional logistic/probit model- ling directly relate to the original (untransformed) scale can account for shifts in both location and spread. We also provide suggestions on sample size estimation. P450 P450 Robust methods for improving power in group sequential randomized trial designs, by leveraging prognostic baseline variables and short-term outcomes Tianchen Qian, Michael Rosenblum, Huitong Qiu Johns Hopkins University Correspondence: Tianchen Qian Trials 2017, 18(Suppl 1):P450 50 Robust methods for improving power in group sequential randomized trial designs, by leveraging prognostic baseline variables and short-term outcomes Tianchen Qian, Michael Rosenblum, Huitong Qiu Johns Hopkins University Correspondence: Tianchen Qian Trials 2017, 18(Suppl 1):P450 The results of the RMST method when applied to analyse the PFS data in ADMIRE will be presented and compared to the results from the Cox proportional hazard model, which is inappropriately applied when the proportional hazards assumption fails to hold. Trials 2017, 18(Suppl 1):200 Page 170 of 235 Page 170 of 235 In group sequential designs, adjusting for baseline variables and short-term outcomes can lead to increased power and reduced sam- ple size. We derive simple formulas for the efficiency gain from such variable adjustment using semiparametric estimators. The formulas reveal the impact of the prognostic value in the variables and how the impact is modified by the proportion of pipeline participants, analysis timing, and enrollment rate. While strongly prognostic base- line variables are always valuable to adjust for, the added value from prognostic short-term outcomes is limited. For example, if at least 2/ 3 of the enrollees have primary outcome observed, the equivalent sample size reduction from prognostic short-term outcomes is at most half of the reduction from an equally prognostic baseline variable. The added value from prognostic short-term outcomes is generally smallest at later interim analyses which are the ones that tend to impact power the most. A practical implication is that in trial planning one should put priority on identifying prognostic baseline variables. Our results are corroborated by simulation studies based on data from a real trial, using the class of readily implemented semi- parametric estimators. patient/clinician assessments. For factor 4, we collapsed some categor- ies into single categories. P453 The design and analysis of early phase ii trials with naturally bounded continuous fractional outcomes 1University of Minnesota, School of Public Health, Division of Biostatistics; 2George Washington University School of Medicine; 3Kirby Institute, University of New South Wales; 4Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Departamento de Microbiología I/Inmunología, Facultad de Medicina, Universidad Complutense de Madrid; 5National Institute of Allergy and Infectious Disease, Biostatistics Research Branch; 6UT Southwestern Medical Center, Department of Internal Medicine; 7CRG, Infection and Population Health, UCL and MRC CTU at UCL, University College London 1University of Minnesota, School of Public Health, Division of Biostatistics; 2George Washington University School of Medicine; 3Kirby Institute, University of New South Wales; 4Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Departamento de Microbiología I/Inmunología, Facultad de Medicina, Universidad Complutense de Madrid; 5National Institute of Allergy and Infectious Disease, Biostatistics Research Branch; 6UT 7 Results Deviations from proportional odds reduced power at most from 80% to 77% given the same overall treatment effect as specified in the FLU-IVIG trial protocol. Misclassification and collapsing categories can reduce power by over 40 and 10 percentage points, respectively, when they affect categories with many patients and a discernible treatment effect. But, collapsing categories that contain no treatment effect can raise power by over 20 percentage points. Differences in the distribution of the placebo group can raise power by over 20 percentage points or reduce power by over 40 percentage points depending on how patients are shifted to portions of the ordinal endpoint with a large treatment effect. g Methods We illustrate with both real and simulated datasets that the use of log transformed data when the data naturally bounded is not al- ways appropriate. Particularly in randomised studies, it is often the case that a log transformation may be suitable for one, but not both arms of the study. Further, interpretations of the data are typ- ically dependent on differences between means and may ignore changes in variation. P453 The design and analysis of early phase ii trials with naturally bounded continuous fractional outcomes Paul Silcocks, Richard Jackson University of Liverpool Correspondence: Paul Silcocks Trials 2017, 18(Suppl 1):P453 Ross Peterson1, David M. Vock1, John H. Powers III2, Sean Emery3, Eduardo Fernández-Cruz4, Sally Hunsberger5, Mamta K. Jain6, Sarah Pett7, James D. Neaton1 Analysis of an ordinal endpoint for use in evaluating treatments for severe influenza requiring hospitalization 1 1 2 3 P453 The design and analysis of early phase ii trials with naturally bounded continuous fractional outcomes Paul Silcocks, Richard Jackson University of Liverpool Correspondence: Paul Silcocks Trials 2017, 18(Suppl 1):P453 Background The FDA suggests that participants who discontinue or otherwise deviate from randomised treatment should continue to be followed up in order to facilitate the estimation of the de facto treatment effect in superiority trials. Conclusions l f Analysis of phase II trials that use continuous fractional outcomes should reflect the underlying nature of the data recorded. We hope to have increased researchers’ awareness of better methodology that will enhance comparative analysis, and to provide suggestions for statistical colleagues who may be asked to perform such work. Analysis of phase II trials that use continuous fractional outcomes should reflect the underlying nature of the data recorded. We hope to have increased researchers’ awareness of better methodology that will enhance comparative analysis, and to provide suggestions for statistical colleagues who may be asked to perform such work. We would also encourage researchers to provide summary data such as distributional shape, mean and variance along with their main results. Where a transformation is used, justification for choice and fit of chosen transformation should be provided. We would also encourage researchers to provide summary data such as distributional shape, mean and variance along with their main results. Where a transformation is used, justification for choice and fit of chosen transformation should be provided. Trials 2017, 18(Suppl 1):200 Page 171 of 235 Methods An investigation of the factors which influence children with asthma having unscheduled medical contacts around the start of the new school year in England and Wales: a mixed methods study Rebecca Simpson, Steven A. Julious, Wendy O. Baird University of Sheffield, UK Correspondence: Rebecca Simpson Trials 2017, 18(Suppl 1):P454 An investigation of the factors which influence children with asthma having unscheduled medical contacts around the start of the new school year in England and Wales: a mixed methods study Rebecca Simpson, Steven A. Julious, Wendy O. Baird University of Sheffield, UK Correspondence: Rebecca Simpson Trials 2017, 18(Suppl 1):P454 We consider several alternative multiple imputation methods that can be used. The methods vary in their use of earlier outcomes and treatment discontinuation time in the mean part and in their use of treatment discontinuation in the variance part. Different methods make different assumptions about the missing data, specifically about what observed data to condition on in order to justify a missing at random (MAR) assumption, and whether or not treat- ment discontinuation is considered to represent a treatment failure outcome; they also make different demands on the observed data. We explore the performance of the methods in a simulation study, aiming to quantify the impact of different MAR assumptions and different variance assumptions. Evidence shows that there is an increase in the number of unsched- uled medical contacts amongst school-aged children with asthma at the beginning of the school year (September). It has been suggested that this is caused by a viral challenge influenced by the return to school. It is hypothesised that this challenge is exacerbated as some children may stop taking their medication over the summer holiday. The aim of this research is to identify factors that can be used to pre- dict which children are more likely to have an unscheduled medical contact in September. The SALVO study: a retrospective take on an interim analysis of futility in a randomised trial The SALVO study: a retrospective take on an interim analysis of futility in a randomised trial Lee Beresford1, Richard Hooper2, Khalid S. Khan3, Philip Moore4, Matthew Wilson5, Shubha Allard6, Ian Wrench7, Jane P. Daniels8, Matthew Hogg9, Doris Lanz3 The first stage of the qualitative study was conducted in June/July 2016 and the second stage will have been conducted in Oct/Nov 2016. In the first stage there were 17 interviews with children aged 5–14, with a mixture of boys and girls. 1Queen Mary University of London; 2Pragmatic Clinical Trials Unit, Queen Mary University of London; 3Women’s Health Research Unit, Barts and the London School of Medicine and Dentistry, Queen Mary University of London; 4Birmingham Women’s Hospital; 5School of Health and Related Research (scharr), University of Sheffield; 6NHS Blood and Transplant; 7Sheffield Teaching Hospitals NHS Foundation Trust; 8Birmingham Clinical Trials Unit, University of Birmingham; 9 The information collected from the qualitative studies will be used to identify any possible subgroups that could be incorporated into the quantitative analysis. Subsequently, a quantitative analysis will be performed to identify the subgroups for which the PLEASANT inter- vention could have been most effective. 9Royal London Hospital, Barts Health NHS Trust Correspondence: Lee Beresford Trials 2017, 18(Suppl 1):P456 This is one of the first studies using a mixed method design with children that have asthma. The findings can be used to propose a possible intervention that can be targeted at those who are most likely to have an unscheduled medical contact in September. Recruitment in clinical trials can often be problematic and marred by unforeseen circumstances. This often leads to requests from trial teams to the funders for extensions to their recruitment period, so that planned sample sizes can be reached. While numerous factors will play a role in deciding the future of an under-recruiting trial, futility analyses are a method sometimes used to assess whether there is hope for a significant result in a trial, should recruitment be allowed to continue. A funder may ask investigators to conduct such an analysis to determine whether an extension should be granted. P455 Using off-treatment data to estimate the de facto estimand in a randomised trial 1 2 1 2 Joseph Royes 1, Juan J. Abellan2, Ian R. White1, Dawn Edwards2, Oliver Keene2, Nicky Best2 1MRC Biostatistics Unit; 2Glaxosmithkline Correspondence: Joseph Royes Trials 2017, 18(Suppl 1):P455 This was the case for the SALVO trial - an evaluation of the effect of intra-operative cell salvage during caesarean section on the need for donor blood transfusion. The funding body requested that the trial team conduct an analysis to assess the probability of obtaining a sta- tistically significant result at the end of the study, given the data col- lected by that time. We proposed an approach to the futility analysis based on stochastic curtailment and predictive power, with the idea to evaluate the conditional power i.e. The probability of obtaining a statistically significant result at the end of the trial, given the data that had already been collected. There is no absolute cut-off for con- ditional power in deciding whether to continue a trial; instead it must be considered alongside other factors. Results The proposed imputation methods are shown to be valid when treat- ment discontinuation is not at random, provided that subsequent loss to follow-up after treatment discontinuation (i.e. Failure to pro- vide off-treatment data) is at random. We show that the loss of per- formance due to making simpler assumptions when only the more complex assumptions are true must be balanced against the gain of performance due to making simpler assumptions when they are true. Optimal choice of model depends on likely assumptions and on the number of treatment discontinuations. A mixed methods approach is being used to investigate the factors that affect children having unscheduled medical contacts at the be- ginning of a new school year. The quantitative data comes from the PLEASANT (Preventing and Lessening Exacerbations of Asthma in School-age children Associated with a New Term) cluster intervention study. PLEASANT investigates whether a simple letter intervention reminding children to take their asthma medication during the sum- mer holidays reduces unscheduled contacts. The quantitative compo- nent includes daily data over a two year period from approximately 12,000 children aged 5–16 with asthma. The qualitative data comes from a study which will be done in two stages, before and after the summer holidays. This qualitative research will explore why children may not take their medication and what factors the children think trigger their asthma symptoms. The first stage of the study will be used to inform the quantitative data analysis and the second stage will be used to validate the results. The two stages will also be used to investigate any differences from the children’s responses before and after the summer holidays. Conclusions The proposed methods provide a framework for choosing a suitable imputation model in this setting, and the simulation results were used to support the choice of sensitivity analysis methods included in the statistical analysis plan for the pharmaceutical trial. The SALVO study: a retrospective take on an interim analysis of futility in a randomised trial Lee Beresford1, Richard Hooper2, Khalid S. Khan3, Philip Moore4, Matthew Wilson5, Shubha Allard6, Ian Wrench7, Jane P. Daniels8, Matthew Hogg9, Doris Lanz3 1Queen Mary University of London; 2Pragmatic Clinical Trials Unit, Queen Mary University of London; 3Women’s Health Research Unit, Barts and the London School of Medicine and Dentistry, Queen Mary University of London; 4Birmingham Women’s Hospital; 5School of Health and Related Research (scharr), University of Sheffield; 6NHS Blood and Transplant; 7Sheffield Teaching Hospitals NHS Foundation Trust; 8Birmingham Clinical Trials Unit, University of Birmingham; 9 participan Methods We tackled these issues using a validation and calibration process. Firstly, participants were divided into three categories according to their completeness of these outcomes. Secondly, time gaps between the urine sample and self-report dates were assessed over different thresholds. Thirdly, we examined the feasibility of inferring partici- pants’ reporting behaviours at follow-up stage by their baseline outcomes. Background Building Blocks was a pragmatic randomised controlled trial asses- sing the effectiveness of giving the Family Nurse Partnership (FNP) home-visiting programme to teenage first-time mothers on infant and maternal outcomes up to 24 months after birth (Robling et al., 2016). One of the primary outcomes was to investigate the effective- ness of the intervention in reducing smoking during pregnancy. At baseline and late pregnancy, we collected a large amount of self- reported data on smoking habits from each participant during a face-to-face and telephone interview respectively. It is well known that self-reported smoking can be inaccurate and therefore some participants are likely to report smoking fewer or more cigarettes than they actually do. In the Building Blocks study, we collected urine samples at the same time as the baseline interview and at follow-up in late pregnancy. The cotinine levels within the urine sample were used to supplement the participants’ self-reported behaviour and then further calibrate their number of cigarettes smoked per day (Dukic et al., 2007). However, this calibration approach requires complete and well-synchronized collection of self-reports and urine samples. The main challenge of our study lies in the collection of urine samples, particularly at follow-up stage. Some urine samples were collected at different time points from their interview and some were missing for a variety of reasons, which cause incompleteness in participant’s data and potentially lead to bias in the results. M th d y Correspondence: Giuseppe de Vito Trials 2017, 18(Suppl 1):P457 A validation and calibration process on self-reported tobacco with participants: Cotinine levels in building blocks Giuseppe de Vito1, Ileana Baldi2, Annamaria Nuzzo3, Filippo Montemurro3, Paola Berchialla4 p p Chao Huang Cardiff University Trials 2017, 18(Suppl 1):P458 p p Chao Huang Cardiff University Trials 2017, 18(Suppl 1):P458 1European Laboratory for Non-Linear Spectroscopy; 2Unit of Biostatistics, Epidemiology and Public Health, Department of Cardiac, Thoracic and Vascular Sciences, University of Padova); 3Department of Investigative Clinical Oncology, Fondazione del Piemonte per l’Oncologia, Candiolo Cancer Institute; 4Department of Clinical and Biological Sciences, University of Torino Results 870 participants with different levels of non-contemporary outcomes collection at follow-up stage were sub-grouped and investigated over their consistency in reporting behaviours. We further validated 222 participants with incomplete data at follow-up stage and calibrated their self-reported tobacco accordingly, which strengthened the power for the main analysis. Introduction Recent studies investigated the possible role of Human Epidermal Growth Factor Receptor 2 (HER2)-targeting compounds as first-line, single-agent therapy for HER2-over-expressing Breast Cancer (BC) with promising results. In particular, for a subgroup of patients the observed disease control duration was similar to that reported for the commonly-used anti-HER2 and chemotherapy combination treatment. In order to gather further insights about the biomarkers that character- ise the patients that can benefit from anti-HER2 single-agent therapy and to evaluate the efficacy of this therapy in patients not previously treated for HER2-positive metastatic BC, two clinical trials were initiated: HERLAP I and HERLAP II, both testing two anti-HER2 agents: trastuzu- mab and lapatinib. However HERLAP I was prematurely terminated, also due to the slow accrual of patients. We devised to measure the Progression Free Survival (PFS) for pa- tients in single-agent therapy from the HERLAP trial data, in order to compare it to the combination treatment. However, the small sample size makes it difficult to apply frequentist statistical ap- proaches and calls for an integration of the information derived from the two trials. In this regard, the Guidance for the Use of Bayesian Statistics in Medical Device Clinical Trials, issued by the Food and Drug Administration, states the opportunity to use a Bayesian approach to combine prior information with new observa- tions, suggesting to base this information on empirical evidences. Using this approach, we generated prior distributions from the data of the early-stopped HERLAP I trial, devising to use them in the ana- lysis of the HERLAP II trial results. Objective We set out to explore how to perform the analysis where data collec- tion is continued in some, but not all, patients after discontinuation of randomised treatment: we call this off-treatment data. The work was motivated by the problem of writing a statistical analysis plan for a pharmaceutical trial. We also sought advice from the independent Data Monitoring Commit- tee (DMC) for the trial, and sent them results from our futility analysis, generated by an independent statistician. In open correspondence the Page 172 of 235 Page 172 of 235 Trials 2017, 18(Suppl 1):200 Page 172 of 235 DMC raised questions about the need for a futility analysis, and follow- ing a closed meeting they recommended that the funder extend the study recruitment period. The SALVO trial recruited to completion after a 13 month extension was granted. obtained by deriving the posterior distributions from the HERLAP I data. In particular, we observed that the median survival times in days (and the extremes of their 95% credible intervals) for the biological PFS and the total PSF are 190 (96; 355) and 333 (172; 672), respectively. If we take into account only the trastuzumab-treated patients, then these values become 335 (139; 893) and 442 (162; 1304); whereas considering only the lapatinib-treated patients they become 99 (46; 232) and 250 (102; 805). It is interesting to note that these survival times are similar to those reported for the combination treatment. Conclusions DMC raised questions about the need for a futility analysis, and follow- ing a closed meeting they recommended that the funder extend the study recruitment period. The SALVO trial recruited to completion after a 13 month extension was granted. We present the methods used and results of the futility analysis that was conducted, as well as final results of the primary analysis and other findings of the study for comparison. We discuss inter- pretations that could have been drawn from the futility analysis and provide a discussion of the pros and cons of conducting futility analysis with the help of hindsight and with particular reference to the events which occurred in the SALVO trial. These data, albeit very preliminary, represent an additional sugges- tion for the efficacy of the single-agent therapy for HER2-positive metastatic BC. Single agent trastuzumab or lapatinib to treat her2-overexpressing breast cancer: combining past and current evidence in a Bayesian reanalysis y Methods We planned to employ a hierarchical Bayesian Weibull survival model to characterise both the ‘Biological PFS’ (i.e. Taking in consideration only the period of exclusive administration of anti-HER2 agents) and the ‘Total PFS’ (regardless of protocol failures). In particular, using non- informative prior distributions, we derived posterior distributions for the parameters of the Weibull model based on the HERLAP I data, and we have planned to use them in turns as prior distributions to derive the posterior distributions for the parameters based on the HERLAP II data, thus ‘borrowing strength’ from the first trial to the second. Results Integrating continuous stratification variables into a dynamic adaptive randomisation algorithm p g Nia Goulden, Zoe Hoare Bangor University Correspondence: Nia Goulden Trials 2017, 18(Suppl 1):P459 Method Final study reports, published protocols, and (where available) grant applications from the NIHR HTA and NIHR EME programmes for trials evaluating tests were identified. The theoretical approach used for computation of sample size was identified and classified according to (i) the study outcome to which it related, (ii) whether it was based on consideration of statistical power to test a hypoth- esis or precision to estimate a parameter, and (iii) whether it was judged an appropriate method to compute sample size in compari- son with the established literature. Estimates of key parameters de- scribing the baseline scenario (such as disease prevalence and progression, the performance of comparative tests, the correlation between tests) were identified from the protocol and their sources identified. Assumed values for key parameters in each sample size calculation were compared with the estimates observed in the tri- als. Details of any sample size revisions undertaken during the study were identified and reported. All assessments were initially undertaken independently in duplicate and consensus reached through team discussion. Discussion We will discuss the challenges that researchers across the NIHR pro- grammes have faced in identifying methods and computing sample size calculations for test evaluation studies, and assess the import- ance of considering planning sample size revision processes in test evaluation studies. Method Method Firstly we test a method that utilises the rank information of the covariates (Hu & Hu, 2012). Using a computationally effi- cient search the method finds the maximum possible difference resulting from assigning a new participant. Secondly we test a method that minimises the Kullback-Leibler divergence (Endo, Nagatani, Hamada, & Yoshimura, 2006). This method is based on probabilities of assigning a new participant to a group and there- fore needed to be adapted in order to be integrated. A trial of 332 participants was simulated, using centre (6 centres recruiting 72, 66, 66, 62, 34, and 32 participants, respectively) and age (continuous 18–65 inclusive) as stratification variables. Comparisons of the methods were based on the resulting differences in means of the variable in two groups, results of t-tests and f-tests of the final allocations, sequence length and the imbalance. g Results Results are displayed in for parameters total = 0.5, centre = 0.5, age = 0.5 and stratum = 0.5, which are set to control the amount of imbalance allowed for each variable. Reducing these parame- ters lessens the control on imbalance while increasing them will increase predictability. We have also tested different parameters to assess the effect of increasing and decreasing the stratum and strata. Increasing the weight for age decreases the difference be- tween means overall, but increases the difference between means within centre for method 1, because the imbalance within the strata are not as well controlled. The 1st percentile for the t and f tests increase for both methods. Increasing the weight for the strata decreases the difference between means overall for both methods and within centre for method 1. Method 1 requires searching the randomised data so it does take longer to compute the result than for method 2. Despite this method 1 can still pro- duce a randomisation result in a few seconds, even with 300 par- ticipants randomised. g Results Background Trials of tests may evaluate their role as screening, diagnostic, staging, monitoring or prognostic tests. The National Institute of Health Research (NIHR) programme has over 20 years experience in commissioning trials of tests for these clinical roles through the Health Technology Assessment (HTA) and Efficacy and Mech- anism Evaluation (EME) Programmes. Trialists often struggle to identify appropriate methods for computing sample size for test evaluation trials, and there is often little data available to inform the assumptions made in sample size calculations. Objective Stratification variables are confounding variables which could po- tentially influence the outcomes being measured within a trial. The aim of this work is to extend a dynamic adaptive randomisation al- gorithm to be able to accept continuous stratification variables, such as age. Many randomisation algorithms categorise such vari- ables, however specifying a measure of imbalance with an aim to minimise imbalance should improve the sensitivity of stratification schemes. From the literature two methods have been tested to in- tegrate into the algorithm published previously by the North Wales Organisation for Randomised Trials in Health (NWORTH), Bangor University (Russell, Hoare, Whitaker, Whitaker, & Russell, 2011). To review the methods used for sample size calculation for trials of tests and assess the evidence base for the assumptions made in the original sample size calculation and assess their validity in compari- son with the experience of the trial. P460 How accurately do trialists pre-specify sample sizes for test evaluation trials? The experience in NIHR funded trials in the HTA and EME programmes Jonathan Deeks, Lucinda Archer, Kelly Handley, Catherine Hewitt, Natalie Marchevsky, Samir Mehta, Laura Quinn, Alice Sitch, Yongzhong Sun, Yemisi Takwoingi University of Birmingham Correspondence: Jonathan Deeks Trials 2017, 18(Suppl 1):P460 How accurately do trialists pre-specify sample sizes for test evaluation trials? The experience in NIHR funded trials in the HTA and EME programmes Jonathan Deeks, Lucinda Archer, Kelly Handley, Catherine Hewitt, Natalie Marchevsky, Samir Mehta, Laura Quinn, Alice Sitch, Yongzhong Sun, Yemisi Takwoingi University of Birmingham Correspondence: Jonathan Deeks Trials 2017, 18(Suppl 1):P460 g Results 62 reports of test evaluation studies were identified from the NIHR HTA and NIHR EME published monographs. Their evaluation is cur- rently ongoing, and we will report on the aspects detailed above. We are considering whether it is possible to predict particular scenarios in which sample size estimates are most challenging and least likely to be valid. Discussion It is not rare that difficulties arises when collecting data at follow-up stages, especially in populations that may be vulnerable and often mobile as in this study. Rather than losing those participants for key analyses, this proposed process could further validate and calibrate self-reported tobacco of participants for public health studies with similar settings. Because of the costings and challenges in urine sam- ple collections, investigating the participants’ reporting behaviours After describing the statistical method in details and presenting the data, in this contribution we shall discuss preliminary results that we Page 173 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 by some associated factors, such as social and demographic factors, has become one of our follow-up research topics. How accurately do trialists pre-specify sample sizes for test evaluation trials? The experience in NIHR funded trials in the HTA and EME programmes Jonathan Deeks, Lucinda Archer, Kelly Handley, Catherine Hewitt, Natalie Marchevsky, Samir Mehta, Laura Quinn, Alice Sitch, Yongzhong Sun, Yemisi Takwoingi University of Birmingham Correspondence: Jonathan Deeks Trials 2017, 18(Suppl 1):P460 P459 Integrating continuous stratification variables into a dynamic adaptive randomisation algorithm Nia Goulden, Zoe Hoare Bangor University Correspondence: Nia Goulden Trials 2017, 18(Suppl 1):P459 p Discussion l h h Although activated in July 2009, planning for the study began two years earlier in July 2007. Around this time, data from multiple stud- ies suggested that the efficacy of EGFR tyrosine kinase inhibitors was likely concentrated in patient with tumors harboring EGFR mutations. As EGFR mutation status and EGFR expression by FISH are correlated, this development could have affected accrual to this first line study and may have reduced the proportion of EGFR FISH+ observed. Background Background A linear mixed model incorporating a random cluster effect is the most commonly used model for analysis of complete stepped wedge designs with Gaussian outcomes and a repeated cross-sectional sampling structure. It is recognised that the max- imum likelihood estimator of the treatment effect in this model is a combination of horizontal (within cluster) and vertical (be- tween cluster) comparisons. However, the precise nature of this combination has not previously been clearly articulated for these designs. Jane Dennis1, Vikki Wylde1, Andrew D. Beswick1, Julie Bruce2, Christopher Ecclesto3, Nicholas Howells4, Timothy J. Peters1, Gooberman-Hill1 1University of Bristol; 2University of Warwick; 3University of Bath; 4North Bristol NHS Trust Conclusions The aim of our systematic review was to synthesise data on the man- agement of chronic pain after surgery. Chronic pain is difficult to treat and combination treatments matched to patient characteristics are advocated. In this review, the majority of studies evaluated pharmacological interventions and we found no studies investigating multidisciplinary or individualised interventions for management of pain after surgery. The results of our systematic review highlight the need for further evidence to inform recommendations about care provision for pa- tients with chronic post-surgical pain. We are now addressing this gap through a multi-centre randomised controlled trial evaluating the clinical and cost-effectiveness of a care pathway for patients with chronic pain after knee replacement. chronic pa Methods The protocol for the review was registered on PROSPERO in 2015. PICO criteria were: patients aged 18 years, with 90% of participants reporting chronic post-surgical pain; interventions for pain delivered a minimum of three months after surgery: control patients receiving placebo, usual care or alternative pain management intervention. Searches were con- ducted in MEDLINE, EMBASE, CINAHL, psycinfo, The Cochrane Library, and opensigle. Screening was performed by a single assessor with 10% of records double-screened. The primary effectiveness outcome was pain; that for harm was serious adverse events. Risk of bias was assessed using the Cochrane Risk of Bias tool. Background Total knee replacement is conducted to relieve pain and improve func- tion, most commonly as a treatment for osteoarthritis. Over 90,000 op- erations take place annually in the NHS, and knee replacement provides pain relief for most people. However, at three months or more after surgery, around 20% of patients report moderate to severe pain. To inform the design of an intervention to improve management of chronic pain after knee replacement, we conducted a systematic review that identified only one small randomised controlled trial assessing an intervention to treat chronic pain following knee replacement. Given Methods All patients were required to submit a paraffin-embedded tissue block or at least 10 unstained slides. In addition to the EGFR FISH assay, if additional tissue remained, secondary objectives included an investigation of the efficacy of cetuximab in patents whose tumor expressed EGFR by immunohistochemistry (IHC) and in patients whose tumor harbored a KRAS mutation, with priority given to IHC if tissue was limited. The FISH assay was performed at University of Colorado and results reported to the SWOG stat center on a monthly basis. IHC was performed at University of Colorado and KRAS per- formed at UC Davis. Results Searches run in March 2016 yielded 17,027 records. 66 trials with data from 3,149 participants were included. Most trials included patients with chronic pain after spinal surgery (23 trials) or phantom limb pain (21 trials). Interventions were predominantly pharmacological, including anti-epileptics, capsaicin, epidural steroid injections, local anaesthetic, neurotoxins, N-methyl-D-aspartate receptor antagonists and opioids. Other interventions included acupuncture, exercise, limb liner after am- putation, spinal cord stimulation, further surgery, laser therapy, mag- netic stimulation, mindfulness-based stress reduction, mirror therapy and sensory discrimination training. Opportunities for meta-analysis were limited by heterogeneity. For all interventions, there was insuffi- cient evidence to draw conclusions on effectiveness. Conclusions Background the fact that chronic post-surgical pain is multifactorial with surgical, biological and psychological contributions, we undertook a broader systematic review to evaluate the evidence for the management of chronic pain after any surgery type. g SWOG S0819 is a phase III trial evaluating both the value of cetuxi- mab in the treatment of advanced non-small cell lung cancer (NSCLC) and epidermal growth factor receptor (EGFR), as measured by FISH, as a predictive biomarker for cetuximab efficacy in NSCLC. The design of the study incorporated co-primary objectives to assess cetuximab in both the overall study population and among EGFR FISH-positive (FISH+) patients. Activated July 15, 2009, it was one of the first trials in SWOG requiring tissue to evaluate a primary object- ive in a biomarker-defined population. We will outline how methods for obtaining adequate tissue, and monitoring results from the FISH assay in comparison with design assumptions impacted the conduct of the study. Results A total of 1333 patients were registered to S0819. Usable tissue specimens were obtained from 1208 patients, of which, 1046 were adequate for FISH. Of these, 406 were FISH+. Comparisons between the study design assumptions and the observed proportions were monitored on a monthly basis. The proportion of FISH+ patients was lower than anticipated as was the assay success rate. This monitoring resulted in the following interventions in the study conduct: efforts to improve these numbers included development of a form that re- quired the local pathologist to review and confirm that the tissue contained at least 100 tumor cells prior to submission. An automated email notification system to prompt sites for additional tissue if their initial submission was deemed to be inadequate when the FISH assay was attempted at Colorado. The study design was modified in June 2015 as a result of the lower than expected number of EGFR FISH+ patients. p Conclusions Both methods produce similar acceptably balanced results how- ever method 1 has been chosen as the best option to integrate into the current algorithm. Method 1 directly produces a measure of imbalance which is more easily integrated, whereas method 2 needed to be adapted to allow integration. In summary, inclusion of continuous stratification variables in randomisation schemes without the need to categorise allows more sensitivity to the vari- able and has indirect impact on the analysis. We advocate the use of stratification variables within models of analysis, if continuous then these should be utilised as such. S0819: lessons learned from conduct of a cooperative group phase III trial with a biomarker defined subset co-primary objective j James Moon, Mary Redman SWOG Statistical Center Correspondence: James Moon Trials 2017, 18(Suppl 1):P461 Trials 2017, 18(Suppl 1):200 Page 174 of 235 Page 174 of 235 Decomposition of the treatment effect estimator in stepped wedge trials: understanding the horizontal and vertical contributions 1 2 Decomposition of the treatment effect estimator in stepped wedge trials: understanding the horizontal and vertical contributions Andrew Forbes1, John N. S. Matthews2 1Monash University; 2Newcastle University Correspondence: Andrew Forbes Trials 2017, 18(Suppl 1):P463 Andrew Forbes1, John N. S. Matthews2 1Monash University; 2Newcastle University Correspondence: Andrew Forbes Trials 2017, 18(Suppl 1):P463 P462 P462 A systematic review to inform a trial of comprehensive pain management for patients with chronic pain after total knee replacement: the star experience Jane Dennis1, Vikki Wylde1, Andrew D. Beswick1, Julie Bruce2, Christopher Ecclesto3, Nicholas Howells4, Timothy J. Peters1, Gooberman-Hill1 1University of Bristol; 2University of Warwick; 3University of Bath; 4North Bristol NHS Trust Correspondence: Jane Dennis Trials 2017, 18(Suppl 1):P462 A systematic review to inform a trial of comprehensive pain management for patients with chronic pain after total knee replacement: the star experience Background Wolfram Syndrome (OMIM 222300) is an ultra-rare, monogenic, neurodegenerative disorder of children and young adults. Prevalence is approximately 1:700,000. The prognosis is poor as premature death and severe neurological disabilities are not uncommon. The natural history of Wolfram Syndrome includes progressive optic and brain- stem atrophy. Many children are registered blind by the age of 18 years. There is no effective treatment. In several clinical trial settings, it is difficult to recruit the overall sample provided at the design stage, and different problems may occur in pa- tient’s enrolment. The amount of information conveyed by a trial termi- nated prematurely for poor accrual may be minimal. A Bayesian analysis of such a trial may salvage this information, by providing a framework in which to combine prior with current evidence. In this work we propose a Bayesian analysis of a trial candidate for termination due to poor accrual. RESCUE trial is a randomized controlled trial evalu- ating the effect of adjunctive oral steroids to prevent renal scarring in young children with febrile urinary tract infections. Primary outcome is the difference in scarring proportion between standard antibiotic ther- apy versus standard therapy + corticosteroids. By study protocol, a fre- quentist approach to sample size calculation require 92 randomized patients per arm, considering 20% lost follow-up. After 2 years, only 8 patients completed the follow up to determine the study outcome (3 in corticosteroids therapy group and 5 in control group). The sample size was recalculated with the Bayesian Worse Outcome Criter- ion for differences in proportions (length = 0.3 and coverage = 0.9) ap- plying a 0.5% down-weight. An informative prior on scar proportions was derived from literature considering a scar probability of 0.33 and 0.66 respectively in treatment and control group (Huang YY, 2011). An interim Bayesian analysis on recruited patients has been performed; having a few data to estimate the likelihood, inference was expected to be seriously conditioned by the prior. To assess robustness of conclu- sion a sensitivity analysis on prior definition has been performed considering 1) informative Beta prior as in sample size estimation 2) informative Beta with 0.5% down-weight 2) uninformative Beta (1,1) prior. Results are compared in term of posterior probability. Design of a practice-changing trial in the ultra-rare condition of Wolfram Syndrome 1 1 1 2 Kristian Brock1, Lucinda Billingham1, Zsuzsa Nagy1, Tamara Hershey2, Holly Smith1, Darren Barton1, Timothy Barrett1 1 2 Methods We apply standard results using partitioned matrices to derive a simple expression for the weighted combination of the horizontal and vertical components of the treatment effect estimator, each presented as linear combinations of cluster-period means. We ex- tend the mixed model to incorporate random effects appropriate for a closed cohort design and derive the analogous results under this design. Trials 2017, 18(Suppl 1):200 Page 175 of 235 P468 Handling poor accrual in adaptive trial setting: Bayesian interim analysis of rescue trial y Danila Azzolina, Ileana Baldi, Silvia Bressan, Paola Berchialla, Valentina di Leo, Liviana Da Dalt, Dario Gregori 1University of Padua Correspondence: Danila Azzolina Trials 2017, 18(Suppl 1):P468 Danila Azzolina, Ileana Baldi, Silvia Bressan, Paola Berchialla, Valentina di Leo, Liviana Da Dalt, Dario Gregori Results units requires 60 evaluable patients in total to provide 80% power and 10% significance. The weights assigned to the horizontal and vertical comparisons involve a simple expression depending on the number of periods in the design, the cluster size and the intra-cluster correlation. We use this result to describe scenarios in which the treatment effect estimator is dominated heavily by the horizontal comparisons. We provide explicit expressions for the horizontal and vertical components of the treat- ment effect estimator in a number of example designs and ?A3B2 show $132#?>explain the intuition behind them. We also describe how the decomposition provides a basis for the construction of randomisation tests. The extension to the closed cohort design involves identical hori- zontal and vertical components as the cross-sectional sampling design, the only difference being in the construction of the weights. C l i and 10% significance. We assess VPV every year for 3 years. To investigate a 70% reduction in the annual rate of degradation from 81.6 to 24.5 mm3 requires 60 evaluable patients to provide 81% power and 7% significance. We infer operating characteristics by simulation. The equivalent non- longitudinal analyses would require approximately 120 patients in total. Conclusion We assess VPV every year for 3 years. To investigate a 70% reduction in the annual rate of degradation from 81.6 to 24.5 mm3 requires 60 evaluable patients to provide 81% power and 7% significance. We infer operating characteristics by simulation. The equivalent non- longitudinal analyses would require approximately 120 patients in total. Conclusion This efficient design, which uses a repeated-measures analysis of the primary outcome, will achieve conventional statistical error rates, thereby enabling a potentially practice-changing clinical trial in this ultra-rare condition. The decomposition into horizontal and vertical components en- ables a better understanding of the explicit linear combinations of cluster-period means underlying the treatment effect estimator. It also describes where the maximal information resides in these designs, leading to suggestions for optimal incomplete designs. This abstract is not included here as it has already been published. P464 Design of a practice-changing trial in the ultra-rare condition of Wolfram Syndrome Kristian Brock1, Lucinda Billingham1, Zsuzsa Nagy1, Tamara Hershey2, Holly Smith1, Darren Barton1, Timothy Barrett1 1University of Birmingham; 2Washington University Correspondence: Kristian Brock Trials 2017, 18(Suppl 1):P464 Background The esti- mated Bayesian sample size is 41 infants per arm, leading to a reduc- tion of 51 patients compared with frequentist oneThe Bayesian inference is a flexible tool, compared to frequentist one, taking into account of a-priori knowledge about treatment effect. The informative inference, on small sample, may be weakly influenced by data. How- ever, sensitivity analysis lead to consider the inference robustness. Nevertheless, we advocate to choose beforehand a Bayesian design and not to switch to a Bayesian analysis method that produces a more favourable outcome after observing the data. Sodium Valproate is classed as an anticonvulsant and is currently approved for use in the treatment of epilepsy and bipolar disorder. The cell cycle regulator p21cip1 has been identified as a therapeutic target for Wolfram Syndrome and one of the mechanisms through which sodium valproate is expected to mediate its effect is by in- creasing p21cip1 expression levels. We investigate the hypothesis that it slows the progression of symptoms. Methods g y Methods We present a randomised, double-masked, placebo-controlled, multi- centre, international clinical trial to investigate whether sodium valpro- ate halts the progression in clinical symptoms of Wolfram Syndrome. We propose the dual primary outcomes: (i) Visual acuity (VA), measured on the logmar scale using standard charts; and (ii) Ventral pons volume (VPV), measured in mm3 by MRI scan. These continuous outcomes are chosen because they are clinically meaningful and associated with dis- ease progression. VA is very important to patients and their families, and any reduction in sight deterioration will be welcome. Recruitment is severely constrained in this ultra-rare condition. We in- crease statistical power by conducting longitudinal analyses of the pri- mary outcomes. This is feasible in Wolfram Syndrome because the symptoms under study tend to deteriorate linearly over time. Justifica- tion for this claim is given. Mean outcome trajectories are modelled using linear mixed effects regression, allowing the average rates of change to be different in each arm, and each patient to have their own intercept. This method allows the study of serially-correlated outcomes. Treatment effect is tested by likelihood-ratio test using an alternative, nested model with no fixed effects for treatment arm. Treatment will be considered suc- cessful if it is associated with a significant, clinically-relevant reduc- tion in the rate of degradation. target popu Discussion This work provides a summary of the factors known to affect when studies with hypothetical elements might be expected to agree with real world decisions. Based on a range of related literatures, our framework will aid investigators who are interested in understanding whether the design of their pilot study will allow them to draw con- clusions about the real world. This initial work will help us to pilot our health services trials more effectively, making the ultimate inter- ventions more efficient and effective. P472 Rituximab for the treatment of neuromyelitis optica: an application of individual patient data meta-analysis in a rare disease Siobhan Bourke1, Catrin Plumpton1, Catrin Tudur Smith2, Anu Jacob3, Dyfrig Hughes1 1Center for Health Economics and Medicines Evaluation, Bangor University; 2Institute of Translational Medicine, University of Liverpool; 3The Walton Centre, Liverpool Correspondence: Siobhan Bourke Trials 2017, 18(Suppl 1):P472 When can hypothetical pilot data predict real-world trial results? A systematic concept review and framework 1 1 2 1 Jamie Brehaut1, Tavis Hayes1, Doug Coyle2, Ian Graham1 1Ottawa Hospital Research Institute; 2University of Ottawa Correspondence: Jamie Brehaut Trials 2017, 18(Suppl 1):P471 Biomarker validation as a clinical trial endpoint: what works and what doesn’t D id R i David Raunig ICON Clinical Research Trials 2017, 18(Suppl 1):P470 Methods Our research question was: ‘What are the factors that affect the asso- ciation between hypothetical and real-world decisions?’ A systematic, peer-reviewed search strategy was developed based on keywords, ?A3B2 show $132#?>titles, and MESH headings related to (i)decision making or behaviour (and related concepts e.g. Reasoning, risk taking); (ii) hypothetical situations (e.g. Uncertainty, proxy), and (iii) real world situations (e.g. Reality, everyday), and applied to psychinfo and Medline in December 2015. Two coders extracted study spe- cifics, as well as quotations describing the relevant factor associating hypothetical and real outcomes. Factor wordings were standardized, collated, and organized into themes. p Methods Standardized statistical methods that are globally recognized by me- trology standards agencies, including the Bureau of International Weights and Measures (BIPM) and the National Institutes of Stan- dards and Technology (NIST) are used to define reliability in terms of repeatability, reproducibility and linearity. Standard metrics include statistical estimation of the variance components that eventually de- fine how reliable the imaging biomarker would be in a clinical trial setting, and a linear relationship to the truth. An additional compo- nent to validation is the ability of the imaging biomarker to predict clinical outcome. Results Two case studies, one with a quantitative and one with a semi-quantitative imaging biomarker evaluation of medical imaging will be examined for what would comprise a complete dossier for validation or qualification. From these case studies, we will summarize a standard protocol for a quantitative im- aging biomarker validation study, risks to the successful completion of these trials and methods to incorporate biomarker validation into the drug development process. P470 Biomarker validation as a clinical trial endpoint: what works and what doesn’t David Raunig ICON Clinical Research Trials 2017, 18(Suppl 1):P470 appropriate to different human biological mechanisms. At the moment, studies involving hypothetical elements are discounted in the literature (i.e. Considered not clinically relevant, excluded from systematic reviews), often because of implicit and unsupported objections that such data cannot predict real-world outcomes. While many literatures have explored predictors of the association between hypothetical and real-world decisions, none has summarized these in a manner that would help health care intervention developers know when hypothet- ical pilot data are likely to agree with the real world. Obj i appropriate to different human biological mechanisms. At the moment, studies involving hypothetical elements are discounted in the literature (i.e. Considered not clinically relevant, excluded from systematic reviews), often because of implicit and unsupported objections that such data cannot predict real-world outcomes. While many literatures have explored predictors of the association between hypothetical and real-world decisions, none has summarized these in a manner that would help health care intervention developers know when hypothet- ical pilot data are likely to agree with the real world. Obj ti , Results A total of 1846 studies captured by our search strategy ultimately yielded 59 studies that contributed at least one factor. Contributing articles addressed issues of behavioural economics(80%), psychology of reasoning (31%), social psychology (17%), health behaviours (12%), and neuroscience(5%). A total of 42 factors were grouped into 5 categories, including Personal Characteristics (9 factors e.g. Age, cognitive ability, personal relevance); Presentation Characteristics (8 factors; e.g. Framing effect, time for reflection, issue salience); Cogni- tive Factors (17 factors; e.g. Discounting, normative beliefs, social de- sirability); and Participant Characteristics (1 factor; samples match target population). Background As medical imaging technology advances, analysis methods mature and scanners become more globally available, there is an increasing interest to use advanced or novel imaging biomarkers as clinical tri- als endpoints. MRI, PET, high resolution CT and even ultrasound have demonstrated unique abilities to measure diseases closer to the mechanism of action. Many novel biomarkers are able to show both structural and functional changes and validation studies provide good evidence that imaging may provide both the sensitivity and specificity that have eluded the assessment of these diseases and their absence may actually be at least partly responsible for the failure to develop effective therapeutics. However, many of the pub- lished studies that declare biomarkers to be validated for use fall far short of demonstrating fitness for use. In 2015, the Quantitative Imaging Biomarker Alliance published the results of a two-year col- laborative effort to standardize the statistical and technical methods and metrics to validate a biomarker for use as an endpoint in a clinical trial. Since then, these methods have been used to validate several imaging biomarkers for study-specific use as primary and sec- ondary endpoints by providing statistically and clinically rigorous study designs to sufficiently demonstrate that these biomarkers are reliably acquired and analyzed and that there is reasonably good prediction of a clinically accepted outcome. To conduct a systematic concept review of the factors affecting the association between hypothetical and real-world decision-making. Methods Results We assess VA every 6 months for 3 years. To investigate a 60% re- duction in the annual rate of degradation from 0.075 to 0.03 logmar Page 176 of 235 Trials 2017, 18(Suppl 1):200 Page 176 of 235 Results There was a significant increase in the number of criteria that were fully met (M (SD) pre-2008 = 15.0 (1.0) vs. Post-2008 = 20.0 (2.7), F = 9.75, p = .008) and fully or partially met (M (SD) pre-2008 = 15.7 (1.2) vs. Post-2008 = 20.2 (2.6), F = 8.50, p = .012). There was also a signifi- cant reduction in the number of criteria that were not met or were ambiguous (M (SD) pre-2008 = 11.3 (1.2) vs. Post-2008 = 6.8 (2.6), F = 8.50, p = .012). However, it should be noted that even with the im- proved reporting many checklist items were still not being included (e.g., adverse event reporting, representativeness of the sample, blinding). We included all experimental and observational study types that assessed rituximab for the treatment of NMO patients. We performed a literature search using MEDLINE, EMBASE, Web of Science, and Cochrane. Risk of bias was assessed for each study. The primary out- come was time until relapse; other outcomes of interest included ?A3B2 show $132#?>Expanded Disability Status Scale (EDSS), patient demographics, annualized relapse rate, NMO igg status, disease dur- ation, number of relapses (before and after treatment), number and timing of rituximab doses. The authors of each study were contacted to obtain individual patient data. Where data were not forthcoming, data was extracted electronically by digitising figures presented in published papers. Conclusions h d This study is ongoing and these preliminary results are susceptible to change. It is hoped that constructing a robust evidence based review can lead to more efficient RCTs. Bayesian design RCT have been sug- gested as a solution to small population trials, incorporating prior in- formation on efficacy, can increase the possibilities for other RCT designs i.e. Non- inferiority or adaptive designs that previously were not feasible for a rare disease trial. Objectives To update the systematic review by Barnard et al. (2010) to identify new methods to predict recruitment in clinical trials and determine whether these new methods address the limitations of methods pre- viously identified. Identify perceived barriers to implementing these models in clinical trials. P473 The effect of the non-pharmacological extension of consort in quality of reporting of behavioural weight loss RCTs Simon Bacon1, Christina Kazazian1, Ariane Jacob2, Kim L. Lavoie2 1Concordia University & CIUSSS-NIM HSCMl; 2UQAM & CIUSSS-NIM HSCM Correspondence: Simon Bacon Trials 2017, 18(Suppl 1):P473 The effect of the non-pharmacological extension of consort in quality of reporting of behavioural weight loss RCTs Simon Bacon1, Christina Kazazian1, Ariane Jacob2, Kim L. Lavoie2 1Concordia University & CIUSSS-NIM HSCMl; 2UQAM & CIUSSS-NIM HSCM Background g Trials of complex health services interventions often lack detailed preparatory work explicating the mechanisms by which the interven- tion is supposed to work. This lack of preparatory work contrasts sharply with drug trials, which can be the culmination of many years of preclinical work. The UK Medical Research Council provides guid- ance that underscores this issue, and highlights the need for better theory development and modeling to support, justify, and optimize trials of complex interventions. We propose that this requires an under- standing of when ‘hypothetical’ Elements (e.g. Using healthy partici- pants instead of patients; piloting interventions on physicians outside their clinical practice) can be used to predict ‘real-world’ Outcomes, analogous to our extensive understanding of which animal models are Neuromyelitis optica (NMO) is a rare, autoimmune disease of the cen- tral nervous system that affects approximately 700 patients in the United Kingdom. It is characterised by relapses of the optic nerves and spinal cord. To reduce the severity and frequency of these attacks, pa- tients are treated with immunosuppressants, including rituximab which is a second line therapy for NMO. Rare diseases pose unique challenges for clinical trials, including difficulties in recruiting sufficient numbers. Many studies are observational and prone to bias. In the absence of high quality randomised controlled trials, the use of individual patient data (IPD) meta-analysis to synthesise the results of existing studies whilst accounting for confounders provides an opportunity to summar- ise the available evidence to inform treatment decision making. Trials 2017, 18(Suppl 1):200 Page 177 of 235 Trials 2017, 18(Suppl 1):200 Page 177 of 235 Page 177 of 235 reporting of 15 RCTs that were published before (3 trials) and after (12 trials) the publication of the 2008 CONSORT NPT. Background Successfully recruiting the pre-specified number of patients to time and target within clinical trials remains a difficult challenge that negatively impacts all stakeholders in a clinical trial. Current methods to monitor recruitment in practice appear limited to the usual com- parison of the predicted and actual recruitment curves and the size of discrepancy. In 2010 a systematic review of methods to predict re- cruitment was conducted, which identified five classes of models and their limitations. P474 Improving the planning and monitoring of recruitment to clinical trials 1 2 3 Efstathia Gkioni1, Roser Rius2, Carrol Gamble3 1University of Liverpool, Polytechnic University of Catalonia, Paris Descartes University; 2Polytechnic University of Catalonia; 3University of Liverpool Efstathia Gkioni1, Roser Rius2, Carrol Gamble3 1University of Liverpool, Polytechnic University of Catalonia, Paris Descartes University; 2Polytechnic University of Catalonia; 3University of Liverpool Correspondence: Efstathia Gkioni Trials 2017, 18(Suppl 1):P474 Methods The project will update the systematic review of Barnard et al. (2010). This update will include methods identified and published from August 2008 until present. The Online Resource for Recruit- ment Research in Clinical Trials database (ORRCA) will be used to identify relevant literature. Newly identified methods will be assessed for eligibility. Methods will be assigned to existing proposed classifications of unconditional, conditional, Poisson, Bayesian and Monte Carlo Simulation Markov Model with new clas- sifications as appropriate. The assumptions made by each method will be identified and compared between models. Levels of infor- mation required to implement the models, will be considered and applied to real examples of ongoing or recently completed clinical trials. Conclusions This study showed that, although there seems to be some improve- ment with the publication of the CONSORT NPT Statement, its effects are still less than ideal. CONSORT should be more widely and strongly endorsed, and enforced, in order to have complete and understandable behavioural RCT reports. Background Increased quality of reporting of randomized controlled trials (RCTs) has been associated with the publication of the main CONSORT Statement. Over time there have been a number of extensions to the CONSORT Statement, such as the Non-Pharmacological Trial [NPT] ex- tension, yet we have little data on how these have changed the reporting practices of investigators. p Results Thirty-five studies involving 393 patients have been included. Of these, 30 were case studies and the remaining 5 were only available in abstract form, no RCT were identified. IPD for 186 patients were extracted from papers. Variable quality of the data has been noted with some papers not reporting key outcome information. All studies were poor quality with no study adjusting for confounders. Authors of selected studies have been contacted to share their data,re- sponses have been positive, however no disclosure of data have been made at this time. There were 131 (70%) women, 13 (7%) men and 42 (23%) unknown participants, with a mean age of 37 years and disease duration of 41 months. The most-frequently used rituxi- mab regimen was two 1 g doses separated by 14 days in 37 cases (28%).The average EDSS score before (after) treatment was 5.3 (4.3). The average number of relapses before (after) treatment was 5 (1). We will be using a Cox-(proportional hazards) regression model to predict the time to relapse rate whilst adjusting for important confounders. l i Objective reporting of 15 RCTs that were published before (3 trials) and after (12 trials) the publication of the 2008 CONSORT NPT. reporting of 15 RCTs that were published before (3 trials) and after (12 trials) the publication of the 2008 CONSORT NPT. Results The aim of this paper is to review all available information to evalu- ate the effectiveness of rituximab in NMO. Methods Methods We distributed the INVEST survey during the two-day International Clinical Trials Methodology Conference in November 2015, and pro- vided access to an online version for one month following the con- ference. All respondents were asked to indicate their views on use of evidence synthesis in trial design and analysis and to rank what they considered to be the three greatest barriers to such use. Respon- dents who indicated that they had been involved in trial design and/ or analysis were asked additional questions about whether and how they have used evidence synthesis in practice. Among these respon- dents we contrasted their views on whether evidence synthesis methods should be used versus actual use. Expected results The results of this systematic review will explore the advances in methodology to predict recruitment in clinical trials. It will highlight limitations of existing methods and barriers to implementation highlighting direction for further developments. In this way we can provide more reliable predictions of recruitment based on each dif- ferent trials recruitment needs. The benefits of more accurate predic- tions will be the reduction of the deviation between observed and expected recruitment curves. The aim of this paper was to assess the change in quality of report- ing of RCTs for behavioural weight loss programs using CBT with the 2008 publication of the CONSORT NPT extension. Methods A systematic review was conducted to identify randomised controlled trials that assessed the efficacy of cognitive behavioural therapy-based weight loss interventions on eating behaviour or psychological vari- ables. The Downs and Black checklist was used to score the quality of Page 178 of 235 Trials 2017, 18(Suppl 1):200 Page 178 of 235 P475 Graphical display techniques for subgroup analysis Yi-Da Chiu1, Franz Koenig2, Martin Posch2, Thomas Jaki1 1Medical and pharmaceutical statistics research unit, department of Mathematics and Statistics, Lancaster University; 2Center for Medical Statistics, Informatics, and Intelligent Systems, Medical University of Vienna Correspondence: Yi-Da Chiu Trials 2017, 18(Suppl 1):P475 Venous thromboembolism and cancer trials evidence synthesis: dealing with both complex knowledge and unexplained heterogeneity Martin Adamcik Assumption University of Thailand Trials 2017, 18(Suppl 1):P476 Investigatin g the use of evidence synthesis in the design and analysis of clinical trials 1 2 3 3 Gemma Clayton1, Isabelle Smith2, Hayley E. Jones3, Julian P. T. Higgins3, Benjamin Thorpe2, Duncan Wilson2, Robert Cicero2, Kusal Lokuge4, Julia Forman5, Borislava Mihaylova4 Gemma Clayton1, Isabelle Smith2, Hayley E. Jones3, Julian P. T. Higgins3, Benjamin Thorpe2, Duncan Wilson2, Robert Cicero2, Kusal Lokuge4, Julia Forman5, Borislava Mihaylova4 1 2 1University of Bristol; 2Clinical Trials Research Unit, University of Leeds; 3School of Social and Community Medicine, University of Bristol; 4Health Economics Research Centre, Nuffield Department of Population Health, University of Oxford; 5Cambridge Clinical Trials Unit, University of Cambridge Correspondence: Gemma Clayton Trials 2017, 18(Suppl 1):P477 Evidence Synthesis Fixed-effect meta-analysis is a powerful instrument for combining related studies but such a combination is considered flawed if studies use different methods or investigate different populations. If differences are merely statistically detected then techniques of random-effects meta-analysis are employed to combine them. On the other hand, complex knowledge is difficult to interpret and although Bayesian methods are currently being developed they are unable to deal with complex knowledge when heterogeneity is statistically detected. Background h d When designing and analysing clinical trials, using previous relevant information, perhaps in the form of evidence syntheses, can reduce research waste. We conducted the INVEST (investigating the use of Evidence Synthesis in the design and analysis of clinical Trials) survey to summarise current evidence synthesis use in trial design and ana- lysis, to capture the opinions of trialists and methodologists on such use, and to identify any barriers. Results Of approximately 638 people attending the conference, 106 (17%) completed the survey, half of which were statisticians. Support was generally high for using a description of previous evidence, a sys- tematic review or a meta-analysis when designing a trial. Fewer participants indicated support for use of network meta-analyses, decision models and value of information analyses. Only about 5% felt that external evidence should not be used in the analysis of a trial, with an additional 20% being unsure. Among respondents in- volved in trial design and/or analysis, fewer indicated that they had used evidence syntheses to inform design or analysis during the last 10 years than indicated that these methods should be used. For example, only 6% (5/81) had used a Value of Information ana- lysis to inform sample size calculations, compared with 22% (18/81) feeling that this was desirable. The greatest perceived barrier to using evidence synthesis methods in trial design or analysis was time constraints, followed by a belief the new trial was the first in the area. Venous Thromboembolism and Cancer According to a large meta-analysis from 2008, around 10% of pa- tients having acute unprovoked venous thromboembolism are ex- pected to be diagnosed with cancer within a year. Nevertheless, several recent clinical studies indicate a lower incidence of diagnosis of cancer in such patients and also a lower sensitivity of extensive screening for cancer than the large meta-analysis suggests. Despite similarities, heterogeneity was statistically detected between those studies. The variability in screening designs requires a method that can deal with complex knowledge to combine them. Results P475 Graphical display techniques for subgroup analysis Yi-Da Chiu1, Franz Koenig2, Martin Posch2, Thomas Jaki1 1Medical and pharmaceutical statistics research unit, department of Mathematics and Statistics, Lancaster University; 2Center for Medical Statistics, Informatics, and Intelligent Systems, Medical University of Vienna Correspondence: Yi-Da Chiu Trials 2017, 18(Suppl 1):P475 Our meta-analytical findings indicate the following: The incidence of diagnosis of cancer in patients with unprovoked venous thrombo- embolism is somewhere between 6.97% and 9.79%. Routine evalu- ation detects between 36.59% and 49.61% of those cancers while the combination of routine and extensive screening methods detects between 74.99% and 83.25% of those cancers. Therefore, the inci- dence of cancer diagnosis is still relatively high and the combined screening is superior to routine evaluation in detecting such an oc- cult cancer. Subgroup analysis has received extensive attention in recent clinical research for the development of stratified medicine. This tendency reflects the advance of genetic testing and the potential exploitation of heterogeneity in subgroups. It also emphasises the identification of medical interventions to suitable subpopulations (as defined by bio- markers) for efficacy and against the others for safety. Graphical ap- proaches are routinely employed in subgroup analysis, typically for describing effect sizes of subgroups. Such visualisation encapsulates subgroup information and greatly boosts the clinical decision-making process. However, existing approaches still have inherent drawbacks and their use may lead to misinterpretations to subgroup effect sizes. For instance, forest plots provide no insight on the overlap of different subgroups; additionally, whether or not a subgroup’s confidence inter- val crosses the no-effect point does not necessarily imply a lack of ef- fect or contribute an effect to the subgroup. It is therefore crucial to correctly depict the effect sizes and information, particularly in order to prevent overstating effects. To develop an optimal visualization ap- proach, we assessed graphical approaches for subgroup analysis under a synthetic dataset. Several techniques (such as level plots, barcharts, Venn diagrams, tree plots, forest plots and matrix plots) were applied to exhibit certain subgroup information. Some have been further im- proved by mitigating their original demerits. In final, we summarise the general strengths and failings of the graphical approaches and outline potential visualisation techniques. Conclusions By presenting these top priorities we will have the foundations of a global health trials methodological research agenda which we hope will instigate further methodology research in specific areas in order to increase and improve trials in LMICs. Development of the rob 2.0 tool has led to supplementary guid- ance aimed at clinical trialists. The work around performance bias presented here is part of the wider initiative to cover all biases that might arise in clinical trials. This initiative recognises that clinical trials and evidence synthesis are part of the same continuum of effectiveness research and aims to ensure that method development in Performance bias in trials that cannot blind participants and healthcare providers to assigned interventions: implications for trial conduct Roy Elbers, Jelena Savovic, Natalie Blencowe, Julian P. T. Higgins, Jonathan A. C. Sterne University of Bristol Correspondence: Roy Elbers Trials 2017, 18(Suppl 1):P478 What might a global health trials methodology research agenda look like? Anna Rosala-Hallas1, Paula R. Williamson2, on behalf of The Global Health Trials Methodology Research Agenda Steering Committee 1Clinical Trials Research Centre, University of Liverpool; 2North West Hub for Trials Research Methodology, Clinical Trials Research Centre, University of Liverpool [1] Tudur Smith C, Hickey H, Clarke M, Blazeby J, Williamson PR. The Trials Methodological Research Agenda: Results from a priority setting exercise. Trials 2014; 15:32 doi:10.1186/1745-6215-15-32. Results A list of the top priorities for trials methodology research in LMICs countries will be presented. Common priorities to those in high in- come countries will also be noted. Background Successful blinding of participants, healthcare providers and trial personnel prevents knowledge of assignment from influencing ad- herence to intended interventions. However, blinding in nonphar- macological trials is difficult, and these trials are often considered to be at high risk of performance bias. The revised Cochrane risk of bias tool for randomized trials (rob 2.0) differentiates between the effect of assignment to intervention and the effect of starting and adhering to intervention. The former is the effect of interest in an intention-to-treat analysis and the latter is the effect of interest in a per-protocol analysis. Issues of blinding, implementation and adher- ence to intended interventions differ importantly between these two effects. Correspondence: Anna Rosala-Hallas Trials 2017, 18(Suppl 1):P479 Correspondence: Anna Rosala-Hallas Trials 2017, 18(Suppl 1):P479 Conclusion Th INVEST A new method for meta-analysis that deals with both complex know- ledge and unexplained heterogeneity was developed. The method is thus applicable to synthesise a wide range of related medical trials in different fields. The method uses propositional probabilistic logic to represent complex findings and merges them using an operator that was shown to be appropriate in the presented setting by an argu- ment related to the maximum entropy principle. The INVEST survey indicates that, generally, trial teams are using evidence synthesis in trial design and analysis less than they think is desirable. Since evidence syntheses can be resource-intensive, we ad- vocate additional research and training on ways to undertake them efficiently. Investment in adequate resources and training at this stage could lead to cost savings in the long term. Trials 2017, 18(Suppl 1):200 Page 179 of 235 one area is maximally integrated with applications in the other area to ensure optimal trial conduct and reporting. one area is maximally integrated with applications in the other area to ensure optimal trial conduct and reporting. P478 Performance bias in trials that cannot blind participants and healthcare providers to assigned interventions: implications for trial conduct Roy Elbers, Jelena Savovic, Natalie Blencowe, Julian P. T. Higgins, Jonathan A. C. Sterne University of Bristol Correspondence: Roy Elbers Trials 2017, 18(Suppl 1):P478 Aim To identify priorities for methodological research to assist the design, conduct, analysis and reporting of clinical trials in low and middle in- come countries (LMICs). To identify priorities for methodological research to assist the design, conduct, analysis and reporting of clinical trials in low and middle in- come countries (LMICs). Background g Research into methods used to design, conduct, analyse and report clinical trials is essential to ensure that clinical decisions made are de- rived from robust and reliable evidence. In a previous study [1] the key stakeholder group of Directors of UK Clinical Research Centre (UKCRC) registered clinical trials units (CTUS) identified the most important methodology research topics. However, it cannot be assumed that these research priorities reflect those in LMICs. There is a need for re- search to come from LMICs countries and it has been stated, in the 2013 World Health Report, that LMICs must become the generators and not the recipients of research data in order for improvements in public health outcomes in these most undeserved regions of the world. In order for any progress to be made in LMICs there is a critical need for this research. This is to ensure that particular methodological issues are identified and communicated to health care workers in these re- gions so that they might optimise future designs for trials. Methods Objective To provide guidelines for trialists to reduce bias due to deviations from intended interventions in nonpharmacological trials in the con- text of an intention-to-treat analysis. Methods Within the development of the rob 2.0 tool, one working group was tasked with the development of signalling questions, criteria for reaching a judgment and full guidance on the domain ‘bias due to deviation from intended interventions’. The new tool pro- vides a more nuanced judgement of performance bias in non- pharmacological trials. In trials that aim to assess the effect of assignment to intervention, deviations from intended interven- tions that reflect usual care do not lead to bias. In the current pro- ject we extended the insights acquired during development of the rob 2.0 tool to propose guidelines for clinical trialists. These guidelines aim to inform trial conduct from planning through to reporting, with the aim of minimizing performance bias in non- pharmacological trials. An online survey will be conducted November 2016 to March 2017 with members of the Global Health Network, globalsurg, The Clinical Research Initiative, Cochrane, Evidence Aid and other clinical trials re- searchers with LMIC experience. The first round will be an online sur- vey in relation to the design, conduct, analysis, reporting and interpretation of a trial. Participants will be asked to list up to three topics they feel are important priorities for trials methodology re- search. Topics identified will be independently reviewed and cate- gorised by two members of the research team and split into two separate lists for the second round. The primary list will consist of topics identified by more than one respondent and the second list will consist of topics identified by a single individual. In the second round the participants will rank the topics in order to identify prior- ities within both the primary and secondary lists. p Results The guidance includes three components. First, interventions should be clearly articulated in the protocol, including any plans to stop or modify interventions in response to clinical events. In particular, trialists should define in advance any co-interventions that would be administered as part of usual care. Second, during the trial, all deviations from the protocol interventions that do not reflect usual care should be monitored and recorded. These deviations from the intended interventions might include co- interventions, contamination, switches to other interventions, non-adherence, or failure to implement some or all of the inter- vention. The important consideration is that these deviations occur because of the trial context rather than as a reflection of routine care. Third, the departures identified should be reported fully and clearly to facilitate risk of bias judgements by trialists themselves, peer reviewers and systematic review authors. C l i P480 Implementation in dental trials: an exploration of trial meta-processes The protocol revision requiring reconsent and addition of new case report forms (CRFs) was distributed to the sites on 10/1/2016. Local institutional review board (IRB) review was required as this study was initially reviewed by such prior to formation of the CIRB. A CRO was hired and trained on the SWOG SDMC systems in September 2016 to facilitate onsite monitoring of nearly 100 physical sites beginning in November 2016. Data are to be complete and every data point veri- fied by the CRO by February 2017 in preparation of a final data trans- fer in March 2017 to the pharmaceutical company. p Paul Brocklehurst, Beth Hall Bangor University Correspondence: Paul Brocklehurst Trials 2017, 18(Suppl 1):P480 As highlighted by a recent report for the National Institute for Health Research's Health Services and Delivery Research funding stream, bridging the implementation gap is increasingly being recognised as an intransigent challenge for complex interventions in health services research. Patient and Public Involvement (PPI), process evaluation and the use of theoretical frameworks have all been highlighted as being important ‘meta-processes’ in trial conduct and design to re- duce research waste and improve implementation of trial evidence. In addition, early consideration of an interventions pathway to impact has been advocated. The aim of this exploratory study was to examine the Cochrane Database of Trials over the last six years to determine the level of utilisation of PPI, process evaluation and theoretical frameworks alongside dental trials, whilst concurrently ex- ploring whether the pathway to impact and implementation was be- ing considered. An overview of all systems used during the conduct of this trial highlighted challenges including implementation of an online EDC system and subsequent capabilities for amending data online. Required updates in adverse event reporting (CTCAE 3.0 to 4.0) over the tenure of this trial also complicated the monitoring efforts. Drug company and CRO staff education of both legacy and current systems was necessary in order to evaluate and query all data for regulatory submission. The membership structure and alignment of participating sites chan- ged as the NCTN and NCORP networks replaced the cooperative group structure in 2014. With the configuration of the network mem- bership shifting, it is vital to identify updated site contacts, as well as track and communicate with the cross-network membership. Sites have experienced staff turnover and some no longer participate in cooperative group research. Lessons learned A hi bl l Achievable goals, concise training, communications, and sufficient timelines are critical to prepare sites and monitors for extensive data verification. The SDMC staff has evaluated over 4500 inquiries gener- ated by the company after initial review of clinical data, posting and resolving the relevant queries to sites (only 1350/3600 remain out- standing) while reviewing incoming data generated by additional CRFs and other site-initiated questions. Sufficient staffing and dy- namic data management systems are vital. The clinical data management system (CDMS), Medidata Rave®, pro- vided by the NCI will benefit future endeavors similar in nature, both in communication and monitoring efforts. We will continue to iden- tify additional challenges and lessons learned as well as strive to compare potential key outcomes based on standard data collection and review compared to the intense retrospective review. 582 of 932,577 records had the term ‘dental’, ‘oral’ and ‘trials’ in the Title, Abstract or Keyword. 56 studies related to psycho-social inter- ventions or had psycho-social pathways to implementation. The pro- portion of trials that reported PPI, process evaluation, theoretical framework or mentioned implementation 0%, 21.7%, 43.5% and 4.3% respectively, whilst the proportion of protocols was higher (46.7%, 60.0%, 73.3% and 6.7%). P483 Design and coordination of the DECAAF II randomized internatio trial Richard Holubkov, Tom Greene, Leonie Morrison-de Boer, Russell Telford, Tyler Bardsley, Molly McFadden, Alicia Peterson, Christina Pacchia, Jeffrey Yearley, Ashley Snyder University of Utah Correspondence: Richard Holubkov Trials 2017, 18(Suppl 1):P483 The use of ‘meta-processes, in trial design and conduct has im- proved, although considerations about pathway to impact and the implementation of the research evidence, once generated, appears to remain poor. P480 Implementation in dental trials: an exploration of trial meta-processes The Cochrane Database of Trials was searched for reports on Rando- mised Controlled trials (RCTs) and protocols of RCTs over a five year period (2010–2016). As the aim of this exploratory study was to get a ‘snap-shot’ of current activity, other subscription databases, open access databases and the grey literature were not searched. Any den- tal intervention that would have utilised psycho-social mechanisms explicitly or implicitly was included, whilst any intervention that acted through a pharmacological mechanism was excluded. Included studies were assessed to determine whether they reported on any of the ‘meta-processes’ detailed above. Titles and abstracts identified by the electronic search were downloaded to a reference management database and duplicates were removed. Reference Trials 2017, 18(Suppl 1):200 Page 180 of 235 P480 Implementation in dental trials: an exploration of trial meta-processes Paul Brocklehurst, Beth Hall Bangor University Correspondence: Paul Brocklehurst Trials 2017, 18(Suppl 1):P480 P485 By the end of this period, participating physicians are encouraged to dis- continue patients from treatment with anti-arrhythmic medica- tions, whose use is considered a confounding factor for primary outcome assessment. According to ICH GCP guidelines, investigators and research staff with delegated trial-related duties should be “Qualified by education, training, and experience” (ICH E6 GCP, 1996) to maintain integrity and quality in clinical trials. Training documentation is essential to demonstrate compliance of the investigator and research staff of these guidelines. Nonetheless, many researchers and sponsors, in particular in multicenter trials, find it difficult to adequately and ac- curately document the staff training requirements. When multicenter trials are conducted within a network, it is important to develop a sustainable level of standardization in training requirements across sites and studies that demonstrate the competency of the individuals being trained. g The Training Documentation Form (TDF) is a comprehensive docu- ment that tracks all training requirements for each study staff mem- ber correlated to their study role(s). The TDF clearly defines the training expectations and requirements from various stakeholders (e.g., the Sponsor, Institutional Review Board) as they relate to the re- sponsibilities for each study role, and consistent with the Study Train- ing Plan (STP) and Site Delegation of Responsibilities Log. When completed, the TDF demonstrates that staff members are qualified and fully trained for their study role(s) prior to performing delegated study activities. The TDF developed for the National Drug Abuse Treatment Clinical Trials Network (NIDA-CTN) by the Clinical Coordin- ating Center at The Emmes Corporation is a user-friendly modifiable electronic document that includes these basic elements critical to a TDF. It captures each staff member’s name, research site, and dele- gated study role(s). The TDF lists all training outlined in the STP and maps the minimal required training and certification prescribed per study role in a grid, based on the staff’s assigned role (e.g., study physician,) and assigned tasks (e.g., prescribe medication, data entry) for the study. The TDF efficiently organizes the training curriculum in accordance with the investigative team’s predetermined decisions as to the various roles and associated training requirements. It includes both general training (e.g., Human Subjects Protection) and protocol- specific training requirements (e.g., administration of investigational product, conduct of study assessments). P485 compared to previous trials, which employed conventional recur- rence assessment methods such as standard ECGs that are adminis- tered much less frequently. However, adherence to this self- administered approach must be aggressively monitored. While the smartphone-based application generates regular reminders, the Coordinating Center must be aware of centers that have substand- ard patient compliance. The primary recurrence outcome will in- corporate all available testing performed on the patient, including standard of care ECGs and Holter monitors, to eliminate depend- ence on full compliance with daily self-assessment. compared to previous trials, which employed conventional recur- rence assessment methods such as standard ECGs that are adminis- tered much less frequently. However, adherence to this self- administered approach must be aggressively monitored. While the smartphone-based application generates regular reminders, the Coordinating Center must be aware of centers that have substand- ard patient compliance. The primary recurrence outcome will in- corporate all available testing performed on the patient, including standard of care ECGs and Holter monitors, to eliminate depend- ence on full compliance with daily self-assessment. P485 The training documentation form – Going beyond the basics for the national institute on drug abuse (NIDA) national drug abuse treatment clinical trials network (CTN) Tracee Williams, Radhika Kondapaka, Dikla Shmueli-Blumberg, Matthew Wright, Dagmar Salazar, Kayla Williams, Julia Collins, Eve Jelstrom, Robert Lindblad National Drug Abuse Treatment Clinical Trials Network for the NIDA CCTN Correspondence: Tracee Williams The training documentation form – Going beyond the basics for the national institute on drug abuse (NIDA) national drug abuse treatment clinical trials network (CTN) The training documentation form – Going beyond the basics for the national institute on drug abuse (NIDA) national drug abuse treatment clinical trials network (CTN) Tracee Williams, Radhika Kondapaka, Dikla Shmueli-Blumberg, Matthew Wright, Dagmar Salazar, Kayla Williams, Julia Collins, Eve Jelstrom, Robert Lindblad Correspondence: Tracee Williams Trials 2017, 18(Suppl 1):P485 Third, the trial design includes a 90-day post-intervention “blank- ing period” That allows atrial tissue targeted during the ablation procedure to respond to treatment and heal. Therefore, the suc- cess of the procedure is more appropriately assessed excluding any arrhythmias observed during this period. In the primary analysis, “time zero” for counting arrhythmia recurrence events therefore begins at 90 days after initial intervention (any repeat ablations after this time will also count as primary outcomes). This “blanking period” is potentially advantageous for training pa- tients in the habitual daily use of the monitoring device. P485 The TDF also captures the dates that staff completed each required training task and the final date of overall training completion, the latter of which is docu- mented on the Site Delegation of Responsibilities Log to serve as the staff’s starting date on the study. When all required training has been completed, the TDF is signed by the staff member and endorsed by both the site’s Principal Investigator and the research center’s train- ing representative, who collectively confirm that staff members are ready to begin study responsibilities. Assuming that MRI-guided ablation reduces relative risk of atrial arrhythmia recurrence by 25%, DECAAF II must observe 517 events in the two equally sized treatment arms to yield 90% power for a logrank test to detect a treatment effect with re- spect to recurrence time. With enrolled patients followed up to 18 months after index procedure, various realistic event rate scenarios indicate that from 750 to 1100 (best estimate: 900) patients will need to be recruited. The first patient was random- ized in July 2016. We will present details of the DECAAF II study design and implemen- tation, and issues encountered in the initial rollout of the trial at vanguard study centers. P481 P481 Retrospective preparation of trial results for regulatory submission: challenges and lessons learned Cathryn Rankin, Rachael Sexton, Evonne Lackey, Sarah Basse, Antje Hoering, Michael LeBlanc SWOG Statistics and Data Management Center Correspondence: Cathryn Rankin Trials 2017, 18(Suppl 1):P481 The Efficacy of Delayed-Enhanced MRI-Guided Fibrosis Ablation vs. Conventional Catheter Ablation of Atrial Fibrillation (DECAAF) II trial is evaluating catheter-based ablation, guided by 3-dimensional MRI highlighting areas of fibrosis, as treatment for atrial arrhythmia. The ob- servational DECAAF I cohort study found an association of atrial fibrosis burden with arrhythmia recurrence. Therefore, DECAAF II is comparing ablation specifically targeting visualized fibrosis to standard-of-care ablation performed without MRI guidance, among atrial arrhythmia patients undergoing first-time ablation. Upon release of primary results from a positive SWOG-coordinated Phase III myeloma trial, a pharmaceutical company supplying study drug plans to retrospectively use the results in a regulatory filing exactly two years after presentation of results. The trial accrued 525 patients from 2008 to 2012 with patients being followed for six years after randomization. After an initial meeting between the SWOG Statistical and Data Management Center (SDMC) and the company in May 2016, the company outlined an aggressive plan to reconsent all living patients in order to extend follow-up, capture additional data and initiate site monitoring including 100% source data verification (SDV) for all registered patients. All patient data as of December 2016 needs to be complete, query-free and verified by March 2017 in order meet the timeline for submission to the FDA in December 2017. Design and execution of DECAAF II are complex for several reasons. First, before a clinically eligible patient can be randomized, the baseline 3-dimensional MRI must be obtained, confirmed to be of adequate quality for study use, and processed by a central core laboratory, all with rapid turnaround to have these images available during interven- tion for patients assigned to the MRI-guided strategy. The core labora- tory also determines extent of coronary fibrosis, a stratification factor for trial randomization. Second, the primary outcome of (time to) atrial fibrillation recurrence is mainly determined by a smartphone-based ap- plication that enrolled patients must implement daily. This technology is expected to increase statistical power to detect a treatment effect Page 181 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Implementation of CTMS functionality for remote monitoring of informed consents informed consents Catherine Dillon Medical University of South Carolina Trials 2017, 18(Suppl 1):P484 Informed consent is a high-risk activity that should be monitored. The FDA’s ‘Oversight of Clinical Investigations- A Risk-Based Approach to Monitoring’ encourages alternative approaches to traditional mon- itoring procedures to improve sponsor oversight of human subject protection. It specifically permits the use of internet portals where sites can upload signed consent (IC) forms or other records for re- mote verification by designated monitors. Functionality was inte- grated into the Clinical Trial Management System (CTMS) for DEFUSE 3, a NINDS-funded Stroke Trial Network study, to facilitate remote monitoring of informed consents. This presentation will explore the implementation strategy utilized which links IC submissions directly to the subject ID and ecrf submission, while storing Protected Health Information (PHI) on a separate server. This strategy allows for IC sub- missions to be processed in a similar way as other clinical trial data, while allowing access to PHI only to designated remote monitors. While the potential benefits of this technique include reducing costs, increasing efficiency, and early detention of errors, serious privacy and confidentiality issues had to be addressed. Implementation strat- egy, advantages, challenges, lessons learned from the Defuse 3 Trial, and other applications of this monitoring strategy will be discussed. The NIDA CTN has implemented this standardized TDF on seven studies since 2013, aiding in setting up expectations for training documentation across studies while minimizing the difficulty of pre- paring and tracking the training completed by research staff. The TDF has been welcomed by the quality assurance monitors and the research management teams and has lead to more efficient study start up as well as provided a valuable tool for documentation of re- search staff competency in delegated study activities. CTN Contract # HHSN271201500065C Trials 2017, 18(Suppl 1):200 Page 182 of 235 P486 Good order is the foundation of all things: a strong project management structure underpins successful research at Keele CTU Sarah Lawton, Kris Clarkson, Ruth Beardmore, Irena Zweirska, Martyn Lewis, Nadine E. Foster Keele Clinical Trials Unit Correspondence: Sarah Lawton Trials 2017, 18(Suppl 1):P486 P486 Good order is the foundation of all things: a strong project management structure underpins successful research at Keele CTU Sarah Lawton, Kris Clarkson, Ruth Beardmore, Irena Zweirska, Martyn Lewis, Nadine E. Background 97% of Keele University’s research was deemed to be world-leading, or of international importance in the REF 2014 and Keele CTU, ?A3B2 show $132#?>providing support for the design, delivery, analysis, reporting and dissemination of applied clinical research is contribut- ing to this success. Specialist expertise in the areas of trial design, intervention development, biostatistic approaches, and regulatory ?A3B2 show $132#?>coordination have been developed and de- ployed. This expertise requires a strong project management struc- ture to conduct and deliver clinical research to the highest quality standards. Evaluating the effectiveness of remote versus on-site initiation visits: an embedded randomised controlled feasibility cluster trial within the SWIFFT trial 1 2 2 2 within the SWIFFT trial Caroline Fairhurst1, Laura Jefferson2, Stephen Brealey2, Liz Cook2, Garry Tew3, Catherine Hewitt2, Ada Keding2, Izzy Coleman2, Matt Northgraves2, Amar Rangan4 1University of York; 2York Trials Unit, University of York; 3Department of Sport, Exercise and Rehabilitation, Northumbria University; 4South Tees Hospitals NHS Foundation Trust, The James Cook University Hospital Correspondence: Caroline Fairhurst Trials 2017, 18(Suppl 1):P488 g Results Employing this project management structure results in a transparent and auditable flow of information and processes within Keele CTU. Dedicated project management forges strong communication links within research teams and with collaborators and participants. Over 25 projects are presented at Clinical Studies Think Tank meetings per year and Keele CTU is currently supporting a portfolio of over 40 re- search projects, each managed efficiently and effectively within the resources available to secure the delivery of projects to time and target. The main analyses used intention to treat. Site-level, time to event outcomes were compared between the trial arms using Cox propor- tional hazards regression, and recruitment outcomes were analysed via Mann-Whitney U tests. Return of questionnaires and time to re- turn were analysed at the participant-level using logistic and Cox re- gression as appropriate accounting for clustering by site using robust standard errors (logistic) or a shared frailty (Cox). Methods A structural flow beginning at project conception through to suc- cessful dissemination of results for each CTU supported research project is employed. From conceptualisation, early research ideas are presented and discussed at a Clinical Studies Think Tank, attended by specialist and generalist clinicians as well as methodol- ogists, resulting in research design improvements. Prior to grant ap- plication, projects seeking CTU collaboration are considered against CTU adoption criteria including; strategic fit, sponsorship, expertise, capacity and funding. Next follows project feasibility and visualisa- tion of project operationalisation, then review by a team of re- search partners that includes the NIHR Clinical Research Network, before moving into the business of a CTU Operations Group. From here, projects are allocated to CTU Trials Managers and by employ- ing the skills from a variety of integrated working groups, the re- search is delivered. Working groups, such as the Health Informatics and Standard Documents working groups, provide invaluable sup- port for project delivery. Within each working group, innovative and effective methodologies are developed, that include techno- logical advances and standardised resources. This process is under- pinned by a Quality Management System (QMS) implemented from the Quality Assurance office, ensuring consistency and adherence to regulatory obligations. Background Delays in site set-up are a common problem in multi-centre rando- mised controlled trials. The frequency and format of contact with po- tential sites could play a role in reducing delays. Preliminary contact, prior to sites submitting for R&D approval, may involve liaising with healthcare professionals at the site to discuss the trial rationale and design, responding to queries, finalising local arrangements, and obtaining agreement to participate in the study. Such contact can be conducted as face-to-face, on-site meetings, or remotely via email, web or telephone correspondence. We sought to compare the effect- iveness of remote versus face-to-face initiation, followed by a final on-site set-up meeting, on recruitment to the SWIFFT trial, and to in- form the feasibility of undertaking such a comparison across other trials. Implementation of CTMS functionality for remote monitoring of informed consents Foster Keele Clinical Trials Unit Correspondence: Sarah Lawton Trials 2017, 18(Suppl 1):P486 Going green whilst maximising questionnaire response rates - Does size matter? Tracey Davidson, John Norrie, Alison McDonald, Gramem MacLennan, Mohamed Abdel-Fattah University of Aberdeen Correspondence: Tracey Davidson Trials 2017, 18(Suppl 1):P487 Sarah Lawton, Kris Clarkson, Ruth B Martyn Lewis, Nadine E. Foster Keele Clinical Trials Unit Correspondence: Sarah Lawton Trials 2017, 18(Suppl 1):P486 g Conclusions l g Conclusions trials. Methods This cluster randomised, feasibility trial was a study within a trial (SWAT) embedded within the SWIFFT surgical trial. The primary outcome was the number of patients recruited per site. Secondary outcomes included: time to (i) submission of R&D applica- tion, (ii) receipt of R&D approval, (iii) final on-site set-up meeting prior to starting recruitment, and (iv) first randomised participant; number of patients screened; the proportion of hospital forms and participant questionnaires returned; and the time to return these forms. No formal power calculation was conducted, as the sample size was restricted by the number of sites approach to take part in the host trial. All sites were randomised (except the site of the Chief Investiga- tor), and blinded to their involvement. Allocation was 1:1 via mini- misation balancing on (i) whether the Principal Investigator had previous experience of working on a multi-centre surgical RCT, (ii) whether the site had a research nurse in place, and (iii) the size of catchment area (< vs > 500,000). Introduction This abstract is not included here as it has already been published. Keele Clinical Trials Unit (CTU) is a UKCRC registered CTU based within the Faculty of Medicine and Health Sciences at Keele Univer- sity. It specialises in the development and delivery of both feasibility and definitive multicentre clinical trials, an increasing portfolio of Clinical Trials of Investigational Medicinal Products (CTIMPs) and epi- demiological studies, in primary care and at the primary-secondary care interface. An effective project management structure is essential for the delivery of high quality research. Results Keele CTU is increasing its portfolio of research projects whilst making strides with innovative and effective methodologies. This all needs to be carried out within a robust and supportive QMS to ensure successful project delivery. Good order is key to the foundations of any project. Our strong project management structure has allowed us to work col- laboratively, integrating all specialties and expertise required, transpar- ently, in order to achieve the successful delivery of research. Thirty-seven sites were included (20 face-to-face and 17 remote), of which 33 (89%) opened to recruitment. The median number of par- ticipants recruited from sites allocated to receive on-site initiation was higher than from those allocated to remote initiation (10 (inter- quartile range 1.5 – 17) vs 6 (5 – 23)), though this difference was not statistically significant (p = 0.79). No statistically significant differences Trials 2017, 18(Suppl 1):200 Page 183 of 235 Page 183 of 235 were observed in any of the secondary outcomes. There were four crossovers: 3 on-site to remote, and 1 remote to on-site. Conclusion Challenges faced during implementation of a surgical clinical trial Julie Crof, Neil Corrigan, Vicky Liversedge University of Leeds Correspondence: Julie Croft Trials 2017, 18(Suppl 1):P490 In this feasibility trial, we found no evidence that face-to-face prelimin- ary initiation of sites recruited to take part in a multi-centre RCT is more effective than remote contact on reducing set-up time, or improving recruitment or data collection. The cost of the two approaches will be explored. Safari is a UK multi-site, parallel-group, randomised controlled, unblinded surgical trial investigating the use of the FENIX MSA (magnetic sphincter augmentation) device, as compared to the current standard treatment of SNS (sacral nerve stimulation) for adult faecal incontinence. Whilst the trial team were aware of the chal- lenges and complexities associated with the design and implementa- tion of surgical trials, a number of unanticipated issues arose during the set-up of safari which severely impacted the trial timelines to the point where funding of the trial was at risk. The issues experienced were related to funding, associated training and supply of the new FENIX device within the trial. Reducing attrition: the communication of retention and withdrawal within patient information sheets 1 2 2 Anna Kearney1, Anna Rosala-Hallas2, Naomi Bacon2, Anne Daykin3, Alison J. Heawood3, Athene Lane3, Jane Blazeby3, Mike Clarke4, Paula R. Williamson1, Carrol Gamble1 Anna Kearney1, Anna Rosala-Hallas2, Naomi Bacon2, Anne Daykin3, Alison J. Heawood3, Athene Lane3, Jane Blazeby3, Mike Clarke4, Paula R. Williamson1, Carrol Gamble1 1North West Hub for Trials Methodology Research/University of Liverpool; 2Clinical Trials Research Centre/University of Liverpool; 3conduct-II Hub for Trials Methodology Research/University of Bristol; 4Centre for Public Health, Queen’s University of Belfast The new intervention, FENIX, provided a cost saving compared to SNS. For non-commercial research within the UK, treatment costs should be met through the normal NHS commissioning process. SNS is funded at a national level through NHS England as a specialised treatment, therefore FENIX should have been funded in the same manner. At the time of safari set-up, NHS England was a relatively new entity. It transpired there was no formal route for approving funding of FENIX other than the lengthy NHS England formal adop- tion process which could take up to 2 years, with no guarantee of success. Although a quicker alternative route was eventually identi- fied and funding for FENIX confirmed, this caused significant delays as sites were not willing to proceed with set-up until this confirm- ation had been received. Correspondence: Anna Kearney Trials 2017, 18(Suppl 1):P489 y Conclusions Withdrawal and retention is poorly described within PIS and address- ing this might positively impact levels of patient attrition, reducing missing data. Consent information is unbalanced, focusing on pa- tient’s rights to withdraw without accompanying information that promotes robust consent and sustained participation. Future research is needed to explore the whether the lack of retention information given at consent is impacting on attrition and if so, how retention can be described to patients to avoid concerns of coercion. Using graphical displays to monitor start-up and recruitment in clinical trials Saams Joy, Robert Henderson, Nancy Prusakowski, El Janet T. Holbrook Johns Hopkins Bloomberg School of Public Health Correspondence: Saams Joy Trials 2017, 18(Suppl 1):P491 p Aim To assess how withdrawal, retention and the value of outcome data collection is described within Patient Information Sheets (PIS). Methods To assess how withdrawal, retention and the value of outcome data collection is described within Patient Information Sheets (PIS). 50 adult or parent PIS from a cohort of 75 National Institute of Health Research Health Technology Assessment (NIHR HTA) programme funded trials that started between 2009–2012 were obtained from protocols, websites or by contacting trialists. A checklist of PIS content developed from UK Health Research Authority and ICH GCP Guidelines was supplemented with retention specific questions. Corresponding trial protocols were obtained and evaluated to cross reference trial specific procedures with information communicated to patients. Finally, an alternative process had to be implemented for supply of the FENIX device. FENIX is available in 7 different sizes with the required size only confirmed at the time of surgery. A set of different sized FENIX devices are therefore required at the time of each operation. Participat- ing sites were reluctant to purchase a full set devices given that only one would be used during each operation. We secured an agreement from the device manufacturer that a set of devices would be provided to each site and remain the property of the device company, with pay- ment only required for a successfully implanted device. p Results PIS frequently reiterated the patient’s right to withdraw at any time (n = 49, 98%), without having to give a reason and without penalty (n = 45, 90%). However, few informed patients they may be asked to give a withdrawal reason where willing (n = 6, 12%). Statements about the value of retention were infrequent (n = 8, 16%). Consent documents failed to include key content that might mitigate with- drawals, such as the need for treatment equipoise (n = 3, 6%). Nearly half the trials in the cohort (n = 23, 46%) wanted to continue to col- lect outcome data if patients stopped trial treatment. However, in 70% (n = 33) of the trials using prospective consent, withdrawal was described in generic terms leaving patients unaware of the differ- ence between stopping treatment and all trial involvement. Nineteen (38%) trials offered withdrawing patients the option to delete previ- ously collected data. Further details on the issues faced during set-up of the safari trial and how these were resolved will be presented, in addition to reflecting on lessons learnt. Background The recruitment and retention of patients are significant methodo- logical challenges for trials. Whilst research has focused on recruitment, the failure to retain recruited patients and collect outcome data can lead to additional problems of biased interpretation of results. Research to identify effective retention strategies has focused on influencing pa- tient behaviour through incentives, reminders and alleviating patient burden, but little attention has been giving to exploring how retention is explained to patients at consent. A baseline level of experience for surgeons was set for both proce- dures to minimise any potential learning curve effect or bias. This was not an issue for SNS as this is an established procedure, however most participating surgeons did not have the required FENIX proced- ure experience at the outset. The trial set-up period was extended to allow time for surgeons to gain this required experience, in addition to incorporating a registration phase within the protocol to facilitate local approvals for use of a new device. Identification of dates for FENIX ‘training cases’ took much longer than anticipated and last mi- nute cancellations were experienced e.g. Due to lack of beds or pa- tients being unfit on the day of surgery. Background Policies regarding publication of articles that describe design features of an individual trial in journals of the Society for Clinical Trials have varied by editor. In 2004, the editor of Clinical Trials specified that such manuscripts must be “instructional” in order to be considered for publication in the Society’s new journal. Subsequently, authors of design manuscripts submitted to the journal frequently received a letter that referred to the editorial, reviewed the requirements, and provided good examples. As review and publication of design articles typically require expenditure of as much or more resources as other types of articles, a question of interest is whether the frequency of citation of design articles has been similar to frequency of citation of articles of other types. Biological sample collection: considerations and lessons learnt at ICR-CTSU Sarah Kernaghan, Leona Batten, Lynsey Houlton, Christy Toms, Claire Snowdon, Emma Hall, Judith Bliss The Institute of Cancer Research Clinical Trials & Statistics Unit (ICR-CTSU) Correspondence: Barbara Hawkins Trials 2017, 18(Suppl 1):P493 Background g Multi-center clinical trials are often plagued with slow starts due to de- lays getting sites started and slow recruitment. Frequently we assume we know the cause of the problems, e.g., slow reviews by multiple Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 184 of 235 Institutional Review Boards (IRBs) or restrictive eligibility requirements. However, there are often other factors, which we can control, that con- tribute to the delays. Information on performance can be presented in tables, but graphical displays are often useful to show patterns, as it is with outcome and safety data, to help identify modifiable factors that contribute long start-up times and slow recruitment as well as to plan for drug packaging, monitoring meetings, and close of recruitment. Methods Liaison with sites in advance through feasibility assessment is import- ant to determine their capability and resources to perform sample collection. During site set-up, training on sample collection proce- dures should be provided to research teams. If biopsy collection is required, engagement with relevant staff, e.g. Surgeons/radiologists, is key. Early engagement with the central laboratory is important to ensure robust procedures are in place at the laboratory to maintain sample and trial integrity, through training and SOP development. A labora- tory manual for sites is developed in collaboration with the central laboratory to ensure that instructions for sample processing and handling at sites and shipment methods are adequate. We present a range of graphical methods for monitoring clinical center start-up and recruitment in clinical trials that may be useful for recog- nizing patterns in performance. Examples are provided from displays we have developed in two clinical trials consortium, the American Lung Associations Airways Clinical Research Centers (ALA-ACRC) and the Multicenter Uveitis Research group (MUST), as well as displays devel- oped by others. g p q Patient information sheets should describe provision, storage and fu- ture use of samples to ensure that the informed consent provided by patients is sufficient to cover future translational work. I d l ti p y Results We have developed and utilized a number of graphical tools that have been effective for identifying key logjams in the conduct of trials, some of which can be addressed by investigators once they have been iden- tified. For example, a display of the stages of trial start-up along with the duration for each individual center identified that training and certi- fication of clinic personnel contributed as much or more to delay in opening trials as IRB reviews. By distributing and discussing this graphic with investigators, we were able decrease start-up times at many sites. We also use a display of recruitment information that integrates infor- mation on two characteristics, total number of participants per site and the time since the last participant was recruited at each site. This display differentiates among sites that have recruited well in the in the past but have either stopped recruitment efforts or have run into diffi- culties finding more eligible patients versus sites that have fewer pa- tients but are actively recruiting. Typically the graphics reporting recruitment emphasize totals without examining recent recruitment ac- tivity. Displays make the information easily accessible to all investiga- tors and can serve to motivate investigators. To maximise participation in sample collection studies, it is crucial that patient advocates are involved in study design and in develop- ing sample collection schedules. This will ensure that sample dona- tion is acceptable to patients and as simple as possible, for example using generic consent to collect research samples at the same time as diagnostic samples. Where possible diagnostic samples should be used, with bespoke additional research biopsies or other samples taken as required. Sample quality may be compromised by delays in processing and lack of resources at site and an adaptable approach is often required to resolve these issues with sites, e.g. Re-training on collection and processing procedures if necessary, provision of additional resources if feasible. To optimise potential recruitment in multi-centre trials, ICR-CTSU acts as the key point of contact between the site and central laboratory when real-time testing of samples is required. Discrepancies between information provided by sites and the central laboratory regarding samples collected do occur, and sample reconciliation is conducted throughout the duration of a trial to identify issues at an early stage. Conclusion Key considerations Sample collection should be considered at an early stage and funding applications should include sufficient funds for consumables, shipment and storage, and trial management time for central coordination. The principle use of samples and mandatory/optional collection require- ments should be considered when developing procedures for ensuring sample integrity and evaluability. Prospective use (e.g. For eligibility in biomarker-driven trials) and type of sample required (e.g. Tumour biopsy vs blood sample) can add additional complexities and time constraints. Biological sample collection: considerations and lessons learnt at ICR CTSU Citation of articles published in clinical trials: design articles vs. others Barbara Hawkins1, Roberta W. Scherer2 1The Johns Hopkins University; 2The Johns Hopkins Bloomberg School of Public Health Background With advancements towards precision medicine and development of novel targeted agents to actionable mutations, collection of bio- logical samples is a key component of many oncology trials. The use of samples for biomarker characterisation, to determine eligibility or as primary/secondary endpoints emphasises their integral nature to the outcome of the trial. Ensuring the quality and integrity of these is vital and at ICR-CTSU the development of procedures to improve sample collection, processing, transfer and storage is ongoing. Key considerations Conclusions Assuming that we know what the obstacles to clinical trials start-up and recruitment without examining the actual data on these metrics, can be counterproductive. We have found that graphical displays can facilitate identification of problems, many of which can be ad- dressed once investigators are made aware of the barriers. Sample collection in clinical trials is becoming increasingly important, but introduces logistical issues. ICR-CTSU constantly works to resolve these issues, and best practice is shared across the unit to minimise repeating problems. P492 Biological sample collection: considerations and lessons learnt at ICR-CTSU Sarah Kernaghan, Leona Batten, Lynsey Houlton, Christy Toms, Claire Snowdon, Emma Hall, Judith Bliss The Institute of Cancer Research Clinical Trials & Statistics Unit (ICR-CTSU) Correspondence: Sarah Kernaghan Trials 2017, 18(Suppl 1):P492 An investigation of the methods used to design, analysis and quantify non-inferiority margins in four medical journals in a 12 month time period Enass M. Duro University of Sheffield Trials 2017, 18(Suppl 1):P494 p Results After recruiting 3% of the participants, we realized that cord blood gases were missed for 20% of deliveries. Missed, incomplete, or er- roneous results occurred even in centers where pre-trial collection was routine. Three successive strategies were implemented to im- prove this rate. The first was alerting the staff. With each center aware of its individual rate and receiving regular updates, unique center-initiated strategies were implemented. Research staff made collection a higher priority, often being present in the delivery room, hand-carrying specimens to the laboratory, troubleshooting with laboratory staff, and occasionally drawing the blood them- selves. A video was distributed showing proper technique for draw- ing the sample. Next, centers were offered a point-of-care blood testing device, which allowed research staff to perform the test with a smaller volume and without involving the laboratory. It also facilitated repeat testing if needed. Lastly, additional funds were provided for after-hours staffing, making it more likely research staff could be present at delivery and facilitate sample collection. After each time point, improvements were observed. The missed rate decreased to 11%, then 9%, and 7.5%, respectively, after each of the three strategies were implemented. Conclusion Most of published NI trials in the four journals did not follow the regulatory guidelines regarding conduct and interpretation of NI trials. There is a need to improve the conduction, interpretation and inference of published NI trials. Methods All issues of Clinical Trials published from 2004 through 2015 were searched to identify design and all other articles. We excluded papers published as part of proceedings of meetings, letters, editorials, invited commentaries, columns, etc. For our preliminary estimates of citation frequency, we randomly selected 20 articles of each type. We searched the Web of Science and Google Scholar databases to determine the number of citations per article as of October 31, 2016. We summarized Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 185 of 235 Page 185 of 235 the distribution of number of citations for each article type and each citation database by the median and interquartile range (IQR). Interim Results the distribution of number of citations for each article type and each citation database by the median and interquartile range (IQR). the distribution of number of citations for each article type and each citation database by the median and interquartile range (IQR). Interim Results calculated by the investigators based on previous studies, in 12(32.4%) trials the NI margins Based on both clinical judgment and historical tri- als, in 5(13.5%) the NI margins based on the regulatory guidelines, and in 2(5.4%) trials the NI margin based on clinical judgment only. Regard- ing the conclusion, non-inferiority established in 24(64.9%) trials, 8 (21.6%) trials failed to establish the non-inferiority, 5(13.5%) trials con- clude the superiority of the tested drug compared with the active control. Of the 598 articles published in Clinical Trials in the 12-year period, 80 (13.4%) were design articles. The 20 design articles selected ran- domly had been cited from 0 to 58 times per Web of Science for a median of 11.0 citations per article (IQR: 7–20) and 5 to 91 times per Google Scholar for a median of 17.0 (IQR: 9–41). The 20 “other” articles selected randomly had been cited 9 to 49 times per Web of Science (median: 17.0; IQR: 4–18) and 2 to 93 times per Google Scholar. (median: 24.6; IQR: 7–30). Interim Conclusion Based on findings for the random samples of 20 articles of each type, design articles published in Clinical Trials during 2004 to 2015 have been cited as frequently as other articles published in the journal during the same time period based on citations in the Google Scholar database but have been cited somewhat less frequently according to citations in the Web of Science database. Final conclu- sions await completion of data collection and analysis of all 598 articles. Background Studies with a non-inferiority (NI) objective have become more popular since the 1990s. A NI study is designed are used to demonstrate that the new treatment is not worse than the proven active comparator. There are methodological and regulatory challenges associated with the planning, conducting and interpretation of these studies and there are a number of regulatory guidelines. The main aim of this review was to investigate the design, analysis; interpretation and reporting of non- inferiority trials in the four top medical journals (Lancet, BMJ, JAMA, The New England Journal of Medicine) in accordance with CONSORT statement. M h d P495 Implementation of strategies to improve collection of a challenging laboratory outcome in the delivery room Steven Weiner George Washington University Trials 2017, 18(Suppl 1):P495 Implementation of strategies to improve collection of a challenging laboratory outcome in the delivery room Steven Weiner George Washington University Trials 2017, 18(Suppl 1):P495 Background In a multi-center obstetric randomized trial, a key outcome was a la- boratory value not routinely collected for clinical reasons at most centers. We determined early in recruitment that a substantial pro- portion of these results were being missed. We describe the steps implemented to decrease these missing results, and some measures of their effectiveness. An investigation of the methods used to design, analysis and quantify non-inferiority margins in four medical journals in a 12 month time period Enass M. Duro University of Sheffield Trials 2017, 18(Suppl 1):P494 Current Status We have begun to search for citations in the Scopus database for comparison with citation counts from the other two citation data- bases for all 598 articles. We also plan to compare the two types of articles for self-citation patterns, distributions of time from publica- tion to citation, and other citation metrics. P495 Implementation of strategies to improve collection of a challenging laboratory outcome in the delivery room Steven Weiner George Washington University Trials 2017, 18(Suppl 1):P495 An investigation of the methods used to design, analysis and quantify non-inferiority margins in four medical journals in a 12 month time period Enass M. Duro University of Sheffield Trials 2017, 18(Suppl 1):P494 This trial required collection of paired umbilical cord blood samples (artery and vein) for measurement of gases. Collection in the delivery room and testing by the hospital laboratory are time-sensitive, logis- tically challenging, and require the cooperation of clinical staff with competing priorities. Drawing blood from the correct vessel is chal- lenging, testing must be completed promptly, and there is little chance for a second opportunity if there is any error. Meanwhile, clinical staff are focused on two patients – mother and baby. There- fore, our strategies focused on minimizing non-research staff partici- pation where possible. p Results Out of the 658 unique trials that were found within the ISRCTN data- base, 456 (69%) of these were labelled as pilot studies, 196 (30%) as feasibility studies and six (1%) considered both a pilot and feasibility study. The overall prevalence of all pilot or feasibility studies does not seem to be showing any particular trend over time. Initially, pilot studies were much more common; however, in recent years feasibil- ity studies have increased in popularity and are now on a par with pilot studies. The median number of target participants for each trial was 50 (range: 5–130,000). When considered separately, pilot studies had a target of 44 (range: 5–130,000) with feasibility studies being slightly larger with a target of 60 (range: 6–1,600). Out of the 658 unique trials that were found within the ISRCTN data- base, 456 (69%) of these were labelled as pilot studies, 196 (30%) as feasibility studies and six (1%) considered both a pilot and feasibility study. The overall prevalence of all pilot or feasibility studies does not seem to be showing any particular trend over time. Initially, pilot studies were much more common; however, in recent years feasibil- ity studies have increased in popularity and are now on a par with pilot studies. The median number of target participants for each trial was 50 (range: 5–130,000). When considered separately, pilot studies had a target of 44 (range: 5–130,000) with feasibility studies being slightly larger with a target of 60 (range: 6–1,600). On average, the trials ran for a period of 18 months with pilot studies reporting a slightly shorter time (17 months) than feasibility studies (18.5 months), which aligns with the larger sample size target. Mental and behavioural disorders accounted for the highest propor- tion of studies (20%), followed by cancer studies (13%). The majority of the studies within the data were interventional (96%) and defined as randomised controlled trials (85%). A hospital envir- onment is the most likely setting for the studies (45%) and in most cases, the recruitment was in a single country (97%). This is consist- ent between the pilot and feasibility studies within the database. Conclusions In the out-of-hospital trial, a treatment difference was noted among 141 children with 1-year outcomes available, but the treatment com- parison (nominal p-value = 0.03) did not approach the conservative stopping boundaries. Enrollment continued, with n = 250 target sur- passed at 2012 year-end. p Results A second interim analysis in August 2013 (n = 213) found that the previously observed treatment difference di- minished (nominal p = 0.12). Results of the ‘negative’ out-of-hospital trial were published in 2015. The in-hospital trial encountered enrollment difficulties due to below- expected patient volumes. During the November 2012 interim analysis, for which 74 children had one-year outcomes available, no treatment difference was noted. Due to the slow accrual, the DSMB skipped effi- cacy monitoring during the 2013 meeting. The second efficacy analysis, in March 2014 (n = 149), again did not approach statistical significance. By late 2014, despite extended enrollment and addition of centers, only 254 eligible patients had been enrolled, indicating final enrollment would be substantially below target. Given these circumstances, the DSMB elected to consider stopping for futility during their January 2015 meeting. Conditional power for the primary efficacy outcome was found to be very limited under a range of assumed treatment effects and realistic final enrollment numbers ranging from 300–400. However, the DSMB decided to continue enrollment and reconvene after add- itional information about potential utility of the trial was available. In a supplemental February 2015 meeting, the DSMB reviewed conditional power estimates for exploratory outcomes, some ad hoc with postu- lated treatment effects based on out-of-hospital trial trends, and subse- quently recommended stopping enrollment. In-hospital trial results are under journal review as of November 2016. g y g g g On average, the trials ran for a period of 18 months with pilot studies reporting a slightly shorter time (17 months) than feasibility studies (18.5 months), which aligns with the larger sample size target. Mental and behavioural disorders accounted for the highest propor- tion of studies (20%) followed by cancer studies (13%) On average, the trials ran for a period of 18 months with pilot studies reporting a slightly shorter time (17 months) than feasibility studies (18.5 months), which aligns with the larger sample size target. Mental and behavioural disorders accounted for the highest propor- tion of studies (20%), followed by cancer studies (13%). The majority of the studies within the data were interventional (96%) and defined as randomised controlled trials (85%). A hospital envir- onment is the most likely setting for the studies (45%) and in most cases, the recruitment was in a single country (97%). This is consist- ent between the pilot and feasibility studies within the database. Methods A search for Non-inferiority trials in Pub Med database that published between 1/1/2015 and 31/12/2015 was performed. The inclusion cri- teria were; Non-inferiority trials that were randomised clinical trials, done on adult humans, published in English and with the full text available. From this search, 387articles were retrieved. Only 45 arti- cles published in the Lancet, BMJ, JAMA and NEJM, 37 of them were analysed. Of 37 articles included in analysis, 15 were published in The Lancet, 12 in the New England Journal of Medicine, 5 in BMJ and 5 in JAMA. Ac- cording to the source of funding: 18(48.6%) of the trials were publicly funded, pharmaceutical companies funded 15(40.5%), and in 4(10.8%) trials, the funding was a combination of public and private sectors. All of the trials were multicentre trials. With respect to the blinding; 24 (64.9%) of the studies were open label studies (no blinding); in these open-label trials, 14 (58.3%) blinding was not possible, no specific rea- son was giving for non-blinding in the other 10(41.7%) trials. Only 8 (21.6%) of the trials were double blinded with 5(13.5%) single blinded. Phase III trials were the most common types of trials - 24 of the 37 (64.9%) while 3(8.1%) were phase IV trials, and 1(2.7%) was a phase II trial. The phase of the trial was not provided in 9 (24.3%) trials. All the trials reported their NI margin. The methods for determining NI margin were not clear in 5(13.55%) trials. In 13(35.1%) trials the margin Not all centers implemented these strategies, nor implemented them at the same time. However, we will describe the rates before and after, including a subset who did not use the point-of-care device nor extended their staffing coverage. Results varied; however in the final year when all strategies were implemented, centers that used both a point-of-care machine and extended hours achieved 3 per- centage points fewer missed results than those who implemented neither. The improvement was also noted beyond the trial. Trial- collected quality control data were provided to the clinical depart- ments, which encouraged them to improve collection procedures for all patients. Trials 2017, 18(Suppl 1):200 Page 186 of 235 Trials 2017, 18(Suppl 1):200 Page 186 of 235 DSMB monitoring of the therapeutic hypothermia after pediatric cardiac arrest (THAPCA) trials 1 1 2 3 Review of pilot and feasibility studies from a registry website; an overview of recent practice Nicola Totton, Andrew Brand, Rachel Evans, Zoë Hoare, Nia Goulden, Paul Brocklehurst Bangor University Correspondence: Nicola Totton Trials 2017, 18(Suppl 1):O2 Richard Holubkov1, Amy Clark1, Andrew M. Atz2, David Glidden3, Beth S. Slomine5, James R. Christensen5, Angie Webster1, Kent Page1, J. Michael Dean1 1University of Utah School of Medicine; 2Medical University of South Carolina; 3University of California at Los Angeles; 4University of California San Francisco; 5Kennedy-Krieger Institute Correspondence: Richard Holubkov Trials 2017, 18(Suppl 1):O1 1University of Utah School of Medicine; 2Medical University of South Carolina; 3University of California at Los Angeles; 4University of California San Francisco; 5Kennedy-Krieger Institute Correspondence: Richard Holubkov Trials 2017, 18(Suppl 1):O1 p Methods An NIH-appointed DSMB monitored safety and efficacy of both trials. A charter specified meetings approximately twice yearly. Symmetric O’Brien-Fleming boundaries were used for monitoring treatment superiority, with an informal <20% conditional power criterion specified for declaring futility. Data on pilot and feasibility studies have been collected from the International Standard Randomised Controlled Trial Number (ISRCTN) registry website. As the website does not provide a downloadable database, a web scraping methodology was adopted and imple- mented using the R programme. Pilot and feasibility studies were identified using a keyword search on the main title. Unique trial re- cords were extracted using regular expressions and collated in an Excel spreadsheet. Recruitment began in September 2009. The first safety-only DSMB review in 2010 combined adverse outcome data across trials as event numbers were small. Subsequent study-specific, safety-only DSMB reviews occurred until the first interim efficacy look for both trials in November 2012. Background Pilot and feasibility studies are increasing in popularity, as demon- strated by the launch of the focussed Journal of Pilot and Feasibility Studies in January 2015. Pilot studies are defined as a mini replica- tion of a proposed full study, used to identify any potential issues. A feasibility study is similar but considered as more exploratory and is used to judge the suitability of a proposed study by assessing the viability of each of the elements individually. In this review, we aim to assess the prevalence of pilot and feasibility studies in recent prac- tice and determine whether the two differ on key characteristics. We envisage this work will then guide future research into the use of pilot and feasibility studies to better inform full studies. M h d The NIH-funded THAPCA randomized trials compared efficacy of therapeutic hypothermia (target temperature 33 degrees C) to normothermia (temperature 36.8 degrees) after cardiac arrest in children >48 hours to <18 years of age. One trial enrolled children who sustained out-of-hospital arrest (target n = 250 analysis-eligible), and a second trial enrolled children with unplanned in-hospital arrest (target n = 504 due to smaller expected hypothermia benefit). The primary effi- cacy outcome was survival with favorable neurological outcome 1 year post arrest. Due to key differences in arrest etiology and post-arrest outcomes, the parallel trials were monitored separately. Conclusion The THAPCA trials provide an interesting study of long-term DSMB review of two parallel studies with differing enrollment patterns. For example, the DSMB elected to remain masked to treatment arm identity in both trials throughout all reviews, recognized the mar- ginal benefit of repeated efficacy monitoring under slow enroll- ment, and considered implications of futility stopping beyond the primary trial outcome. Our experience may help optimize strategies for successful DSMB involvement in randomized trials with long- term follow-up. When laboratory outcomes in a hospital setting are essential to a clinical trial, systems that rely less on clinical staff are advantageous. These strategies could apply to other settings in clinical research, such as emergency departments or remote-care locations. Further- more, the systematic collection and review of data quality measures within a trial can provide useful information for clinical care outside the research setting. When laboratory outcomes in a hospital setting are essential to a clinical trial, systems that rely less on clinical staff are advantageous. Oral Presentations O1 DSMB monitoring of the therapeutic hypothermia after pediatric cardiac arrest (THAPCA) trials Richard Holubkov1, Amy Clark1, Andrew M. Atz2, David Glidden3, Beth S. Slomine5, James R. Christensen5, Angie Webster1, Kent Page1, J. Michael Dean1 1University of Utah School of Medicine; 2Medical University of South Carolina; 3University of California at Los Angeles; 4University of California San Francisco; 5Kennedy-Krieger Institute Correspondence: Richard Holubkov Trials 2017, 18(Suppl 1):O1 DSMB monitoring of the therapeutic hypothermia after pediatric cardiac arrest (THAPCA) trials DSMB monitoring of the therapeutic hypothermia after pediatric cardiac arrest (THAPCA) trials Richard Holubkov1, Amy Clark1, Andrew M. Atz2, David Glidden3, Beth S. Slomine5, James R. Christensen5, Angie Webster1, Kent Page1, J. Michael Dean1 Conclusions The re-randomisation design can increase the recruitment rate compared to parallel group designs, which could reduce costs and make trials more feasible to conduct. If used appropriately, it can provide unbiased esti- mates of treatment effect, control the type I error rate, and maintain or even increase power compared to a parallel group design. O5 Adaptive non-inferiority margins under observable non-constancy Brett Hanscom1, Deborah Donnell1, Brian Williamson2, James P Hughes2 1Fred Hutchinson Cancer Research Center; 2University of Washington Correspondence: Brett Hanscom Trials 2017, 18(Suppl 1):O5 Background A central assumption in the design and conduct of non-inferiority (NI) trials is that the active-control therapy will have the same degree of effectiveness in the planned non-inferiority trial as it had in the prior placebo-controlled trials used to define the non-inferiority mar- gin. This is referred to as the ‘constancy’ assumption. If the constancy assumption fails, the chosen non-inferiority margin is not valid and the study runs the risk of either approving an inferior product or fail- ing to approve a beneficial product. The constancy assumption is unlikely ever to be met completely in practice, and it cannot be validated in a trial without a placebo arm. However, it is often the case that there exist strong, measurable predictors of constancy, such as dosing and adherence. Such predictors can be used to identify appropriate non-inferiority margins during the planning phase, as well as adjust the margin during the monitoring and analysis phases. Methods However, limitations with accuracy of coding, confidentiality, owner- ship and access have previously been identified as significant barriers to accessing routinely recorded data for prospective research. This study has assessed the feasibility of accessing both clinical and non-clinical routinely recorded data within the context of a RCT and will assess the agreement and additional benefits of routinely recorded data compared to data collected using standard prospective methods in a RCT. We propose using meta-analysis regression to model the association between population characteristics and the effectiveness of an active-control therapy, and assume that the model provides an un- biased estimate of effectiveness. Together with expected population characteristics, the fitted model parameters are used to specify a non-inferiority margin targeted to the planned study population. During interim and final analyses, observed population characteristics are used in combination with the initial meta-regression results to adjust the margin. Two methods of adjustment are proposed: one that maintains a pre-planned minimal clinically important difference (MCID) over an inferred placebo, and a second that maintains a fixed proportion of the estimated active-control benefit over placebo. In the second scenario, sample size adjustment may be necessary. Results Using re-randomisation in clinical trials to increase patient recruitment Brennan Kahan Queen Mary University of London Trials 2017, 18(Suppl 1):O4 Brennan Kahan Brennan Kahan Queen Mary University of London Trials 2017, 18(Suppl 1):O4 q Methods We describe some properties of the re-randomisation design, such as the conditions required to obtain unbiased estimates of the treatment effect and control type I error rate. We also evaluate the likely impact of re-randomisation on the recruitment rate is several clinical areas. Results The re-randomisation design can provide unbiased estimates of treat- ment effect and control the type I error rate under some very simple conditions. Furthermore, in some instances this design will have the same or even higher power than a parallel group with an equivalent number of observations. Based on a modelling study across three dif- ferent clinical areas, we estimated that re-randomisation could reduce the time to complete recruitment between 19-45%. Routinely recorded data used in clinical research is potentially cost and resource-use effective. Clinical sources of data such as HES and CPRD are commonly accessed to provide data for retrospective observational and record-linkage studies and provide a valid dataset for this purpose. Routinely recorded data may also present advan- tages to prospective clinical research such as randomised controlled trials (RCTs). For example, routinely recorded data have been used to inform judgements about the feasibility of sample size and recruit- ment targets and measure participant outcomes. Pragmatic cluster RCTs have even been conducted through routine data sources in- cluding patient recruitment, randomisation, administration of inter- vention and trial assessments, such as through The Clinical Practice Research Datalink (CPRD). The majority of RCTs incur health service costs as clinicians assess participants, record outcomes and complete Case Report Forms (CRFs) - hence using routinely recorded data may provide an efficient alternative method for data collection in addition to reducing the burden on participants. Furthermore, data from non- clinical routine sources may inform outcomes beyond the standard RCT assessments of clinical efficacy and effectiveness. For example, cost data (such as use of healthcare resources) and socio-economic data (such as employment and means-tested benefits data) may in- form health economic analyses and the assessment of the broader societal impact of healthcare interventions. O3 Using routinely recorded data in a clinical trial: the feasibility, agreement and additional benefits compared to standard prospective data collection methods Graham Powell1, Laura Bonnett1, Catrin Tudur-Smith1, Dyfrig Hughes2, Tony Marson1, Paula Williamson1 1University of Liverpool; 2Bangor University Correspondence: Graham Powell Trials 2017, 18(Suppl 1):O3 An alternative approach is the re-randomisation design. This design al- lows patients to be re-enrolled and re-randomised for each new treat- ment episode. The number of times each patient is re-randomised is determined by the number of treatment episode each patient ex- periences, rather than by the trial design. Because each patient can contribute multiple treatment episodes, this design can facilitate an increased recruitment rate, potentially making trials easier and quicker to conduct. Graham Powell1, Laura Bonnett1, Catrin Tudur-Smith1, Dyfrig Hughes2, Tony Marson1, Paula Williamson1 1University of Liverpool; 2Bangor University Correspondence: Graham Powell Trials 2017, 18(Suppl 1):O3 There are a number of administrative datasets that routinely record information on individuals in the UK. Such routine or ‘Big Data’ sources record data for a specified primary purpose and are regu- lated for security, confidentiality and disclosure by The Data Protec- tion Act 1998 and Freedom of Information Act 2000. Access for secondary uses such as clinical research is permitted when health and social care benefit can be demonstrated. Routine sources include clinical datasets such as Hospital Episode Statistics (HES) and the Clinical Practice Research Datalink (CPRD) and non-clinical datasets such as data recorded by the Department of Work and Pensions (DWP), HM Revenue and Customs (HMRC) and The Driving and Vehicle Licensing Authority (DVLA). Background Patient recruitment is often a major challenge for randomised trials. Reviews of publicly funded UK trials have found that 45 to 69% fail to recruit to target. This increases costs, delays results, and adversely impacts on the feasibility of conducting trials for conditions where there is a limited patient pool. p Results Conclusions In recent years, the prevalence of feasibility studies has begun to match that of pilot studies but the characteristics found for the two are very similar. Due to their similar nature, there could be an argu- ment that pilot and feasibility studies should considered as one. Alternatively, if these studies are to be separate more education is required to outline the differences and provide guidelines for each to help shape future research projects. Trials 2017, 18(Suppl 1):200 Page 187 of 235 undergo multiple treatment cycles until they become pregnant. The current norm for trials in these conditions is for patients to be enrolled for only one treatment episode. O3 Using routinely recorded data in a clinical trial: the feasibility, agreement and additional benefits compared to standard prospective data collection methods Graham Powell1, Laura Bonnett1, Catrin Tudur-Smith1, Dyfrig Hughes2, Tony Marson1, Paula Williamson1 1University of Liverpool; 2Bangor University Correspondence: Graham Powell Trials 2017, 18(Suppl 1):O3 Conclusion O7 Simulation of various strategies for optimal selection of randomization methods in multicenter clinical trials Zhibao Mi, Rebecca A. Horney, Eileen M. Stock, Kousick Biswas VA Cooperative Studies Program Coordinating Center Correspondence: Zhibao Mi Trials 2017, 18(Suppl 1):O7 If prior placebo-controlled trials provide evidence of an association between population characteristics and the effectiveness of an active- control therapy, non-inferiority margins can be adjusted based on ob- served population features, effectively maintaining pre-specified levels of Type-I error and power. The random allocation and masking of participants to treatment are procedures in a study design essential to minimizing bias and the suc- cess of a clinical trial. The essence of the randomization process is to ensure an equal probability for each participant to be assigned to ac- tive or control treatment groups, which naturally leads to three proper- ties of the randomization procedure: balance in sample size and constitution between treatment and control groups, unpredictability in the allocation of a participant to a certain group, and simplicity for an investigator to implement without compromising the randomization principle. With recent advances in biomedical and statistical method- ologies, the area of clinical trial design is evolving rapidly with varying opinions on an optimal randomization method. Randomization methods have now expanded into more advanced approaches beyond the classical assignment to treatment groups (e.g. covariate adjustment in the randomization procedure, changing probability of assignment as in adaptive designs). Thus, the traditional randomization method faces new challenges, both theoretically and pragmatically. With increasingly complex trial designs, it becomes more challenging to determining the most appropriate randomization method. To help select an optimal randomization method, we performed numerical simulations to assess various randomization strategies in a centralized randomization system for multicenter clinical trials, by considering varying values for several design parameters including trial sample size, covariate strata, clinical sites, treatment arms, and allocation schema. For each sce- nario of simple, permuted block, stratified permuted block, and adaptive randomization strategies, imbalance and predictability were estimated through numerical simulations (repeated 10,000 times). Simulation results are tabulated in a series of tables serving as a useful reference for choosing an appropriate randomization method given a particular trial design. Results For some conditions, patients may require treatment on multiple occa- sions. For example, patients with sickle cell disease require pain relief for each new pain crisis, and women using fertility treatment may We consider a hypothetical NI trial of an experimental HIV Pre-exposure Prophylaxis (PrEP) drug versus a standard PrEP drug (active control) Page 188 of 235 Page 188 of 235 Trials 2017, 18(Suppl 1):200 Page 188 of 235 Conclusions designed to provide one-sided alpha equal to 2.5%, and 90% power. If, due to lack of adherence to the standard drug, the constancy assump- tion fails and the active-control therapy is 10% less effective than planned, the probability of a false non-inferiority finding rises from 2.5% to 16%. If the active control therapy is 10% more effective than planned (for example, if adherence were higher than planned), power falls from 90% to 52%. By revising the NI margin according to the pre- specified meta-regression model, and maintaining the pre-specified MCID, both alpha and power can be corrected to planned levels with- out modification to the planned sample size. If the allowable effective- ness of the experimental therapy is permitted to vary depending on the estimated active-control effect, alpha and power can be partially corrected by updating the margin, and fully corrected by updating both the margin and the sample size. We believe the new tool will offer considerable advantages over the existing tool. Once programmed into software, we expect the tool will be easier to use than the first version. Some issues remain to be re- solved, however, such as how many results should be assessed for each study, and how best to integrate the assessment into the data extrac- tion process. This presentation will provide an introduction to the tool. Further details of RoB 2.0 will be available from riskofbias.info. Conclusion The goal of this study is to provide data support for identifying an optimal randomization method, accounting for the trade-off between pre- cision of randomization balance and simplicity of implementation since more complex methods may lead to a greater likelihood of randomization schedule or allocation algorithm errors during im- plementation and human errors during the trial. A revised tool for assessing risk of bias in randomized trials (RoB 2.0) Jalena Savovic1, Matthew Page2, Roy Elbers2, Asbjorn Hróbjartsson3, Isabelle Boutron4, Barney Reeves2, Jonathan Sterne2, Julian Higgins2 1University of Bristol; NIHR CLAHRC West University Hospitals Bristol NHS Foundation Trust; 2University of Bristol; 3University of Southern Denmark; 4University Paris Descartes Correspondence: Jalena Savovic Trials 2017, 18(Suppl 1):O6 Correspondence: Jalena Savovic Trials 2017, 18(Suppl 1):O6 Funding Funding MRC Network of Hubs for Trials Methodology Research (MR/L004933/ 1- N61); MRC ConDuCT II (MR/K025643/1), NIHR CLAHRC West. u d g MRC Network of Hubs for Trials Methodology Research (MR/L004933/ 1- N61); MRC ConDuCT II (MR/K025643/1), NIHR CLAHRC West. Background The Cochrane risk of bias tool for randomized trials seeks to determine whether the findings of a randomized trial can be believed. First released in 2008, and revised slightly in 2011, it is the most widely used risk of bias tool in both Cochrane and non-Cochrane reviews on the effects of interventions. However, evaluations of the tool have highlighted some problems. Objective: To introduce a revised tool to assess risk of bias in randomized trials (RoB 2.0), which builds on the established Cochrane risk-of-bias tool as well as the thinking behind the recently developed tool for non-randomized studies (ROBINS-I). Methods Over the last year, we assembled collaborators from across the world to develop RoB 2.0. We held an initial development meeting in Au- gust 2015 where the main structure of the tool was agreed. Working groups were formed and tasked with developing signalling ques- tions, criteria for reaching a judgment and full guidance. Working groups’ contributions were collated and the draft version of the new tool was extensively piloted by individuals with varying degrees of experience, at a three-day event held in Bristol in February 2016 and remotely. The piloting feedback was considered at a second develop- ment meeting in April 2016, where refinements to the tool and to the written guidance that accompanies it were made. The working groups were further tasked with developing algorithms for reaching a domain-level judgment and creating worked examples. Further pre-release piloting took place in September 2016. p p g p p Results O8 Should consort do more to improve the quality of missing data reporting in trials? Jamilla Hussain1, Martin Bland2, Dean Langan3, Miriam J Johnson4, David C Currow5, Ian R White6 1Hull York Medical School, University of York; 2Health Sciences Department, University of York; 3University College London; 4SEDA, University of Hull; 5Palliative and Supportive Services, Flinders University; 6MRC Biostatistics Unit, University of Cambridge Correspondence: Jamilla Hussain Trials 2017, 18(Suppl 1):O8 Results h Key changes in RoB 2.0 compared with the 2011 version of the tool are: −simplification of issues into fewer (mandatory) bias domains; − clearer focus on risk of bias in a specific result from the randomized trial; −introduction of signalling questions - which are reasonably fac- tual in nature - to facilitate risk-of-bias judgements; −algorithms to reach risk of bias judgements; −clarification of differences between the review team's interest on the effect of assignment to intervention (the intention-to-treat effect) versus the effect of starting and adhering to intervention: issues of blinding, implementation and adherence differ importantly between these; −clarification that selective reporting should be assessed only when a result is available (whereas selective non-reporting should be assessed at meta-level); −separate templates for parallel group trials, cluster-randomized trials and cross-over trials. Page 189 of 235 Trials 2017, 18(Suppl 1):200 Page 189 of 235 Page 189 of 235 Background in realistic clinical settings. Accomplishing pragmatism requires better summaries of the totality of the evidence that allow for informed bene- fit:risk decision-making and in a way that clinical trials consumers, pa- tients, physicians, insurers find transparent. The current approach to the analysis of clinical trials is to analyze efficacy and safety separately and then combine these analyses into a benefit:risk assessment. Many assume that this will effectively describe the impact on patients. But this approach is suboptimal for evaluating the totality of effects on pa- tients. We describe a broad vision for the future of clinical trials consist- ent with increased pragmatism. Greater focus on using outcomes to analyze patients rather than patients to analyze outcomes particularly in late-phase/stage clinical trials is an important part of this vision. We discuss partial credit, a strategy for design and analysis of clinical trials based on benefit:risk assessment that has greater pragmatism than standard methods. The strategy involves utilizing composite benefit:risk endpoints with a goal of understanding how to analyze one patient be- fore trying to figure out how to analyze many. With a desire to measure and weigh outcomes that are most important from the patient’s perspective, we engage patients as a resource to inform analyses. We discuss partial credit within the context of antibiotic clinical trials. Transparent reporting of missing data is crucial to the critical ap- praisal of trial results. This is particularly important in palliative care trials where large amounts of missing data and truncated data due to death occur. Although the CONSORT 2010 statement recommends the impact of missing data on the validity of intention-to-treat ana- lyses be reported, it does not provide specific guidance on how to report: methods to handle missing data, assumptions about the miss- ing data mechanism and missing data sensitivity analyses. Several other groups have provided further missing data reporting recom- mendations that include such criteria. Whether trials report missing data according to the recommendations by CONSORT and other groups however is not known. Objectives Assess (i) the quality of reporting and handling missing data in pallia- tive care trials against current guidance, (ii) any differences in the complete reporting of criteria specified by the CONSORT 2010 state- ment compared to those not specified by CONSORT, (iii) the association between the quality of missing data reporting and journal impact factor and CONSORT endorsement status, to explore how implementation of reporting guidance may be optimised. Conclusion The rigorous methods, evolving nature, and wide recognition of the CONSORT statement make it ideally placed to facilitate better reporting of missing data. Further development and implementation of the CONSORT missing data reporting guidance is likely to improve the quality of reporting. Specific suggestions for CONSORT will be discussed. O9 Using outcomes to analyze patients rather than patients to analyze outcomes: partial credit, pragmatism, and benefit: risk evaluation p Results 08 i l 108 trials (15,560 participants) were included. In general missing data reporting was incomplete and not handled in accordance with current guidance. Reporting criteria specified by the CONSORT state- ment were better reported than those not specified by CONSORT (proportion of trials reporting CONSORT criteria: account for all par- ticipants who enter the study 69%, data completeness 94%, reason for missing data 71%). However item-level (15%) and secondary out- come (9%) missing data were poorly reported, so the proportion of missing data stated is likely to be an underestimate. Provided rea- sons for missing data were unclear for 54% of participants. 48% of trials clearly reported their method to handle missing data, of the trials with missing data (n = 93): 60% used complete case analysis alone and 16% reported a missing data sensitivity analysis. Only one trial used a recommended method to handle truncated data due to death. As the journal impact factor doubled the odds of reporting the flow of participants (odds ratio (OR) 1.54, 95% CI 1.20, 1.97), data completeness (OR 1.39, 95% CI 1.15, 1.69), comparison of baseline characteristics of those with and without missing data (OR 1.50, 95% CI 1.20, 1.87), and method of handling missing data (OR 1.40, 95% CI 1.13, 1.73) were statistically significantly increased. There was insuffi- cient evidence that the criteria specified by CONSORT were more likely to be reported in journals that endorsed the CONSORT statement. p g g Methods S i Systematic review of palliative care randomised controlled trials. An information specialist searched CENTRAL, MEDLINE, and EMBASE (2009–2014) with no language restrictions. A random sample of iden- tified trials were screened, selected and had data extracted by two independent reviewers. O10 Regression discontinuity for replication of randomized controlled trial results: an application to the mycotic ulcer treatment trials Catherine Oldenburg1, N. Venkatesh Prajna2, Tiruvengada Krishnan2, Revathi Rajaraman2, Muthiah Srinivasan2, Kathryn J. Ray1, Kieran S. O’Brien1, Travis C. Porco1, Nisha R. Acharya1, Jennifer Rose-Nussbaumer1 1University of California, San Francisco; 2Aravind Eye Hospitals Correspondence: Catherine Oldenburg Trials 2017, 18(Suppl 1):O10 Methods The Mycotic Ulcer Treatment Trials (MUTT-I & MUTT-II) were two con- temporaneous randomized controlled trials with identical outcome assessments designed to compare strategies for the treatment of fungal corneal ulcers. MUTT-I, which enrolled patients with better best spectacle-corrected visual acuity (BSCVA) (<20/400), compared topical voriconazole to topical natamycin. MUTT-II enrolled patients with worse BSCVA (20/400) and compared adding oral voriconazole versus placebo to topical voriconazole. We estimated the RD effect of natamycin versus voriconazole on 1) 3-month BSCVA and 2) odds of perforation and/or requiring a therapeutic penetrating kerato- plasty (TPK), and compared these results to those estimated in the trial. We utilized enrollment visual acuity as a clinical decision rule to replicate the results of MUTT-I, using the natamycin arm from MUTT-I and the placebo arm of MUTT-II, and 20/400 as the threshold for re- ceiving natamycin (<20/400) or voriconazole (20/400), representing an RD design. The RD model included terms for being above or below the threshold and a term for baseline visual acuity above and below the threshold. Conclusion Background Regression discontinuity (RD) is a quasi-experimental method that utilizes threshold rules for determining treatment assignment to esti- mate causal effects when randomization is not available. Examples of clinical threshold rules include CD4 count for determining antiretro- viral therapy eligibility in HIV-infected individuals or blood pressure determining eligibility for hypertension treatment. However, the val- idity of RD has not been established via direct comparison to effects estimated via the gold standard randomized controlled trial (RCT). Here, two concurrent RCTs allow us to directly compare an effect size from an RCT to that of RD. We utilize a continuous enrollment criter- ion in the RCTs to test if regression discontinuity achieves similar re- sults to the intention-to-treat (ITT) effect from the trial itself. Methods Methods Each month, we searched PubMed (between May 2014 and April 2015) to identify primary reports of randomised trials published in six high impact general and 12 high impact specialty journals. The corresponding author of each trial publication was then contacted by email asking them to complete an online survey investigating changes made to their manuscript as part of the peer review process. Our main outcome was the nature and extent of changes made to manuscripts by authors as part of the peer review process, in relation to reporting of the primary outcome(s) and/or primary statistical analysis. We also assessed how often authors follow these requests and whether this was influenced by specific journal or trial characteristics. Surrogate endpoints are often used in randomized clinical trials in- stead of well-established hard endpoints for practical convenience: they are usually cheaper, more rapid, or less invasive to measure. The meta-analytic approach relies on two measures of individual level surrogacy (R_indiv^2) and trial level surrogacy (R_trial^2) [1]. This approach was extended to the survival data case [2], with a first step using copulas to measure individual level surrogacy in terms of Kendall's tau and a ?A3B2 show $132#?>second step using weighted regression to compute R_trial^2. Despite being the refer- ence method for survival data today, this approach can suffer from convergence problems in the second step, which is the one which computes R_trial^2. In the present work, we considered a bivariate survival model with (i) an individual random effect shared between the two endpoints to measure individual level surrogacy (Kendall's tau) and (ii) correlated treatment-by-trial interactions to measure R_trial^2. We used auxiliary mixed Poisson models to jointly esti- mate the parameters of such model with piecewise constant base- line hazards. To reduce the computational complexity, we also considered reduced Poisson models, accounting for only individual- or only trial-level surrogacy. We studied via simulations the operat- ing characteristics of this mixed Poisson approach as compared to the two-step copula approach, with Clayton, Plackett and Hougaard copulas and with or without adjustment of the second-step regres- sion for measurement error. The Clayton copula model was the most robust and reliable of the copula models compared; the Pois- son model with both individual- and trial-level random effects out- performed its reduced equivalents. We also applied the methods to an individual patient data meta-analysis in advanced/recurrent gas- tric cancer (4069 patients from 20 randomized trials). Objective l j Selective reporting of outcomes in clinical trials is a serious problem. We aimed to investigate the influence of the peer review process within biomedical journals on the reporting of primary outcome(s) and statistical analyses of reports of rando- mised trials. Conclusion Overall there was little evidence of a negative impact of the peer review process in terms of selective reporting of the primary out- come. Most changes requested resulted in improvements to the manuscript, improving clarity of statistical methods used, and providing more cautious conclusions. However, some changes re- quested by peer reviewers were deemed inappropriate and could have a negative impact on reporting of the final publication, such as the adding of unplanned additional analyses. Results Nine hundred eighty-three corresponding authors were invited to take part in the online survey, of which 258 (29%) responded. The majority of trials were multicentre (n = 191; 74%), parallel group (n = 225; 86.5%); median sample size = 325 (IQR 138 to 1010). Half assessed drug interventions (n = 127; 49%), over half were non- industry funded (n = 159; 62%) and the primary outcome was clearly defined in 92% (n = 238), of which the direction of treatment effect was statistically significant in 48%. y g The majority of authors responded (1–10 Likert scale) they were satisfied with the overall handling (mean 8.6, SD 1.5) and quality of peer review (mean 8.5, SD 1.5) of their manuscript by the journal. Only 3% (n = 8) said the editor or peer reviewers asked them to change or clarify the trial’s primary outcome. However, 27% (n = 69) reported they were asked to change or clarify the statistical analysis of the primary outcome; most responded they fulfilled the request, the main motivation being to improve the statistical methods (n = 38; 55%) or avoid rejection (n = 30; 43.5%). Overall there was no dif- ference between authors being asked to make this change and the type of journal, intervention, significance of the primary outcome or funding source. 36% (n = 94) responded that they were asked to include additional analyses that had not been included in the ori- ginal manuscript; in 77% (n = 72) these were not pre-specified in the protocol. 23% (n = 60) were asked to modify their overall con- clusion, in most cases (n = 53; 88%) to provide a more cautious interpretation. [1] Buyse, M, Molenberghs, G, Burzykowski, T, Renard, D and Geys, H. (2000). The validation of surrogate endpoints in meta-analyses of randomized experiments. Biostatistics 1(1), 49–67. [2] Burzykowski, T, Molenberghs, G, Buyse, M, Geys, H and Renard, D. (2001). Validation of surrogate end points in multiple randomized clinical trials with failure time end points. J Roy Statist Soc C Appl Statist 50(4), 405–422. O11 Evaluation of failure time surrogate endpoints in individual patient data meta-analyses of randomized clinical trials. A poisson approach O Evaluation of failure time surrogate endpoints in individual patien data meta-analyses of randomized clinical trials. A poisson approach Federico Rotolo1, Xavier Paoletti1, Toasz Burzykowski2, Marc Buyse2, Stefan Michiels1 1Gustave Roussy/INSERM; 2I-Biostat U Hasselt/IDDI Correspondence: Federico Rotolo Trials 2017, 18(Suppl 1):O11 approach Federico Rotolo1, Xavier Paoletti1, Toasz Burzykowski2, Marc Buyse2, Stefan Michiels1 1Gustave Roussy/INSERM; 2I-Biostat U Hasselt/IDDI Correspondence: Federico Rotolo Trials 2017, 18(Suppl 1):O11 pp Federico Rotolo1, Xavier Paoletti1, Toasz Burzykowski2, Marc Buyse2, Stefan Michiels1 1Gustave Roussy/INSERM; 2I-Biostat U Hasselt/IDDI Correspondence: Federico Rotolo Trials 2017, 18(Suppl 1):O11 Influence of peer review on the reporting of primary outcome(s) and statistical analyses of randomised trials Influence of peer review on the reporting of primary outcome(s) and statistical analyses of randomised trials Sally Hopewell1, Claudia M. Witt2, Klaus Linde3, Katja Icke2, Olubusola Adedire4, Shona Kirtley4, Douglas G. Altman4 1Oxford Clinical Trials Research Unit; 2Institute of General Practice, Technical University Munich; 3Institute for Social Medicine, Epidemiology and Health Economics, Charité Universitätsmedizin Berlin; 4Centre for Statistics in Medicine, University of Oxford Correspondence: Sally Hopewell Trials 2017, 18(Suppl 1):O12 Influence of peer review on the reporting of primary outcome(s) and statistical analyses of randomised trials Sally Hopewell1, Claudia M. Witt2, Klaus Linde3, Katja Icke2, Olubusola Adedire4, Shona Kirtley4, Douglas G. Altman4 1Oxford Clinical Trials Research Unit; 2Institute of General Practice, Technical University Munich; 3Institute for Social Medicine, Epidemiology and Health Economics, Charité Universitätsmedizin Berlin; 4Centre for Statistics in Medicine, University of Oxford Correspondence: Sally Hopewell Trials 2017, 18(Suppl 1):O12 Influence of peer review on the reporting of primary outcome(s) and statistical analyses of randomised trials Sally Hopewell1, Claudia M. Witt2, Klaus Linde3, Katja Icke2, Olubusola Adedire4, Shona Kirtley4, Douglas G. Altman4 1Oxford Clinical Trials Research Unit; 2Institute of General Practice, Technical University Munich; 3Institute for Social Medicine, Epidemiology and Health Economics, Charité Universitätsmedizin Berlin; 4Centre for Statistics in Medicine, University of Oxford Correspondence: Sally Hopewell Trials 2017, 18(Suppl 1):O12 Influence of peer review on the reporting of primary outcome(s) and statistical analyses of randomised trials Sally Hopewell1, Claudia M. Witt2, Klaus Linde3, Katja Icke2, Olubusola Adedire4, Shona Kirtley4, Douglas G. Altman4 1Oxford Clinical Trials Research Unit; 2Institute of General Practice, Technical University Munich; 3Institute for Social Medicine, Epidemiology and Health Economics, Charité Universitätsmedizin Berlin; 4Centre for Statistics in Medicine University of Oxford While RD and RCT results were similar, the RD effect was larger, although not significantly so (P = 0.52 for BSCVA). These results sug- gest that the use of threshold rule in an RD design may be useful for estimation of causal effects under conditions where trials are not possible, or for replication of trial results. Correspondence: Sally Hopewell Trials 2017, 18(Suppl 1):O12 Methods As the conver- gence rate and the estimation results may vary substantially between models, we encourage the user to carefully evaluate the convergence of each alternative approach and to report the results of different models. We implemented the methods presented here in the R package surrosurv (https://cran.r-project.org/package=surrosurv). [1] Buyse, M, Molenberghs, G, Burzykowski, T, Renard, D and Geys, H. (2000). The validation of surrogate endpoints in meta-analyses of randomized experiments. Biostatistics 1(1), 49–67. [2] Burzykowski, T, Molenberghs, G, Buyse, M, Geys, H and Renard, D. (2001). Validation of surrogate end points in multiple randomized clinical trials with failure time end points. J Roy Statist Soc C Appl Statist 50(4), 405–422. Results Trials 2017, 18(Suppl 1):O9 In the MUTT-I RCT, patients randomized to natamycin had a nearly 2- line improvement in BSCVA at 3 months (logMAR −0.18, 95%CI −0.30 to −0.05) and reduced odds of perforation and/or TPK (OR = 0.42, 95%CI 0.22 to 0.80) compared to voriconazole. In the RD model, In the future, clinical trials will have an increased emphasis on pragma- tism, providing a practical description of the effects of new treatments Trials 2017, 18(Suppl 1):200 Page 190 of 235 natamycin was associated with a three-line improvement in BSCVA at 3 months (logMAR −0.29, 95%CI −0.50 to −0.08) and reduced odds of perforation/TPK (OR = 0.35, 95%CI 0.15 to 0.82). Results were ro- bust to multiple sensitivity analyses, including flexible functional forms of visual acuity and restricting the analysis to narrower band- widths around the cutoff (20/400). Conclusions natamycin was associated with a three-line improvement in BSCVA at 3 months (logMAR −0.29, 95%CI −0.50 to −0.08) and reduced odds of perforation/TPK (OR = 0.35, 95%CI 0.15 to 0.82). Results were ro- bust to multiple sensitivity analyses, including flexible functional forms of visual acuity and restricting the analysis to narrower band- widths around the cutoff (20/400). Conclusions Opportunistic trial recruitment during routine primary care consultations for acute conditions: a mixed methods evaluation of recruitment performance and barriers Opportunistic trial recruitment during routine primary care consultations for acute conditions: a mixed methods evaluation of recruitment performance and barriers Jeremy Horwood, Niamh M. Redmond, Christie Cabra, Emer Brangan, Petra Manley, Sophie Turnbull, Jenny Ingram, Patricia Lucas, Alastair D. Hay, Peter S. Blair University of Bristol Correspondence: Jeremy Horwood Trials 2017, 18(Suppl 1):O14 p Results For CHICO, 32 practices were randomised and 501 children were re- cruited one month ahead of schedule. More children were recruited to the intervention (292, 58%) than the control (209, 42%) arm. There was a difference in clinician type (higher proportion of nurses) and more unwell children in the intervention arm. Although just over a quarter of clinicians were nurses, they recruited more frequently, recruiting 220 (44%) of the children. Interviews revealed that many clinicians prioritised dealing with the cough first and only afterwards attempted to recruit children. This meant that clinicians, particularly in the control arm, reported they preferentially recruited less unwell children, because these were quicker and it was easier to ‘fit in’ the research on top of the normal consultation. O15 Designing clinical trials with age-related multiple morbidity outcomes p Jeremy Horwood, Niamh M. Redmond, Christie Cabra, Emer Brangan, Petra Manley, Sophie Turnbull, Jenny Ingram, Patricia Lucas, Alastair D. Hay, Peter S. Blair University of Bristol p Jeremy Horwood, Niamh M. Redmond, Christie Cabra, Emer Brangan, Petra Manley, Sophie Turnbull, Jenny Ingram, Patricia Lucas, Mark Espeland1, Jill P. Crandall2, Stephen Kritchevsky1, Eileen M. Crimmins3, Brandon R. Grossardt4, Judy Bahnson1, Michael E. Miller1, Jamie Nicole Justice1, Nir Barzilai2 Mark Espeland1, Jill P. Crandall2, Stephen Kritchevsky1, Eileen M. Crimmins3, Brandon R. Grossardt4, Judy Bahnson1, Michael E. Miller1, Jamie Nicole Justice1, Nir Barzilai2 2 Correspondence: Jeremy Horwood Trials 2017, 18(Suppl 1):O14 1Wake Forest School of Medicine; 2Albert Einstein College of Medicine; 3University of Southern California; 4Mayo Clinic Correspondence: Mark Espeland Trials 2017, 18(Suppl 1):O15 Background Evaluating the effectiveness of interventions for acute conditions in primary care often necessitates clinicians opportunistically recruiting patient during time-pressured consultations. References Trials 2017, 18(Suppl 1):200 Page 191 of 235 O13 Agreeing outcomes that matter to patients? What are the challenges? Heather Bagley, Bridget Young University of Liverpool Correspondence: Heather Bagley Trials 2017, 18(Suppl 1):O13 feasible to clinicians and patients/parents. However, the requirement for individual patient/parent consent during the consultation was a barrier to recruitment and may have introduced bias. Given the na- ture of the interventions and the views expressed it is viable and valid that future trials of both interventions should not require indi- vidual consent providing the choice to opt out is provided and fol- low up procedures maintain patient anonymity. Trials evaluating the effectiveness of interventions for acute conditions in primary care should avoid recruitment processes that add burden to routine prac- tice. The study highlights the value of conducting mixed method evaluations of recruitment performance and barriers during feasibility trials to inform future trial design. This abstract is not included here as it has already been published. Background and Objectives g j The incidence of age-related chronic diseases rises exponentially with age. This parallels the exponential increases with age in rates of major disease-specific deaths tracked by the US National Center for Health Statistics, including those for heart disease, cancer, stroke, type 2 diabetes mellitus, and Alzheimer’s disease. It has re- peatedly been shown that the major, and by far the most potent, risk factor cutting across age-related chronic diseases is age itself. There is growing evidence for a biologic construct underlying aging, leading to the potential that interventions may be devel- oped to slow its progression. The primary goal is not to increase the number of years lived, but to increase the number of years lived with better health and function. The NIA Interventions Testing Program has been established to organize research towards this goal across model organisms. As interventions emerge from this program as candidates for human intervention, clinical trials will be mounted to assess their efficacy. We discuss design and analytical issues, including the choice of outcomes, eligibility criteria, monitor- ing rules, and analytical strategies. We present projections of rates at which outcomes occur, as benchmarks for estimating the statis- tical power for future trials. To describe the performance of, barriers to, and implications of clinicians recruiting trial participants during consultations within two primary care feasibility cluster randomised controlled trials, CHICO and IMPACT-PC. p Methods Parallel analyses were conducted using data from three large cohorts of older individuals: the Rochester Epidemiology Project, the Health and Retirement Study, and the Women’s Health Initiative Observa- tional Study. These allowed us to contrast outcomes, evaluate poten- tial eligibility criteria, and project incidence rates. R lt Methods For the CHICO trial GP practices were randomised to a within con- sultation web-based intervention to reduce antibiotic prescribing for children with acute cough and respiratory tract infection, or usual care. For the IMPACT-PC trial GP practices were randomised to a nurse-led telephone based management service for patients testing for Chlamydia trachomatis (CT) and Neisseria gonorrhoea (NG), or usual care. Performance data analyses were conducted and 44 clinicians and 26 trial participants (patients/parents) were interviewed post recruitment and analysed thematically to explore their experiences. g Results The incidence rate of composite multi-morbidity outcomes and the rate that they accumulate over time are attractive clinical trial out- comes. Rates increase with age and, for cohorts at suitably in- creased risk due to choice of eligibility criteria, are sufficiently great enough to support the development of tractable (4–6 years; N = 3,000) multi-center clinical trials. To provide evidence that interven- tions target aging and health span, rather than individual compo- nents of the composite outcome measure, nuanced approaches to monitoring and analysis are required, which we describe. The benchmarks and methods that we present support the feasibility of designing efficient clinical trials for interventions targeting aging. As an example, we describe the design of the Targeting Aging with Metformin (TAME) multicenter clinical trial. Conclusions For IMPACT-PC, 11 practices were randomised, 1154 patients were re- cruited (60% of eligible patients) and 30 (2.6%) patients tested posi- tive for CT, 9 (0.8%) tested positive for NG and 3 (0.3%) tested positive for both. CT positivity was higher (4.3%) amongst individuals’ eligible but not recruited to the study in intervention practices. Inter- views revealed the main reason for failure to recruit eligible patients was insufficient time to undertake consent procedures. Despite pa- tient consent being recorded, patients were sometimes unclear that they were participating in a research study. However, patients found both the intervention and the use of their medical records in evalu- ation acceptable, as long as their anonymity was maintained. Conclusions Recruitment to both trials was successful in terms of numbers re- cruited and timescales and the interventions were acceptable and Clinical trials targeting aging are feasible, but require careful design consideration and monitoring rules. Trials 2017, 18(Suppl 1):200 Page 192 of 235 Page 192 of 235 O16 Improving the testing of treatment effect in clinical trials with time to event outcomes Song Yang1, Ross Prentice 1National Heart, Lung, and Blood Institute, NIH; 2Fred Hutchinson Cancer Research Center Correspondence: Song Yang Trials 2017, 18(Suppl 1):O16 variables used in analyses, annotated forms, and biospecimen linking files. To date, over 600 publications are known to have resulted from requestors using BioLINCC resources. Conclusion Efficient preparation of study data and documents is essential to maximizing the scientific utility of study resources. O18 Patient preferences for outcomes in clinical trials: implications for medicines optimization p Emily Holmes1, Anthony G. Marson2, Dyfrig A. Hughes1 1Bangor University; 2University of Liverpool Correspondence: Emily Holmes Trials 2017, 18(Suppl 1):O18 y Leslie Carroll1, John Adams1, Corey Del Vecchio1, Karen Mittu1, Kevin Zhou1, Jane Wang1, Carol Giffen1, Elizabeth Wagner2, Sean Coady3 1Information Management Services, Inc.; 2Translational Blood Science and Resources Branch, Division of Blood Diseases and Resources, National Heart, Lung, and Blood Institute; 3Epidemiology Branch, Prevention and Population Sciences Program, Division of Cardiovascular Sciences, National Heart, Lung, and Blood Institute Results The rank order of AEDs based on trial data for remission: lamotrigine, carbamazepine, topiramate, oxcarbazepine, then gabapentin, chan- ged when patient benefit-risk preference was considered. The prob- ability of patients with partial epilepsy preferring each AEDs was, in descending order: carbamazepine (0.29), lamotrigine (0.26), oxcarba- zepine (0.24), gabapentin (0.15), topiramate (0.07). Women with the potential to become pregnant, had a preference probability of: lamo- trigine (0.31), oxcarbazepine (0.21), gabapentin (0.20), carbamazepine (0.19), topiramate (0.09). Comparable results were found for patients with generalised or unclassified epilepsy. Changes to rank ordering are explained by patients’ stronger preferences for reducing the risk of AEs than for improving treatment benefit. In return for a 1% im- provement in 12-month remission, the maximum acceptable risk of adverse events was: depression 0.31%, memory problems 0.30%, ag- gression 0.25%. The maximum acceptable risk of adverse event in ex- change for a 1% improvement in 12-remission was, for women with the potential to become pregnant was: depression 0.56%, memory problems 0.34%, and foetal abnormality 0.20%. Conclusions Background Drug choices for given therapeutic indications are often guided by clinical trial evidence, however, patients may consider outcomes beyond those measured as primary endpoints within trials in their decision to adhere to medication. Discrete choice experiments (DCEs) are a valid method that has been used to quantify patient prefer- ences for drug outcomes. Data from DCEs may be combined with the results of clinical trials to provide a more patient-orientated per- spective on drug choice. Correspondence: Leslie Carroll Trials 2017, 18(Suppl 1):O17 Correspondence: Leslie Carroll Trials 2017, 18(Suppl 1):O17 Introduction The National Heart, Lung, and Blood Institute (NHLBI) established the Biologic Specimen and Data Repositories Information Coordinating Center (BioLINCC) www.biolincc.nhlbi.nih.gov in 2008 to provide online access to NHLBI data and biospecimen resources. To assist non-study investigators’ use of the datasets, each study’s BioLINCC webpage provides information on the study design and results, including docu- ments that provide insight into the study data. Given the recent inter- est by journal editors in the rapid release of publication data, the need for efficient curation methods is becoming more important. The proce- dures that have been developed by BioLINCC to review and prepare study datasets and documents for sharing with secondary users are one example of how this can be accomplished. p g Objective p Methods Data packages submitted to BioLINCC undergo review for secondary usability. Data dictionaries are examined for ease of use by researchers outside of the original study group. Reviews are performed to find any data elements that are considered personally identifiable information (PII) which are then redacted or recoded in order to de-identify the data for distribution. An informed consent questionnaire is completed to discern if there are any restrictions related to wide data sharing. A comparison of the data with a publication representative of the study as a whole, such as a primary outcome manuscript, is conducted. The population included in the analysis as well as key statistics are repro- duced and deviations identified. Key variables used in the analysis (e.g. inclusion criteria, adjudicated variables, outcomes) are noted and the documentation is examined to ensure these variables are well anno- tated. If study biospecimens are being transferred to the NHLBI Biorepository, the link between clinical data and those specimens is verified. Additional documentation including the study protocol, in- formed consent templates, MOP/MOOs, annotated forms, codebooks, and a publications list are collected to provide a useful context for the data and biospecimens. g Results Preparing data for release to the general scientific community requires a significant commitment of time and effort to ensure investigators, not affiliated with the original study, have sufficient information to effectively con- duct secondary analyses. This abstract is not included here as it has already been published. p Objective To demonstrate the impact of incorporating patients’ benefit-risk preferences into the results of clinical trials, using a case study of preferences for anti-epileptic drugs (AEDs). Preference weights for outcomes of AEDs (12-month remission, fewer seizures, depression, memory problems, aggression, foetal abnormal- ity) were derived from a web-based DCEs of 414 adult patients with epilepsy. Rates for each of these outcomes were extracted from a large randomised controlled trial comparing the effectiveness of new and standard AEDs (SANAD), and from a systematic review of treat- ments of epilepsy in pregnancy. The preference weights were com- bined with the clinical event rates to estimate of patient utility for each AED. The probability of patients preferring each AED was then calculated as the ratio of exponentiation of the utility of each individ- ual AED to the sum of the exponentiation of the utilities of all AEDs. Results were compared to rankings of AEDs as indicated by clinical trials. O19 Patient and public involvement into the design of a paediatric surgical trial: the ninja study In trials of emergency care interventions standardised pathways for obtaining participant consent can be inappropriate and alternative models are required. Unless specific approval is granted, obtaining par- ticipant consent is an essential prerequisite to research participation. OBS2 was a randomised trial evaluating the effectiveness of using early fibrinogen replacement in the management of complex postpartum haemorrhage. Reflecting the range of potential clinical scenarios in which recruitment could potentially occur, and to meet NHS ethics committee requirements, several consent pathways were identified, each requiring tailored patient information and consent forms. Methods Cushla Cooper1, David Beard1, Abhilash Jain1, Aina Greig2, Adam Sierakowski3, Matthew Gardiner1, Nicola Farrar1, Jonathan Cook1 1 2 3 Matthew Gardiner , Nicola Farrar , Jonathan Cook 1University of Oxford; 2Guys & St Thomas’ Hospital; 3Mid-Essex Hospital Services NHS Trust Correspondence: Cushla Cooper Trials 2017, 18(Suppl 1):O19 Correspondence: Cushla Cooper Trials 2017, 18(Suppl 1):O19 O21 O21 How can incentives be designed and used to improve recruitment and retention in ways that are effective, efficient and ethical? Peter Bower1, Beth Parkinson2, Eleonora Fichera2, Rachel Meacock2, Matt Sutton2, Shaun Treweek3, Nicola Harman4, Katie Gillies3, Nicola Mills5, Gillian Shorter2 1MRC North West Hub for Trials Methodology Research; 2University of Manchester; 3University of Aberdeen; 4University of Liverpool; 5University of Bristol; 6Ulster University Correspondence: Peter Bower Trials 2017, 18(Suppl 1):O21 Due to PPI involvement, the full NINJA study objectives were modi- fied and a follow-up regime and content designed to suit this very specific patient population was developed. Our experience shows that solutions offered by children and parents can be incorporated into trial design at an early stage. The PPI exercise helped address and titrate issues raised in a pilot study and generated design and procedural elements that had not previously been discussed. O20 Consent pathway options in trials of emergency interventions Julia Sanders1, Peter Collins2, Julia Townson3, Nadine Aawar3 1Cardiff University; 2Cardiff University, School of Medicine; 3Cardiff University, Centre for Trials Research Correspondence: Julia Sanders Trials 2017, 18(Suppl 1):O20 O20 Consent pathway options in trials of emergency interventions Julia Sanders1, Peter Collins2, Julia Townson3, Nadine Aawar3 1Cardiff University; 2Cardiff University, School of Medicine; 3Cardiff University, Centre for Trials Research Correspondence: Julia Sanders Trials 2017, 18(Suppl 1):O20 Results Over the first seven years of BioLINCC, data from 139 completed stud- ies were made available through BioLINCC and 666 requests for 1496 data packages were fulfilled. A total of 130 original data packages and updates were processed and shared with an average effort of 75 hours per data package. The level of effort varied, not according to the complexity of the study design, but due to the stage of curation of the submitted data and documentation. Additional effort at both BioLINCC and the parent study’s coordinating center was required in nearly all re- views to prepare and obtain missing information such as algorithms for calculated analysis variables, explanatory data labels, code books, key DCEs represent a robust method for quantifying benefit-risk prefer- ences that can be analysed alongside clinical trial data, to provide a patient-orientated perspective on the optimal choice of treatment. Trials 2017, 18(Suppl 1):200 Page 193 of 235 Background O19 Patient and public involvement into the design of a paediatric surgical trial: the ninja study Cushla Cooper1, David Beard1, Abhilash Jain1, Aina Greig2, Adam Sierakowski3, Matthew Gardiner1, Nicola Farrar1, Jonathan Cook1 1University of Oxford; 2Guys & St Thomas’ Hospital; 3Mid-Essex Hospital Services NHS Trust Correspondence: Cushla Cooper Trials 2017, 18(Suppl 1):O19 Background Patient and Public Involvement (PPI) is increasingly important in the design of research and surgical trials. Evidence on PPI for paediatric trials appears is limited. Using a case study, the NINJA study, the po- tential impact of PPI on the design of a new paediatric surgical trial is highlighted. All women booked to give birth at the six participating maternity units during the recruitment period, were provided with written in- formation about the study during the antenatal period. Five consent pathways were developed: (1) for women at higher risk of postpar- tum haemorrhage, pre-event consent; (2) for women with controlled haemorrhage, written consent at the time of the bleed; (3) for women competent to provide assent during the bleed, but unable to provide written consent, verbal assent at the time of the bleed; and in the event of women lacking capacity to provide assent, pathways utilising a personal (4) or professional representatives (5). All women, regardless of the pathway followed, once well enough following their bleed were also required to provide written consent for use of their collected data. NINJA study: Nail bed injury is the commonest hand-related cause of emergency paediatric consultation. After nailbed repair, there is de- bate whether the nail should be replaced or not. There is controversy and uncertainty around whether replacing the nail is beneficial in causing or preventing infection. A 60 patient pilot study (NINJA-P) was conducted to demonstrate the viability of a large multicentre paediatric surgical trial comparing infection rates in patients with re- placed and discarded fingernails. Patients were recruited in just over 4 months at 4 sites and followed-up for 4 months. The paediatric population created some unique aspects and challenges, especially with retention and completion of patient-reported assessments. Methods The issues raised by the pilot were put to a youth group - the Young Person’s Executive (YiPpEe) group based at Oxford University Hospitals NHS Foundation Trust and also to a focus group of parents (and one toddler). Information from both groups was collated to inform the de- velopment of the definitive study. The issues discussed were: Choice of the primary outcome measure and how to administer this; Retention of this study population; Presentation of study information. Results Regarding the outcome, the appearance of the nail was overwhelm- ingly the most important variable. This was in contrast to the clinicians’ choice of outcome; the incidence of infection. The NINJA study now has co-primary outcome measures of appearance and infection rates. The groups shared ideas for how children (or parents) could measure their satisfaction with their nail appearance. A simple 3 point scale showing facial expressions was developed. This was favoured over the 5 point scale used in the pilot. Both groups were clear about the method of collection for follow up data. This population includes busy working parents. They suggested moving away from postal question- naires and clinical visits, if not necessary, and employing mobile tech- nology i.e.: ‘apps’ to upload photographs and complete questionnaires. The parent group felt the option to complete follow up requirements in this manner would improve the retention rate. Both groups had spe- cific ideas regarding patient information presentation. The use of tech- nology, videos, and comic-strips showing real people was supported. Collaboration with YiPpEe will continue to help develop information portals for the study. p Conclusions Conclusions Discussion Appropriate recruitment and consent pathways are an essential com- ponent in the design of all trials. Trials of emergency intrapartum care bring particular challenges as they combine a known population, women booked to give birth at participating units, with unknown eli- gible potential participants, in the case of OBS2, women who went on to have a postpartum haemorrhage of >1,000 ml. The requirement to provide all potentially eligible women with antenatal information was intensive of professional time and resources. The pathways of consent available to staff in the recruitment of women were identified to have strengths and weaknesses, and these were reflected in the utilisation of each. Based on the experience of the OBS2 trial, the legal and logistic complexities of consent in emergency trial settings will be presented and discussed. Results The study recruited 663 women who experienced a moderate to se- vere postpartum haemorrhage, with a minimum of 1,000 ml blood loss. Data relating to the mode of consent were captured on the site screening logs for 511 participants. No participants were recruited using the pathways developed for written consent provided at the time of the bleed, nor for the pathway utilising a professional legal representative. Antenatal (pre-bleed) consent was obtained from 15 (2.9%) recruited women; verbal assent was provided by 473 women (92.5%) during the haemorrhage, and for 23 women (4.5%) assent was provided by a personal representative, a relative or friend present. All women, once well enough following their bleed, provided written consent to the use of collected data. Methods The review was underpinned by a conceptual framework drawn from microeconomics, agency theory and behavioural economics to help us determine which elements of incentive design to consider. We also explored potential intended and unintended effects of incentives. We will describe the key considerations that influenced the design of the DSMS, whilst focussing on scalability, as the DSMS was intended to be a cost-effective solution that could be used by a number of tri- als within the unit. We also ran interactive sessions with experts in the field (trials and be- havioural economics), principal investigators, regulatory representatives, and patients, to better understand stakeholder views. The key activity was building a forecasting-and-site-refill algorithm to minimise drug wastage whilst optimising shipment quantities, there- fore reducing shipping costs. To achieve the required scalability, the first consideration was introduction of flexibility into the algorithm. It was developed with many inputs/parameters to cater for variation across future trials and sites in terms of number of patients, recruitment rates, site capacity and location. Each shipment request is reviewed and approved by the trial team, which further allows the process to be fine-tuned for each study. Synthesising these two forms of data, we developed guidance for the design and delivery of incentives in trial recruitment and retention. Results We searched PubMed and Econlit, securing 963 eligible studies, of which 123 were included. Some of the core recommendations from the review are as follows: the review are as follows: 1. When designing an incentive system it is vital to consider the current incentives already operating Integration with internal and external contract research organization (CRO) systems was fundamental to building a successful system. The DSMS is a web-based system (C#/MVC.NET/SQL Server database) and has been integrated with an existing randomisation system and study database. The system allows for pack/kit numbers to be used in blinded trials, therefore integration with CRO systems is vital to an efficient pack selection process for site shipments. 2. The evidence is mixed about who incentives should be directed towards (patients, recruiters, clinicians or a combination) 3. Incentivising processes (such as invitations to a trial) is likely to in- duce more effort than incentivising outcomes (e.g. recruitment and retention). Scaling the drug supply management mountain: a case study of the add-aspirin trial 2 2 2 2 the add-aspirin trial Kenneth Babigumira1,2, Nancy Tappenden1,2, Fay Cafferty1,2, Marta Campos1,2, Carlos Diaz-Montana1,2, Keith Fairbrother1,2, Samuel Rowley1,2, Mary Rauchenberger1,2, Ruth E. Langley1,2 1MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL, London, UK; 2MRC London Hub for Trials Methodology Research, London, UK Correspondence: Kenneth Babigumira T l (S l ) O Background Increasingly drugs are being developed with consideration to a bio- marker defining a sub-population where the drug will demonstrate increased clinical benefit. However, frequently the prior evidence of the necessity of the marker isn’t overwhelming or the exact defin- ition of the biomarker cannot be specified in advance either in terms of a cut-off and/or the optimal assay or property of the biomarker that will be used to define the sub-population. In these cases a study will typically be run in an unselected population, and the analysis performed in both the whole study population as well as biomarker defined sub-populations. In these situations, although many separate biomarker hypotheses may be tested, it is desired to maintain an overall type-1 error rate control for testing the single hypothesis that the drug has an effect within a patient population. p Kenneth Babigumira1,2, Nancy Tappenden1,2, Fay Cafferty1,2, Marta Campos1,2, Carlos Diaz-Montana1,2, Keith Fairbrother1,2, Samuel Rowley1,2, Mary Rauchenberger1,2, Ruth E. Langley1,2 1 gy Correspondence: Kenneth Babigumira Trials 2017, 18(Suppl 1):O22 Conclusion In the Add-Aspirin trial to date, over 1,500 participants from 128 UK sites have been dispensed 3,000 treatment packs using the DSMS. The number of shipments created so far is in line with what was pro- jected using simulation before the start of the trial. The shipments have been optimised for low, medium and high recruiting sites. The DSMS is a platform that continues to evolve as new functionality is required for the Add-Aspirin trial and other trials within the MRC CTU at UCL portfolio. We will present our findings in full, and explore incentives schemes which illustrate different features, advantages and disadvantages. There is a need to consider the role of incentives in enhancing re- cruitment and retention, taking account of the ethical issues and thinking creatively about design to maximise benefit and minimise harm. Equally, there is a need to test their effectiveness and effi- ciency using appropriate randomised and non-randomised methods to ensure that any systems are a good use of public funds. Decision making in the face of biomarker uncertainty Chris Harbron Roche Trials 2017, 18(Suppl 1):O23 Background Recruitment and retention is critical for trials, yet both remain sig- nificant problems. Very little evidence exists on effective methods to boost recruitment and retention. There is increasing interest in exploring financial and non-financial incentives. Trials 2017, 18(Suppl 1):200 Page 194 of 235 We asked the question: “How can incentives best be designed and used to improve recruitment and retention in ways that are effective, efficient and ethical?” MS Access databases). However, there are challenges integrating these systems with internal study databases to work seamlessly. Add-Aspirin is a phase III double-blind, randomised trial with over 180 sites in the UK, aiming to recruit 10,000 patients. The size of the trial and the use of double-blinded drug as part of the trial design led to a decision to develop an in-house DSMS. M h d We conducted a structured scoping review to explore the current lit- erature on the use of incentives in health care (inside and outside of trials). Methods However, there is a danger that changes in process do not translate to increased recruitment and retention, or lower the overall suitability of recruits Another important consideration to facilitate scalability was ensuring ease of setting up new trials and studies, which has been enabled by how the DSMS integrates with internal systems which house both trial and site data. In addition to this, a metadata template was cre- ated to expedite the process of gathering new requirements from new trials. This has been successfully implemented for Add-Aspirin and its use has already been extended to the FOCUS4 biomarker- stratified platform trial. 4. Complex payment schemes can better direct incentives to increased activity, limiting costs. However, they will take more time and effort to implement, and may fail to induce increased effort 5. Monetary incentives are likely to have a larger direct price effect, but may have negative psychological effects (e.g. crowding out altruism) 5. Monetary incentives are likely to have a larger direct price effect, but may have negative psychological effects (e.g. crowding out altruism) 6. Other unintended consequences of incentives may include effects on the types of patients recruited and research integrity Finally another important component was the ease of use, supported by the provision and implementation of system training. Training has been developed using different formats, webinars, user guide, slides and videos. The training materials are available from within the system and on the trial website; they can easily be adapted by new trials. Conclusion The impact of incentives will be influenced by many features, such as the setting of the trial, the risk inherent in trial procedures, and the social and demographic characteristics of patients. Patients dis- cussed the importance of the language used in offering incentives, and the impact on the professional-patient relationship. Patients re- ported less concerns over the use of incentives for retention com- pared to recruitment. Background Managing drug supply for large clinical trials presents significant logis- tical challenges. The MRC CTU at UCL has used either an external drug supply management system (DSMS) or in-house tools (spreadsheets or Trials 2017, 18(Suppl 1):200 Page 195 of 235 Page 195 of 235 Methods 18% (a 6% increase) based on the 358 participants who had reached the 8 week time point by October 2016. Completion of the sub-study later in 2016 will reveal how effective phone reminders were com- pared to SMS reminders. The cost of an SMS reminder is approxi- mately $0.18AUD with negligible staff time required per person reminded. The cost of a phone reminder is approximately $0.48AUD with an average of 4 minutes of staff time required per person reminded. Spiessens-Dubois (2010) provide an approach for controlling overall type-1 error rate when all biomarker hypotheses are nested by consid- ering the correlation between different tests using an analogous approach to group sequential analysis. This describes the situation of a single biomarker being investigated at multiple cut-offs. In this presen- tation, this is extended to the more general case of non-nested tests representing the situation of multiple biomarkers which may be corre- lated but not ordered, still using the intrinsic correlation from overlap- ping populations to construct an efficient test. This solution generates sets of significance boundaries all maintaining an overall type-1 error rate which can be optimized according to a variety of different optimal- ity criteria based upon different characteristics of the study including functions of power, effect size and significance levels. R lt g Aim In this sub-study we aimed to assess the impact of phone and SMS (text message) reminders on lab screening rates while also maximiz- ing lab screening uptake in the lead up to recruitment close in December 2016. O24 Using phone, SMS and email screening reminders to improve clinical trial recruitment: results from a sub-study of the t4dm diabetes prevention study Karen Bracken1, Wendy Hague1, Gary Wittert2, Anthony Keech1, Kristy Robledo1 1NHMRC Clinical Trials Centre, University of Sydney; 2University of Adelaide Correspondence: Karen Bracken Trials 2017, 18(Suppl 1):O24 There has been a surge in designing clinical trials based on the as- sumption that a biomarker is predictive of treatment response. Pa- tients are stratified by their biomarker signature, and one tests the null hypothesis of no treatment effect in either the full population or the targeted subgroup. However, in order to directly verify the pre- dictability of a biomarker, it is essential that hypothesis be tested in the non-targeted subgroup too and within a randomised controlled trial [1]. In a Phase IIB oncology trial with progression free survival (PFS) endpoint, the data obtained can inform the Phase III design aimed at establishing overall survival whether to restrict recruitment to just the targeted subgroup or not. Correspondence: Karen Bracken Trials 2017, 18(Suppl 1):O24 Conclusion This sub-study will establish the extent to which phone and SMS screening reminder strategies increase participant follow-through with the screening process. It will also assess the relative costs of each approach in terms of cost per notification, cost per participant screened and cost per participant enrolled. These findings have the potential to inform the choice of screening reminder strategy in future prevention clinical trials. We present and compare the results of optimising the significance boundaries of a study using different optimality criteria and link this to the properties of the biomarkers being investigated. We give guid- ance as to how this approach may be implemented in practice and the beneficial discussions within clinical teams that adopting these approaches will facilitate. O25 Adaptive enrichment design for randomised clinical trials with predictive biomarkers Deepak Parashar, Iliana Peneva, Nigel Stallard University of Warwick Correspondence: Deepak Parashar Trials 2017, 18(Suppl 1):O25 Method [1] Mehta C, Schafer H, Daniel H, Irle S. Biomarker driven population enrichment for adaptive oncology trials with time to event outcomes. Statist. Med 2014; 33: 4515–4531. Between June and October 2016, 709 participants who did not at- tend lab screening within 4 weeks of online registration were ran- domized to receive either an SMS or a phone reminder to attend for lab screening. This was in addition to an automated email reminder that all registered participants receive at 4 weeks. Participants were followed to determine whether they attended lab screening by 8 and 12 week time points. The sub-study completed enrollment in October 2016 with all data collection to be complete by the end of 2016. The quality of reporting of pilot and feasibility cluster randomised trials: a systematic review 1 2 1 Claire Chan1, Leyrat Clémence2, Eldridge M. Sandra1 1Queen Mary University of London; 2London School of Hygiene and Tropical Medicine Preliminary results O25 Adaptive enrichment design for randomised clinical trials with predictive biomarkers Deepak Parashar, Iliana Peneva, Nigel Stallard University of Warwick Correspondence: Deepak Parashar Trials 2017, 18(Suppl 1):O25 Background Successful and timely participant recruitment is a key aspect of clinical trial conduct. Failure of potential participants to complete screening is often reported as an issue in prevention studies. The T4DM diabetes prevention study, being conducted at 6 sites around Australia, employs a step-wise screening process. Potential participants first complete an online study registration questionnaire and, if eligible, the online sys- tem generates consent and laboratory forms. Participants are then re- quired to attend for lab screening before being allocated to the nearest study site. After the first 12 months, only 58% of men who registered online had attended the lab for screening. Given the cost and difficulty associated with attracting men to register, a quarterly email reminder and ad-hoc phone reminders were introduced to improve uptake of lab screening. After a further 24 months, we observed that the number of men attending for screening late (more than 12 weeks after registra- tion) had grown but the overall lab screening rate remained largely un- changed at 60%. We propose a new two-stage adaptive randomised Phase II popula- tion enrichment trial design, with PFS as the primary endpoint and comparing an experimental drug with a control treatment. We adap- tively test the null hypotheses of hazard ratios in both the targeted as well as the non-targeted subgroups, with strong control of the familywise error rate. It is assumed that the hazard ratio of the tar- geted subgroup is much less than that of non-targeted, since the drug is expected to be more beneficial for the biomarker-positive subpopulation. Simulations for an example trial in non-small cell lung cancer show that the probability of recommending an enriched Phase III trial in- creases significantly with the hazard ratio in the non-targeted sub- group. We compare our decision rules with [1] and illustrate the efficiency achieved. Our adaptive design testing first in the non- targeted subgroup followed by testing in the targeted subgroup for a randomised controlled trial constitutes part of the proof of a bio- marker’s predictability. Preliminary results y Prior to the introduction of the reminder sub-study, only 12% of men who didn’t attend lab screening within 4 weeks had done so by 8 weeks. To date, the introduction of reminders has increased this to p Correspondence: Claire Chan Trials 2017, 18(Suppl 1):O26 Page 196 of 235 Trials 2017, 18(Suppl 1):200 Page 196 of 235 Treatment Clinical Trials Network (CTN) Clinical Coordinating Center (CCC) and Data and Statistics Center (DSC), both at the Emmes Cor- poration, collaboratively developed a series of processes and tools, some of which are incorporated in the electronic data system, to ensure an efficient and controlled chain of custody and process be- ginning from initial supply distribution through dispensing proce- dures at the research sites and final reconciliation and destruction. The CCC and DSC consider several factors when determining the process for study drug management, including treatment blinding, drug type, quantity and packaging, frequency of distribution, expir- ation dating, and the number of sites. Based on these parameters, the CCC assists the study teams in development of clear and thor- ough drug management logs as well as defining drug storage and temperature monitoring requirements. To remedy last minute re- quests, supply hoarding, and waste at the sites, the coordinating centers have developed a centralized inventory tracking and reor- dering process to monitor drug supply and distribution. In this process, research staff report inventory weekly directly in the Elec- tronic Data Capture (EDC) system, and the data is pulled into re- ports, which identify reorder needs based on thresholds and usage. Before shipping initial supplies to each site, all regulatory docu- ments are collected and training is provided to research sites on the importance of drug accountability and consequences for par- ticipant safety if inaccurately reporting drug dosing and disposal. Site monitors review the drug logs, medication storage, and regula- tory documentation throughout the trial (remotely or on-site) in order to identify and resolve any improper practices, discrepancies and errors. The Emmes Corporation has supported substance use treatment interventions implemented in the CTN for over 11 years, and throughout that time have developed best practices including using systematic, clear and precise processes for study drug pro- curement, distribution, and monitoring. Over 14 clinical trials across 105 clinical sites have involved study drug, including 4 double- blinded studies and 4 Investigational New Drugs and 6 studies using controlled substances. O27 O27 Best practices for study drug management and accountability throughout the study lifecycle in multi-site randomized controlled trials Dikla Blumberg1, Patricia Novo2, Beth Jeffries1, Lauren Yesko1, Abigail G. Matthews1, Julia Collins1, Dagmar Salazar1, Eve Jelstrom1, Matthew Wright1, Radhika Kondapaka1 1The Emmes Corporation; 2NYU School of Medicine Correspondence: Dikla Blumberg Trials 2017, 18(Suppl 1):O27 Preliminary results Effective communication between the CCC/DSC, central pharmacy, third-party vendors, research sites, and all other stakeholders allows for efficient planning and prompt resolution to problems that arise. Supporting this communication with real time data collection and reporting allows for the proper maintenance of a comprehensive and accurate study drug manage- ment system. This presentation will emphasize best practices for achieving an organized and controlled chain of custody throughout the life of a trial. g Results Eighteen pilot CRTs were identified, with most (56%) published in the UK. 44% did not have feasibility as their primary objective, and many performed formal hypothesis testing for effectiveness/efficacy despite being underpowered (50%). Most pilot CRTs (83%) reported the term “pilot” or “feasibility” in the title, and discussed implications for progression from the pilot to the future definitive trial (89%), but less than half gave reasons for the randomised pilot trial (39%), re- ported a rationale for the sample size (44%), reported criteria used to judge whether or how to proceed with the future definitive trial (17%), or reported where the pilot trial protocol could be accessed (39%). Most pilot CRTs defined the cluster (100%), and reported the number of clusters randomised (94%) and assessed for the primary objective (82%). Items reported least well included how clusters were consented (11%), the cluster design during the description of the ra- tionale for numbers in the pilot (17%), who enrolled clusters (17%), the number of exclusions for clusters after randomisation (18%), a table showing baseline characteristics for the cluster level (11%), and from whom consent was sought (11%). Background There are an increasing number of studies described as pilot and feasibility studies. A pilot or feasibility trial conducted in advance of a future definitive trial is a study where part or all of a future trial is carried out on a smaller scale to see whether it can be done and whether we should proceed with it. Reporting of pilot and feasibility studies is poor, and these studies are particularly important when de- signing cluster randomised trials (CRTs), which bring with them extra complications. p Objectives T i To systematically review the quality of reporting of pilot and feasibility CRTs. In particular, to identify 1) The number of pilot CRTs conducted between 01/01/2011 and 31/12/2014, 2) Whether pilot CRTs have ap- propriate objectives and methods, and 3) The extent to which the qual- ity of reporting of pilot CRTs is sufficient. y p Methods We searched PubMed (2011–2014) for CRTs with “pilot” or “feasibility” in the title/abstract, that were assessing some element of feasibility and showing evidence the study was in preparation for a main effectiveness trial. Quality assessment criteria were based on the CONSORT extension for CRTs, and the CONSORT extension for pilot trials which was in the final stages of development. Conclusions The identification of just eighteen pilot CRTs highlights the need for increased awareness of the importance of carrying out and publish- ing pilot CRTs and good reporting. It is possible that some pilot CRTs were missed because they did not include “pilot” or “feasibility” in the title/abstract. Pilot CRTs should primarily be assessing feasibility, with methodology reflecting this focus. Improvement is needed in reporting reasons for the pilot, rationale for the sample size, progres- sion criteria, and where the protocol can be accessed. Cluster level items also need better reporting, since these are important for asses- sing feasibility. We recommend adherence to the new CONSORT ex- tension for pilot trials, in conjunction with continued adherence to the CONSORT extension for CRTs. O28 A look at the future of data standardization and sharing in clinical research Derk Arts1, River Wong2, Nidal Amenchar2 1Department of medical informatics, Academic Medical Centre (AMC), Amsterdam; 2Castor Electronic Data Capture Correspondence: Derk Arts Trials 2017, 18(Suppl 1):O28 Correspondence: Derk Arts Trials 2017, 18(Suppl 1):O28 Sharing collected data from trials has the potential to exponentially increase the efficiency and accuracy of research and reduce research waste through repeated trials. Unfortunately, barriers to do so still exist. These include the difficulty to find, access and use previously collected research data sets because they are not centrally indexed or standardized. Best practices for study drug management and accountability throughout the study lifecycle in multi-site randomized controlled trials The European Commission unveiled its plans in April earlier this year to create a new European Open Science Cloud that will offer Europe's 1.7 million researchers and 70 million science and technol- ogy professionals a virtual environment to store, share and reuse their data across disciplines and borders. The aim is to make all data derived from EU-funded research projects Findable, Accessible, Interoperable and Reusable (FAIR). The European Union has fully embraced the FAIR principles, which are created to ensure high data quality, shareability, and usability. Dikla Blumberg1, Patricia Novo2, Beth Jeffries1, Lauren Yesko1, Abigail G. Matthews1, Julia Collins1, Dagmar Salazar1, Eve Jelstrom1, Matthew Wright1, Radhika Kondapaka1 1The Emmes Corporation; 2NYU School of Medicine Correspondence: Dikla Blumberg Trials 2017, 18(Suppl 1):O27 Managing study drug throughout a trial is a complex, vital task further compounded when there are multiple research sites participating. Adherence to good clinical practice (GCP) requirements and all applic- able regulatory requirements is paramount. The National Drug Abuse We will discuss the benefits of FAIR data, explain what is required for FAIR data, and give guiding principles on how to create FAIR data. Trials 2017, 18(Suppl 1):200 Page 197 of 235 We will also go further into the challenges that we face as we move towards the worldwide implementation of FAIR. Conclusions Our experiences of participant involvement have demonstrated that trial participants can add insight to the studies they are involved in. Participant involvement in clinical trials is feasible and seems to offer significant benefits in some circumstances. We recommend that current INVOLVE guidance on PPI should be updated to include participant in- volvement as a valid and potentially useful approach to PPI. Participant involvement can complement other forms of PPI in clinical trials in ap- propriate circumstances. We are developing plans and strategies to fur- ther explore its potential. These challenges include: Ensuring all research data is of high quality Standardization of research data at the source Provide everyone with the ability to make FAIR data (FAIRification) We will discuss how to deal with these challenges and present our solution to make capturing FAIR data accessible for every researcher worldwide. By making these data available in environments like the European Open Science Cloud, the world will experience a major increase in the quality and efficiency of research. This in turn will help to improve healthcare in the long run, by ensuring better qual- ity of evidence to base our medical guidelines on. O30 Administering patient-reported outcome questionnaires in Australian cancer trials: the roles, experiences, training received and needs of site coordinators Rebecca Mercieca-Bebber1, Derek Kyte2, Melanie Calvert2, Martin Stockler1, Madeleine King1 1The University of Sydney; 2University of Birmingham Correspondence: Rebecca Mercieca-Bebber Trials 2017, 18(Suppl 1):O30 Administering patient-reported outcome questionnaires in Australian cancer trials: the roles, experiences, training received and needs of site coordinators Background Background In clinical trials, patient-reported outcome (PRO) questionnaires offer information about the impact of disease and treatment from the patients’ perspective. The ‘Clinical Research Coordinator (CRC)’ is typically responsible for PRO data collection. Recent evidence suggests CRCs are not offered adequate PRO-specific trial guidance. As PROs are increasingly being valued in the interpretation of can- cer trials, the need to scrutinise current practice has become ever more important. The present study explored the experiences of Australian CRCs responsible for PRO assessment in cancer trials. Methods Correspondence: Claire Vale Trials 2017, 18(Suppl 1):O29 Results Twenty participants (19 female) were interviewed (mean 9.3 years’ experience) with professional training in nursing (n = 12), science/re- search (n = 4) or both (n = 4)). Participants worked in medical oncology (n = 10), haematology (n = 5), radiotherapy (n = 4), and endocrinology (n = 1) departments. Skills and responsibilities: All CRCs described organisational and communication skills, the ability to multi-task and work around patient needs. Differences included whether CRCs explained the purpose of PRO assessments to patients, which may result in bias if patients alter their responses if patients believe it will impact their care. There were also differences in assist- ance provided to patients; some CRCs read questions aloud and re- corded patient responses, some paraphrased questions, others excluded patients who could not independently self-complete. This may lead to bias as a result of missing data from sicker patients, or differences in explanations of question meaning. Some CRCs pursued responses for accidentally missed questions; potentially leading to differences in data quality across sites. Some CRCs checked for con- cerning data or general outcome profile, whereas others felt ques- tionnaires should be kept confidential and not checked, which may lead to bias if these CRCs adapted procedures of care in response to PRO data. Challenges: CRCs described challenges with electronic PRO assessment, non-English-speaking patients, dealing with patients’ rel- atives who inappropriately attempted to complete questionnaires, and patient unwillingness to complete questionnaires. Inconsisten- cies in data collection and the nature of challenges experienced supports the need for increased PRO-specific training. Training: PRO- specific training received varied considerably; ranging from dedi- cated PRO training (study-specific or general); PROs being addressed Australian Methods Cancer trial CRCs at approved Australian sites with 12+ months PRO experience were eligible. Interested CRCs provided informed consent. Semi-structured interviews were audio-recorded and transcribed ver- batim. Interviewees discussed their PRO-specific skills, responsibilities, challenges, procedures, PRO training received and training needs. Re- cruitment continued until data saturation. Transcripts underwent content analysis; codes were applied to organise interview content inductively and deductively by RMB and 20% were checked by DK. The study team agreed on the final code structure. p Methods Two workshops were held at the MRC CTU at UCL to discuss: defini- tions; rationale; potential advantages and disadvantages; models; and appropriateness of participant involvement in clinical trials. We considered how participant involvement might overlap with, or differ to, involvement of other patients and the public. Workshops were attended by two patient representatives and seven staff members, each of whom has experience of PPI. Staff members from studies that had actively involved participants shared details of that work to inform discussions. Participant involvement as a form of patient and public involvement in clinical trials: experience, reflections and recommendations Claire Vale, William Cragg, Ben Cromarty, Bec Hanley, Annabelle South Richard Stephens, Kate Sturgeon, Mitzy Gafos MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL, and MRC London Hub for Trials Methodology Research, London, UK Background P d Patient and public involvement (PPI) in clinical trials describes a variety of activities ensuring that research is carried out collabora- tively with patients and/or members of the public. Traditionally, the patients and public involved have not been taking part in the study in question and in the UK, guidance from INVOLVE suggests that it is not appropriate to involve clinical trial participants in PPI activities. However, as part of a study exploring PPI in randomised controlled trials conducted by the MRC CTU at UCL, we identified 3 studies (2 trials and 1 cohort study) where participants had been in- volved. In the light of this we reviewed the concept of participant involvement, setting out to develop guidance based on our experience. Background Correspondence: Patricia Healy Trials 2017, 18(Suppl 1):O32 g The Clinical Data Interchange Standards Consortium (CDISC) data standards for clinical trials (http://www.cdisc.org/) are widely used in the pharmaceutical industry and are now mandatory for FDA submis- sions of studies started from December 2016. Relatively few trials run by academic groups are submitted to regulators for marketing authorisation, and the adoption of CDISC standards by them is sub- stantially lower than in industry. We present the advantages and dis- advantages of using CDISC standards in the light of the experience of the CTSU in the University of Oxford across multiple phase II to IV trials ranging from 400 to 30,000 participants. Experience f CTSU first used CDISC when providing Study Data Tabulation Model (SDTM) data for an FDA submission after the main analyses had been developed without using CDISC. The resulting discrepancies between CDISC and non-CDISC analyses took considerable effort to resolve. In subsequent studies, whether or not regulatory submissions are planned, a more systematic approach has been used. Collected data is mapped to SDTM datasets, from which Analysis Data Model (ADaM) datasets are derived. All analyses and complex reports, both during and at the end of the trial, are performed on these ADaM datasets. This approach has been successfully employed for a num- ber of large trials at different stages (completed, currently undergo- ing analysis and ongoing), which together have randomized over 70,000 participants, and more recently to partially convert some leg- acy studies to CDISC for particular analyses. Objectives To identify unanswered questions around trial recruitment, and then prioritise those that stakeholder groups including members of the pub- lic, recruiting clinicians and researchers, agree are the most important. Background The PRioRiTy study - Priority Setting Partnership (PSP) included stake- holders involved in all aspects of clinical trial recruitment; members of the public approached to take part in a randomised trial or who have sat on randomised trial steering committees, health profes- sionals and research staff with experience of recruiting to rando- mised trials, people who have designed, conducted, analysed or reported on randomised trials and people with experience of rando- mised trial methodology. O32 Prioritising recruitment in randomised trials: the priority study- an Ireland and UK priority setting partnership 1 2 3 4 O31 Why use cdisc for trials not submitted to regulators? Lessons from the experience of an academic clinical trial unit Karl Wallendszus, William Stevens, Martin Landray University of Oxford Correspondence: Karl Wallendszus Trials 2017, 18(Suppl 1):O31 y Benefits (1) CDISC standards eliminate the need for an organisation to de- velop its own data standards, and, since they are developed by a wide range of stakeholders over many years, are more comprehen- sive and coherent than any single organisation is likely to achieve. (2) Analysis and validation tools are available which support CDISC standards. (3) CDISC-compliant datasets are well documented, so statisticians, data analysts and researchers can easily switch between studies without having to learn a new data schema. (4) The effort re- quired to respond to queries about CDISC-compliant data is consist- ently less than that for non-compliant data. (5) CDISC standards provide a useful common framework for data sharing and long term data and metadata storage. (6) The large CDISC user community is a valuable source of support. A total of 1,880 questions were extracted from 790 survey respondents, which after merging duplicate questions, was reduced to 496 ques- tions. Merging appropriate questions together and excluding questions asked by fewer than 15 people and/or fewer than 6 of the 7 stake- holder groups resulted in 31 unique research questions. All questions were retained after confirming a lack of relevant, up to date research evidence addressing the question. Methods This partnership involved eight key stages: (i) formulating the PSP idea and identifying a unique, relevant prioritisation area within clin- ical trial methodology (ii) establishing an oversight Steering Group (iii) identifying and engaging with partners and stakeholders (iv) for- mulating an initial list of uncertainties from a stakeholder survey (v) collating the uncertainties into research question format (vi) checking existing research evidence to confirm that the questions are a current recruitment challenge (vii) shortlisting questions in an interim priority setting exercise through another survey of stakeholders and (viii) final prioritisation through a face to face workshop with stake- holders to agree a top 10 list of priorities of methodological uncer- tainties around trial recruitment. Both surveys were open to all stakeholders and were disseminated through national clinical trial re- search networks, patient groups, funding bodies and other relevant stakeholder channels including social media and direct emails. Results Results l Trial participants were defined as individuals taking part in the study in question, irrespective of whether or not they have completed their trial treatment and follow-up. Their direct experience of taking part in the trial may be especially useful in studies of new interventions or procedures, where they may be the only people who have experi- ence of the interventions, or where it is hard to identify patient or community groups that include or speak for the study population, for example in prevention trials. Participant involvement is possible at all stages of a trial, except identifying the research question and trial design (when, there are no participants to involve). Participants can be involved in trials through a range of models, with managerial, oversight or responsive roles (as for PPI). The only specific role identi- fied as being inappropriate for trial participants was involvement in data safety and monitoring committees, because of the likelihood of obtaining information about the arm of the trial they are in and the potential for unblinding. Involvement of participants can benefit tri- als by improving the trial experience for participants; optimising study procedures; and improving the communication of key mes- sages and results. Specific challenges to involving participants in- cluded managing confidentiality; practicalities around payments; and ethical concerns around recruitment for involvement. Page 198 of 235 Trials 2017, 18(Suppl 1):200 Page 198 of 235 in good clinical practice or nursing training; informal, on-the-job training from colleagues; and no PRO training. Many agreed that additional training was needed to improve current practices. Conclusion view of the benefits which are seen later. Because of the reuse of metadata and controlled terminology, costs are lower for subsequent studies. The benefits of CDISC for data analysis and reporting are maximised when their needs are built into the systems used to capture and process the data. Differences between trials in PRO administration are expected, but the described differences between CRCs regarding communication, patient assistance and checking are concerning as they may lead to various forms of bias and poor data quality. PRO training received varied considerably between CRCs and may be a key reason for these differences. Our findings highlight the importance of providing clear, PRO-specific guidance to CRCs. Results l O32 Prioritising recruitment in randomised trials: the priority study- an Ireland and UK priority setting partnership Patricia Healy1, Sandra Galvin2, Shaun Treweek3, Caroline Whiting4, Beccy Maeso4, Paula Williamson5, Derek Stewart6, Derick Mitchell7, Joan Jordan8, Mary Clarke-Moloney9 114NIHR National Dental & Oral Health Speciality, Clinical Research Network/University of Leeds; 2HRB Trials Methodology Research Network (Ireland) ; 3TrialForge, University of Aberdeen; 4James Lind Alliance; 5University of Liverpool/MRC Trial Methodology; 65NIHR Clinical Research Network Associate Director for Patient and Public Involvement; 7Irish Platform for Patients Organisations, Science and Industry (IPPOSI) ; 8EUPATI trainee/IPPOSI; 9Health Research Institute, University of Limerick O32 Prioritising recruitment in randomised trials: the priority study- an Ireland and UK priority setting partnership Patricia Healy1, Sandra Galvin2, Shaun Treweek3, Caroline Whiting4, Beccy Maeso4, Paula Williamson5, Derek Stewart6, Derick Mitchell7, Joan Jordan8, Mary Clarke-Moloney9 114NIHR National Dental & Oral Health Speciality, Clinical Research Network/University of Leeds; 2HRB Trials Methodology Research Network (Ireland) ; 3TrialForge, University of Aberdeen; 4James Lind Alliance; 5University of Liverpool/MRC Trial Methodology; 65NIHR Clinical Research Network Associate Director for Patient and Public Involvement; 7Irish Platform for Patients Organisations, Science and Industry (IPPOSI) ; 8EUPATI trainee/IPPOSI; 9Health Research Institute, University of Limerick Objectives The Ottawa Statement, published in 2012, provides researchers and research ethics committees (RECs) with specific guidance for the eth- ical design and conduct of CRTs. Our objectives are to: (1) review crit- ically the FIRST trial controversy; (2) apply the Ottawa Statement to the FIRST trial; and (3) identify issues not adequately addressed by the Ottawa Statement, thus requiring further analysis and guidance. Results y AspECT is a phase III RCT investigating the use of aspirin and Proton Pump Inhibitors to prevent oesophageal cancer and death in Barrett’s Oesophagus patients. Trial follow-up will end in May 2017, 14 years since design and set-up began in 2003. Many challenges have been encountered and addressed by the trial team over this time. With the trial’s close, we reflect on its set-up and management, to identify lessons learned and discuss these issues in relation to future trials. Methods Objective 1: Controversy erupted following publication of the FIRST trial in New England Journal of Medicine in 2016. Critics accused the investigators of “egregious ethical and regulatory violations,” arguing that the flexible duty-hour intervention knowingly exposed residents and their patients to increased risks of serious harms. They decry the decision by Northwestern University’s REC exempting the trial from human subjects research regulation, calling it a “colossal failure” of all participating RECs. Critics also denounce the resultant consent waiver as a violation of the ethical principle of respect for persons. Defenders of the FIRST trial argue that the flexible duty-hour inter- vention did not pose a greater risk to participants, and conditions for the waiver obtained. We critically review the FIRST trial controversy, finding that commentators fail to identify the relevant ethical issues systematically. Objective 2: We examine the utility of the Ottawa Statement for CRTs of policy interventions involving healthcare pro- viders. We find that the Ottawa Statement provides much-needed clarity by identifying systematically the ethical issues common to all CRTs, including: justifying the cluster design, identifying research par- ticipants, consent, gatekeepers, benefit-harm analysis, and vulnerable participants. Objective 3: We show how the FIRST trial raises unique We conducted semi-structured interviews with researchers involved with the set-up and running of the trial at any stage of its 14 years, such as trial coordinators, trial statisticians, and clinicians. Background h l b l g The Flexibility in Duty Hour Requirements for Surgical Trainees (FIRST) trial was a pragmatic cluster randomized trial (CRT) involving 117 surgery residency programs in the United States. It evaluated non-inferiority of flexible duty-hour policies compared to standard restricted duty-hour policies with respect to surgical resident well- being and patient safety. Investigators concluded that flexible duty- hour policies were non-inferior to standard duty-hour policies. The ethics of the FIRST trial have been vehemently debated. One com- mentator describes it as “among the most unethical research studies [he has] ever seen.” Another argues that it was “not just ethical but laudable to comparatively evaluate duty-hour policies. The FIRST trial illustrates the complex ethical challenges posed by CRTs of policy in- terventions involving healthcare professionals. 1The Centre for Statistic in Medicine, University of Oxford; 2University of Oxford; 3School of Medicine, Pharmacy and Health, Durham University ; , y , y Correspondence: Gavin Reilly Trials 2017, 18(Suppl 1):O34 Correspondence: Gavin Reilly Trials 2017, 18(Suppl 1):O34 Costs Th Currently (Nov 2016), the partnership is undergoing the interim pri- oritisation process in which stakeholders are shortlisting the top 10 questions they regard as important uncertainties. The top 10 prior- ities of methodological uncertainty around trial recruitment will be agreed at a final prioritisation stakeholder workshop scheduled for 1st December 2016. Full results will be available for presentation at ICTMC 2017. The most significant cost of using CDISC standards is staff training, both technical and on the value of using CDISC. When processing data, extra effort is required to ensure compliance with the stan- dards. Where data are not collected using CDISC, a labour-intensive mapping stage is required, which is more onerous the later in the study life cycle it is done. Conclusion Discussion We find the considerable investment required for CDISC at the start of a study, particularly when CDISC is first used, to be worthwhile in Despite the global problem of inadequate recruitment to randomised trials, there is little evidence to guide researchers on decisions about Page 199 of 235 Trials 2017, 18(Suppl 1):200 Page 199 of 235 Page 199 of 235 how patients are recruited. A comprehensive, rigorous and inclusive process has been undertaken, with participation from key stake- holders, including members of the public. Priority areas of focus in trial recruitment methodology have been identified by those for whom it matters most. The Top 10 list should inform the scope and future activities of funders and researchers in the area of trial recruit- ment methodology. clinical world, and adapting to the changing processes for obtaining national regulatory and local R&D approval and multicentre trial set-up. Also, the trial management had to repeatedly react to poor quality epi- demiology claims about drug reactions or side effects, or unproven benefits. We will also discuss issues around the evolving world of meth- odology, including placebo blinding costs today compared to during trial set-up and the potential for Studies Within A Trial, an emerging re- search movement to make better use of trial data. clinical world, and adapting to the changing processes for obtaining national regulatory and local R&D approval and multicentre trial set-up. Also, the trial management had to repeatedly react to poor quality epi- demiology claims about drug reactions or side effects, or unproven benefits. O33 Valuing the effect sizes hypothesized in phase 3 trials of targeted therapies in oncology 1 2 2 2 Correspondence: Nicola Lawrence Trials 2017, 18(Suppl 1):O33 An ethical analysis of the first trial: addressing ethical challenges in pragmatic cluster randomized trials of policy interventions targeting healthcare providers Austin Horn1, Cory E. Goldstein2, Monica Taljaard3, Charles Weijer2 1Western University; 2Western University, Rotman Institute of Philosophy; 3University of Ottawa, Ottawa Hospital Research Institute Correspondence: Austin Horn Trials 2017, 18(Suppl 1):O35 This abstract is not included here as it has already been published. Background The past two decades have seen dramatic changes in clinical trial conduct and methodology. From trial regulation to data analysis, the rapid rise of randomised control trials (RCTs) has introduced many new techniques. Some methods of designing and conducting RCTs have been widely adopted, whereas other ideas have been used sparingly, despite their promise. Large-scale phase III trials are no dif- ferent. We present a case study to highlight the changes experienced in designing, setting up, and conducting a large-scale multicentre phase III trial over 14 years. Costs Th We will also discuss issues around the evolving world of meth- odology, including placebo blinding costs today compared to during trial set-up and the potential for Studies Within A Trial, an emerging re- search movement to make better use of trial data. Sponsorship O34 The changing world of clinical trials 2003–2017: a view from the aspect trial 2 3 2 Gavin Reilly1, Adelyn Wise2, Stephen Attwood3, Claire Scudder2, Sharon Love2 Gavin Reilly1, Adelyn Wise2, Stephen Attwood3, Claire Scudder2, Sharon Love2 Conclusions This project was funded by the Health Research Board (Ireland) Knowledge Exchange and Dissemination Scheme Award 2015 and was supported by the James Lind Alliance and NIHR. Many changes have occurred since the set-up of AspECT in 2003. Some of these changes have made trials more transparent and safer for the patients involved, benefiting the medical research world. However, some changes may deter and slow good research, inhibiting the emergence of new treatments. Our experiences over a 14-year phase III trial highlight the issues experienced by the trial research community and are presented to inform the design and conduct of similar future trials. O33 Valuing the effect sizes hypothesized in phase 3 trials of targeted therapies in oncology Nicola Lawrence1, Felicia Roncolato2, Andrew Martin2, Martin Stockler2 1University of Sydney; 2NHMRC Clinical Trials Centre, University of Sydney Correspondence: Nicola Lawrence Trials 2017, 18(Suppl 1):O33 Objectives The inter- view structure ensured each individual was asked to address the same key topics and also allowed them to provide their personal views of the changes in trial methodology and set-up. Thematic ana- lysis identified the major challenges experienced by the respondents. Results Interviews conducted with the current trial coordinator, current trial statistician, and a clinician involved throughout the trial’s history re- vealed the 14-year lifespan of the trial and regulation changes in this time to be the main challenge. AspECT was begun before the current clinical trials regulations were published as Statutory Instrument 2004/1031. The regulations are set to change again soon. Themes reported included the difficulty in maintaining knowledge of the trial with changing PI’s and study nurses in the hospital sites, main- taining and auditing a high quality database over the trial lifespan, handing over study roles, dealing with an ever-evolving and sceptical Interviews conducted with the current trial coordinator, current trial statistician, and a clinician involved throughout the trial’s history re- vealed the 14-year lifespan of the trial and regulation changes in this time to be the main challenge. AspECT was begun before the current clinical trials regulations were published as Statutory Instrument 2004/1031. The regulations are set to change again soon. Themes reported included the difficulty in maintaining knowledge of the trial with changing PI’s and study nurses in the hospital sites, main- taining and auditing a high quality database over the trial lifespan, handing over study roles, dealing with an ever-evolving and sceptical Trials 2017, 18(Suppl 1):200 Page 200 of 235 ethical issues not adequately addressed by the Ottawa Statement. For instance, does clinical equipoise obtain when a novel policy is compared to an existing policy that has little or no evidence-base? How should researchers and RECs conceptualize healthcare providers targeted by policy interventions? Are they obligated to participate in research? If so, what are the implications for consent? Alternatively, should healthcare providers be conceptualized as vulnerable partici- pants? A power-differential often exists between healthcare providers and their superiors, particularly when providers are trainees or em- ployees. Does this relationship undermine the validity of their con- sent? If so, what safeguards might be implemented to ensure protection of healthcare providers, while at the same time ensuring that important research proceeds both feasibly and expeditiously? Mediation analysis to explore causal mechanisms in trials of complex interventions Mediation analysis to explore causal mechanisms in trials of complex interventions Deborah DiLiberto, Charles Opondo, Diana Elbourne, Elizabeth Allen London School of Hygiene and Tropical Medicine Correspondence: Deborah DiLiberto Trials 2017, 18(Suppl 1):O36 p Deborah DiLiberto, Charles Opondo, Diana Elbourne, Elizabeth Allen London School of Hygiene and Tropical Medicine Correspondence: Deborah DiLiberto Trials 2017, 18(Suppl 1):O36 Results Results on the progress of this project, in terms of the classification of COS outcomes within the COMET database and development of a standardised outcome taxonomy for the classification of COS out- comes, will be reported, along with any conclusions drawn during discussions which took place during the ‘Outcome Classification’ ses- sion at COMET VI (November 2016). Materials and methods The PRIME intervention was designed to attract patients to seek care and to improve the quality of care, including for the diagnosis and treatment of malaria, delivered at public health centres. The complex, multi-component intervention focused on ensuring access to appro- priate treatment and diagnostic tests at health centres through a range of components to improve provider behaviour and health centre operations. Following the MRC guidance, the impact of the PRIME intervention was comprehensively evaluated including a rigor- ous outcome evaluation; a cluster Randomised Controlled Trial (cRCT) with data from community cross-sectional surveys, and a parallel mixed-methods ‘process’ study. Here we explore the use of the ‘po- tential outcomes framework’ to undertake a mediation analysis of the PRIME intervention theory of change. Background There is increasing enthusiasm for the use of mediation analysis in the secondary analysis of complex interventions with the aim of iso- lating the causal mechanisms through which an intervention pro- duces the outcome of interest. Recent guidance from the Medical Research Council (MRC) on evalu- ating complex interventions suggests that Randomised Controlled Trials (RCTs) should be complemented by process evaluations which might provide evidence about the possible causal mechanisms that produce intervention effects. Process evaluations often include the development of an intervention theory of change - a description of how the intervention inputs, change mechanisms and context are hypothesised to produce the intended outcomes. It is recommended that these intervention theories are represented and evaluated using ‘logic models’ which visually demonstrate the pathway of effect between intervention inputs and intended outcomes. Mediation frameworks are potentially useful here as they can generate tests of the logic model and hence the intervention theory of change. The traditional framework for mediation analysis applies structural equation modelling (SEM). While SEM has been valuable because of its relatively simple approach to analysing mediators, recent ad- vances in mediation theory have shown that the SEM approach has theoretical limitations which make it insufficient for more complex applications. An alternative nonparametric approach is based on the ‘potential outcomes framework’ and applies the logic of counterfac- tuals in an attempt to identify causal pathways. p // Methods g Methods The COS outcome classification project involves the extraction of all core outcomes/domains from existing COS through the COMET database and reviewing the systematic reviews of outcomes in the COMET database to determine how outcomes were classified. Exist- ing conceptual models (including The International Classification of Functioning, Disability and Health (ICF), Patient-Reported Outcome Measurement Information System (PROMIS) and the Wilson and Cleary framework) will be reviewed for suitability, with a view to- wards developing a standardised ontology for classification of re- search outcomes. O37 Standardised taxonomy for the classification of trial outcomes within core outcome sets and cochrane reviews 2 3 Susanna Dodd1, Paula R. Williamson1, Jane Blazeby2, Mike Clarke3 1University of Liverpool; 2University of Bristol; 3Queen’s University, Belfast Correspondence: Susanna Dodd Trials 2017, 18(Suppl 1):O37 Susanna Dodd1, Paula R. Williamson1, Jane Blazeby2, Mike Clarke3 1University of Liverpool; 2University of Bristol; 3Queen’s University, Belfast Correspondence: Susanna Dodd Trials 2017, 18(Suppl 1):O37 Objectives approach in the design process of the UPAVAN trial- a three-year, four arm cRCT to assess the impact and cost-effectiveness of three variants of an innovative intervention to improve agricultural and nutrition out- comes, with an integrated theory of change. ethical issues not adequately addressed by the Ottawa Statement. For instance, does clinical equipoise obtain when a novel policy is compared to an existing policy that has little or no evidence-base? How should researchers and RECs conceptualize healthcare providers targeted by policy interventions? Are they obligated to participate in research? If so, what are the implications for consent? Alternatively, should healthcare providers be conceptualized as vulnerable partici- pants? A power-differential often exists between healthcare providers and their superiors, particularly when providers are trainees or em- ployees. Does this relationship undermine the validity of their con- sent? If so, what safeguards might be implemented to ensure protection of healthcare providers, while at the same time ensuring that important research proceeds both feasibly and expeditiously? O37 Standardised taxonomy for the classification of trial outcomes within core outcome sets and cochrane reviews Susanna Dodd1, Paula R. Williamson1, Jane Blazeby2, Mike Clarke3 1University of Liverpool; 2University of Bristol; 3Queen’s University, Belfast Correspondence: Susanna Dodd Trials 2017, 18(Suppl 1):O37 Background Background The COMET (Core Outcome Measures in Effectiveness Trials, http:// www.comet-initiative.org.uk) Initiative brings together people inter- ested in the development and application of agreed standardised sets of outcomes, known as “core outcome sets” (COS). These sets represent the minimum that should be measured and reported in all clinical trials of a specific condition, and are also suitable for use in clinical audit or research other than randomised trials. One of the successes of COMET has been the development of a publicly avail- able searchable database of completed and ongoing projects in COS development. This database is currently searchable by population, intervention and condition, but as yet has not been categorised ac- cording to outcome (the fourth of the essential elements that should be defined for a trial, according to the PICO model). Similarly, out- comes in trials registries (including the EU Clinical Trials Register, ClinicalTrials.gov and ISRCTN registry) can be entered as free text only, leading to inconsistencies. Ninety percent of queries related to requests to register a trial relate to outcomes (Alison Cuff, ISRCTN, personal communication). Standardised terminology to describe out- comes is starting to come into use in pre-clinical research (Robinson et al. “The Human Phenotype Ontology: A Tool for Annotating and Analyzing Human Hereditary Disease” (2008) The American Journal of Human Genetics 8: 610–615), but there is currently no consensus on how trial outcomes should be classified. The lack of a standard taxonomy relating to trial outcomes impedes the ability to efficiently and effectively search the literature. A standard classification system for trial outcomes would facilitate literature searches to identify the use of a particular COS, as well as being of use to reviewers when annotating Cochrane Reviews according to outcome, as part of the PICO review description (via the Cochrane Linked Data Project, http://linkeddata.cochrane.org/). O38 How might patient and public involvement (PPI) improve recruitment and retention in surgical trials? A qualitative study exploring the views of trial staff and PPI contributors 1 2 2 3 How might patient and public involvement (PPI) improve recruitment and retention in surgical trials? A qualitative study exploring the views of trial staff and PPI contributors 1 2 2 3 Joanna Crocker1, Keira Pratt-Boyden2, Jenny Hislop2, Sian Rees3, Louise Locock1, Sophie Petit-Zeman4, Alan Chant5, Shaun Treweek6, Jonathan A. Cook7, Nicola Farrar8 1 Joanna Crocker1, Keira Pratt-Boyden2, Jenny Hislop2, Sian Rees3, Louise Locock1, Sophie Petit-Zeman4, Alan Chant5, Shaun Treweek6, Jonathan A. Cook7, Nicola Farrar8 Participants proposed a variety of ways in which PPI contributors might improve recruitment and retention in surgical trials, also giving examples of when PPI might be unhelpful or even harmful. Trialists should carefully consider how to involve patients and members of the public most effectively. 1NIHR Biomedical Research Centre and Nuffield Department of Primary Care Health Sciences, University of Oxford; 2Health Experiences Research Group, Nuffield Department of Primary Care Health Sciences, University of Oxford; 3Health Experiences Institute, Nuffield Department of Primary Care Health Sciences, University of Oxford; 4NIHR Oxford Biomedical Research Centre and Unit; 5Patient Partner; 6Health Services Research Unit, University of Aberdeen; 7Surgical Intervention Trials Unit, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford and MRC ConDuCT-II Hub for Trials Methodology Research, School of Social and Community Medicine, University of Bristol; 8Surgical Intervention Trials Unit, Nuffield Introduction Long stay in hospital and poor mobility put people at risk of devel- oping pressure ulcers (PU) at a number of areas of the body (but- tocks, heels etc.). PUs result in admission to hospital, prolonged hospital stay, impaired quality of life, significant cost to the NHS and have been described as a key quality indicator for the Department of Health. Motivation PUs are classified on a 4 point ordinal scale from 1–4 with 4 the most severe category. In RCTs skin assessment for on- set or progression of PUs is scheduled to take place at a number of fixed time points, resulting in serial measurements of PU categories at up to 14 skin sites. Thus each patient typically has 50–100 PU as- sessments during trial follow-up. However, due to administrative and patient-related events, scheduled measurements may be missed or only partially completed. This results in observation times that are different for different patients and different skin sites, and intervals between assessments may vary. Moreover, the reasons for missing data may not be independent of the PU category. Typically, the pri- mary outcome for PU prevention trials is the time from randomisa- tion to the first category 2 PU at any skin site, so that the 50–100 assessments per patient are reduced to a single outcome measure- ment. This outcome is inefficient in that it ignores the information from serial measurements and multiple skin sites; it may also be biased due to the interval censoring between observations and the missed assessments. Thus sample sizes for PU prevention trials may be larger than necessary, resulting in delays in getting effective treat- ments into practice, or in ruling out ineffective treatments. Aim The aim of this study was to investigate the use of multi-state models of PU onset and progression, in order to provide less biased and more efficient estimates of treatment effects. M h d Methods In this study we show how to design a PU prevention trial and analyse resulting data. Specifically, multi-state models that incorporate both the sampling process (availability and completeness of follow-up) and the observed PU categories at all skin sites are developed. The assumptions that are required for different models, their implications and their valid- ity in this context are presented. Methods for estimation of commonly used outcome measurements within this framework are presented. Through re-analysis of an existing serial measurement from a PU pre- vention study we demonstrate how fixed covariates (e.g. treatment group and stratification factors) can be incorporated into the analysis. Efficiency is explored using simulation studies based on the example trial to demonstrate potential influence on sample size estimates, of using more informative designs and analyses. Drawing on their experiences, participants proposed several ways in which PPI contributors could improve recruitment to trials: improving the relevance of the research question; informing trial design includ- ing the benefits and burdens for participants, recruitment process (where, when, who) and participant information sheets; assessing pa- tients’ willingness to take part; directly recruiting participants; and publically endorsing the trial Suggested ways in which PPI contributors could improve retention in trials included: changing which outcomes are collected and how; assessing the burden or acceptability of follow-up methods to poten- tial participants; suggesting appropriate incentives; communicating with participants during the trial (e.g. newsletter updates, explaining why it is important to stay in the trial); challenging regulatory barriers to adopting new data collection methods. Methods Participants were recruited via surgical and PPI networks and organi- sations. 6 focus groups (4 with surgical trial staff and 2 with PPI con- tributors) were facilitated at 4 sites across the UK. PPI contributors unable to attend focus groups were offered a one-to-one interview in person or by telephone. All participants as well as those unable to attend focus groups were invited to submit additional comments in writing. Verbatim transcripts and textual data were analysed themat- ically by three researchers who identified emerging themes. Results Fifty-four people took part, of whom 31 were surgical trial staff (15 trial managers/coordinators, 7 investigators, 7 research nurses, 1 clin- ical trial administrator and 1 research associate), 21 were PPI contrib- utors and 2 were PPI coordinators. Staff took part in focus groups at surgical research centres in Oxford (N = 7), Aberdeen (N = 8), Bristol (N = 9) and Birmingham (N = 7), while PPI contributors took part in one of two focus groups at the Library of Birmingham (N = 6 and N = 8) or a one-to-one interview (N = 7). Eleven people submitted written contributions. O39 Maximising information in pressure ulcer prevention trials using multi-state modeling g Linda Sharples, Isabelle Smith, Jane Nixon University of Leeds Correspondence: Linda Sharples Trials 2017, 18(Suppl 1):O39 g Linda Sharples, Isabelle Smith, Jane Nixon University of Leeds C d Li d Sh l g Linda Sharples, Isabelle Smith, Jane Nixon University of Leeds Correspondence: Linda Sharples Trials 2017, 18(Suppl 1):O39 Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences University of Oxford y Correspondence: Joanna Crocker Trials 2017, 18(Suppl 1):O38 Introduction Clinical trials are commonly affected by slow recruitment, leading to prolonged study duration and increased cost, and also attrition, which weakens trials. It has been suggested that patient and public involvement (PPI) in designing and/or conducting trials could help to alleviate these problems, yet PPI is often implemented with little planning or thought as to the role of PPI contributors and how their input might benefit the trial. We are developing a PPI intervention aimed at improving recruitment and retention in surgical trials, which can be particularly difficult to recruit to. As part of this process we explored surgical trial staff and PPI contributors’ views regarding how PPI might achieve such improvements. Conclusions y g Results, conclusions and future research The ultimate aim of this project is to agree on standardising termin- ology and definitions through consensus among different stake- holders, including patients, clinicians and methodologists. Progress made to date on achieving this aim will be presented. We demonstrate the challenges and limitations of mediation analysis in this context and suggest a cautious approach for incorporating the ideas of mediation analysis into evaluations of complex interventions. Building on this experience, we discuss the utility of the suggested Trials 2017, 18(Suppl 1):200 Page 201 of 235 However, it was also suggested that PPI contributors could be un- helpful in some circumstances, for example if involved too late (e.g. only in developing informed consent documents), if their literacy level is too high, or if they are not from the trial’s target population. Conclusion However, it was also suggested that PPI contributors could be un- helpful in some circumstances, for example if involved too late (e.g. only in developing informed consent documents), if their literacy level is too high, or if they are not from the trial’s target population. Conclusion Results The primary themes identified were the role of the CTU in trial over- sight and power issues within trial oversight. The central role of the PI in the MRC Guidelines was not reflected in our data. Instead, the clinical trials units (CTUs) supporting the trials took on the responsi- bilities of the PI outlined in the Guidelines. We observed CTUs per- forming additional roles such as advising the PI on research methodology, being the main channel of communication for the trial and arbitrating between the PI and other trial oversight groups. The perceived power of individual oversight groups over trials was influ- enced by the behaviour of funding bodies. For example, by appoint- ing their own TSC members, funders were viewed as reducing the power of TSCs and trial sponsors to make independent decisions. This could lead trial teams to fear their funder’s power and be guarded in their communication with the funder. Trial oversight groups had differing views regarding who has the power to stop tri- als. The sponsors, independent TSC members, TSC chairs and funders all believed they had the power to terminate the trial and that the buck stopped with them. Quality control of the mapping process is partially automated. The proposed automated QC report algorithm reduces the amount of work involved in validating the mapping against a discrete set of rules (e.g. every variable is mapped, no cell in the mapping tool is left blank, each required variable in the domain is mapped, compare mapping to a gold standard - a protocol that was tested and can serve as a template, all fields in the mapping entries start with the form code, all subjects belong to the protocol). O42 Outcome-adaptive randomization: some ethical issues J li Si Outcome-adaptive randomization: some ethical is Julius Sim Keele University Trials 2017, 18(Suppl 1):O42 Outcome-adaptive random Julius Sim Keele University Trials 2017, 18(Suppl 1):O42 Changing roles and relationships within trial oversight: an ethnographic study of eight clinical trials facing challenges Anne Daykin1, Lucy E. Selman1, Helen Cramer1, Sharon McCann2, Gillian W. Shorter3, Matthew R. Sydes4, Carrol Gamble5, Rhiannon Macefield6, Alison Shaw1, J. Athene Lane6 1University of Bristol; 2Formerly: Health Services Research Unit, University of Aberdeen; 3Ulster University; 41 MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology and London Hub for Trials Methodology Research; 5MRC North West Hub for Trials Methodology Research, Institute of Translational Medicine, University of Liverpool; 6MRC ConDuCT Hub for Trials Methodology Research, School of Social and Community Medicine, University of Bristol Changing roles and relationships within trial oversight: an ethnographic study of eight clinical trials facing challenges Anne Daykin1 Lucy E Selman1 Helen Cramer1 Sharon McCann2 In a conventional randomized controlled trial (RCT), randomization is in fixed, usually equal, proportions throughout. As judgments of rela- tive treatment superiority are suspended until the end of the study, there is no reason to use accruing data to adjust allocation, other than in planned interim analyses. In trials using outcome-adaptive randomization (OAR), allocation to treatment arms is repeatedly ad- justed, to weight allocation to the hitherto more effective treatment. This has the ethical merit of seeking to maximize the number of pa- tients experiencing a treatment success. However, this apparent eth- ical advantage is offset by other issues concerning equipoise, informed consent and the methodology of the trial. Gillian W. Shorter , Matthew R. Sydes , Carrol Gamble , Rhiannon Macefield6, Alison Shaw1, J. Athene Lane6 1University of Bristol; 2Formerly: Health Services Research Unit, University of Aberdeen; 3Ulster University; 41 MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology and London Hub for Trials Methodology Research; 5MRC North West Hub for Trials Methodology Research, Institute of Translational Medicine, University of Liverpool; 6MRC ConDuCT Hub for Trials Methodology Research, School of Social and Community Medicine, University of Bristol Correspondence: Anne Daykin Trials 2017, 18(Suppl 1):O41 g Methods The Study Data Tabulation Model (SDTM) defines a standard structure for submission of electronic clinical trial data to a regulatory authority, such as the FDA. These electronic listings of individual observations comprise the essential data reported from a clinical trial and are sub- mitted with the analysis datasets. Using an ethnographic study design, 8 TSC and 6 TMG meetings from eight trials were observed and audio-recorded and 65 semi- structured interviews conducted with 51 purposively sampled key in- formants (members of the trials’ TSCs/TMGs and other relevant infor- mants). Selected trials represented a range of clinical topics and were all dealing with challenging scenarios (e.g. recruitment issues, proto- col deviation or amendments). Data were analysed thematically and findings triangulated and integrated to give a multi-perspective ac- count of current oversight practices. R lt The Clinical Data Interchange Standards Consortium (CDISC) team at the Emmes Corporation, developed a novel process to map data col- lected in electronic case report forms (eCRFs) to the SDTM paradigm with these unique advantages: the mapping specifications are devel- oped alongside the design of the CDASH conformant CRFs; Advan- tage eClinicalSM, Emmes' form building and data capture and management suite, provides an intuitive user interface that permits a non-programmer to specify the mapping to SDTM - this process is completed before the initiation of data collection; then the mapping is executed on the production data in an automated fashion at least daily while the trial is accumulating data, and the results are written to a tabulation database. This enables the use of SDTM data struc- tures for oversight and safety reporting. The use of standardized data tables throughout the life cycle of the study yields efficiencies in stat- istical reporting and reduces the timeframe required for delivery of the final databases and code at the end of the study. pp Conclusions The program creates a complete set of reports for the entire proto- col, for each combination of domain and eCRF. Each report has a summary of the failed tests and hyperlinks are utilized so that the tester can easily navigate the report, see the description of the test, the mapping code used, and the relevant data, as well as the reason for failure. This program has been utilized and tested on Emmes plat- forms and has consistently helped to identify errors while saving tes- ters time. It provides all the information for the tester to evaluate the results and relevant code if they want to execute it themselves. The more accurately this QC of the SDTM mapping is done, the more effi- cient subsequent testing will be. The roles and relationships of trial oversight groups have changed since the publication of the MRC Guidelines in 1998. We found that CTUs, and not the PI or TMG, had responsibility for the day to day management of trials, and this should be acknowledged when the MRC Guidelines are revised. The TSC, funder and sponsor all have the power to stop trials, and acknowledging this may be useful to raise the awareness of all the parties concerned, in order to facilitate the constructive collaboration of trial oversight groups. g Conclusion Given the current difficulties in recruiting patients to RCTs it is im- portant to make best use of the rich data that accrue during trials. Important reductions in sample size for PU trials may be possible if all available observations are included in the analysis. Trials 2017, 18(Suppl 1):200 Page 202 of 235 Page 202 of 235 steering committee (TSC) and data monitoring committee. This model is endorsed by several UK funders. According to these Guidelines, the Principal Investigator (PI) has the central role and overall responsibility for the co-ordination and day-to-day management of the trial. How- ever, recent quantitative evidence suggests heterogeneity in trial over- sight and some confusion regarding the diverse roles of stakeholders, indicating the MRC Guidelines may be outdated. Aim: To explore roles and relationships in trial oversight to ascertain current practice and sug- gest recommendations to support an update of the MRC guidelines. Methods O40 Quality control of SDTM domain mappings from electronic case report forms Noga Lewin, Miebi Eradiri, Sheena Aris, Angela Soriano, Gaurav Sharma, Jill Barrett, Heather Hill, Marian Ewell, Noble Shore, Abigail G. Matthews Emmes Corporation Correspondence: Noga Lewin Trials 2017, 18(Suppl 1):O40 Consent At present, 55 and 57 participants (before and after wound closure) have been allocated to no dressing; 52 and 54 to simple dressing; 54 and 54 to glue-as-a-dressing. Nine allocation disclosure deviations were identified. For 5/165 participants randomized to allocation dis- closure AFTER wound closure, system log-on times for obtaining allo- cation were >50 minutes before the manually entered time of wound closure; another 2 participants had first and second log-on times <2 minutes apart. For 2/161 participants randomized to alloca- tion disclosure BEFORE wound closure, the manually entered ‘knife- to-skin’ time preceded the first system log-on time by >90 minutes. Informants were not specifically aware of any attempts to work around the double-randomisation system; some were aware that such behaviours could be detected, and one questioned why one might try to ‘cheat the system’, acknowledging this as a protocol de- viation. Practical issues, such as limited internet access in theatre or no one available to log into the database, were also reported. Feed- back from two centres suggested that theatre staff are ringing a re- search nurse outside theatre to log-on when required. On at least one occasion, a surgeon first logged in after wound closure, to avoid having to log-on twice. Centres have also reported occasional diffi- culties in accessing the database from theatres. Generic usernames for randomization only, accessible using a mobile phone, were of- fered to improve access. l i The moral force of consent depends on information about the trial being adequately understood. Empirical research suggests that this is hard to achieve, but it is likely to be even harder if one has to ex- plain how randomization is continually readjusted in relation to out- comes. This is likely to increase the ‘therapeutic misconception’: participants’ tendency to think that treatment allocation is based on their individual clinical need, rather than being (semi-)random. A further complication is that the information required by new partici- pants will vary over the course of the trial, as it should reflect the ac- cruing outcomes within the trial (rather than just external evidence that may become available). Conveying appropriate information is therefore challenging, and if not achieved, the value of consent will be reduced accordingly. Crucially, simply telling participants that allocation reflects accumu- lating evidence without also indicating which specific treatment is currently favoured may be insufficient for consent to be informed. O44 Priority setting for core outcome set development Sarah Gorst1, Mike Clark2, Paula R. Williamson1 1 2 U e s ty o e poo ; Quee s U e s ty e ast Correspondence: Sarah Gorst Trials 2017, 18(Suppl 1):O44 Success of randomizing trial participants to disclosure of allocation early or late: a methodological study to investigate performance bias Barnaby Reeves1, Rosie A. Harris1, Leila Rooshenas1, Kate Ashton1, David Hutton1, Chris A. Rogers1, Natalie S. Blencowe1, Jane M. Blazeby1, Bluebelle Study Group2 1 2 Background g The Global Burden of Disease Study identified the leading causes of chronic disorders worldwide. If the findings from this study are to guide future health research, it is important to ensure that appropri- ate outcomes are measured in that research. Core outcome sets (COS) will help to achieve this. COS represent an agreed minimum set of outcomes that should be measured and reported, as a mini- mum, in all clinical trials for a specific health condition. The applica- tion of COS allows the results of clinical trials to be appropriately combined, minimising waste and ensuring that usable evidence is made available. If COS were available for the leading causes of chronic disorders, this should accelerate the impact of research and result in improvements in global health. No prioritisation for COS de- velopment has previously been undertaken, therefore this study aimed to identify COS that have been developed in relation to the most prevalent chronic conditions throughout the world, and to highlight areas for future COS development or improvement. Methods 1University of Bristol; 2Universities of Bristol & Birmingham Correspondence: Barnaby Reeves Trials 2017, 18(Suppl 1):O43 Background Equipoise indicates genuine uncertainty as to the relative merit of the treatments being tested. In a conventional RCT this is established at the outset and only revisited if interim analyses occur. Hence, no patient is knowingly disadvantaged by allocation to either treatment. g The Medical Research Council (MRC) 1998 Guidelines for Good Clinical Practice in Clinical Trials recommend that, in the UK, trial oversight is managed by three committees: a trial management group (TMG), trial Page 203 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 203 of 235 In OAR, equipoise is re-examined repeatedly, as it determines alloca- tion. Accordingly, allocation is increased to the treatment showing superiority - but patients are nonetheless still knowingly allocated to the apparently inferior treatment, albeit in smaller numbers, and thereby disadvantaged. Whilst action is taken in response to changes in equipoise, equipoise is not thereby completely restored. that users should log into the randomization system at the begin- ning of surgery. The user is then either given the allocation or asked to log in again after wound closure to obtain the allocation. When logging in again, the user has to enter the time of wound closure. Acceptability of the double randomization is assessed from three sources of information: times for system log-on, knife-to-skin and wound closure from the trial database; in-depth interviews with health care professionals; feedback from participating centres about their ways of working. In OAR, equipoise is re-examined repeatedly, as it determines alloca- tion. Accordingly, allocation is increased to the treatment showing superiority - but patients are nonetheless still knowingly allocated to the apparently inferior treatment, albeit in smaller numbers, and thereby disadvantaged. Whilst action is taken in response to changes in equipoise, equipoise is not thereby completely restored. q p q p y p y Additionally, at the end of the trial, OAR may have required a larger sample than a conventional RCT; whilst the proportion of participants disadvantaged by a poorer outcome may decrease, the number doing so may increase. Background f Performance bias arises in randomized controlled trials (RCTs) if care providers implement co-interventions differentially on the basis of their knowledge of participants’ treatment allocation. It can especially affect surgical trials because it is rarely possible to blind surgeons and randomization within the operating theatre environment (i.e. as close to intervention delivery as possible) poses logistical challenges. This study aimed to measure and assess the influence of performance bias in a surgical RCT. Consent A study is only ethical if it generates methodologically robust find- ings. However, some features of OAR may have undesirable meth- odological implications. Thus, the fact that differing information should be given to patients entering the trial at different times may lead to contamination, or, coupled with the changing allocation ratio, may be a confounder. Additionally, the need to monitor outcomes repeatedly to determine allocation may limit the degree of blinding achievable. Conclusions Timings collected during the trial demonstrate good adherence to the double randomization. Methods adopted by research personnel in order to adhere may not be practicable in a large trial. Generic access for randomization may facilitate theatre personnel doing this task. Conclusion Conclusion Initially, OAR appears to have ethical merit in terms of maximizing the number of participants who receive the superior treatment within the trial, but this claim needs to be tempered by other ethical considerations. Background Most confirmatory clinical trials are designed so as to achieve a spe- cified power, usually 80% or 90%, for a hypothesis test conducted at a given significance level, which is almost invariably set to be 5% for a two-sided test. Licensing decisions by regulatory agencies are then based on the result of such a significance test informally combined with other information to balance the risk of adverse events against the value of the treatment to future patients. In the setting of a rare disease, recruitment of the number of patients required to achieve conventional error rates for clinically reasonable effect sizes may be infeasible or even impossible, suggesting that the decision-making process should reflect the size of the population for whom the treat- ment can be used in the future. Pragmatic integrated randomised controlled trials in screening: experience from a trial in 1.2million women attending breast screening Sian Taylor-Phillips1, David Jenkinson1, Matthew Wallis2, Janet Dunn1, Aileen Clarke1 1University of Warwick; 2Cambridge Universities NHS Foundation Trust Correspondence: Sian Taylor-Phillips Trials 2017, 18(Suppl 1):O45 1University of Warwick; 2Cambridge Universities NHS Foundation Trust Correspondence: Sian Taylor-Phillips Trials 2017, 18(Suppl 1):O45 Randomised controlled trials (RCTs) are expensive, the pragmatic inte- grated randomised controlled trial has been proposed to deliver large scale RCTs at a much reduced cost. In these studies elements of the trial such as recruitment, randomization, intervention, data collection and/or long term follow up are integrated into standard practice to re- duce costs and increase potential sample size. These designs are par- ticularly appropriate for screening where practice is standardized and many centres use the same software systems. We present an example of a pragmatic integrated randomised controlled trial design in breast cancer screening, the Changing Case Order to Optimise Patterns of Per- formance in Screening (CO-OPS) ISRCTN46603370. The study was de- signed to examine whether breast screening radiologists experience a vigilance decrement of decreasing ability to detect cancer in x-rays with time on task, and whether an intervention to change case order could reduce such an effect. The trial was funded as part of an NIHR postdoc- toral fellowship and cost less than £300 k. Of the 80 breast screening centres in England, 46 consented to take part in the trial for 1 year. This included research active centres and those with little experience of re- search. g Conclusion This study describes the first approach to identifying gaps in existing COS, and to priority setting in this area. Important gaps have been identified for at least 12 of the 25 most prevalent conditions. The de- velopment and application of COS in these areas would provide the foundation for ensuring that appropriate outcomes are measured and reported in clinical trials for these most prevalent disorders worldwide. Without such international consensus on the key out- comes for research in these conditions, new studies might not make a full contribution to improving global health and opportunities to reduce waste in research will be lost. A wider range of perspectives, including those of patients, on existing COS are also needed when not otherwise included. Furthermore, it is evident that COS are failing to include a range of international stakeholders within the develop- ment process. Therefore, the inclusion of stakeholders from Asia, South America, Australia, and Africa is an additional gap that future research should aim to address. A value of information approach to optimal design of confirmatory clinical trials Nigel Stallard1, Michael Pearce2, Siew Wan Hee2, Jason Madan2, Martin Posch3, Simon Day4, Frank Miller5, Sarah Zohar6 1Warwick Medical School, University of Warwick; 2University of Warwick; 3Medical University of Vienna; 4Clinical Trials Consulting and Training Limited; 5Stockholm University; 6INSERM Correspondence: Nigel Stallard Trials 2017, 18(Suppl 1):O46 Background Consent was at the centre level rather than the individual woman screened, as both intervention and control groups were consid- ered different versions of standard practice as both were implemented in different parts of the NHS. The trial was implemented through the National Breast Screening Service computer system, which is used at all English breast screening centres. The software was adapted to random- ise women in batches to intervention or control, and display the cases in the desired order. A total of 1,194,147 women were randomised and analysed. A standard Crystal report was designed to extract trial out- comes from the NBSS computer system. Data extraction was delayed Methods We have considered the use of the decision-theoretic value of infor- mation (VoI) method to obtain the optimal sample size and signifi- cance level for definitive randomised controlled clinical trials in a range of settings, focussing particularly on the impact of different population sizes. For simplicity we have assumed the primary end- point to be continuous and normally distributed with unknown mean with some normal prior distribution, the latter representing in- formation on the anticipated effectiveness of the therapy available from sources external to the trial itself. We explicitly specify the gain in terms of improvement in primary outcome for patients treated with the a new therapy and compared this with the costs, both fi- nancial and in terms of risk of potential harm, of treating patients, ei- ther in the trial or in the future if the therapy is approved. Results We have found that as the size of the population that can be treated in the future increases, the optimal sample size for the clin- ical trial also increases. If there is a non-zero cost, whether financial or in terms of potential harmful effects, of treating future patients, stronger evidence is required for approval as the population size in- creases, though this is not the case if the costs of treating future patients are ignored. Results A search of the COMET database identified 33 published and on- going COS that are relevant to 13 of the world’s most prevalent con- ditions. The majority were developed only with the involvement of people from North America and Europe (n = 27/33). Thirty-one COS involved clinical experts in the development and 18 involved pa- tients. No published or ongoing COS have been identified for the remaining 12 of the 25 most prevalent conditions. C l i Methods Participants having general abdominal surgery or caesarean section at five hospital sites are being recruited to a pilot RCT investigating the influence of wound dressings on surgical site infection. They are randomized twice: first, to the type of dressing to be applied (simple wound dressing, glue-as-a-dressing, or no dressing) and, second, to the time of disclosing the allocation (before or after the surgeon closes the wound at the end of operation). The protocol specifies The COMET (Core Outcome Measures in Effectiveness Trials) Initiative promotes the development and application of COS, by including per- tinent individual studies in a publically available online database. The COMET database is a unique inventory containing references of planned, ongoing and completed work relating to COS development. In total, there are more than 300 published and ongoing COS Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 204 of 235 Page 204 of 235 Page 204 of 235 registered in the COMET database. The COMET database was searched to identify published and ongoing COS that might be relevant to the 25 conditions with the highest global prevalence of chronic sequelae identified in the Global Burden of Disease Study. registered in the COMET database. The COMET database was searched to identify published and ongoing COS that might be relevant to the 25 conditions with the highest global prevalence of chronic sequelae identified in the Global Burden of Disease Study. until after each centre completed their annual reports for routine qual- ity assurance, as the datasets are cleaned in preparation for these. Further data cleaning was conducted in collaboration with each centre. As a result there was very little missing data, making up less than 0.1% of the final dataset. This is an example of implementing a successful pragmatic integrated trial in screening. Such trials are effective in situations where some of the following conditions are met: individual informed consent for the trial is not necessary, the intervention itself is inexpensive, trial outcomes are already rou- tinely recorded in a standard way, routine data collection is accur- ate and audited, and management pathways are standardized and the intervention does not require major changes to these. The ad- vantages of this design are the low cost and large sample size, and the opportunity to involve a greater number of hospitals to in- crease generalizability. Statistical considerations for analyzing multiplex biomarker data from prospective studies 1 1 1 1 p p Shun Fu Lee1, Guillaume Paré1, Matthew J. McQueen1, Hyejung Jung1 Sibylle Hess2, Hertzel Gerstein1 1Population Health Research Institute; 2Sanofi Aventis Deutschland GmbH R&D Division Diabetes Correspondence: Shun Fu Lee Trials 2017, 18(Suppl 1):O49 Ethical issues in individual-cluster trials: beyond the Ottawa statement statement Cory Goldstein1, Austin R. Horn1, Monica Taljaard2, Charles Weijer1 1Western University, the Rotman Institute of Philosophy; 2Ottawa Hospital Research Institute Correspondence: Cory Goldstein Trials 2017, 18(Suppl 1):O47 statement Cory Goldstein1, Austin R. Horn1, Monica Taljaard2, Charles Weijer1 1Western University, the Rotman Institute of Philosophy; 2Ottawa Hospital Research Institute Correspondence: Cory Goldstein Trials 2017, 18(Suppl 1):O47 p g Conclusions The results of clinical trials are often summarised by a frequentist hypothesis test conducted at a 5% significance level with the sam- ple size chosen to give specified power of 80% or 90%. These values are arbitrary. We showed how decision-theoretic analysis suggests a more flexible approach with both type I error rate and Trials 2017, 18(Suppl 1):200 Page 205 of 235 Trials 2017, 18(Suppl 1):200 power (or equivalently trial sample size) depending on the size of the future population for whom the treatment under investigation is intended. An annotated guideline to the use of a health economics analysis plan (heap) alongside randomised controlled trial Melina Dritsaki1, Alastair Gray2, Stavros Petrou3, Susan Dutton2, Sarah E. Lamb2 1Oxford Clinical Trials Research Unit, University of Oxford; 2University of Oxford, Nuffield Department of Orthopaedic Rheumatology and Musculoskeletal Sciences, Oxford Clinical Trials Research Unit, Centre for Statistics and Medicine; 3University of Warwick, Division of Health Sciences, Warwick Medical School Correspondence: Melina Dritsaki Trials 2017, 18(Suppl 1):O48 An annotated guideline to the use of a health economics analysis plan (heap) alongside randomised controlled trial Melina Dritsaki1, Alastair Gray2, Stavros Petrou3, Susan Dutton2, Sarah E. Lamb2 1Oxford Clinical Trials Research Unit, University of Oxford; 2University of Oxford, Nuffield Department of Orthopaedic Rheumatology and Musculoskeletal Sciences, Oxford Clinical Trials Research Unit, Centre for Statistics and Medicine; 3University of Warwick, Division of Health Sciences, Warwick Medical School Correspondence: Melina Dritsaki Trials 2017, 18(Suppl 1):O48 O47 Ethical issues in individual-cluster trials: beyond the Ottawa statement Cory Goldstein1, Austin R. Horn1, Monica Taljaard2, Charles Weijer1 1Western University, the Rotman Institute of Philosophy; 2Ottawa Hospital Research Institute Correspondence: Cory Goldstein Trials 2017, 18(Suppl 1):O47 y Methods Guidelines on how to perform economic evaluations based on clinical trials were searched from the literature. HEAPs were also obtained from a few clinical trial units, although there were certain confidentiality is- sues that had to be surpassed. Section headings (domains) and items were extracted on pre-specified schema we have designed for trial- based HEAPs. Objective 1: Systematic review of individual-cluster trials Using an electronic search strategy, we identified a random sample of pub- lished individual-cluster trials in Canada, the USA, UK, France, Australia and Low and Middle Income Countries. Two reviewers inde- pendently extracted details about ethical issues and practices (e.g., justification for the cluster randomized design, prevalence of seeking informed consent, presence and roles of gatekeepers). Practices will be compared over time, between countries, types of clusters and in- terventions, and other descriptors. Objective The aim of this study is to develop agreed guidance for health econo- mists who work on clinical trials on how to pre-plan their analysis in the absence of any data and how to present it in an unambiguous but flex- ible way. Background The conduct of pragmatic randomized controlled trials is increasing due to their societal importance and their role within the Patient- Centered Outcomes Research Institute (PCORI) initiative “to improve the quality and relevance of evidence available to help patients, care- givers, clinicians, employers, insurers, and policy makers make informed health decisions.” Cluster randomized trials (CRTs), in which groups rather than individuals are randomized to intervention and control con- ditions, naturally tend to be more pragmatic. CRTs may be categorized as “individual-cluster trials” where the intervention is delivered directly to individuals, or “cluster-cluster trials” where interventions are not divisible at the individual-level. The Ottawa Statement is the first com- prehensive ethical guidance document specific to CRTs. Whereas the Ottawa Statement generally presumes that informed consent will be sought for individual-cluster trials, such trials’ when used to evaluate usual care interventions’ raise particular ethical issues that require fur- ther analysis and guidance. This paper has three objectives: to (1) de- scribe current practices and reporting of ethical issues in published individual-cluster trials; (2) present an in-depth ethical analysis of an individual-cluster trial randomizing dialysis centres to two different usual care interventions; and (3) identify ethical issues that require fur- ther analysis and guidance. Health economists working on clinical trial based economic evaluations are often asked at a preliminary stages of studies, and sometimes be- fore data are available, to propose a plan for the collection and analysis of information on resource use, costs and quality of life. Questions that frequently arise when designing a Health Economics Analysis Plan (HEAP) for a clinical trial include what information should be included as standard within the HEAP, whether and how a proposed plan can be changed, how health economists and statisticians should split re- sponsibility for data preparation and analyses, how missing data should be dealt with, and whether there are circumstances when a HEAP is not needed (for example in a feasibility study). Results We have identified a lack of guidance or any standardised templates on how health economists should present HEAPs for clinical trials. In the current climate where clinical trials units increasingly rely on standard op- erating procedures (SOPs) that need to be followed, SOPs for economic evaluations should also be considered as good practice. We identified nine main sections that should be presented in a HEAP with a provisional total of 15 items included. We further recommend the analysis be carried out by an identified, appropriately qualified and experienced health economist, who should ensure the integrity of the data during process- ing. Any deviations from the health economics analysis plan should be described and justified in the final report of the trial. Objective 2: An ethical analysis of the TiME trial the optimal duration for individual hemodialysis treatments in chronic renal failure is currently unknown. The Time to Reduce Mortality in End-Stage Renal Disease (TiME) trial is a PCORI funded individual- CRT in which dialysis treatment centres are randomized to one of two hemodialysis durations (usual care or extended) to evaluate their comparative effectiveness. The main outcome measures are mortality, hospitalization, and quality of life. The trial uses an IRB approved “opt out” approach to informed consent. Applying the Ottawa Statement highlights a range of issues, including justifica- tion for the study design, participant identification, informed con- sent, gatekeeper permission, benefit-harm analysis and protection of vulnerable participants. Conclusions Certain items should be included when designing a HEAP for a clin- ical trial, but others require debate and consensus. Senior health economists and principal investigators should be involved in writing, reading and authorising a HEAP. Deviations from or changes to the HEAP should be reported and justified when reporting. Objective 3: Ethical issues that require further analysis and guid- ance While the Ottawa Statement provides a systematic approach to the ethical analysis of CRTs, we conclude that further analysis and guidance is required for individual-cluster trials of treatments adopted as policy at cluster-level. The TiME trial highlights a num- ber of generalizable ethical issues, including (1) whether there is an appropriate justification for the cluster randomized design (e.g., what justifies adoption of cluster randomization if individual randomization is feasible in principle?), (2) the appropriateness of the consent procedure (e.g., can consent be waived due to prag- matic challenges?), and (3) how we should understand gatekeeper permission (e.g., is gatekeeper permission identical to obtaining proxy consent?). O49 Statistical considerations for analyzing multiplex biomarker data from prospective studies Shun Fu Lee1, Guillaume Paré1, Matthew J. McQueen1, Hyejung Jung1, Sibylle Hess2, Hertzel Gerstein1 1Population Health Research Institute; 2Sanofi Aventis Deutschland GmbH R&D Division Diabetes Correspondence: Shun Fu Lee Trials 2017, 18(Suppl 1):O49 Method After preparing a prespecified analysis plan, the three main statistical considerations when using with multiplex biomarkers to predict out- comes based on survival data are, data preparation, model building and validation. Data with multiplex biomarkers require an extensive data cleaning process including handling the missing information, assessing the distribution for each biomarker, and normalizing and standardizing the biomarkers analyzed. During the model building phase, the approaches used to select the variables, to correct Type I error for multiple testing and to handle proportionality hazards assumptions in survival data are critical to identify the sets of bio- markers to predict the outcome. Lastly, both internal validation (sample splitting vs. bootstrap) and external validation from an inde- pendent sample can be used to assess the performance of the model. Biobanked serum samples from 8494 participants with dys- glycemia in ORIGIN trial were assayed for 284 biomarkers. The object- ive of the study was to identify sets of biomarkers that could identify people with dysglycemia at risk for a CV outcome or death when added to well-established clinical predictors of the relevant out- comes. The results of this study will be used for illustration. Conclusion GLMMs with log link and normal random effects can be useful for simulating binary and count data with specified marginal ICCs. Our results indicate that the approach of Hooper et al. [1] can be ex- tended to binary and count outcomes under such models. Background g Biochemical markers of disease have the potential to both enhance our understanding of disease pathophysiology and identify as-yet Trials 2017, 18(Suppl 1):200 Page 206 of 235 unaffected people who are most likely to develop the disease. As the cost of measuring large sets of biomarkers has fallen, a growing number of prospective cohort studies and trials are including bio- marker measurement in their design. However many of these studies are limited by the absence of a systematic, statistically conservative approach to integrating these measures into the reported findings. ICCs assuming a log link and normally distributed random effects. We also describe a method for checking the compatibility of the specification of ICCs with random effects from GLMMs by solving a set of nonlinear equations. unaffected people who are most likely to develop the disease. As the cost of measuring large sets of biomarkers has fallen, a growing number of prospective cohort studies and trials are including bio- marker measurement in their design. However many of these studies are limited by the absence of a systematic, statistically conservative approach to integrating these measures into the reported findings. ICCs assuming a log link and normally distributed random effects. We also describe a method for checking the compatibility of the specification of ICCs with random effects from GLMMs by solving a set of nonlinear equations. q Results y Methods Using the properties of the log-normal distribution we specify the re- lationship between ICCs on the raw data scale and the variance com- ponents from GLMMs. Results are exact when no covariates are present and we describe an approximate method in the presence of covariates. We implement these results in sample size calculations for a parallel cluster randomised trial with multiple baseline measure- ments and multiple followup measurements. We compare power for given sample sizes using specification of ICCs on the ‘raw’ data scale using the formulae in the approach of Hooper et al. [1] with that from numerical simulations of the corresponding GLMMs in which the variance components are computed from the relationships with Background An interactive two-stage workshop was devised for consumers active in the Cochrane Collaboration at an annual Cochrane Colloquium. The aim of the workshop was to gain understanding in how to communi- cate randomisation to the public and to better inform potential participants of clinical trials. In stage 1, workshop members were given magazine adverts promoting purported clinical benefits and asked to design a study that would address the claims made. Subsequent dis- cussion progressed to the concept of randomisation, potential biases that can arise and how these can be minimised with careful study de- sign. With this background, in the second stage of the workshop, the consumers were asked to develop statements describing randomisa- tion to potential study participants of the CORD pilot trial. The con- sumers, working in groups, were specifically asked for ways to explain to women why trial participants would not be able to choose which study group they could go into, and so would not have a say in when their baby’s cord would be clamped. The final statements produced by the different groups were discussed by all workshop members and modifications suggested. This work was later presented at a CORD pilot trial collaborators’ meeting. Sample size calculations for cluster randomised trials (e.g. parallel, stepped wedge, crossover) require specification of one or more intra- cluster correlations (ICC). With Gaussian outcomes, sample size for- mulae can be specified equivalently in terms of ICCs directly, or using variance components of a linear mixed model. However, with binary or count outcomes, recommended methods use one or the other ap- proach due to lack of direct relationships between the values of ICCs on the ‘raw’ (i.e. binary or count) scales and the variance components from generalised linear mixed models (GLMMs). In this presentation we describe an approach for sample size calculations that reconciles the two by employing GLMMs with a logarithmic link function and normally distributed random effects. Reference 1. Hooper R, Teerenstra S, de Hoop E, et al. Sample size calculation for stepped wedge and other longitudinal cluster randomised trials. Statistics in Medicine 2016;35(26):4718–28. O51 Explaining randomisation to potential clinical trial participants Gillian Gyte1, Sally Crowe2, Dell Horey3 1Consumer Editor, Cochrane Pregnancy & Childbirth Group; 2Crowe Associates; 3Senior Lecturer, Department of Public Health, La Trobe University O51 Explaining randomisation to potential clinical trial participants Gillian Gyte1, Sally Crowe2, Dell Horey3 1Consumer Editor, Cochrane Pregnancy & Childbirth Group; 2Crowe Associates; 3Senior Lecturer, Department of Public Health, La Trobe University These statistical considerations provide a reproducible approach for de- veloping clinically useful prognosis model with multiplex biomarkers based on survival data. Correspondence: Gillian Gyte Trials 2017, 18(Suppl 1):O51 Correspondence: Gillian Gyte Trials 2017, 18(Suppl 1):O51 Funded by Sanofi; ORIGIN NCT 00069784 Results For our simulated examples the empirical power from the GLMM- based simulations using ICCs ranging in value up to 0.10 closely matched that of the formulae of Hooper et al. [1]. For binary out- comes, although large variance components can lead to simulated probabilities above unity, this caused only minimal bias in our exam- ples. For count data one needs to be careful that specification of the marginal overdispersion is compatible with the specified ICCs. Conclusion Sample size calculations for cluster randomised trials: reconciling variance components in generalised linear mixed models with marginal intracluster correlations 1 2 1 3 marginal intracluster correlations Andrew Forbes1, James P. Hughes2, Jessica Kasza1, Richard Hooper3 1Monash University; 2University of Washington; 3Queen Mary University of London Correspondence: Andrew Forbes Trials 2017, 18(Suppl 1):O50 Andrew Forbes1, James P. Hughes2, Jessica Kasza1, Richard Hooper3 1Monash University; 2University of Washington; 3Queen Mary University of London Correspondence: Andrew Forbes Trials 2017, 18(Suppl 1):O50 Background g An important challenge in recruiting people to clinical trials is explaining randomised controlled trials, and this is exacerbated in the context of emergency situations. In the pilot phase of a feasibility randomised controlled trial (RCT) involving babies born at less than 32 weeks gestation that aimed to compare delayed and early cord clamping (CORD pilot trial), clinicians found it particularly difficult to explain why mothers in the study could not choose either option and why randomisation was important. We asked healthcare con- sumers, familiar with RCTs, to help us find ways to explain these is- sues in time-limited recruitment situations. Methods Results The process identified expressions that consumers disliked and ex- pressions that consumers preferred when discussing clinical trials. The issues raised will be presented along with the statements the consumers produced explaining the CORD RCT. The feedback from collaborators in the CORD pilot trial will also be reported. Trials 2017, 18(Suppl 1):200 Page 207 of 235 Trials 2017, 18(Suppl 1):200 y Discussion When running a large complex trial involving a diverse range of staff and researchers working across different health care settings, it is im- portant to consider using IT solutions to optimise efficiency and data quality. Database features developed for the Airways-2 trial are now being implemented in other studies. Chris Rogers1, Sangeetha Paramasivan2, Daisy Elliott2, Paul Whybrow2, Sofia Kanavou1, Rosie A. Harris1, Graziella Mazza1, Tim Brush1, Jane M. Blazeby2, Eric Lim3 1 2 1Clinical Trials and Evaluation Unit, University of Bristol; 2School of Social and Community Medicine, University of Bristol; 3The Royal Brompton Hospital Brompton Hospital Correspondence: Chris Rogers Trials 2017, 18(Suppl 1):O54 Background Airways-2 is a cluster randomised trial comparing two airway manage- ment devices for the treatment of out-of-hospital cardiac arrest. Partici- pating paramedics from four UK ambulance trusts are randomised to manage the patient’s airway using either an i-gel device or intubation. We are aiming to recruit 1,500 paramedics, screen approximately 60,000 patients and enrol more than 9,000 patients over a two year period. As patient recruitment takes place in an emergency setting and patients who survive to hospital admission could be admitted to one of 100 hospitals, data collection needs to be as efficient and streamlined as possible. Funding Th Ai The Airways-2 trial is funded by the National Institute for Health Research (NIHR) Health Technology Assessment (project number 12/167/102). The views and opinions expressed are those of the authors and do not necessarily reflect those of the NIHR, NHS or the Department of Health. Background D i i Despite increasing international interest, there is a lack of evidence about the most efficient, effective and acceptable ways to implement patient and public involvement (PPI) in clinical trials. This study aimed to identify the priorities of UK PPI stakeholders for methodo- logical research to help resolve uncertainties about PPI in clinical trials. Maximising trial efficiency in the airways-2 trial using a multi-functional database 1 1 1 Lauren Scott1, Jodi Taylor1, David Hutton1, Barney C. Reeves1, Jonathan R. Benger2, Chris A. Rogers1 Lauren Scott1, Jodi Taylor1, David Hutton1, Barney C. Reeves1, Jonathan R. Benger2, Chris A. Rogers1 1Clinical Trials & Evaluation Unit, University of Bristol; 2Faculty of Health and Applied Sciences, University of the West of England Correspondence: Lauren Scott Trials 2017, 18(Suppl 1):O52 O52 Maximising trial efficiency in the airways-2 trial using a multi-functional database Lauren Scott1, Jodi Taylor1, David Hutton1, Barney C. Reeves1, Jonathan R. Benger2, Chris A. Rogers1 1Clinical Trials & Evaluation Unit, University of Bristol; 2Faculty of Health and Applied Sciences, University of the West of England Correspondence: Lauren Scott Trials 2017, 18(Suppl 1):O52 Methods A modified Delphi process including a two round online survey and a stakeholder consensus meeting. We used snowball sampling to identify and invite PPI stakeholders to take part in the online Delphi. Stakeholders included PPI contributors, lay and non-lay reviewers from funding panels and Research Ethics Committees, PPI coordina- tors, PPI Planners (e.g. Chief Investigators and Trial managers), PPI ad- visors (e.g. Research Design Service members) and PPI researchers. Results Priorities for methodological research on patient and public involvement in clinical trials: a modified Delphi process 1 2 2 Healthcare consumers can contribute to recruitment to RCTs by devel- oping wording to help explain randomisation to potential trial partici- pants. We used an interactive workshop to generate useful insight about consumer views about clinical trial participation. This approach could be extended to other areas to gather further ideas from health- care consumers. Priorities for methodological research on patient and public involvement in clinical trials: a modified Delphi process Kerry Woolfall1, Anna Kearney2, Heather Bagley2, Carrol Gamble2, Jim T. Elliot3, Helen Bulbeck3, Stephen Thomas3, Bridget Young4, Simon Denegri5, Delia Muir6, Natalie A. Simon7, Joanna C. Crocker8, Claire Planner9, Mike Clarke10 1 2 Priorities for methodological research on patient and public involvement in clinical trials: a modified Delphi process Kerry Woolfall1, Anna Kearney2, Heather Bagley2, Carrol Gamble2, Jim T. Elliot3, Helen Bulbeck3, Stephen Thomas3, Bridget Young4, Simon Denegri5, Delia Muir6, Natalie A. Simon7, Joanna C. Crocker8, Claire Planner9, Mike Clarke10 Kerry Woolfall1, Anna Kearney2, Heather Bagley2, Carrol Gamble2, Jim T. Elliot3, Helen Bulbeck3, Stephen Thomas3, Bridget Young4, Simon Denegri5, Delia Muir6, Natalie A. Simon7, Joanna C. Crocker8, Claire Planner9, Mike Clarke10 Kerry Woolfall1, Anna Kearney2, Heather Bagley2, Carrol Gamble2, Jim T. Elliot3, Helen Bulbeck3, Stephen Thomas3, Bridget Young4, Simon Denegri5, Delia Muir6, Natalie A. Simon7, Joanna C. Crocker8, Claire Planner9, Mike Clarke10 1The University of Liverpool; 2Clinical Trials Research Centre (CTRC), North West Hub for Trials Methodology Research, University of Liverpool; 3Patient and Public Involvement partner; 4Department of Psychological Sciences, North West Hub for Trials Methodology Research, University of Liverpool; 5University College London and National Institute for Health Research (NIHR), INVOLVE; 6Leeds Institute of Clinical Trials Research (LICTR), University of Leeds; 7Health Care Research Wales, Public Involvement and Engagement, Castlebridge 4; 8NIHR Biomedical Research Centre and Nuffield Department of Primary Care Health Sciences, University of Oxford; 9Centre for Primary Care, University of Manchester; 10Centre for Public Health, Queen’s University of Belfast Correspondence: Kerry Woolfall Conclusions The prioritised methodological research topics identified by the Delphi process indicate important areas of uncertainty about PPI in trials. Ad- dressing these uncertainties will be critical to enhancing PPI. Our find- ings should be used by those involved in the planning and funding of PPI in clinical trials to help focus research efforts and minimise waste. The Airways-2 database has approximately 300 users undertaking one of five different roles. So far, over 44,000 patients have been screened and nearly 6,000 patients have been enrolled. Data queries, along with regular central monitoring, have enabled successful pa- tient tracking and over 99% data completeness for the primary out- come. User feedback has been positive. The system is intuitive and easy to use. Methods We designed a multi-functional database to track patients’ progress through the trial, as well as capture the trial data. Data collection has been organised in sections reflecting the different stages of the pa- tient journey. Access to the different sections is dependent on the outcome of the previous section, e.g. it is not possible to access the area of the database that captures hospital admission data if the pa- tient died at the scene of the arrest. Access is also restricted accord- ing to role and location, e.g. hospital based researchers cannot access the sections relating to pre-hospital paramedic data collection. User access is via a secure NHS web server. Data quality is maximised by validation checks on data items; the database allows users to save invalid data or leave items blank but such instances generate queries. Users may correct data entry errors or provide responses to queries within the database, resulting in a complete audit trail of any changes. A “traffic light” system helps users to keep track of outstanding queries. Additionally, the database creates reports to facilitate the coordination team in identifying patients who are due 3 or 6 month post cardiac ar- rest follow-up. Documents for follow-up containing mail merged pa- tient details can be downloaded from the database. In total, 237 stakeholders registered of whom 219 (92%) completed the first round. 187 of 219 (85%) completed the second; 25 stakeholders attended the consensus meeting. Delphi findings were presented and considered at the consensus meeting. 16 of the 42 topics were rated of critical importance by over 70% of stakeholders at the meeting. 96% of stakeholders rated the top three topics as equally important. These were: research on ways to develop strong and productive working rela- tionships between researchers and PPI contributors; exploring PPI prac- tices in selecting trial outcomes of importance to patients; and a systematic review of PPI activity to improve the accessibility and useful- ness of trial information materials for clinical trial participants. Conclusions Background Recruitment to randomised controlled trials can be challenging, par- ticularly when the burden of participation is high (e.g. long and/or Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 208 of 235 intensive follow-up), the interventions under evaluation are very dif- ferent (e.g. surgery versus best medical management) and there are strong preferences amongst clinicians and/or potential participants. To help overcome these challenges, a quintet recruitment interven- tion (QRI) was developed by Donovan and colleagues in the context of the ProtecT trial. The QRI is a complex intervention with several components. These include detailed assessment of screening logs, in-depth interviews, audio-recording of recruitment consultations, and expert review and analysis of the consultations to allow group and individual feedback to recruitment teams on how to optimise in- formed consent. The impact of the individual elements of the QRI have not been previously explored. The aim of this study is to exam- ine the association between trial recruitment and audio-recording of consultations. h d intensive follow-up), the interventions under evaluation are very dif- ferent (e.g. surgery versus best medical management) and there are strong preferences amongst clinicians and/or potential participants. To help overcome these challenges, a quintet recruitment interven- tion (QRI) was developed by Donovan and colleagues in the context of the ProtecT trial. The QRI is a complex intervention with several components. These include detailed assessment of screening logs, in-depth interviews, audio-recording of recruitment consultations, and expert review and analysis of the consultations to allow group and individual feedback to recruitment teams on how to optimise in- formed consent. The impact of the individual elements of the QRI have not been previously explored. The aim of this study is to exam- ine the association between trial recruitment and audio-recording of consultations. might vary within a study according to patient characteristics or between studies due to contextual factors such as the composition of the study population or the study setting. Statistically the investigation of treatment effect moderation amounts to an assessment of interactions between putative moderators and treatment groups. Interaction tests tend to be underpowered when based on individual participant data (IPD) from a single study. Pooling multiple trials aimed at evaluating the same inter- vention offers a way forward here. Aggregate data meta-analytic techniques are routinely used to com- bine results from multiple studies. Results IPD meta-analysis performed by multilevel modelling was able to ac- count for trial design features such as variation in randomisation ra- tios, stratification and clustering; and enabled principled analyses to be carried out in the presence of missing values. It also allowed us to separate trial-level from individual-level moderation effects and to make adjustments. The comparison of the modelling results with the findings from a standard meta-regression showed that IPD meta- analysis can avoid aggregation bias (also known as the ecological fal- lacy), and can produce more powerful inferences when moderation effects do not differ between the trial level and the individual level. Conclusion We will describe the variation in audio-consent rates by centre and recruiting surgeon and the impact of QRI feedback on trial recruit- ment rates. Background Specifically meta-regression ob- tains treatment effect estimates (and associated precision estimates) from different trials and then investigates the association between the trial-level treatment effect estimates and contextual factors using a weighted regression approach. Importantly, meta-regression is only able to assess trial-level variability in treatment effects. In addition, the number of replicate trials tends to be small and hence limits the ability for making adjustments in the analysis. y Methods We pooled IPD from 13 European trials of the Incredible Years (IY) parenting intervention aimed at improving disruptive child behaviour (Eyberg Child Behavior Inventory) in children with conduct problems. We considered a number of pre-specified putative individual-level moderators of the IY intervention effect. We assessed moderation ef- fects using both, meta-regression based on published effect sizes, as well multilevel modelling of the pooled IPD. O55 O55 Assessing treatment effect moderation in trials of psychological interventions: a case for individual participant data meta-analysis of pooled trials Sabine Landau1, Victoria Harris1, Patty Leijten2, Joanna Mann2, Eva-Maria Bonin3, Jennifer Beecham3, Judy Hutchings4, Stephen Scott1, Frances Gardner2 1King’s College London; 2University of Oxford; 3London School of Economics and Political Science; 4Bangor University Correspondence: Sabine Landau Trials 2017, 18(Suppl 1):O55 O55 Assessing treatment effect moderation in trials of psychological interventions: a case for individual participant data meta-analysis of pooled trials Sabine Landau1, Victoria Harris1, Patty Leijten2, Joanna Mann2, Eva-Maria Bonin3, Jennifer Beecham3, Judy Hutchings4, Stephen Scott1, Frances Gardner2 1King’s College London; 2University of Oxford; 3London School of Economics and Political Science; 4Bangor University Correspondence: Sabine Landau Trials 2017, 18(Suppl 1):O55 Working with a vulnerable population on a sensitive topic to better understand neonatal intensive care trials: development of a suite of strategies to meet challenges for qualitative data collection in the bracelet study 1 2 3 4 Assessing treatment effect moderation in trials of psychological interventions: a case for individual participant data meta-analysis of pooled trials 1 1 2 2 Sabine Landau1, Victoria Harris1, Patty Leijten2, Joanna Mann2, Eva-Maria Bonin3, Jennifer Beecham3, Judy Hutchings4, Stephen Scott1, Frances Gardner2 1 2 3 y Claire Snowden1, Peiter Brocklehurst2, Robert Tasker3, Martin Ward-Platt4, Diana Elbourne5 1London School of Hygiene and Tropical Medicine MRC London Hub for Trials Methodology Research, London, UK; 2Birmingham Clinical Trials Unit, Institute of Applied Health Research, University of Birmingham; 3Department of Neurology, and Anaesthesia (Pediatrics), Harvard Medical School; 4Newcastle Neonatal Service, Royal Victoria Infirmary; 5London School of Hygiene and Tropical Medicine Correspondence: Claire Snowden Trials 2017, 18(Suppl 1):O56 Claire Snowden1, Peiter Brocklehurst2, Robert Tasker3, Martin Ward-Platt4, Diana Elbourne5 1London School of Hygiene and Tropical Medicine MRC London Hub for Trials Methodology Research, London, UK; 2Birmingham Clinical Trials Unit, Institute of Applied Health Research, University of Birmingham; 3Department of Neurology, and Anaesthesia (Pediatrics), Harvard Medical School; 4Newcastle Neonatal Service, Royal Victoria Infirmary; 5London School of Hygiene and Tropical Medicine Correspondence: Claire Snowden Trials 2017, 18(Suppl 1):O56 1King’s College London; 2University of Oxford; 3London School of Economics and Political Science; 4Bangor University Correspondence: Sabine Landau Trials 2017, 18(Suppl 1):O55 Methods Data from two on-going “difficult-to-recruit to” multi-centre RCTs in surgery with an integrated QRI were used for this study; one compar- ing the effectiveness and cost-effectiveness of three types of bariatric surgery for severe and complex obesity (By-Band-Sleeve) and the other comparing open versus video-assisted thoracotomy (key hole) surgery for early stage lung cancer (Violet). In both trials, recruiters and eligible participants were invited to have their recruitment con- sultation audio-recorded. We examined the rate of consent amongst patients who did and did not agree to audio recording. R l In order to investigate treatment-effect moderation by individual participant characteristics it is necessary to retrieve and analyse the IPD. Two strategies for IPD meta-analysis exist: A two-stage ap- proach analyses the IPD for each trial separately, generates appro- priate trial-level summaries and then combined these in a second step using aggregate data meta-analysis. In contrast multilevel modelling of the pooled IPD from all trials assesses moderation in a single analysis. Importantly, the latter approach can assess both, moderation by variables that vary between trials and by variables that vary within trials. Over the two trials, approximately two-thirds of eligible patients agreed to their consultation being audio-recorded (By-Band-Sleeve 1209/1806, 67%; Violet 149/185, 80%), with some variation in audio-consent rates across centres. In both studies the proportion of patients agreeing to randomisation is higher amongst the group who consented to audio-recording and the pattern is consistent across the two studies (By-Band-Sleeve 39% vs 9%, Violet 76% vs 16%). The probability of consenting to randomisation is four-times higher (risk ratio 4.45, 95% CI 3.2-6.1) when the consultation is re- corded. The overall randomisation rate is 29% in By-Band-Sleeve and 50% in Violet. Withdrawal rates following consent are low in both trials (By-Band-Sleeve 4%, Violet 6%). Discussion f This is the first study to examine the association between consent for audio-recording and trial participation. Whilst we have demonstrated an association, the reasons for this are unclear and require further in- vestigation. They may be patient-related, recruiter-related, or both. For example, a patient who consents to audio-recording may be naturally more predisposed to research, or they may feel they should agree to randomisation because the consultation could be listened to (although we have no evidence of coercion). Similarly, recruiters who consent to audio recording may be more comfortable with and better at conduct- ing recruitment consultations. They may also “try harder” to recruit the patient when the consultation is being audio-recorded. It is important to explore and better understand the factors that might underlie this association to further support trial recruitment. We encourage the use of the one-stage multilevel modelling ap- proach to IPD meta-analysis. A two-stage IPD meta-analysis does not require explicit assumptions regarding the variability of effects across trials and tools are increasingly becoming available. While there is a lot to be said for simplicity, we prefer the more complex one-stage approach as it offers the flexibility to address all the moderation questions with optimal efficiency. Results Patient eligibility for different treatments may change throughout their journey, mirroring the choices that are made in clinical care. At any one time, umbrella trials provide an infrastructure for screening patients and directing them to an appropriate treatment, and these can be combined with a platform trial which evaluates various differ- ent treatment options for each subgroup. However, numbers soon become vanishingly small and even large national collaborative groups can struggle to find sufficient patients. We present therefore an extension of these approaches called the Patient Journey Planner. The combined design allows different national collaborative groups to maintain their own trials infrastructure while contributing to mul- tiple evaluations of targeted therapies. Evaluations will typically take place at different stages through a patient’s “journey” with a condi- tion; at each stage (e.g. induction, maintenance, relapse), a number of different options will be available based upon genetic characteris- tics or response to prior therapies. The design of the evaluation of each of these is appropriate to the stage of development of the therapy, and the aspirations for the treatment or the patient group. Such an approach is proposed and illustrated for the treatment of Acute Myeloid Leukaemia, and also in solid tumours such a skin can- cer, where there is a similar degree of heterogeneity, and the re- quirement to evaluate novel targeted therapies at different disease stages. The approach also allows the integration of supportive care and prevention studies. The BRACELET Study involved bereaved parents whose babies died subsequent to enrolment in five neonatal intensive care trials. A suite of strategies was developed to: maximise collection of good quality data, ensure data collection processes were sensitive and supportive to contributors, and to facilitate involvement of hard-to-reach be- reaved parents. Data collection and support strategies were devel- oped in response to the sensitivities involved. BRACELET involved face to face interviews with bereaved parents. Recognising that some parents would not wish to be interviewed, two alternative participa- tion routes were developed, an online comments option and short questionnaires. A detailed Code of Practice was developed to guide the conduct of lone and joint interviews which took into account challenges of discussing bereavement and trials with potentially vul- nerable individuals. The patient journey planner: an integrated approach to trials of targeted therapies with application to cancer and haematological malignancies 1 2 1 3 1 Robert Hills1, Nigel Russell2, Kerry Hood1, Girlish Patel3, Richard Adams1 1Cardiff University; 2University of Nottingham; 3University Hospital of Wales Correspondence: Robert Hills Trials 2017, 18(Suppl 1):O57 Background The investigation of treatment effect moderation is a common research objective in psychological studies. The benefit or harm of an intervention Page 209 of 235 Trials 2017, 18(Suppl 1):200 Page 209 of 235 Page 209 of 235 Results The Code was organised around five key princi- ples: the interviewees should be given control of the physical environment; the interviewee should be given control over the pro- gress of the interview; the distinction between counselling and re- search should be clear; all interviewees will be treated with courtesy and respect; and the interviewee should have access to information and support after the interview. Each principle had multiple dimen- sions which were explored and operationalised in the Code. To meet the final principle, dedicated support systems were developed in collaboration with local and national support providers to ensure par- ticipants could access help and information post-interview should it be needed. A post-interview questionnaire was used to monitor reac- tions to the interviews. Background entity. However, while these insights provide valuable evidence on dis- ease mechanisms, the fragmentation of conditions leads to challenges in trial design. Instead of a classical Phase 3 mega-trial, with wide eligi- bility, the evaluation of targeted therapies requires the identification of subgroups of patients, some of which might be very small. Agents may be at different stages of development; and further still, clonal evolution can mean that agents are not equally efficacious at different stages of disease. There is growing understanding of the value of qualitative research to elucidate the conduct and impact of randomised trials, and to guide fu- ture trial conduct. Some of the most challenging trials assess interven- tions aimed at improving care for seriously ill patients and so can take place in the context of major life events. For qualitative research to offer insights into such trial processes and settings, researchers must engage with potentially vulnerable populations to ask often difficult questions. As researchers, we must take responsibility for the proper care and treatment of those who take part in our research and engage with those questions, and so need appropriate research tools and sup- port systems to work with challenging trials. p Conclusion Relying on appropriate exchange of data the Patient Journey Planner allows the evaluation of therapy, using modern trial designs, across multiple collaborative groups, not only speeding up drug development but enabling the assessment of treatments in groups which otherwise would be too small for formal trials run by a single centre or collabora- tive group. Aims To design trials to accelerate the evaluation of new targeted therap- ies which may be at different stages of development or relevant to different stages within the patient pathway. Background Background Initial evaluations of treatment activity are typically performed in Phase 2 clinical trials. Historically, such trials used either one or two- stage single-arm designs, such as the Gehan, A’Hern, or Simon ap- proaches. Such designs rely on identifying the level of outcome (e.g. response rate) which merits further investigation, and the design of such a trial would specify a minimum number of responses to proceed to further evaluation. The choice of threshold depends on the condition being studied, which is often informed by historical data. By contrast, randomised Phase 2 trials do not specify an abso- lute threshold of response, but rather a level of improvement that is required to be seen. Results Results Fifty-one bereaved parents were interviewed, and the online com- ments option and short questionnaires yielded valuable extra data from 8 parents which offered new insights and confirmed themes arising from interviews. Thirty-nine post-interview questionnaires confirmed that, although the process was deeply emotional for many of the parents, it was perceived as valuable both for themselves and for parents in the future. Correspondence: Ian Thomas Trials 2017, 18(Suppl 1):O58 Correspondence: Ian Thomas Trials 2017, 18(Suppl 1):O58 Correspondence: Ian Thomas Trials 2017, 18(Suppl 1):O58 The importance of randomisation in evaluation of treatments for acute myeloid leukaemia: lessons from the UK NCRI aml16 and li-1 trials The BRACELET study enabled parents to elucidate their bereavement experiences in a trial context, and to offer guidance on good conduct for future trials where mortality is anticipated. The strategies devel- oped for the BRACELET study were crucial factors in its success and ethical conduct, and may be adapted by others exploring sensitive issues for trials with vulnerable populations. While our strategies in- cluded elements tailored to our specific challenges, they are readily adaptable for other trial-related sensitive research areas. Ian Thomas1, Robert K. Hills1, Nigel Russell2, Mike Dennis3 1Cardiff University; 2University of Nottingham; 3Christie Hospital NHS Trust Background The poster themes varied greatly in the number of comprising abstracts but the “Use of Innovative Methods in Active Trials” garnered the most average views (median 91), and “Involving Research Partners” least (median 60). Background Background Good reporting of research findings in primary papers is crucial to allow meta-analyses to include all available studies on the topic of interest. However, key information in trial results is still commonly missing (Alt- man, 2015), such as standard deviations (SD) of continuous outcomes. Specific recommendations in the Cochrane Handbook and PRISMA statement on handling missing data include retrieving all connected in- formation and performing sensitivity analysis to challenge the missing data assumptions. These are not frequently followed, nor is there a sim- ple method to evaluate the impact of omitting a study from a meta- analysis. We propose a statistic that would help reviewers to assess the influence that a study without an SD estimate may have on the pooled effect meta-analysis and calculate this statistic across multiple Cochrane meta-analyses. We also report on whether reviewers could have de- duced or approximated the missing SD in primary papers or imputed the SDs by those reported in other studies in the same meta-analysis. Methods Correspondence: Andrew Embleton Trials 2017, 18(Suppl 1):O59 y Potential Impact We can draw together conclusions from the previous conference, compare and contrast the abstract views from the two conferences and highlight any changes or consistent themes. Also, just as cru- cially, we will be tracking abstracts views over time beyond the end of the conference and ensuring access is available to both authors and external researchers, both on the website and in dataset format. This will ensure that interested parties from the 2017 conference can clearly gauge the impact of their own work and any promotional ef- forts they make. Our results demonstrate the need for randomisation in phase 2 trials in this group of patients, and indeed in other heterogeneous condi- tions, to ensure interpretability of results. Background Background Recent advances in the understanding of disease at a genetic level have demonstrated that many conditions are not a single homogeneous We present here the results from the control arm of the UK National Cancer Research Institute Pick-A-Winner programme for older patients Page 210 of 235 Page 210 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 210 of 235 into an expected hierarchy over time. Though focusing specifically on the top-viewed 5% abstract (n = 16): at Day 3, 12 of these were poster presentations, including all the top 10; by the latest data capture at Day 55, the top four abstracts were oral presentations, although posters still occupied 11 out of the 16. The most viewed theme from oral ab- stracts was “Adapting Trials Based on Internal Data” (median 124 views by Day 55), with Trial and Project Management least popular (median 68 views by Day 55). The poster themes varied greatly in the number of comprising abstracts but the “Use of Innovative Methods in Active Trials” garnered the most average views (median 91), and “Involving Research Partners” least (median 60). with acute Myeloid Leukaemia not suitable for intensive treatment. The programme uses a seamless Phase2/Phase 3 design to evaluate treat- ments, with an early assessment for futility. New treatment options can be brought in at any time to replace options which are closed, and evaluation only takes place between novel therapy and control among contemporaneously randomised patients. Therapies which pass the ini- tial futility hurdles proceed seamlessly to Phase 3 evaluation within the programme. The programme has been running for over 10 years, and over 1500 patients have entered the randomised comparisons for 11 different novel therapies. into an expected hierarchy over time. Though focusing specifically on the top-viewed 5% abstract (n = 16): at Day 3, 12 of these were poster presentations, including all the top 10; by the latest data capture at Day 55, the top four abstracts were oral presentations, although posters still occupied 11 out of the 16. The most viewed theme from oral ab- stracts was “Adapting Trials Based on Internal Data” (median 124 views by Day 55), with Trial and Project Management least popular (median 68 views by Day 55). Potential Results Outcomes of the control arm of low-dose ara-C show a wide vari- ation in complete remission rates from comparison to comparison, ranging from less than 10% in some cases to more than 30%. Response rates can fluctuate by comparator drug based upon the eli- gibility criteria for the treatments; but even when eligibility criteria remain constant, wide variation is seen in remission rates, which may be explained by differing referral patterns and the perceived attract- iveness or toxicity of the novel therapies. The wide variation in re- sults shows the issues associated with using a single arm phase 2 trial in a heterogeneous condition: setting an ‘average’ target of 20% response rate (similar to that seen in the original trial of low-dose ara-C), some treatments which showed a remission benefit would have failed to pass the hurdle, while others closed for futility would have proceeded to further evaluation. We propose presenting both short- and long-term findings from ICTMC 2015 plus initial, short-term results from ICTMC/SCT 2017 con- ference, possibly as a penultimate talk in the final session. We will provide an on-line searchable resource for this conference using at least daily views of abstract, during and beyond the conference. Potential Impact Methods We used the whole Cochrane Database of Systematic Reviews issued in January 2008 (Davey et al., 2011) to identify studies omitted with a missing SD from 6672 continuous-outcome meta-analyses. A sub- sample of these, published since 2006, was selected and enabled hand-searching and extracting to assess whether SDs could have been deduced or approximated and to calculate the influence statis- tic. We report on fixed effect meta-analyses. Abstracts were available online in a supplement to BioMed Central’s Trials from conference Day 1. Initially motivated in tracking interest ICON6 (#P184), we used an algorithm applied to the journal website to collect basic article view metrics from Trials giving a snapshot of cumulative online views. The impact on abstract views of presenta- tion type, submission category and title form can be explored. Results Background bl Co-publication of papers to coincide with conferences is not yet standard for clinical trials, although improving e.g. a selection of plenary abstracts at European Society of Medical Oncology 2016 were co-published in high-impact medical journals. For clinical trial methodology, publication usually lags presentation (if achieved); the conference abstract is the key written information available. The International Clinical Trials Methodology Conference (ICTMC) 2015 presented 3 invited talks with 94 oral and 238 poster presentations. We are interested in their online attention. If a tree falls in a forest: abstract view statistics as a measure of research impact Joana Vasconcelos1, Rebecca M. Turner2, Toby A. Prevost1 1Imperial College London; 2Medical Research Council Biostatistics Unit Correspondence: Joana Vasconcelos Trials 2017, 18(Suppl 1):O60 p Andrew Embleton1, Deborah Ashby2, Ella Flemyng3, Peter Langhorne4, William J. Meurer5, Annabelle South1, Matthew Sydes1 1MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL, London; MRC London Hub for Trials Methodology Research, London; 2Imperial Clinical Trials Unit, Imperial College London; 3BioMed Central; 4University of Glasgow; 5University of Michigan Correspondence: Andrew Embleton Trials 2017, 18(Suppl 1):O59 p Andrew Embleton1, Deborah Ashby2, Ella Flemyng3, Peter Langhorne4, William J. Meurer5, Annabelle South1, Matthew Sydes1 1MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL, London; MRC London Hub for Trials Methodology Research, London; 2Imperial Clinical Trials Unit, Imperial College London; 3BioMed Central; 4University of Glasgow; 5University of Michigan Correspondence: Andrew Embleton Trials 2017, 18(Suppl 1):O59 p Andrew Embleton1, Deborah Ashby2, Ella Flemyng3, Peter Langhorne4, William J. Meurer5, Annabelle South1, Matthew Sydes1 1MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL, London; MRC London Hub for Trials Methodology Research, London; 2Imperial Clinical Trials Unit, Imperial College London; 3BioMed Central; 4University of Glasgow; 5University of Michigan Andrew Embleton1, Deborah Ashby2, Ella Flemyng3, Peter Langhorne4, William J. Meurer5, Annabelle South1, Matthew Sydes1 1MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL, London; MRC London Hub for Trials Methodology Research, London; 2Imperial Clinical Trials Unit, Imperial College London; 3BioMed Central; 4University of Glasgow; 5University of Michigan tic. We re Results The results describing the views over time for every accepted ab- stract from ICTMC 2015, along with a summary of wider themes, are available at http://andyembleton.me/ICTMC/ The abstract addressing “Longitudinal mediation in the PACE trial” was consistently the most popular abstract from Day 4 onwards (i.e. post-conference). This ab- stract gained 527 views between Days 4 and 8, the largest uptick in views, perhaps due to its posting on a popular Twitter account and discussion on a patient forum. There was little overall difference be- tween those oral and poster abstracts initially, but this manifested Amongst the 6672 sampled meta-analyses, 396 (6%) were affected by omitted studies. The hand-searched sub-sample comprised 52 studies, from 28 meta-analyses within 18 Cochrane reviews, including 33 subgroup meta-analyses. In 5 (28%) reviews, reviewers did not at- tempt to contact original publication authors to retrieve missing in- formation and only one meta-analysis reported sensitivity analysis to assess the impact of imputing SDs. For 12 (36%) subgroup meta- analyses, the SDs could have been externally imputed from other Trials 2017, 18(Suppl 1):200 Page 211 of 235 studies’ SDs. In 19 (37%) of omitted studies, the SDs could have been internally retrieved through either deducing (8%) or approximating (29%) from available information. Developed from the inverse- variance weighting formula for fixed effect models, we proposed an ‘influence statistic’ that tells us whether, and by how much, a study that does not report the precision of an effect could influence the overall meta-analysis estimate, defined as the Z-statistic ratio of the distance between the omitted study effect from the pooled effect and the standard error of this distance. This statistic was calculated and its distribution displayed across 16 studies that were omitted from 12 subgroup meta-analyses. Nineteen percent of these had a high influence (Z > 2) on the subgroup meta-analysis pooled effect. Conclusions A web-based system for the management and storage o metadata for multi-center clinical trials Kevin Wilson, Michale Ham, Brenda Hair, Eugene Turner RTI International Correspondence: Kevin Wilson Trials 2017, 18(Suppl 1):O62 A web-based system for the management and storage of metadata for multi-center clinical trials Kevin Wilson, Michale Ham, Brenda RTI International Correspondence: Kevin Wilson Trials 2017, 18(Suppl 1):O62 p Results Data elements were imported from the EDC systems of two research networks. A total of 8,620 unique data elements, derived from 291 unique CRFs, were imported from 4 clinical research trials. Initial searches of the repository revealed interesting patterns in the data elements and clearly showed inconsistencies between similar vari- ables collected across the trials. Examples included the same variable collected in multiple studies with different variable names, different response options for the same variable in different studies, and the same variable names used for different variables. After curation, the system was used to generate formatted CRFs and the associated EDC system. y In the sensitivity analysis we do not want to lose any of the valuable information collected in the trial. A natural principle, which we refer to as the information anchoring principle, is to preserve the informa- tion loss due to missing data in the primary analysis in the sensitivity analysis. However, it is also possible to have much less information in the sensitivity analysis than in the primary analysis. Moreover, a naïve sensitivity analysis may insert more information than we would have had if we actually observed the data. Conclusions from the primary analysis can then be overturned. Methods The system design and methods encompassed several components, including: (1) an automated tool to import study metadata from previously implemented studies into a centralized repository; (2) a tool to standardize key study metadata elements to support curation and output to multiple EDC systems; (3) an efficient search engine to identify and group elements into new case report forms; and (4) the ability to output metadata to define a new study in the EDC system along with PDF versions of the case report forms. Study metadata for an EDC system is typically defined in a structured CSV or XML-based format and includes definitions of individual data elements, case re- port form (CRF) structures, and the study event structure. These metadata include the name of the variable, question text, data type, allowable values if categorical or range if numeric, and whether the variable is required. In addition, data elements are grouped into elec- tronic case report forms, where related variables are collected to- gether (e.g., demographics, medications, etc.). These elements were automatically extracted using a connector program and formatted for importing into a central repository. Unfortunately, there will often be uncontrollable developments in clinical trials which result in the occurrence of missed patient visits or unrecoverable outcome measures. However primary ana- lysis is approached, we must make an untestable assumption about the distribution of the unobserved data. It is then import- ant to perform sensitivity analysis to explore the robustness of the conclusions to alternative plausible assumptions about the missing data. In primary analysis it is most often reasonable to assume that con- ditional on any covariates included in the analysis, data is Missing at Random (MAR). Or equivalently, that the conditional distributions of the outcome data for observed and unobserved patients are equal. For sensitivity analysis it may then be of interest to explore the impact of unobserved patients having a poorer/better response than those observed. The so called `Delta- method’ is an accessible pattern-mixture approach which enables such investigation using multiple imputation. Imputations are conducted under MAR, then edited by a postulated amount, Delta, to reflect a worse or better response. The primary analysis model is retained in the sensitivity analysis to asses purely the impact of alternative behaviour among the unobserved on the primary outcome. Background The use of common data elements (CDEs) to standardize data collec- tion in clinical trials is widely viewed as a mechanism to improve effi- ciency in trial design and conduct and to improve the quality of research. CDEs have the potential to enhance the comparability of data from different clinical trials, reduce development time for clinical trial protocols, and significantly reduce the time needed to develop electronic data capture (EDC) systems. There are several challenges to leveraging CDEs effectively on large clinical trial net- works. Such networks typically comprise several collaborating institu- tions that conduct multiple trials, typically over a 5-year funding cycle. Each protocol is developed by a group of investigators in col- laboration with staff from a data coordinating center. Due to the na- ture of this work flow with individual trials developed over a period, it is common for the definitions of individual data elements to evolve over different clinical trials. M th d Incomplete reporting of variance estimates in continuous outcomes by study authors still remains a reality. We proposed a tool that would help authors to determine the influence of a missing trial in the pooled meta-analysis estimate. Current work includes develop- ment of the influence statistic to include random effects models and to the influence of multiple omissions per meta-analysis. References Altman, 2015. doi:10.1186/s13063-015-0575-7 Davey et al., 2011. doi:10.1186/1471-2288-11-160 O61 Sensitivity analysis for missing data using the delta method: the impact of incorporating a prior distribution on delta Suzie Cro1, Michael Kenward2, James Carpenter3 1Imperial College London; 2The London School of Hygiene and Tropical Medicine; 3MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL Correspondence: Suzie Cro Trials 2017, 18(Suppl 1):O61 y Conclusion Our previous efforts to standardize data collection in clinical trials fo- cused on standardization at the CRF level but this approach was often unsuccessful. In contrast, standardization at the data element level, al- lows flexibility across studies while collecting variables in a standard- ized way. The collection of standardized metadata also enables the development of EDC systems for new trials extremely quickly. We show how an information anchored variance estimate is obtained for the treatment estimator when a fixed Delta adjustment is used. However, when uncertainty surrounds the value of Delta and we incorporate a prior distribution on Delta in imputation, there will be less information in sensitivity analysis than primary analysis. Re- sults from a peer review trial demonstrate the consequences sur- rounding the choice of Delta in a real life setting and why careful elicitation is required. A systematic review of measures of informed consent for randomised controlled trials Katie Gillies, Alex Duthie, Marion Campbell University of Aberdeen Correspondence: Katie Gillies Trials 2017, 18(Suppl 1):O63 Trials 2017, 18(Suppl 1):200 Page 212 of 235 g Results The search identified 6669 citations. After screening titles and abstracts, 16 complied with our pre-specified inclusion criteria. The included stud- ies report 16 separate instruments whose aim is to measure an aspect of informed consent for RCTs. Four of the included instruments report development of a tool to assess competence of research subjects to consent to participation in RCTs i.e. was set amongst participants who may have diminished decisional capacity (e.g. early stage Alzheimer’s, schizophrenia, etc.). Of the 16 instruments, 3 explicitly reported a theor- etical or conceptual framework underpinning their development, a fur- ther 3 implicitly refer to the “conceptual dimensions of informed consent” or “principles of research ethics” as informing their develop- ment and 10 reported no guiding theoretical framework. Linked to this, some instruments were explicit with regard to which constructs they were measuring while others were more vague. Finally, only 3 of the 16 studies reported patient or public involvement in the development of the tool. Findings from the narrative synthesis of individual constructs will also be presented. Conclusions SWT d i SWT designs are gaining popularity, but statistical methods for economic evaluation conducted alongside SWTs have not been suffi- ciently explored and used. The use of appropriate methods that ac- count for time, clustering, and correlation between costs and outcomes is an important part of SWT health economics analysis. Thus study aims to demonstrate how such models perform and can be implemented in the context of economic evaluation alongside SWTs. What are the methodological challenges in the design and conduct of orthopaedic randomised controlled trials comparing surgery and non-operative interventions? A systematic review Loretta Davies, Jonathan Cook, Andrew Price, David Beard University of Oxford Correspondence: Loretta Davies Trials 2017, 18(Suppl 1):O65 What are the methodological challenges in the design and conduct of orthopaedic randomised controlled trials comparing surgery and non-operative interventions? A systematic review Loretta Davies, Jonathan Cook, Andrew Price, David Beard University of Oxford Correspondence: Loretta Davies Trials 2017, 18(Suppl 1):O65 Background Objectives 1. To systematically review economic evaluations alongside SWTs to examine statistical methods used to adjust cost and outcome variables for clustering and time effects inherent to the stepped wedge design. 2. To explore theoretical models that combine existing ap- proaches to the joint (bivariate) modelling of cost and effectiveness in cluster randomised trials with multi-level models that have been pro- posed for univariate outcomes in stepped wedge trials. Methods The subjective assessment of informed consent for clinical trials, and the potential difficulties associated with it, has led several studies to develop objective measures of informed consent for clinical trials. These objective measures of informed consent are often specific to a particular population or clinical condition and largely focus on under- standing of (some or all of) the key elements of informed consent, namely: capacity, disclosure, understanding, voluntariness and permis- sion. Many of the developed tools are study-specific, but some vali- dated measures exist. Whether these objective tools conceptualize and measure informed consent in the same way is not known. As such, it is not clear whether meta-analyzing data from studies reporting different tools is worthwhile. The aim of this systematic review was to critically appraise the evidence on the conceptualization and item content of validated questionnaire based measures of informed consent for rando- mised controlled trials. An ad-hoc search strategy of Medline/PubMed, DARE, NHS-EED, HTA and the Cochrane Library was used to identify economic evaluations alongside SWTs or related methodological studies. Abstracts were inde- pendently screened by two investigators using the following inclusion criteria: (i) use of the SWT design and (ii) economic evaluation as part of the research question(s) in the study. Modelling the cost- effectiveness alongside SWTs We propose in which the multi-level model for effectiveness (on a continuous scale) mirrors the multi-level model for cost, with corresponding random effects and error terms in the two models assumed to follow bivariate normal distributions. Results Economic evaluation alongside stepped wedge trials: a systematic review and a proposal of analysis Gian Luca Di Tanna, Vladislav Berdunov, Richard Hooper Pragmatic Clinical Trials Unit, Queen Mary University of London Correspondence: Gian Luca Di Tanna Trials 2017, 18(Suppl 1):O64 the two m Results A total of 100 abstracts identified by the electronic search were screened. A total of 18 papers were included in our review: 15 were study protocols, 1 gave insights on the methods for economic evalu- ation alongside SWTs and 2 papers were trial results. Methods used (or intended to be used) in studies can be grouped into three main categories: mixed/multilevel models, generalized estimating equa- tions and (generic) bootstrap, but there is an underlying vagueness in the models (to be) used given also the limited guidance available. We present the results of a set of simulations exploring the perform- ance of the proposed method under different assumptions related to the number of participants per cluster, number of clusters in each trial arm and values of the ICC. Background Background Surgical randomised controlled trials (RCTs) often present additional challenges in design, conduct and analysis to those evaluating pharma- cological treatments. This is especially evident when the interventions being evaluated are quite different, for example, the comparison of sur- gery with a non-operative intervention such as physiotherapy. Specific methodological issues have been reported such as the impact of treat- ment preferences of both patients and participating clinicians on re- cruitment, difficulties with blinding and standardising interventions. The aim of this study was to systematically review the methodological challenges and limitations in musculoskeletal RCTs which compare a surgical and non-operative interventions (e.g. drug treatment or physiotherapy). M th d Methods A systematic search of the literature was conducted to identify rele- vant articles that described the development, and/or validation, of measures of informed consent for RCTS. General data extraction cat- egories were split into those relating to the context of the included study and those relating to items included in the instrument. Data was synthesized by coding of the items identified into domains and sub-domains which were determined by nomenclature defined in in- cluded studies. Both for descriptions of included studies and of the instruments reported in those studies, descriptive statistics were used to describe general information and instrument detail. A narrative synthesis of the instruments and their inter-related domains and sub domains was conducted to identify areas of both convergence and divergence. Discussion This presentation will discuss the key issues relevant for this work specifically relating to the issues surrounding the heterogeneity of existing measures of informed consent to RCTs. The results from the narrative synthesis will be discussed with explicit considerations re- garding the conceptualization of informed consent and inclusion of constructs and items that matter to potential trial participants. O66 Gatekeeping amongst allied health professionals: recruitment in stem cell trial of recovery enhancement-3 (STEMS-3) feasibility study Claire Diver1, Rebecca O'Connor2, Nikola Sprigg3, Louise Conell4, Marion Walker5, Philip Bath3 1Associate Professor; 2Nottingham University Buisiness School, University of Nottingham; 3Stroke Trials Unit, University of Nottingham; 4Allied Health Research Unit, University of Central Lancashire; 5Division of Rehabilitation and Ageing, University of Nottingham Correspondence: Claire Diver Trials 2017, 18(Suppl 1):O66 Background A search of the MEDLINE and CENTRAL databases between 2010 and 2015 was conducted. Randomised orthopaedic clinical trials which had a surgical and non-operative comparison such as physiotherapy were included. Data were extracted and summarised on the study characteristics such as target and actual recruitment, adherence to randomisation allocation, and in addition reported challenges to The stepped wedge trial (SWT) design has gained popularity in recent years and may have practical and methodological advantages over the classic parallel group design especially in the context of health services research. The use of statistical methods in economic evaluations con- ducted alongside SWTs have hitherto not been systematically explored. Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 213 of 235 successfully conducting the trial amongst other details. A quality as- sessment was also conducted using the Cochrane Risk of Bias tool. R l successfully conducting the trial amongst other details. A quality as- sessment was also conducted using the Cochrane Risk of Bias tool. Results Background g Frequent data collection is desirable in randomised controlled trials (RCTs) where time-to-event is the primary outcome, in order to ac- curately capture the event of interest with reduced risk of recall bias. However, an inevitable problem with repeated data collection is missing data caused by participants’ loss to follow-up prior to the oc- currence of the event or the end of the planned follow-up period. This undermines the Intent-To-Treat (ITT) principle, leads to reduced power, and possible bias. Analytical strategies for drawing inferences from incomplete data, including the non-informative censoring for time-to-event data, rely on untestable assumptions about the miss- ingness mechanism. Therefore, it is key to minimise the chance of dropouts at the design stage. Postal or electronically-sent question- naires are not ideal for collecting frequent follow-up data as they often yield poor response rates even after reminder mailings. Short message service (SMS) using mobile telephones might offer a new way to enhance real-time outcome data collection. We describe the use of SMS to obtain weekly data on recovery of sciatica patients in the SCOPiC trial, describe the response patterns in the short and lon- ger term and consider the implications for data analysis. Method Results Six NHS staff were recruited: 3 physiotherapists, 1 Occupational ther- apist, 1 speech and language therapist and 1 specialist mental health nurse. 3 main themes were identified: information, eligibility and intervention. AHPs felt informed about the trial and professional re- sponsibility to recruit to clinical trials including STEMS-3; they consid- ered the regular presence of a member of the trial team a valuable resource. Despite clear and broad inclusion criteria, AHPs were reluc- tant to approach eligible stroke survivors with communication, cogni- tive and low mood problems. A strong sense of duty of care to patients stemmed from perceptions that: the intensive physiotherapy intervention provided in the trial contradicted CST goals of self- management and independence; randomisation to the control group with no physiotherapy could not be blinded and might distress pa- tients with expectations of further physiotherapy; patients might have misplaced expectations of G-CSF providing a 'miracle cure’; and wanting to identify patients that would benefit from further treatment. In total, 6484 records were identified and 54 trials were included in the review. The majority of included studies were of fracture man- agement (28, 52%). Twenty three (43%) were multi centre studies, with a sample size of 85 (median, IQR [50,150]). The main interven- tion comparisons of the included studies were of surgery and physio- therapy interventions (44, 81%). Methodological problems identified included a high proportion of participant crossover from their rando- mised intervention and difficulties reaching target sample size. Chal- lenges reported by the authors included the impact of both patient and clinician preference for particular treatment on the number of potentially eligible patients recruited. In addition, various practical challenges were reported related to the nature of the interventions evaluated and health care systems in which the research was being conducted. This included difficulties implementing blinding of patients, care providers, and outcome assessors and post-randomisation delays in receiving an intervention. O67 The use of regular text messaging over one year to collect primary outcome data in a randomised controlled trial Reuben Ogollah, Martyn Lewis, Kika Konstantinou, Sarah Lawton, Jamie Garner, Nadine E. Foster Keele University Claire Diver1, Rebecca O'Connor2, Nikola Sprigg3, Louise Conell4, Marion Walker5, Philip Bath3 Claire Diver1, Rebecca O'Connor2, Nikola Sprigg3, Louise Conell4, Marion Walker5, Philip Bath3 1Associate Professor; 2Nottingham University Buisiness School, University of Nottingham; 3Stroke Trials Unit, University of Nottingham; 4Allied Health Research Unit, University of Central Lancashire; 5Division of Rehabilitation and Ageing, University of Nottingham Correspondence: Claire Diver Trials 2017, 18(Suppl 1):O66 1Associate Professor; 2Nottingham University Buisiness School, University of Nottingham; 3Stroke Trials Unit, University of Nottingham; 4Allied Health Research Unit, University of Central Lancashire; 5Division of Rehabilitation and Ageing, University of Nottingham Correspondence: Reuben Ogollah Trials 2017, 18(Suppl 1):O67 g Conclusion Gatekeeping was evident throughout the recruitment process and re- flects possible tensions between research and clinical priorities. AHPs act to promote patient autonomy in their transition from acute stroke care and rehabilitation to self-management. This contrasts with protective behaviours evidenced in identifying potential pa- tients for clinical trials and denying some eligible patients the oppor- tunity for participation. The motivation for this was “acting” in the patient’s best interests: this manifested itself in presenting the trial as the potential opportunity for further treatment when patients were discharged or not discussing the trial when they thought patient ex- pectations of treatment were too high, or patients would not under- stand the implications of no active treatment allocation. Training of AHPs in study design issues should be considered especially when they have an active role in recruitment and delivery. This review demonstrated that trials which compare a surgical and non-surgical interventions have been successfully conducted for a number of conditions, however, several methodological issues were identified that have the potential to impact on the design, conduct and analysis of these trials. If not adequately ad- dressed, these issues may introduce significant bias and threaten the validity of the trial results. The planning and design of future studies should take into account and consider the specific chal- lenges associated with the evaluation of surgical and non-surgical interventions. Background Randomised controlled trials (RCTs) of rehabilitation in the chronic stroke population are complex and challenging. Patient-therapist relationships within a rehabilitation setting can be strong and powerful. This is associated with treatment satisfaction in the trad- itional healthcare setting but it is unclear if this could impact on re- cruitment to clinical trials. The aims of clinical practice and clinical research can be different: clinical practice is focused on individual care and research on population benefit. Allied Health Professionals (AHPs) are known to be reluctant to recommend oncology and pal- liative care patients to clinical trials through well intentioned as- sumptions that it might not be in the best interests of the individual patient. This attitude has not been explored in chronic stroke rehabilitation trials. STEMS3 (ISRCTN16714730) was a feasibil- ity, 2x2 factorial randomised controlled trial of granulocyte colony stimulating factor (G-CSF) vs placebo, and community stroke physiotherapy vs none, in stroke survivors discharged from rehabili- tation services. ISRCTN1671470. The aim of this study was to ex- plore recruitment by AHPs to STEMS-3. Gatekeeping amongst allied health professionals: recruitment in stem cell trial of recovery enhancement-3 (STEMS-3) feasibility study O67 The use of regular text messaging over one year to collect primary outcome data in a randomised controlled trial Reuben Ogollah, Martyn Lewis, Kika Konstantinou, Sarah Lawton, Jamie Garner, Nadine E. Foster Keele University Correspondence: Reuben Ogollah Trials 2017, 18(Suppl 1):O67 p y Conclusion Collecting frequent follow-up outcome data with SMS is feasible in an RCT and provides high response to both short and longer term follow-up. This could be an additional and/or alternative strategy to collecting data in large pragmatic trials, and is particularly useful for collecting regular primary outcome data, which is key to time-to- event and pragmatic ITT-evaluation. Methods Sonia Davis, Hengrui Sun, Kwanhye Jung University of North Carolina Correspondence: Sonia Davis Trials 2017, 18(Suppl 1):O70 Inverse Probability-of-Treatment Weighting (IPTW) is a general meth- odology for removing treatment-adjustment bias. Working under the hypothesis of *No Unmeasured Confounding*, it creates a pseudo- population by weighting each patient with the inverse probability of observing a certain treatment administration given the past treat- ment and toxicity history. However, a review of data collected from RCTs on osteosarcoma suggests that treatment side-effects may not be sufficiently well-documented. The compass study - dynamic generation of patient-specific ecare plans in a pragmatic trial p p g Scott Rushing, Pamela W. Duncan, Rica M. Abbott Wake Forest Baptist Health Correspondence: Scott Rushing Trials 2017, 18(Suppl 1):O69 p p g Scott Rushing, Pamela W. Duncan, Rica M. Abbott Wake Forest Baptist Health Correspondence: Scott Rushing Trials 2017, 18(Suppl 1):O69 p p g Scott Rushing, Pamela W. Duncan, Rica M. Abbott Wake Forest Baptist Health Correspondence: Scott Rushing Trials 2017, 18(Suppl 1):O69 Causal inference with randomised clinical trials of chemotherapy: the importance of well-documented treatment side-effects Carlo Lancia1, Cristian Spitoni2, Jakob Anninga3, Jeremy Whelan4, Matthew R. Sydes5, Gordana Jovic5, Marta Fiocco1 1Leiden University Medical Center; 2University Medical Center Utrecht; 3Radboud University Medical Center; 4University College London Hospital; 5MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL Causal inference with randomised clinical trials of chemotherapy: the importance of well-documented treatment side-effects Carlo Lancia1, Cristian Spitoni2, Jakob Anninga3, Jeremy Whelan4, Matthew R. Sydes5, Gordana Jovic5, Marta Fiocco1 1Leiden University Medical Center; 2University Medical Center Utrecht; 3Radboud University Medical Center; 4University College London Hospital; 5MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL The COMPASS Study is a pragmatic, cluster-randomized trial of 41 hospitals in North Carolina designed to determine the effectiveness of a comprehensive model of post-acute stroke care (i.e. the COM- PASS intervention) compared with usual care (Control). p The COMPASS intervention includes activities such as: a follow-up telephone call two days after having been discharged; a 7–14 post- acute stroke clinic evaluation and take a patient-reported functional assessment; and generation of a individualized patient-specific care plan that is clearly discussed by the clinical provider with the patient and caregiver; and follow up phone calls 30, 60, and 90 days post discharge. Correspondence: Carlo Lancia Trials 2017, 18(Suppl 1):O68 Background Opposite to the *intention-to-treat approach*, a statistical analysis based on actual treatment data might be problematic due to the presence of the so-called *treatment-adjustment bias*. Exposure to chemotherapy agents may in fact be reduced and/or delayed as a consequence of previous-treatment side-effects. In particular, both reductions and delays contribute to lowering the value of the so-called Received Dose Intensity. Introduction Randomised Controlled Trials (RCTs) are universally considered as the most reliable way to demonstrate and assess causal relationships between treatments and outcome; science-based medicine is rooted in them. Spurious relationships between the outcome and a time- fixed treatment-variable are eliminated by randomising patients over two or more arms of the trial. Hence, the randomisation procedure initiates the process by which treatment and outcomes of interest should be interpreted in a causal way. However, treatment is not al- ways administered as intended, not least because of the occurrence of side effects and adverse events. In RCTs of chemotherapy, for ex- ample, the treatment administered may differ from the intended one because of the application of either cycle delays or dose reductions. In order to efficiently implement COMPASS in participating hospitals, Wake Forest Baptist Health developed a web-based application that manages assessment collection, generates a series of helpful reports for the clinical provider and most importantly, uses a series of propri- etary decision algorithms to dynamically in real time generate a pa- tient specific care plan that identifies current health concerns for a patient, coaches the patient and family to access resources and to self manage health behaviors to improve recovery, independence, and health. In this presentation, we will describe and demonstrate the COMPASS web-based application including the dynamic generation of patient specific care plans. Implementation of this care plan is a model of value based clinical care, meeting numerous CMS quality metrics. p p Results In total, 90% (n = 283) of participants opted for SMS follow-up and 10% (n = 31) for phone calls. There have been 4,299 valid responses via SMS out of the expected 4,787 (90% response rate), compared to 74% (417/567) telephone call responses. The median (IQR) weekly re- sponse rate over the first 16 weeks was 93% (90%, 95%) and 75% (71%, 77%) for texts and calls, respectively. There was no evidence of decrease in weekly response rate over time (i.e. pattern of missing- ness was intermittent). The median response rate for months 5 to 12 was 70% (62%, 80%) for SMS and 67% (63%, 82%) for telephone calls. 190/283(67%) and 3/31(10%) participants completed 100% of the expected texts and calls, respectively. 243/283 (86%) and 20/31 (65%) participants completed 80% texts and telephone calls, respectively. Using data from Medical Research Council trial BO06 (European Organisation for Research and Treatment of Cancer trial 80931) we will illustrate the use of IPTW and Marginal Structural Models (MSMs) for estimating the causal effect of dose reductions on Event-free Survival (EFS). The use of IPTW and MSMs allows to move beyond intention-to-treat and unbiasedly estimate the effect of treatment modifications on EFS. Conclusions We demonstrate that, even with complex and entangled data such as those collected in a RCT of chemotherapy, constructing and estimating a causal model is possible, provided that side-effects are well docu- mented. When this is not the case, the removal of treatment- adjustment bias via IPTW might be problematic, if not prevented at all by the presence of unmeasured confounder. As such, good-quality toxicity data should be regarded as important enablers of causal mod- elling in RCTs of chemotherapy. p Method Q li i Qualitative interview study underpinned by theoretical perspectives of pragmatism and critical reflection. We used a purposive sampling strategy to identify AHP’s working in a community stroke team (CST) involved in participant identification. Semi-structured interviews were conducted, recorded and transcribed verbatim. Thematic analysis using an iterative and emergent approach was adopted. We used data from 314 participants randomised to date (target recruit- ment of 470) to a trial that is testing the clinical and cost-effectiveness of stratified care versus usual care for patients with sciatica in primary care (SCOPiC Trial; HTA 12/201/09) in order to evaluate weekly response rates to SMS. The primary outcome measure is patient-reported time to resolution of sciatica symptoms (defined as completely recovered/ Trials 2017, 18(Suppl 1):200 Page 214 of 235 much better) measured on a six-point global perceived change scale, collected using regular SMS (with the alternative of brief phone calls for those where text messaging is not possible or missed) for the first 16 weeks for all participants, and thereafter monthly up to month 12, or until stable resolution of symptoms (defined as two consecutive SMS responses of patient-reported resolution). much better) measured on a six-point global perceived change scale, collected using regular SMS (with the alternative of brief phone calls for those where text messaging is not possible or missed) for the first 16 weeks for all participants, and thereafter monthly up to month 12, or until stable resolution of symptoms (defined as two consecutive SMS responses of patient-reported resolution). The pseudo-population created by IPTW has the following two properties: The pseudo-population created by IPTW has the following two properties: 1. Pseudo-patients’ past toxicity-history no longer predicts exposure to chemotherapy in the next cycle; 2. The causal effect of treatment modifications on outcome is the same in both original and pseudo- populations. g y Conclusions Guidance is required to improve the consistency of selection, analysis and reporting multidimensional harm data in clinical trials. The use of online supplementary appendices for journals provide an oppor- tunity to include more detailed information on AEs but this should not negate the inclusion of a useful harm profile summary in the main paper. Continue or stop: the rationale for early closedown of a clinical trial as a result of stopping rules within the data monitoring committee charter Lisa Woodhouse1, Jason P. Appleton1, Lelia Duley1, Ian Ford2, Didier Leys3, Stuart Pocock4, Nikola Sprigg1, Catherine Sudlow5, Matthew Walters2, Philip M. Bath1 Background g Data Monitoring Committees (DMCs) are charged with evaluation of accumulating patient safety data in ongoing clinical trials. Periodic review involves scrutinizing rates of adverse event (AE) coded terms, Trials 2017, 18(Suppl 1):200 Page 215 of 235 as well as trends and rates of abnormal values of clinical laboratory, ECG, vital sign and other assessments. DMC members of varying backgrounds complete their review in a brief time prior to and during the DMC meeting. Missing data from early drops-outs due to adverse event in the active treatment group or lack of efficacy in pla- cebo group, plus missing at random (MAR) data caused by ongoing participants who have not yet had an assessment or experienced an event can impede data interpretation. Data reports that accurately and clearly display trends are essential for the identification of poten- tial safety concerns. Both reviewability and statistical rigor are crucial, yet most common reporting formats used in manuscripts or study re- ports for completed studies lack both. Lessons to learn from the reporting of adverse events (AEs) in randomised controlled trials published in high impact journals Rachel Phillips1, Victoria Cornelius2 1University of Nottingham; 2University of Glasgow; 3University of Lille; 4London School of Hygiene & Tropical Medicine; 5University of Edinburgh Correspondence: Lisa Woodhouse Trials 2017, 18(Suppl 1):O72 1University of Nottingham; 2University of Glasgow; 3University of Lille; 4London School of Hygiene & Tropical Medicine; 5University of Edinburgh Results The reporting methods were implemented and refined through an unblinded independent statistical analysis center (ISAC) for a DMC of a program of phase II/III pharmaceutical clinical trials. Reviewability is substantially improved by maximizing the use of graphical displays at both the treatment group and patient level. Adverse events rates are accurately portrayed through incidence densities. We present best practices for graphical displays of disposition, AE rates, individ- ual event profiles, assessment trends and abnormal values. All dis- plays provide transparency of the ongoing or early discontinuation status of the participant and are producible by SAS. DMC members have assisted with further display refinement and have enthusiastic- ally received these methods. Results We identified 189 eligible trial reports (Lancet n = 62; BMJ n = 4; NEJM n = 85; and JAMA n = 38). AEs that cause patients to discon- tinue treatment or withdraw from the study are useful indicators of severity and impact to patients. We found that 56% reported the number of withdrawals/discontinuations due to AEs by treatment arm but only 14% included information on which AEs caused with- drawal. Only a quarter of reports included information on the sever- ity of specific AEs, with several studies instead using severity to select events to report e.g. only reporting those of grade 3–5. Assess- ment of disproportionalities of AEs between arms was predominantly undertaken by means of tabulations, including frequencies (90%) and percentages (80%), allowing for informal comparisons. Despite a lack of power to undertake formal hypothesis testing of event counts, 39% of studies reported p-values and many studies included statements such as “no statistical differences found” to summarise the harm profile. Several studies only included AE data in online sup- plementary material. Continue or stop: the rationale for early closedown of a clinical trial as a result of stopping rules within the data monitoring committee charter 1 1 1 2 Lisa Woodhouse1, Jason P. Appleton1, Lelia Duley1, Ian Ford2, Didier Leys3, Stuart Pocock4, Nikola Sprigg1, Catherine Sudlow5, Matthew Walters2, Philip M. Bath1 Lisa Woodhouse1, Jason P. Appleton1, Lelia Duley1, Ian Ford2, Didier Leys3, Stuart Pocock4, Nikola Sprigg1, Catherine Sudlow5, Matthew Walters2, Philip M. Bath1 Background The international Triple Antiplatelets for Reducing Dependency after Ischaemic Stroke (TARDIS) trial assessed the safety and efficacy of in- tensive (combined aspirin, dipyridamole and clopidogrel) versus guideline (aspirin/dipyridamole, or clopidogrel alone) antiplatelets given for one month in patients with acute stroke or transient ischae- mic attack (TIA). The primary outcome was the incidence and severity of any recurrent stroke or TIA within 90 days. Recruitment com- menced on 7 April 2009 but was halted on 18 March 2016, with a total enrolment of 3096 participants (of a planned target of 4100, 75.5%), on the advice of the independent Data Monitoring Commit- tee (DMC). h d The harm profile of a drug is developed through a cumulative process incorporating information from preclinical through to post-marketing studies. An integral part of this process is the information elicited from randomised clinical trials (RCTs), as while RCTs are often not large enough to signal rare adverse drug reactions, they provide unbiased estimates of harm effects and provide a controlled comparison allow- ing causality to be evaluated. Many studies have demonstrated that the reporting of adverse events (AEs) in published trial reports is often inadequate, this is despite the gathering of high quality AE data re- quired by regulators. We undertook a systematic review to ascertain current approaches to the collection, selection, analysis and presentation of AEs in RCTs published in high impact journals. We identify examples of good practice and provide recommendations for future practice. Methods p Methods Methods In this talk we present data reporting strategies that best help a DMC evaluate safety signals through (a) clear graphical displays, and (b) identification and adjustment for potentially differential rates of missing data caused both by early discontinuations and ongoing study participants. A simulation of typical DSMB data is used to dem- onstrate the impact of differential discontinuation on adverse event incidence rates compared to incidence densities. We will also present exemplary examples of AE reporting, pre- specified analysis methods for emerging AEs, criteria used to select which AEs to report and classification used, if any, to group AEs e.g. organ system. Background There is limited research and literature on the trial management challenges encountered in running adaptive platform trials. This trial design allows both (i) seamless addition of new research compari- sons when compelling clinical and scientific research questions emerge, and (ii) early stopping of accrual to individual comparisons that do not show sufficient activity without affecting other active comparisons. FOCUS4 (colorectal cancer) and STAMPEDE (prostate cancer), run by the MRC CTU at UCL, are two leading UK examples of clinical trials implementing adaptive platform designs with biomarker stratified and non-stratified comparisons all within one protocol. g In 2012 National Institute of Health Research funded the By-Band- Sleeve study (BBS), a multi-centred, pragmatic trial comparing the ef- fectiveness and cost effectiveness of three surgical interventions for weight-loss: the gastric bypass, the adjustable gastric band and the sleeve gastrectomy (Byrne et al. 2015). This was anticipated to be a “hard to recruit” study with the goal of recruiting 1,341 patients. The QuinteT Recruitment Intervention (QRI), a method of understanding recruitment and responding to trial recruitment difficulties (Donovan et al., 2016), was integrated with BBS. Over the last three years the QRI has informed recruitment by identifying issues with patient path- ways, improving patient information leaflets and education sessions and providing regular feedback to surgeons and nurses. With 570 pa- tients randomised, BBS is at an advanced stage of recruitment. Methods Adaptive platform design trials offer many potential benefits over traditional trials, from faster time to accrual to contemporaneously recruiting multiple research comparisons, added flexibility to focus on more promising research comparisons via pre-planned interim analyses and potentially shorter time to primary results. To date, STAMPEDE has added four new research comparisons, closed two following pre-planned interim analysis (lack-of-benefit) and com- pleted recruitment to six research comparisons. FOCUS4 has closed one research comparison following pre-planned interim analysis (lack-of-benefit) and added one new research comparison, with a number of further comparisons in the pipeline. The QRI methods were employed by a) incorporating key findings from the internal pilot phase into the (pre-recruitment) training pro- vided for the new centres in the main phase and b) concurrently ini- tiating the QRIs in the new centres to identify challenges specific to each centre. Discussion Discussion The DMC reviewed unblinded data in confidence every 6 months. Overall, the DMC met on 13 occasions and recommended trial con- tinuation for all but the last data review. The recommendation to stop was based on a combination of three observations: (i) although intensive antiplatelet therapy was associated with a non-significant reduction in the primary outcome, a conditional power futility ana- lysis suggested the trial was highly unlikely to ever demonstrate a significant difference in the primary outcome; (ii) the presence of a significant increase in major (including fatal) bleeding in participants randomised to intensive antiplatelet therapy (this increase was not present during the start-up phase as per the pre-defined stopping rules); and (iii) overall neutral findings with no difference in the net balance of death, stroke, myocardial infarction and major bleeding. The Trial Steering Committee (TSC) reviewed the same data (and subsequent additional analyses) and agreed that the trial should stop recruitment and complete follow-up on the basis of futility. Specific operational challenges in these platform trials, additional to those in traditional two-arm trials, were identified that should be considered when setting up an adaptive platform trial. During set-up of an adaptive platform trial, it is important to carefully consider the practicality of the protocol structure (modular versus single protocol), the longevity and continuity of trial oversight com- mittees and having clear clinical and scientific criteria for the addition of new research comparisons. The common challenges in introducing a new research comparison were linked to the need for fast development timelines and a continuous training and communi- cation programme with sites to ensure the protocol is clearly under- stood and followed. Early stopping of a research comparison due to lack-of-activity also pre- sents operational challenges, specifically when planning for stopping to recruitment is balanced with management of ongoing comparisons and sometimes the development of new research comparisons. Conclusions Even though the DMC charter for TARDIS did not include stopping rules for futility, it was agreed by both the DMC and TSC to stop the trial on this basis. DMCs may need to stop such trials in certain cir- cumstances, as seen here. In our experience, the benefits arising from rapid acceleration of re- cruitment and shortened time to primary results outweighs the oper- ational challenges. O74 Maintaining recruitment and informed consent in the advanced stages of a trial: the by-band-sleeve study MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL; MRC London Hub for Trials Methodology Research, London, UK g y y Paul Whybrow, Sangeetha Paramasivan, Jane M Blazeby, Chris Rogers, Jenny Donovan, on behalf of By-Band Sleeve U i it f B i t l Correspondence: Francesca Schiavone Trials 2017, 18(Suppl 1):O73 y Correspondence: Paul Whybrow Trials 2017, 18(Suppl 1):O74 ( ) Methods The DMC for the TARDIS trial followed a pre-defined charter, which included stopping rules for safety and efficacy. The DMC charter did not include any stopping rules for futility. With respect to safety, the DMC charter listed the following reasons for recommending early stopping of the trial: the primary outcome favoured the control group, P < 0.01 (nominal, 2-sided); the combined outcome of fatal or non-fatal stroke or major bleeding favoured the control group, P < 0.01 (nominal, 2-sided); the overall rate of symptomatic intracranial haemorrhage exceeded 2%; that during the start-up phase, major bleeding favoured the control group, P < 0.01 (nominal, 2-sided). For efficacy, the DMC was to conduct formal interim analyses, after 40% and 70% of the target number of participants had had their 90 day outcome assessed, based on whether the combined outcome of fatal or non-fatal stroke or major bleeding event favoured the control group, P < 0.001 (2-sided). Before making any decision, the DMC was Original phase II-IV studies looking at the efficacy/effectiveness of an intervention published in the Lancet, British Medical Journal (BMJ), New England Journal of Medicine (NEJM) and Journal of the Ameri- can Medical Association (JAMA) in the period September 2015 to September 2016 (inclusive) were included. RCTs where the interven- tion was non-medicinal and where harm/s was the primary outcome were excluded. We manually searched the archives of each journal for original reports of RCTs. Using a standardised, pre-piloted, 60-point checklist we extracted data on trial characteristics, methods of collection, assessment of se- verity and causality, selection criteria for reporting, analysis methods and presentation of harm data. Descriptive analysis was performed. Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 216 of 235 Page 216 of 235 asked to consider the overall internal and external evidence, the multiplicity of testing and the possibility that trends in the data might be reversed with longer follow-up or increased recruitment. Results the addition of new research comparisons and stopping recruitment following pre-planned interim analysis were considered in our evaluation. This is a platform alteration: a trial management perspective on the operational aspects of adaptive platform trials O74 Maintaining recruitment and informed consent in the advanced stages of a trial: the by-band-sleeve study Paul Whybrow, Sangeetha Paramasivan, Jane M Blazeby, Chris Rogers, Jenny Donovan, on behalf of By-Band Sleeve University of Bristol Correspondence: Paul Whybrow Trials 2017, 18(Suppl 1):O74 Background This involved thematic analysis of a purposive sample of 198 audio-recorded consultations from 11 centres, observations of education sessions where details of the surgical procedures were ex- plained to patients and analysis of screening log data. Findings were used to deliver group and individual feedback on recruitment across the BBS sites. Centre reviews, similar to those used in previous stud- ies (ProtecT trial), were undertaken for the centres with particularly low recruitment. We share here our experiences from a trial management perspective, highlighting the challenges and successes. (Note: our lessons learnt from a central data management perspec- tive are reported in our companion abstract: “Changing platforms without stopping the train: A Data Management Perspective on the operational aspects of adaptive platform trials”). h d Discussion Careful planning is paramount when making all changes to ensure that day-to-day running is not affected and imple- mentation of all changes is time-effective and efficient. Adaptive platform trials offer an efficient model to run randomised controlled trials and we are continuing to work to reduce further the effort re- quired from an operational perspective. O73 This is a platform alteration: a trial management perspective on the operational aspects of adaptive platform trials Francesca Schiavone, Riya Bathia, Krishna Letchemanan, Lindsey Masters, Louise Brown, Claire L. Amos, Anna Bara, Mahesh KB Parmar, Matthew R. Sydes MRC Clinical Trials Unit at UCL, Institute of Clinical Trials and Methodology, UCL; MRC London Hub for Trials Methodology Research, London, UK Correspondence: Francesca Schiavone Trials 2017, 18(Suppl 1):O73 Methods A systematic review was undertaken in seven electronic bibliographic databases including MEDLINE and CENTRAL, using terms relating to the device and assessment of adherence. Included were all types of trial, observational study, and case study/series prescribing a removable de- vice after trauma or surgery. Devices were eligible for the review if they were removable by the patient, applied to a bone or joint of the appen- dicular skeleton, and provided full or partial immobilisation. Studies with children or animals published before 1990 were not included. Screening and data extraction were conducted with standardised online forms developed by the researchers. Screening was conducted in full by two reviewers and discrepancies were resolved by two further reviewers. Data were summarised by study design for injury type, device, and methods for assessment of adherence. We scrutinised the papers to understand how authors valued adherence within the context of their study. The value of the methods to future research was considered. Results A systematic review was undertaken in seven electronic bibliographic databases including MEDLINE and CENTRAL, using terms relating to the device and assessment of adherence. Included were all types of trial, observational study, and case study/series prescribing a removable de- vice after trauma or surgery. Devices were eligible for the review if they were removable by the patient, applied to a bone or joint of the appen- dicular skeleton, and provided full or partial immobilisation. Studies with children or animals published before 1990 were not included. Data are remotely entered into the EDC system by staff at participat- ing sites and are immediately available for review at ICR-CTSU. How- ever, only single forms can be opened in the EDC system at a time, making checks across several forms difficult. The data review soft- ware can be programmed to extract data from several forms for mul- tiple trial visits across different participants. Reports are created, validated and saved to be run at required time points to extract live data, so irregularities can be identified and queried. This is now the principle method of data verification and validation for EDC trials at ICR-CTSU. A data management plan (DMP) is developed for each trial and in- cludes a comprehensive list of the data points which require verifica- tion. Data management checks can be grouped and programmed into reports produced using data review software. Background Wi h h i With the introduction of remote electronic data capture (EDC) at ICR- CTSU in 2012, a new process for data management was required to detect data discrepancies that would previously have been identified during central data entry. Use of a data review program was intro- duced in 2015. This software is now in use for several EDC trials, ?A3B2 show $132#?>necessitating a standard approach. Implementation Use of data review software for data management Use of data review software for data management Leona Batten, Deborah Alawo, Lisa Jeffs, Charlotte Friend, Joanna Illambas, Sharon Ereira, Carolyn McNamara, Claire Snowdon, Judith Bliss, Emma Hall The Institute of Cancer Research Correspondence: Leona Batten Trials 2017, 18(Suppl 1):O75 Use of data review software for data management Leona Batten, Deborah Alawo, Lisa Jeffs, Charlotte Friend, Joanna Illambas, Sharon Ereira, Carolyn McNamara, Claire Snowdon, Judith Bliss, Emma Hall The Institute of Cancer Research Correspondence: Leona Batten Trials 2017, 18(Suppl 1):O75 Judith Bliss, Emma Hall The Institute of Cancer Research Correspondence: Leona Batten Trials 2017, 18(Suppl 1):O75 O76 Understanding methods for the assessment of patient adherence: a case study in removable devices for the improvement of trial outcome data The By-Band-Sleeve study is a complex, multi-centred and pragmatic trial within a changeable clinical environment. The value of the QRI in the By-Band-Sleeve RCT has been in transforming recruitment in a difficult surgical RCT. More importantly, the flexible, iterative and re- sponsive approach has helped to sustain recruitment and address emergent challenges. Background and Aims Patient adherence to treatment is a key determinant of outcome for healthcare interventions. Nonadherence likely influences thera- peutic outcomes and, if unmeasured, introduces uncertainty into trial data. Whilst nonadherence has been well evidenced in settings such as drugs therapy, there is a paucity of information regarding patient adherence to rehabilitative or “removable” devices, such as splints and braces, whereby unscheduled removal may lead to irre- versible deterioration of the injured structure. The aim of this study was to identify and summarise the methods reported in the litera- ture for assessing patient adherence to removable devices to inform further research in this area and improve the quality of outcomes. O76 Understanding methods for the assessment of patient adherence: a case study in removable devices for the improvement of trial outcome data Zoe Tolkien1, Shelley Potter1, Dan Yeomans2, Abhilash Jain3, James Henderson4, Jane M. Blazeby1 1University of Bristol; 2John Radcliffe Hospital; 3University of Oxford; 4North Bristol NHS Trust Correspondence: Zoe Tolkien Trials 2017, 18(Suppl 1):O76 O76 Understanding methods for the assessment of patient adherence: a case study in removable devices for the improvement of trial outcome data Zoe Tolkien1, Shelley Potter1, Dan Yeomans2, Abhilash Jain3, James Henderson4, Jane M. Blazeby1 1University of Bristol; 2John Radcliffe Hospital; 3University of Oxford; 4North Bristol NHS Trust Correspondence: Zoe Tolkien Trials 2017, 18(Suppl 1):O76 Methods Higher priority checks, such as those related to eligibility criteria, safety data and endpoint data, are programmed first. A total of 1655 de-duplicated records were screened for eligibility, resulting in 28 studies in the analysis (RCT = 7; Comparative study = 4; Non-comparative study = 17). The most frequently employed methods of adherence assessment were non-validated participant self-completion questionnaires and attendance at follow up appoint- ments, with four studies using multiple methods (RCT = 3; Non- comparative = 1). More complex methods were used by one study only, i.e. sensors. The association between patient adherence and pa- tient outcomes was evaluated by 13 studies (RCT = 2; Comparative = 1; Non-Comparative = 10) with the majority of studies reporting poorer outcomes with poorer adherence. Three studies inferring an association between outcome and adherence in the discussion pre- sented neither methods nor data for the assessment of adherence. Reasons for non-adherence were reported by only nine studies (32%), most commonly citing discomfort as the cause. Conclusions Filters can be built into reports to select specific patient subsets such as treatment cohorts. Reports can be programmed so that cells or lines are highlighted, to identify data that are outside of an expected range. Reports are saved within the system according to a standar- dised naming convention. Complex reports which filter or extract data from multiple forms are validated to ensure accuracy. Upon a change to the EDC system, reports may need to be altered and re- validated. Report specification details are documented and reports are run ac- cording to priority. A log is completed to track when reports have been run. Report outputs can be exported to Microsoft Excel for further manipulation. Advanced filtering techniques can be used to distinguish new data from previously checked data. Report specification details are documented and reports are run ac- cording to priority. A log is completed to track when reports have been run. Report outputs can be exported to Microsoft Excel for further manipulation. Advanced filtering techniques can be used to distinguish new data from previously checked data. Following the identification of data discrepancies, queries are raised in the EDC system and are available for immediate review by sites. Conclusion Findings Methods g The QRI identified new and unforeseen barriers to recruitment in each centre. Findings show how changes to staff, or changes to the organ- isation of patient care, present unexpected obstacles to recruitment. We systematically evaluated the operational aspects of making changes to these adaptive platform trials and identified both common and trial- specific challenges. The operational steps and challenges linked to both Trials 2017, 18(Suppl 1):200 Page 217 of 235 Recruiters reported perceived changes in patient preferences or shifts in opinion within the broader clinical community. These issues under- mined recruiters’ confidence and ability to approach patients and obtain informed consent, bringing individual discomforts or conflicts about recruitment to the surface. Centre reviews were helpful in identi- fying where training and intervention could be most effective. Conclusion programmed and validated. Once in place the programmed checks are easy to run and streamline data management. programmed and validated. Once in place the programmed checks are easy to run and streamline data management. pp Conclusions Owing to information governance and issues with timeliness of ac- cess associated with electronic datasets, self-reported methods may currently be the preferred option. In particular, patient self-report is necessary if the study perspective encompasses costs borne by the patient themselves. However, hospital data are relatively easy to ob- tain electronically; in trials where secondary care constitutes a major cost driver, detailed electronic data may be considered superior to self-reported methods. Taking the redcap data management tool to the next step. Reducing complexity in study design by smoother integration of external randomisation services thus providing a more streamlined experience for users experience for users Allan Walker, Garry Milne University Of Edinburgh Clinical Trials Unit Correspondence: Allan Walker Trials 2017, 18(Suppl 1):O79 O77 O77 Self-reported and electronic resource use for trial-based economic evaluations: the current state of play in England and considerations for the future Matthew Franklin1, Joanna Thorn2 1University of Sheffield; 2.University of Bristol Correspondence: Matthew Franklin Trials 2017, 18(Suppl 1):O77 O77 Self-reported and electronic resource use for trial-based economic evaluations: the current state of play in England and considerations for the future Matthew Franklin1, Joanna Thorn2 1University of Sheffield; 2.University of Bristol Correspondence: Matthew Franklin Trials 2017, 18(Suppl 1):O77 Background Matthew Franklin1, Joanna Thorn2 1University of Sheffield; 2.University of Bristol Correspondence: Matthew Franklin Trials 2017, 18(Suppl 1):O77 Qualitative research is often used to inform the development of com- plex interventions in health care. However, study designs rarely use a phased approach to fully understand the chain of events in any healthcare process, and tend to rely on a small and predictable set of data collection methods and qualitative analytical frameworks, which may not be best suited to either the research questions or data collected. We used a multimethod qualitative approach, tailored to each phase, research question, and data type to inform the devel- opment of a complex intervention to support patients and their clini- cians when considering treatment options following a diagnosis of advanced lung cancer (The PACT study). Background Randomised controlled trials provide an opportunity to examine patient-level resource-use data to assess the costs associated with an intervention. Combined with outcome data in an economic evalu- ation, these measurements gauge the value for money that a particu- lar intervention represents. Typically, researchers develop a bespoke questionnaire on a trial by trial basis to ask patients about their re- source use. However, the advent of electronic data sources that are routinely collected within the health service has opened up new op- portunities for collecting resource-use data without burdening patients. Results Stages I to IV have been completed and stage V is currently under- way. Data from stages I to IV provided an in-depth analysis of key turning points influencing information sharing, communication and decision-making between patients and clinicians. Based on the find- ings of stages I to IV, an expert consensus group to select the do- mains for decision-making, and mode of intervention followed by cognitive interviews using probing and “think-aloud” protocols with patients, will determine the face-validity of the intervention. The next step will test the intervention in a cluster randomised controlled trial to include QoL and economic outcomes in order to establish the intervention as effective and beneficial. This multimethod approach has enabled a reflection of the complexity of the clinical pathway, and has mapped what happens during the patient-clinician consult- ation. We hope that this example will encourage a broader approach to qualitative study design when developing complex interventions in health care. Methods The PACT study is a five-stage prospective, qualitative and multicentre exploratory study. Data is collected from: I. Non-participant observation and audio recording of multi-disciplinary team (MDT) meetings to determine how patients were allocated to treatment pathways. II. Audio recording of patient-clinician consultations to measure patient’s involvement in decision-making (stage II). III. From the same cohort, separate audio recorded and observed interviews with patients IV. And their clinicians to explore the perception of treatment options and involvement in decision-making. V. An expert consensus meeting to fi- nalise the content and format of the intervention Qualitative analytical frameworks were used according to data type, research question and included: I. Mediated discourse analysis (MDA) II. Thematic Analysis and Option Grid scoring III. Interpretative Phenomenological Analysis (IPA) IV. Framework Analysis V. Consensus methodology and cognitive interviewing. g Discussion g Discussion Patient self-report forms a pragmatic and cheap method of data col- lection that is largely under the control of the researcher. However, there are known issues with the validity of the data collected, loss to follow-up may be high, and questionnaires suffer substantially from missing data. Routinely collected electronic data may be more accur- ate, although the validity of the datasets is not commonly assessed for the purposes of health economic evaluation. The use of routine datasets is likely to be more practical than patient self-report if large numbers of patients are involved. However, access to datasets often incurs a significant cost and researchers are bound by the time for data approval and extraction by the data holders. p Methods We describe current electronic data sources that may be suitable for use in economic evaluations conducted alongside randomised con- trolled trials, including Hospital Episode Statistics (HES) and Second- ary Users Service (SUS) datasets for secondary care in England; the Clinical Practice Research Datalink (CPRD), ResearchOne and in- practice software systems for primary care data; commissioning data- sets and their potential use for research; as well as looking to future developments such as GP Connect and the need to link electronic datasets. We also describe the current status of instruments for measuring resource use via patient self-report, and discuss initiatives to improve the methodology. We assess the risks and benefits associ- ated with each method, and compare the suitability of the two methodologies. Conclusions This study provides novel information regarding methods for the assessment of patient adherence to a removable device. The major- ity of studies in this analysis employed low burden methods easily implemented into a trial protocol. A combination of methods would therefore represent a practical approach for future trials whilst providing both objective and subjective data for increased validity. Following the identification of data discrepancies, queries are raised in the EDC system and are available for immediate review by sites. Conclusion Data review software is now used at ICR-CTSU to facilitate data man- agement in EDC trials. Implementing this new process can be time consuming, and manual checks are necessary while reports are being Page 218 of 235 Trials 2017, 18(Suppl 1):200 Given these findings, expensive and complex methods such as sen- sors may not be required. Developing a validated questionnaire would further increase validity and allow for comparison between studies. The paucity of data regarding reasons for non-adherence calls for a mixed methods approach including qualitative research to identify and explore patient factors. Using multiple qualitative methods to develop a complex intervention: the PACT study Mirella Longo1, Despina Anagnostou2, Stephanie Sivell2, Simon Noble2, Jason Lester3, Antony Byrne2, David Jones4, Lesley Radley4, Catherine Sampson2, Annmarie Nelson2 1Cardiff University, School of Medicine; 2MCPCRC, Cardiff University, School of Medicine; 3Velindre NHS Trust; 4PPI Correspondence: Mirella Longo Trials 2017, 18(Suppl 1):O78 Background O80 Adapting the quintet recruitment intervention (QRI) to optimise recruitment in an ongoing randomised controlled trial Marcus Jepson1, Jenny Donovan1, Rafiyah Khan2, Nikki Cotterill2, Paul Abrams2, The MASTER study group 1University of Bristol; 2Bristol Urological Institute Correspondence: Marcus Jepson Trials 2017, 18(Suppl 1):O80 Two-stage, single-arm designs are commonly employed phase II oncology trials. Typically these designs aim to determine whether the experimental treatment has sufficient activity to warrant further development, without formally testing the safety of the treatment. Bryant and Day (1995) developed a two-stage design to formally evaluate both response and toxicity. This design allows early termin- ation at the interim analysis due to inadequate response rate and/or excessive toxicities. p g Methods b d p g g g p Methods An abridged QRI was applied at two time points, 20 and 30 months into the recruitment phase of the RCT, each culminating in a collabo- rators’ workshop. Semi-structured interviews were undertaken with the CI, 16 recruiting urologists, and 2 research nurses (time point 1) and 5 urologists (time point 2). Interviews, screening and recruitment data were analysed to explore reasons for patient non-participation. Workshops were attended by the majority of recruiting centres and involved facilitation of an interactive discussion session based on emergent findings from the QRI. Attendees discussed the implica- tions of these findings, and considered whether they would be able to amend their subsequent recruitment practices. Simulations were used to implement NSC in stage two of the Bryant and Day design considering a range of scenarios. Response and tox- icity rates between 0.05-0.8 and 0.6-0.95 were investigated, respect- ively. The probability of early termination and expected sample size conditional on making it to the second stage of the trial were calcu- lated, when allowing early stopping for futility or efficacy (simulated under the null or alternative hypotheses respectively). Methods Kunz and Kieser (2012) explained that type I error and power are pre- served when non-stochastic curtailment (NSC) - a form of continuous monitoring - is implemented, as the trial is terminated once the desired result is impossible to achieve or already achieved. We there- fore focus on the impact on probability of early termination and expected sample size. We consider the case where a treatment successfully makes it past stage one and continuous monitoring oc- curs in stage two. Results Under all scenarios investigated, on average 77% of trials were termi- nated early during the second stage for futility, compared to 89% for termination due to efficacy. Without curtailment, the mean total sam- ple size was 29.9 patients, whereas when allowing early stopping for futility during stage two 26.4 patients were expected and for efficacy 26.7. This equates to savings of 12% and 11% respectively. Conclusion Issues that may have hindered recruitment emerged from the ana- lysis: different interpretations of eligibility criteria reduced the poten- tial sample population in some centres, and different positions of equipoise emerged in relation to one very familiar and established procedure, and a newer, less well-established technique. Additionally, there was inconsistency between the reasons for patient preferences as documented in screening data and described in interviews. Prior to the first meeting, 113 patients had been randomised (average 4.7 per month). In the subsequent 10 months, 135 patients were rando- mised (average 13.5 per month). The second collaborators’ meeting Non-stochastic curtailment in phase ii oncology trials Jessica Kandall1, Sarah Brown1, Cornelia Kunz2 1University of Leeds; 2Lancaster University Correspondence: Jessica Kandall Trials 2017, 18(Suppl 1):O81 Non-stochastic curtailment in phase ii oncology trials Jessica Kandall1, Sarah Brown1, Cornelia Kunz2 1 2 Introduction A disadvantage of these designs is early termination can only occur at the end of stage one, after a pre-specified number of patients are re- cruited and followed up. However it may be the case that during the trial, insufficient number of events occur such that early termination of the trial may occur at any point. Exposing more patients to a treatment, which has for example already observed too few responses and won’t be recommended for further development, is unethical. Additionally, this is wasting precious time and resources, which could be used else- where. Implementing continuous monitoring throughout these de- signs, could help overcome this issue. Randomised Controlled Trials (RCTs) are the most rigorous study design to evaluate healthcare interventions. However, their success relies on patient recruitment. The QuinteT Recruitment Intervention (QRI) aims to address recruitment difficulties and enhance informa- tion delivery in RCTs, using qualitative methods. It has been imple- mented in 26 current and completed RCTs, either as part of the initial study design, or brought in part-way through when recruit- ment is particularly challenging. The QRI comprises two-phases: Phase I - investigation of recruitment processes, using interviews, screening logs and recordings of trial consultations; Phase II - devel- oping an action plan in agreement with the RCT’s Chief Investigator. A QRI typically takes 12–18 months. Where trials are experiencing recruit- ment difficulties, and have to respond swiftly to funders it may not be possible to complete a full QRI. We report and reflect on a develop- mental abridged version of the QRI as applied to a UK-based RCT com- paring urological surgical procedures. Here we consider the statistical implications of employing continuous monitoring in the Bryant and Day design, to allow stopping when the desired outcome becomes unachievable or already achieved. M h d Acknowledgments JT acknowledges funding from the ConDuCT-II Hub for Trials Methodology Research. MF acknowledges funding from the CLAHRC Yorkshire and Humber (CLAHRC-YH). Trials 2017, 18(Suppl 1):200 Page 219 of 235 Page 219 of 235 The REDCap data management tool is gaining traction in the wider research community due to its generous licensing terms and its ease of use in setting up and managing research studies. was held in November 2016. If current rates are maintained, the study is now expected to successfully achieve its recruitment target. Conclusions An abridged QRI in the form of interview data and good quality screening information, combined with the accumulated knowledge of the commonly-cited barriers to trial recruitment, appeared to lead to an increase in the average number of monthly randomisations in this RCT. If RCTs require a short-term fix to recruitment challenges, an abridged version of the QRI may be useful. However, without the benefit of a full Phase I of the QRI, there is a limited understanding of recruitment barriers and processes, reducing opportunities to offer tailored suggestions for improving communication which may be ne- cessary in some RCTs. Many randomised clinical trials implement complex treatment alloca- tion algorithms such as minimisation that are not supported by the REDCap tool. This forces study designers into building in additional complexity to the design to enable the study to proceed. An outcome of this is that research nurses often need access to two systems’ REDCap itself, plus an external randomisation system. Additional complexity such as this should be avoided in a well- designed research study. We propose to demonstrate a mechanism where we leverage the REDCap data trigger mechanism, the REDCap API and Edinburgh Clinical Trials Unit’s own Randomisation Service to seamlessly inte- grate an external randomisation service with the REDCap data collec- tion process. The practical outcome of implementing the mechanism is that the end-users - our research nurses - are unaware they are in fact using two separate systems. Our stated aim of reducing complexity is thus achieved. O81 Non-stochastic curtailment in phase ii oncology trials Jessica Kandall1, Sarah Brown1, Cornelia Kunz2 1University of Leeds; 2Lancaster University Correspondence: Jessica Kandall Trials 2017, 18(Suppl 1):O81 O84 Implementing a risk based monitoring approach in the early phase myeloma portfolio at Leeds CTRU Lucy Bailey, Fiona K. Brudenell Straw, Suja E. George University of Leeds Correspondence: Lucy Bailey Trials 2017, 18(Suppl 1):O84 s 2017, 18(Suppl 1):O84 Employing efficient strategies for monitoring clinical trials is para- mount to mitigating risks using limited resources. While effective monitoring is crucial in protecting the well-being of trial participants and maintaining the integrity of data, in recent years there has been a gradual shift towards a risk based monitoring (RBM) approach. In 2011, An MRC/DH/MHRA Joint Project produced a paper entitled “Risk-adapted Approaches to the Management of Clinical Trials of Investigational Medicinal Products”, which aimed to facilitate a risk- proportionate approach in applying the principles of GCP when running clinical trials in the UK. In the same year guidance was also released by the U.S Food and Drug Administration on the expanded use of RBM. Like many clinical trials units, Leeds Institute of Clinical Trials Re- search (LICTR) previously assessed the risk for the trial as a whole and established a relatively inflexible monitoring plan whereby a des- ignated number of visits was performed at each of the sites, with extra visits only being performed at sites that recruited large num- bers of participants. Sites that had a good record of trial conduct and data compliance received the same number of visits as sites that had a poor record. The only monitoring above the standard would be when a triggered monitoring visit was required, such as if a site had breached the protocol and this was reportable to the MHRA. In order to prioritise finite resources, a formal, documented methodology was required for analysing trial-specific risks, and determining when more frequent, targeted monitoring was necessary. Design issues of a non-inferiority trial: prompt panretinal photocoagulation versus intravitreous ranibizumab for proliferative diabetic retinopathy (a tale of two diseases) Isoken Odia Michele Melia Design issues of a non-inferiority trial: prompt panretinal photocoagulation versus intravitreous ranibizumab for proliferative diabetic retinopathy (a tale of two diseases) I k Odi Mi h l M li p p Isoken Odia, Michele Melia Jaeb Center for Health Research Correspondence: Isoken Odia Trials 2017, 18(Suppl 1):O82 Non-inferiority trials are often used to compare a novel treatment with an active treatment in hopes of showing the novel treatment is not clinically worse than the proven standard therapy. Factors such as the choice of non-inferiority margins make these trials difficult to design, but these trials become significantly more complex when the condition being treated is associated with another disease that war- rants additional treatment, making it difficult to tease out treatment effects. The Diabetic Retinopathy Research network designed a non- inferiority trial to determine whether intravitreous ranibizumab (IVR), an anti-VEGF agent, with deferred panretinal photocoagulation (PRP) resulted in outcomes that were non-inferior to prompt PRP alone for the treatment of proliferative diabetic retinopathy (PDR). Diabetic macula edema (DME) can occur concomitant with PDR and is usually treated with an anti-VEGF drug if visual acuity is decreased. In this trial, about 35% of the prompt PRP group received the study drug (IVR) in addition to prompt PRP for the treatment of DME at baseline, and an additional 18% received it during follow up, after developing DME. This presentation will highlight some of the challenges encoun- tered in designing this trial such as choosing an appropriate non- inferiority margin, appropriate statistical methods and approach to treatment of DME in the standard treatment group. We discuss the advantages and disadvantages of the approaches taken to design this trial and discuss alternatives for handling these challenges. Our experience will help to inform investigators on possible solutions to similar situations in the future. O84 Implementing a risk based monitoring approach in the early phase myeloma portfolio at Leeds CTRU Lucy Bailey, Fiona K. Brudenell Straw, Suja E. George University of Leeds Correspondence: Lucy Bailey Trials 2017, 18(Suppl 1):O84 q Conclusion On average, a smaller sample size was required when implementing NSC in scenarios with larger toxicity rates. When allowing stopping for futility, greater savings in expected sample size were seen in the Page 220 of 235 Trials 2017, 18(Suppl 1):200 Page 220 of 235 component to the minimization algorithm can be detrimental to the objectives of minimization and reduce the efficiency with which a trial can be conducted. Unpublished data from case studies of on- going and completed multi-center clinical trials that are using or used minimization with a random component show that imbalance in treatments at study centers often occurs throughout the recruit- ment phase and treatment balance at all study centers is not guar- anteed at the end of the trial. Through simulation studies we show that these problems are less evident when minimization is used without a random component. We discuss the contexts (e.g., medica- tion kits shipped in advance of need and stored at study centers) in which trial inefficiencies and cost overruns occur with the inclusion of a random element to the minimization algorithm. We conclude that con- sideration should be given to not adding a random component to minimization in multi-center clinical trials that use an interactive central allocation system and recommend that decisions about the statistical aspects of trial design, especially decisions about treatment allocation methods, be made in the context of planned procedural and oper- ational aspects of a trial. component to the minimization algorithm can be detrimental to the objectives of minimization and reduce the efficiency with which a trial can be conducted. Unpublished data from case studies of on- going and completed multi-center clinical trials that are using or used minimization with a random component show that imbalance in treatments at study centers often occurs throughout the recruit- ment phase and treatment balance at all study centers is not guar- anteed at the end of the trial. Through simulation studies we show that these problems are less evident when minimization is used without a random component. We discuss the contexts (e.g., medica- tion kits shipped in advance of need and stored at study centers) in which trial inefficiencies and cost overruns occur with the inclusion of a random element to the minimization algorithm. q Conclusion We conclude that con- sideration should be given to not adding a random component to minimization in multi-center clinical trials that use an interactive central allocation system and recommend that decisions about the statistical aspects of trial design, especially decisions about treatment allocation methods, be made in the context of planned procedural and oper- ational aspects of a trial. scenarios with larger toxicity rates. However greater savings were seen in the lower toxicity scenarios, when stopping for efficacy. The probability of early termination was significantly larger when allow- ing early stopping for efficacy compared with futility. Where recruitment is slow or observations may be monitored in a short-time frame, NSC for futility and efficacy may be beneficial. scenarios with larger toxicity rates. However greater savings were seen in the lower toxicity scenarios, when stopping for efficacy. The probability of early termination was significantly larger when allow- ing early stopping for efficacy compared with futility. scenarios with larger toxicity rates. However greater savings were seen in the lower toxicity scenarios, when stopping for efficacy. The probability of early termination was significantly larger when allow- ing early stopping for efficacy compared with futility. Where recruitment is slow or observations may be monitored in a short-time frame, NSC for futility and efficacy may be beneficial. Design issues of a non-inferiority trial: prompt panretinal photocoagulation versus intravitreous ranibizumab for proliferative diabetic retinopathy (a tale of two diseases) Isoken Odia, Michele Melia Jaeb Center for Health Research Correspondence: Isoken Odia Trials 2017, 18(Suppl 1):O82 Design issues of a non-inferiority trial: prompt panretinal photocoagulation versus intravitreous ranibizumab for proliferative diabetic retinopathy (a tale of two diseases) Isoken Odia Michele Melia Use of minimization in multi-center clinical trials - when to not add randomization Elaine Pascoe, Darsy Darssan, Liza A. Vergara The University of Queensland Correspondence: Elaine Pascoe Trials 2017, 18(Suppl 1):O83 Minimization is increasingly recommended as the method of choice for allocating subjects to treatment groups in clinical trials. The method is designed to eliminate chronological bias by guaranteeing similar num- bers of patients in treatment groups throughout the trial recruitment phase and to ensure treatment groups are balanced on prognostic and other baseline characteristics at the end of the trial. Primarily because of its superior capacity to balance treatments across large numbers of baseline variables, minimization has been referred to as the platinum standard for treatment allocation in clinical trials. In multi-center trials, study center is typically one of the minimization variables. In its original form, minimization deterministically (p = 1.0) allocates the next patient to the least common treatment for previously allocated patients with the same combination of baseline characteristics. Subsequent exten- sions to the minimization algorithm included the addition of a random element to the allocation process (p between 0.5 and 1.0) to mitigate the risk of a clinician predicting the next treatment to be allocated. We argue that the risk of selection bias is negligible in multi-center trials that use an interactive central allocation system (web-based or voice response). We present data showing that the addition of a random With this goal in mind, a risk-based monitoring tool was developed for use in the early phase myeloma portfolio being run at the LICTR. Data regarding site performance is collated on a monthly basis. Each metric relating to the safety of participants, data management, regu- latory compliance and adherence to protocol has parameters for low, medium or high risk scores. Sites are given an overall risk rating de- pendant on the number of areas in which they are considered a low, medium or high risk. The risk assessment is examined regularly throughout the course of the trial to ensure that the risk score and, subsequently, the monitoring approach is amended according to the changing quality of site performance. This tool involves collaboration between trial coordinators, data managers and monitors and enables simple and quantitative reporting at meetings and gives closer align- ment of data management, trial management and site monitoring processes. Our monitoring plans still incorporate visits to every site but are now also adaptive, allowing the monitoring strategy for a particular site to evolve as the trial progresses. Preliminary analysis of a new measure of quality of patient decision making about research participation Correspondence: Charlotte Murkin Trials 2017, 18(Suppl 1):O86 1University of York; 2University of Cambridge; 3University of Manchester Correspondence: Peter Knapp Trials 2017, 18(Suppl 1):O85 Objectives h To report the rationale for, and initial performance data of, a measure of quality of patient decision making about research participation. Background To understand patients’ decisions about research participation, including trials, or to evaluate interventions intended to boost re- search recruitment rates, it is useful to assess quality of decision making about participation. It would be equally useful to do this when the patient has opted to participate or has declined. The QuIC measure [Joffe, 2001] has been used in several studies but there are concerns about its completion rate and the relevance of some items to non-drug trials. The ICQ measure [Guarino, 2006] has some useful items but also those that depend on post- study completion. There are established, well-performing mea- sures of patient decision making about treatments [O’Connor, 1995; Parayre, 2014] but both these measures contain items that do not apply to decisions about research participation. Given these problems we devised a new measure and tested it with a small sample of people who had consented to take part in the REFORM study [Cockayne, 2016]. Background h The importance of pragmatic study design is gaining increasing rec- ognition in research. A distinctive feature of this is the selection of clinically relevant outcomes that are meaningful to patients. Studies, therefore may choose to focus on the symptomatic condition of interest rather than the asymptomatic presence of a condition alone. However, there are challenges associated with this, including the selection of symptoms and their assessment. Here we describe the methods used to design a questionnaire that aims to identify the im- portant symptoms of the relevant condition. In a future study, we in- tend to set a threshold to define participants as symptomatic and use this as part of the definition of the primary outcome. This work highlights how built in feasibility work can be used to develop pa- tient centred symptom-based outcomes to inform a pragmatic trial design. Results Results 280 (93.0%) participants completed at least some of the measure, of whom 98.9% completed all items. The mean total score was 20.03 (SD 2.44; range 12–25; mode 20; median 20). Individual item means ranged from 3.81 (“possible changes to treatment or care”) to 4.24 (“overall informed choice”). Internal consistency was high (Cronbach alpha 0.83) and alpha was not increased by the deletion of any of the 5 items. g Methods The first phase of the project (Phase A) consists of built in feasibility work designed to inform the future study (Phase B). This methodo- logical work, performed as part of Phase A, has three main compo- nents: i) Identification of known and unknown healthcare domains from the literature and qualitative interviews with patients living with and without the condition and interviews with clinicians and special- ist nurses, ii) Development domains into questions within a question- naire for completion by patients, iii) pre-testing the questionnaire in patients living with and without the condition to refine the instru- ment and develop thresholds (green, amber and red) to trigger fur- ther investigation for the condition of interest. Methods y y Methods The measure was intended to be short (to limit patient data burden) with items referring to elements of participant information required under UK research governance. The measure has 5 items (covering: potential benefits of participation; potential disadvantages; likely experience of participation; possible changes to treatments or care; and an evaluation of the overall extent to which choice was in- formed), each using 5 Likert responses (from ‘strongly agree’ to ‘strongly disagree’, scored 5 to 1 respectively) This derives a total score with possible range 5–25, and higher scores indicating greater agreement. The measure was included in the baseline pack posted to participants in the REFORM study [Cockayne, 2016], which used a “trial within a cohort study” design to evaluate the effectiveness of a falls prevention intervention in older people living at home. The measure was posted to 301 people (aged 64–98; 50.8% were female) in 4 centres in northern England. p Conclusion This mixed methods feasibility work developed a patient centred outcome measure to inform a pragmatic study design. It focuses on endpoints relevant to patients (i.e. a symptomatic condition of inter- est). Further work is planned in the main study to validate the clinical findings with symptomatic and asymptomatic patients. Discussion & Conclusions O86 Assessing clinically relevant endpoints in a pragmatic trial: development of a measure to identify relevant clinical symptoms Charlotte Murkin1, Kerry Avery1, Sian E. Cousins1, Natalie S. Blencowe1, Leila Rooshenas1, Karen Coulman1, Daisy Elliott1, N. J. Smart2, Jane M. Blazeby1, On behalf of the CIPHER study group2 1University of Bristol, School of Social and Community Medicine; 2Exeter Surgical Health Sciences Research Unit (HESRU), Royal Devon & Exeter Hospital O85 Preliminary analysis of a new measure of quality of patient decision making about research participation Peter Knapp1, Jonathan Graffy2, Peter Bower3, Joanna Rick3, Caroline Fairhurst1, Sarah Cockayne1, S. Rogers1, D. Torgerson1 1University of York; 2University of Cambridge; 3University of Manchester Correspondence: Peter Knapp Trials 2017, 18(Suppl 1):O85 Results 169 relevant descriptions of known symptoms were identified from the literature, which were grouped into health domains independ- ently by two researchers, resulting in 19 domains that included pain, body image and social functioning. These domains formed the basis of the topic guides for the semi-structured interviews. 10 healthcare professionals with clinical experience of the condition of interest and 17 patients (9 living with the condition and 8 without, on review of medical records) were interviewed. The topic guides were updated iteratively as new lines of enquiry were uncovered during the inter- views. Interviews were analysed through constant comparison ap- proaches. Transcripts were coded line by line and categorised into themes using NVivo software. The Interviews identified 16 additional symptom domains. Work is ongoing to develop items from the do- mains. Future work will pre-test the tool and establish thresholds to define participants as symptomatic. Use of minimization in multi-center clinical trials - when to not add randomization Page 221 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 This presentation will detail how we adapted the RBM approach and implemented a site monitoring tool for our phase two trials in the myeloma portfolio. Discussion & Conclusions The scale was acceptable to respondents and shows promise as a measure of the quality of decision making about research partici- pation. Mean scores were relatively high, which may indicate the quality of information provision in this study but could also re- flect the positive wording of the measure’s items. Further evalu- ation is needed, particularly in studies with different age groups and lower participation rates. Also needed are consideration of a threshold approach to scoring, defined by minimally acceptable values to indicate that informed consent has been obtained; and evaluation of the measure’s ability to discriminate and its sensi- tivity to change. Results The treatments that those in the intervention and control arms re- ceived were unexpectedly similar. This appeared to represent a mixture of treatment non-receipt in the active arm (non-compliance) and treat- ment receipt in the control arm (contamination). The intention-to-treat analysis suggested little effectiveness of treat- ment. The efficacy analyses, which accounted for non-compliance and contamination, gave estimates that were a little larger and with wider confidence intervals. However, these did not show a statisti- cally or clinically significant effect. Inconsistencies between the participant-reported account and the record linkage report were detected. In an attempt to address this issue contact was made with the hospital site to extract information directly from the patients’ notes. If no response was received, the participant’s record of the event was included in the analyses in both trials. For an event reported by record linkage that could not be veri- fied by the hospital (and was not reported by the participant) the event was excluded from the analyses. Contamination in trials of complex interventions: measuring and accounting for it in the analysis of a trial King’s College London Correspondence: Nicholas Magill Trials 2017, 18(Suppl 1):O88 O87 Trials 2017, 18(Suppl 1):200 Page 222 of 235 O88 Contamination in trials of complex interventions: measuring and accounting for it in the analysis of a trial Results The supplementation of participant-reported clinical outcomes with routine hospital admission data allowed us to better evaluate the key clinical question; approximately 20% of relevant hospital readmis- sions were obtained from routine admission data only. However the exercise also highlighted potential issues with the coding of routine admission data: for 5.8% of index operations, the coding in the rou- tine data did not match that recorded within the trial dataset. Di i Background In mental health trials there is concern that the control treatment might be contaminated, meaning that a participant in the comparator arm receives the active intervention. This can often be avoided by design through the use of cluster randomisation, with clusters defined by the p Method This methodological research was motivated by a trial of nurse- delivered cognitive behavioural therapy and motivational interview- ing for people with poorly controlled type 2 diabetes in primary care. Participants were offered either this (in addition to standard care) or standard care with attention control. The primary outcome was glycated haemoglobin. Contamination was anticipated if a given nurse were asked to deliver both intervention and comparator treatments. Therefore cluster randomisation was used with treatment allocation at the level of the primary care centre. However, in practice both non- compliance and contamination did occur due to variation in nurses’ skills and possibly as an effect of the attention control design. Two surgical RCTs that utilised a dual model of data collection whereby clinical outcomes were reported by the participant and through record linkage were examined. In both trials the use of hos- pital admission data was appropriate as the key clinical outcome was further surgical intervention for reoccurrence of the condition. Con- sent for record linkage was obtained from all participants at the on- set of the trials which allowed the collection of data from those participants who subsequently declined further questionnaire follow- up or who were lost to follow-up (unless their consent to do so was withdrawn). Both trials have reported their primary outcome and one has entered a longer follow-up phase. Treatment fidelity was measured using two fidelity scales, which were then used to construct measures of treatment receipt and subse- quently to account for non-compliance and contamination in the ana- lysis of the trial. One measure was of binary treatment receipt and this was used to perform a CACE efficacy analysis using instrumental vari- able methods. Another was a continuous measure of dose of treat- ment. In addition, existing methodology was developed to allow the estimation of the effect of exposure on the causal treatment effect where exposure is dose of treatment and is measured in control and intervention participants. O89 Successful recruitment to a large online randomised trial: the TIME study O89 Successful recruitment to a large online randomised trial: the TIME study Amy Rogers, Isla Mackenzie, David Rorie, Thomas M. MacDonald University of Dundee Correspondence: Amy Rogers Trials 2017, 18(Suppl 1):O89 The collection of hospital readmission data to supplement participant- reported outcomes was a worthwhile exercise across both trials as it re- sulted in a more complete dataset. However it was a time consuming process and had implications in terms of staff time preparing the appli- cation, analysing the data and resolving any inconsistencies observed. y Conclusions The similarity between the treatment groups may in part explain the lack of a treatment effect in this trial. This research has demonstrated the importance of measuring treatment fidelity for all participants as a post randomisation process variable. Such a measure enables an assessment of fidelity and adjustment for treatment receipt in the re- sults of a trial of a psychotherapy. The motivating trial also highlights the difficulty of training non-clinical psychologists to provide psycho- therapy and possibly the drawback of the use of attention control. Issues surrounding the collection of record linkage data were also iden- tified. This was particularly problematic for data collected through the Health and Social Care Information Centre (now known as NHS Digital). Problems encountered included a lengthy application and approval process, mainly due to changes in the application process, the name of the service provider and to the legal gateway for consent. p g g y We also experienced lengthy applications to extend retention periods be- yond the initial agreement (to comply with sponsor and funders regula- tions) and extensive data security checks of the University’s policies. In addition, there is still ongoing confusion regarding data classification, in particular, what constitutes derived reversible or irreversible source data. Conclusions Background level at which contamination is thought to take place. One alternative to this problem is to account for contamination in the analysis by using complier average causal effect (CACE) estimation. M th d Collection of outcome data within randomised controlled trials (RCTs) can be challenging, especially in trials with a lengthy follow-up phase. If the outcomes of interest are participant-reported, for example, quality- of-life, then participant contact is unavoidable. However, if the out- comes are clinical, for example, re-operation or hospitalisation then rec- ord linkage of trial datasets to hospital admission records (or other routine data-sets) may be an option and could potentially overcome some of the issues associated with participant retention. M th d Background Background The TIME (Treatment in Morning versus Evening) Study is a rando- mised study of the effect of timing of antihypertensive medication administration on cardiovascular outcomes using an online method- ology [1]. This presentation will describe and discuss the methods used to surpass the study recruitment target. Background TIME aims to determine if antihypertensive therapy taken in the evening results in improved cardiovascular outcomes compared with conventional morning dosing. Over 20,000 participants, followed-up over 4 years, will be required to generate the necessary number of cardiovascular events to answer this question. The TIME study is funded by the Brit- ish Heart Foundation and we wanted to develop novel methods of recruitment to maximise cost efficiency. Methods Background Ileana Baldi1, Corrado Lanera1, Clara Minto1, Dario Gregori1, Paola Berchialla2 Response review is a necessary stage in the verification of efficacy endpoints in trials of multiple myeloma (MM) as it provides an indi- cator of quality assurance. UK NCRI Myeloma XI is probably the lar- gest randomised clinical trial in newly diagnosed patients with MM conducted so far, with 4420 recruited patients. A large trial requires a robust method of assessment of response and progression through- out the patient pathway. As Myeloma XI requires response assess- ment after completion of key treatment periods, and progression monitoring throughout the protocol, a semi-automated verification process is considered essential to ensure robust results and manage resources efficiently. 1University of Padova; 2University of Torino Correspondence: Ileana Baldi Trials 2017, 18(Suppl 1):O90 Background Systematic reviews of clinical trials are an important means of synthe- sizing medical evidence. The validity of systematic reviews depends on the ability to fully capture the complete body of evidence through searches of many data sources. Nevertheless, examination of clinical tri- als registry databases is not routinely included in systematic reviews (Jones et al., 2014). A possible explanation is that all databases have their own unique features and creating consistent search strategies across literature and registry databases may be challenging. Methods Prospective TIME participants are invited to visit a secure study website, https://www.timestudy.co.uk/. All study procedures are conducted Trials 2017, 18(Suppl 1):200 Page 223 of 235 Page 223 of 235 online. Several different methods were used to identify potential par- ticipants including: letters sent from Primary and Secondary Care (using the NHS-approved Docmail service), emails sent to partici- pants in biobank projects, pharmacy searches and more traditional advertising by poster and print media articles. the title and condition fields and the DTM was built. A prediction task was then performed by applying the validated classifier to the ICTRP DTM. Methods Response and progression in MM were defined according to the Inter- national Myeloma Working Group (IMWG) Criteria. Myeloma XI collected local laboratory results alongside local response assessments, and for consenting patients - equivalent data from an experienced central labora- tory. All data was stored on bespoke trial databases which could be easily imported into statistical software packages. We developed a suite of SAS programs which apply the IMWG criteria to reported laboratory values and verified local and central response and progression assessments, and validated local and central assessments against each other. If assessments were concordant, these were considered to be validated and required no further review. If results were discordant, the patient file was selected for review by a committee of clinicians. Results In the PubMed training set, the sensitivity of the classifier was good (81.2%) and the specificity was excellent (99.3%). When applied to the ICTRP database it allowed to identify a set of protocols likely pertaining to nursing research. We are currently organizing data and code in a R software package which will be freely available on GitHub. Conclusions Reference 1. Rorie DA, Rogers A, Mackenzie IS, Ford I, Webb DJ, Willams B, et al. Methods of a large prospective, randomised, open-label, blinded end- point study comparing morning versus evening dosing in hypertensive patients: the Treatment In Morning versus Evening (TIME) study. 1. Rorie DA, Rogers A, Mackenzie IS, Ford I, Webb DJ, Willams B, et al. Methods of a large prospective, randomised, open-label, blinded end- point study comparing morning versus evening dosing in hypertensive patients: the Treatment In Morning versus Evening (TIME) study. Results 21,415 participants throughout the UK were randomised between the start of the initial pilot study in 2012 and November 2016. The total cost of recruitment methods was £277,139. Primary care patient identification (PIC) sites sending letters of invitation generated the most participants (n = 16657). An arrangement with UK Biobank invit- ing potential participants by email resulted in 3210 randomisations. Recruitment strategies such as posters and media advertising had lit- tle impact on recruitment despite relatively high costs. Discussion Materials and Methods The main idea is to use the title and abstract text fields of relevant articles, identified by a literature search, for training purposes of a machine learning algorithm. Next, the trained algorithm may be used to identify documents with a comparable word characterization in a registry database. A hypothetical research question concerning nursing research is used to demonstrate the proof-of-concept. The PubMed database was searched for articles published from 2006/01/ 01 to 2016/11/01, tagged with Clinical trial Publication Type and appearing in journals allocated to the Nursing subject area according to the Journal Citation Reports®. To provide a training set with “posi- tives” and “negatives”, we selected also a set of clinical trials run in other research fields. The following text pre-processing procedures were applied to the title and abstract fields of the retrieved records in the following order: conversion to lowercase, removing numbers, removing punctuation, removing stop-words, stemming words, strip- ping white space, and building a sequence of two adjacent words from the text (bigrams). The whole collection was finally tokenized in a document-term matrix (DTM) which was used to train and test a logitBoost MLT classifier (Dettling and Buhlmann, 2002). The classifier was 10-fold cross-validated using 1,000 bootstrap iterations at each step. Next we accessed records of all clinical trials registered in the International Clinical Trials Registry Platform (ICTRP), established by World Health Organization in 2006. After eliminating duplicates, the same above mentioned text pre-processing strategy was applied to Preparing the clinical review data involved collation of all local and central results available. This was achieved using a suite of R and Java programs to produce an MS Excel clinical review file for each patient. The clinical review file contained derived and reported as- sessments, as well as details of key assessment timepoints within the study. The review files were sent to clinicians for review via a Secure File Transfer System (SFTS). The reviewer considered available data to determine the response/progression and for difficult and discordant cases, a clinical committee meeting was convened to determine a consensus or classification of the case as unable to determine (UTD), or to request that more data be sought from site. When review was completed the review file was returned via SFTS. Conclusions Routine examination of registry databases deserves further consider- ation since it may allow a more accurate characterization of publica- tion and outcome reporting biases and improve the validity of systematic reviews. The approach described here provides an auto- mated solution that can be tailored to address a variety of clinical trial-related questions by building a comprehensive search on both literature and registry databases. Successful recruitment to the TIME study was mainly due to a com- bination of efficient study design with the use of IT to facilitate send- ing high numbers of study invitations at low cost. It is hoped that these methods can be used to streamline the recruitment process for future studies. O91 Response and progression endpoint review in a large phase III randomised control trial of multiple myeloma Alina Striha1, David A. Cairns1, Corinne Collett1, Charlotte Pawlyn2, Phillip Best1, Andrea Paterson1, Inga Sakauskiene1, John Jones2, Mark T. Drayson3, Graham H. Jackson4 1University of Leeds; 2Institute of Cancer Research; 3Clinical Immunology Service, University of Birmingham; 4Northern Institute for Cancer Research, Newcastle University Correspondence: Alina Striha Trials 2017, 18(Suppl 1):O91 p Results At final analysis in July 2016, 3894 patients had undergone central review. At this time 1848 subjects had demonstrated progression Page 224 of 235 Trials 2017, 18(Suppl 1):200 Trials 2017, 18(Suppl 1):200 Page 224 of 235 Page 224 of 235 used in ROLARR, the resulting learning effects analyses that were performed and the impact that they had on our understanding of the outcomes that we saw in trial will be presented. Time permit- ting, the limitations of these particular analyses, including areas where further work is required and recommendations for future trials, will also be discussed. and 3795 had a response assessment. After computer-based verifica- tion and validation of progression, 443 patients (24.0%) required sub- sequent clinical review. A similar process for response resulted in 477 patients (12.6%) requiring clinical review. All remaining progressions and responses were considered verified without the need for review by clinician (83.7% of potential response and reviews). In clinician re- view of progression a decision was reached in most cases with only 25 patients (5.6%) classified as UTD. and 3795 had a response assessment. After computer-based verifica- tion and validation of progression, 443 patients (24.0%) required sub- sequent clinical review. A similar process for response resulted in 477 patients (12.6%) requiring clinical review. All remaining progressions and responses were considered verified without the need for review by clinician (83.7% of potential response and reviews). In clinician re- view of progression a decision was reached in most cases with only 25 patients (5.6%) classified as UTD. Exploring and adjusting for potential learning effects in ROLARR - a randomised controlled surgical trial N il C i H l M h ll J li B Exploring and adjusting for potential learning effects in ROLARR - a randomised controlled surgical trial Neil Corrigan, Helen Marshall, Julia Brown University of Leeds Correspondence: Neil Corrigan Trials 2017, 18(Suppl 1):O92 Methods Our motivating data come from a network of IPD from 37 trials com- paring combinations of radiotherapy, chemotherapy and surgery from 5922 patients with cervical cancer (2394 events), where age and disease stage are two potential interaction covariates. We show how the Royston-Parmar NMA model can be fitted using restricted cubic splines, and then extend it to include treatment-covariate inter- actions. We propose two models: (i) two effects separating out the within- and across-trial information and (ii) a single interaction effect (combining within- and across-trial information). We argue for a vis- ual assessment of the consistency of the within- and across-trial in- formation. We also discuss more detailed aspects of meta-analysis interaction modelling, such as common vs trial-specific main effects of the covariate, and suitable approaches for handling missing covar- iate data. This leads us to propose a practical framework for IPD NMA with treatment-covariate interactions. Results However, as alluded to by Cook, Ramsay & Fayers [1], this is only a partial answer to the problem; in principle, statistical exploration of any potential learning effects within a trial is required to more fully address the issue. Following this guidance in ROLARR, data on the number of robotic-assisted and standard laparoscopic rectal cancer operations performed both within and outside of the trial was col- lected periodically throughout the trial. At analysis, for every ROLARR operation performed we had data about how many previous robotic- assisted and standard laparoscopic rectal cancer operations the oper- ating surgeon had performed. This allowed us to perform sensitivity analyses exploring potential learning effects within the trial, in par- ticular addressing the question of whether or not the estimated dif- ference between the arms yielded by our primary analysis changed depending on operating surgeon experience. Conclusions This semi-automated process of central review will increase confi- dence in the robustness of reported results from Myeloma XI. This is an efficient process which has saved man-hours for data manage- ment and clinical reviewers. The software tools developed are being further validated for use in data management and central review of new trials of MM. O93 A framework for identifying treatment-covariate interactions in individual participant data network meta-analysis Suzanne Freeman, David Fisher, Jayne Tierney, James Carpenter MRC Clinical Trials Unit at UCL; MRC London Hub for Trials Methodology Research Correspondence: Suzanne Freeman Trials 2017, 18(Suppl 1):O93 Reference [1] Cook JA, Ramsay CR, Fayers P. Statistical evaluation of learning curve effects in surgical trials. Clinical Trials 2004; 1: 421–427. Background g Stratified medicine aims to identify patients most likely to respond to treatment. However, individual trials are rarely powered to detect in- teractions between treatment effects and participant characteristics. Network meta-analysis (NMA) models potentially have greater power to identify such treatment-covariate interactions, particularly when individual participant data (IPD) are available. The Royston-Parmar model, fitted in a Bayesian framework using WinBUGS, provides a practical and flexible approach for IPD NMA of time-to-event data. Pairwise IPD meta-analysis of treatment-covariate interactions contains so-called “within-trial” and “across-trial” information, where across-trial information is particularly susceptible to confounding and ecological bias. The same is true of IPD NMA; therefore it is important that the within- and across-trial information are reviewed separately, before de- ciding whether to combine them. A further frequent complication is missing patient-level covariate data. We show that using a Bayesian framework for IPD NMA allows this to be handled relatively easily. Methods ROLARR is an international, randomised controlled, confirmatory surgical trial designed to perform a rigorous comparison of robotic- assisted surgery against standard laparoscopic surgery for the cura- tive treatment of rectal cancer. At the beginning of trial recruitment, the standard laparoscopic approach was already well-established, with many centres and expert surgeons proficient in delivering the intervention, whereas the robotic-assisted approach was relatively new, with only several centres having an established track record of delivering it. Proficiency in standard laparoscopic surgery coupled with enthusiasm for, but relatively little experience with, robotic- assisted surgery was a common trait of potential participating sur- geons in ROLARR. This situation is common in surgical trials and must be addressed during the design and analysis, since a naïve comparison of out- comes of patients who underwent a well-established technique in which the operating surgeons were already proficient vs. patients who underwent a new technique which operating surgeons were still learning would be inappropriate. Ideally, comparisons between the two interventions should be made when they are both being per- formed optimally i.e. when surgeons are proficient in both. Tradition- ally, this issue is addressed at the design stage by stipulating that any participating surgeon in the trial must have some minimum level of experience in each intervention before randomising any patients. This was done in ROLARR, with inclusion criteria stipulating that par- ticipating surgeons must have performed at least 30 rectal cancer operations (with at least 10 using each intervention). NMA wit Results The cervical cancer network showed no evidence of a treatment-age interaction, but there was some evidence for a treatment-stage inter- action. A visual assessment highlighted inconsistency between the within- and across-trial information. Following our proposed framework, the within- and across-trial information should not be combined. The remaining within-trial evidence for a treatment-stage interaction is weak; most evidence for an interaction is across-trial, with the NMA providing additional power for this over and above the pairwise evidence. Conclusion Conclusions Formative research findings on the design of an early enrollment clinical trial on hospital-acquired bacterial pneumonia (HABP) and ventilator-associated bacterial pneumonia (VABP) 1 2 3 4 The EE strategy was overwhelmingly accepted by stakeholders. Spon- sors should evaluate whether this strategy improves enrollment rates in prospective HABP/VABP clinical trials. Amy Corneli1, Brian Perry2, Deborah Collyar3, John H. Powers4, Helen K. Donnelly5, Carisa De Anda6, Denise Hinton7, Jonas Santiago7, Sara B. Calvert8, Vance G. Fowler Jr. 1 Amy Corneli1, Brian Perry2, Deborah Collyar3, John H. Powers4, Helen K. Donnelly5, Carisa De Anda6, Denise Hinton7, Jonas Santiago7, Sara B. Calvert8, Vance G. Fowler Jr. 1 Reducing bias in randomised controlled trials involving therapists: to randomise or not to randomise - What are the questions? Alexandra Wright-Hughes, Rebecca Walwyn, Liz Graham, Sadie Reed, Amanda Farrin University of Leeds Correspondence: Alexandra Wright-Hughes Trials 2017, 18(Suppl 1):O95 1Duke University; 2Duke Clinical Research Institute; 3Patient Advocates In Research; 4George Washington University School of Medicine; 5Northwestern University; 6Merck & Co., Inc.; 7Center for Drug Evaluation and Research, U.S. Food and Drug Administration; 8Clinical Trials Transformation Initiative Correspondence: Amy Corneli Trials 2017, 18(Suppl 1):O94 Clinical trials of behavioural and psychological interventions often in- volve therapists or other clinical specialists. This adds further com- plexity to the trial design and leads to considerations relating to clustering effects (i.e. intra-cluster correlation, cluster size), random- isation, contamination, and the impact on analysis. In individually- randomised controlled trials (RCTs) the involvement of more than one therapist per site can introduce selection bias, whereby thera- pists can choose which participants they treat. If therapist selection is based on participants characteristics, for example with the “best” treating the “worst”, the effects of therapists and participant charac- teristics may be confounded. This limits the inferences a trial is able to make, particularly if the therapist effect is of interest in its own right. It also reduces the ability to attribute treatment effects to treat- ment alone, thus impacting a trial’s internal validity. To reduce the overall sample size required, and avoid problems fitting analysis models, trialists may also wish to ensure similar cluster sizes. To avoid this source of selection bias, allocation of therapists to participants can be concealed and random. However, doing so under constraints of therapist capacity, location, and a trial’s relative priorities can be problematic. y Methods We conducted 52 qualitative, in-depth interviews with patients at risk for HABP/VABP (n = 18), caregivers/LARs (n = 12), members of Institu- tional Review Boards (IRBs) (n = 10), investigators (n = 7), and study coordinators (n = 5). We provided stakeholders with an overview of the proposed EE study prior to questions on acceptability and prefer- ences. We also explored the acceptability of two treatment designs: randomization to 1) one of two FDA-approved antibiotics commonly used in hospitals to treat pneumonia (one approved for lower re- spiratory infections and the other for serious infections other than pneumonia), and 2) a new, investigational antibiotic (not FDA- approved) compared to a standard-of-care antibiotic. We used ap- plied thematic analysis to analyze the data. We summarise our experience of randomly allocating therapists in three clinical trials with different designs: (i) SHIFT, an NIHR HTA-funded phase III definitive RCT set in NHS Child and Adolescent Mental Health Services (CAMHS), had teams of Family Therapists (employed for the re- search) working together to deliver therapy across services. Where teams worked across a number of services, the lead therapist was randomised, otherwise participants were assigned to the single lead therapist for a particular service. (ii) TIGA-CUB, an NIHR RfPB-funded feasibility RCT, similarly set in CAMHS, has greater constraints on ther- apist capacity. Trial therapy is provided ‘within’ therapists NHS role, with variable numbers of therapists per service, and complications for ther- apist allocation to primary carer as well as child participants. (iii) OK- Diabetes, an NIHR HTA-funded feasibility RCT recruited community- based participants with diabetes and a learning disability. Diabetes nurse specialists were allocated to participants based on prior contact to ensure consistency in contact and nurse randomisation occurred where there had been no prior contact. None of the patients or caregivers/LARs expressed concern about study staff accessing patients’ medical records to monitor for infection. Nearly all patients (88%) believed they could understand the study and give consent, even though they would not have pneumonia at the time of enrollment. While appreciated by many (56%), some patients (44%) said they may experience anxiety after being informed they are at risk for pneumonia. None of the IRB members raised concerns regarding the EE strategy, although suggestions were made about information to include in the study protocol. Most investigators and coordinators (83%) believed the EE strategy would improve recruitment. Conclusions The success of therapist involvement and implications on randomisation can be further influenced by the context in which they work (e.g. involvement of ‘research’ therapists or those working within the NHS). Background The Clinical Trials Transformation Initiative (CTTI), a public-private partnership established by the FDA and Duke University, is interested in assessing whether an Early Enrollment (EE) strategy can improve enrollment rates for HABP/VABP clinical trials. Using this proposed strategy, patients at high risk for developing HABP or VABP, or their legally-authorized representatives (LARs), would be asked to give their consent to enroll in a trial before the patient develops symp- toms of pneumonia. Patients would agree to being monitored by study staff, and if pneumonia develops, to being randomized to receive the study antibiotics. We conducted formative research to as- sess the acceptability of and preferences for components (e.g., opt- out process) of an EE strategy for a HABP/VABP clinical trial among key stakeholders. p Conclusion A Bayesian approach for NMA using the Royston-Parmar model with splines naturally handles missing covariate data and allows the separ- ation of within- and across-trial treatment-covariate interaction infor- mation. Our proposed framework, incorporating this model, provides practical guidance for researchers, and reduces the risk of unduly optimistic interpretation of treatment-covariate interactions. Collecting this data and performing these analyses allowed us to quantify and adjust for the confounding effect that the difference in proficiencies between the arms had on the primary analysis, which was invaluable to our understanding and interpretation of the out- comes that we observed in ROLARR. Details about the approaches Trials 2017, 18(Suppl 1):200 Page 225 of 235 Page 225 of 235 Methods In response to this new initiative, the AMC developed a two-tiered strategy to select international sites. This began by soliciting applica- tions from 14 sites in which a member of the site staff had a pre- existing relationship with an AMC investigator, and systematically gathering written information about their resources, clinical research experience and active collaborations with other clinical trials organi- zations. After review of these data, the nine top-ranked sites partici- pated in a structured data collection exercise that involved collection of disease-specific patient information about three AIDS-related can- cers, auditing that data, and conducting site visits. Each site visit team included an oncologist and a representative from the AMC Op- erations and Data Management Center (ODMC) and evaluators filled out a site visit worksheet at the end of each visit. We consider three outcome-generating models where the outcome (Y) is dependent on: (1) the treatment arm (A) only, (2) on both treat- ment arm and a predictive baseline covariate (X) through an additive relationship, and, (3) additionally dependent on the interaction of A and X so that there is treatment heterogeneity by levels of the pre- dictive covariate X. For each of these three outcome-generating models, we consider a range of common missing outcome mecha- nisms including: missing completely at random (MCAR), missing at random (MAR), including covariate dependent missingness (CDM), as well as specific examples of missing not at random (MNAR) mecha- nisms. Specifically, we consider the following missing data mecha- nisms, where the probability of an outcome being missing is dependent: (1) on neither A nor X (MCAR), (2) on treatment arm only, (3) on X only, (4) on A and X additively, (5) additionally on an inter- action between A and X so that there is differential missingness by treatment arm (all examples of CDM), (6) on observed levels of the outcome (MAR), and, (7) on unobserved levels of the outcome (MNAR). The information gathered from the sites provided insight into the identifi- cation, diagnosis, and treatment of patients at each of the sites. However, it was the on-site visits that proved most valuable in assessing suitability for participation in AMC trials. Based on the site visit evaluations the four top sites (in Zimbabwe, Uganda, Kenya, and South Africa) were selected for participation. g Conclusions The AMC has developed a detailed and methodical process for selecting international sites that have the clinical research infrastruc- ture, data management operations and the human and material re- source capacity required to successfully participate in clinical trials in HIV-associated malignancies. In our experience, the most essential and valuable part of that process is the on-site visits, which have been successful in vetting research sites that would be able to pro- vide high quality data and contribute to AMC’s mission of investigat- ing new treatment and prevention interventions of malignancies in people living with HIV both in the USA and internationally. Strategies for maximizing the value of these visits and collection of material from the visits will be discussed. An adaptive design for the identification of the optimal dose using joint modelling of efficacy and toxicity in phase I/II clinical trials of molecularly targeted agents 1 2 Paris-Saclay, Villejuif, France Correspondence: Maria Altzerinakou Trials 2017, 18(Suppl 1):O98 Methods Lessons learned from the implementation of the process allowed the ODMC to improve site selection methods by standardizing the site visit worksheets, and asking more targeted questions that better identified particular issues that were critical to study conduct. These changes streamlined the process and allowed for better comparison of the sites in the next round of site selection that occurred in 2014–5, dur- ing which three additional sites were selected. y Methods Half of IRB members believed an opt-out process was necessary and sufficient, while 40% believed it was unnecessary because initial consent was pro- vided and participants can always withdraw from research at any time. Most patients (65%) favored an active opt-out process at the time of randomization. Stakeholders’ views were mixed on the LAR’s role in the opt-out process when patients provide initial consent but later become unable to opt out. While patients and caregivers/LARs did not perceive any physical risks from enrolling early, perceived risks were associated with treatment: using two FDA-approved antibiotics was viewed as low risk, whereas the “unknown” aspects of a new investigational treatment generally heightened risk perceptions. As a result, most patients (67%) and caregivers/LARs (67%) were willing to enroll in a trial with FDA-approved antibiotics but were far less willing to partici- pate if an investigational antibiotic would be used (patients = 28%, caregivers/LARs = 25%). Challenges have included the practicality of randomising therapists within a restricted service, therapist capacity following variable re- cruitment rates, impact on recruitment, waiting lists, and deviations from the allocated therapist. Randomising therapists also has poten- tial implications for the intention-to-treat principle, where partici- pants are analysed as allocated, with consideration required as to how to analyse according to therapist. The context in which therapists work, their capacity and the needs of the trial population all influence the feasibility and appropriateness of randomising therapists. We will discuss the methodological benefits of including therapist randomisation and the implementation challenges this introduces, providing guidance on when it is appropriate and how to ensure it ‘works’ for therapists, participants and trial delivery. Page 226 of 235 Trials 2017, 18(Suppl 1):200 Page 226 of 235 Background To successfully execute clinical trials the study operations team must have an appreciation of the capabilities of the clinical sites. This is particularly vital in developing countries where necessary infrastruc- ture, availability of qualified staff and proper facilities are often scar- cer than in wealthier nations. The AIDS Malignancy Consortium (AMC) is a National Cancer Institute supported multicenter clinical tri- als group founded in 1995 to support innovative trials for AIDS- related cancers. In 2010 the AMC was tasked with expanding opera- tions internationally in sub-Saharan African countries with a high prevalence of HIV, with the goal of identifying and engaging a core group clinical sites that were capable of conducting contextually ap- propriate therapeutic and prevention trials in a variety of HIV- associated cancers and contributing to the AMC’s scientific agenda. Methods Introduction O96 Developing a comprehensive site selection process for a cancer network in a resource-limited settings in sub-Saharan Africa Megan Wirth, Dikla Blumberg, Kimberly Mosby-Griffin, Don Vena Emmes Corporation Correspondence: Megan Wirth Trials 2017, 18(Suppl 1):O96 O96 Developing a comprehensive site selection process for a cancer network in a resource-limited settings in sub-Saharan Africa Megan Wirth, Dikla Blumberg, Kimberly Mosby-Griffin, Don Vena Emmes Corporation O96 Developing a comprehensive site selection process for a cancer network in a resource-limited settings in sub-Saharan Africa Megan Wirth, Dikla Blumberg, Kimberly Mosby-Griffin, Don Vena Emmes Corporation Correspondence: Megan Wirth Trials 2017, 18(Suppl 1):O96 It is common for randomized controlled trials (RCTs) to have some missing outcomes. Yet recent reviews indicate that some authors analyze using only available outcome data (sometimes called a complete records analysis) without explicitly addressing the nature of the missingness. In most cases, this will lead to bias due to selection of a non-representative population. This can throw in to doubt the evi- dence generated from the trial. However, when proper adjustments are made, a complete records approach can provide valid inference under certain conditions. Yet, there are misconceptions and difficulties in de- termining when such an approach is valid. Megan Wirth, Dikla Blumberg, Kimbe Emmes Corporation Correspondence: Megan Wirth Trials 2017, 18(Suppl 1):O96 Emmes Corporation Correspondence: Megan Wirth Trials 2017, 18(Suppl 1):O96 g Background There is a growing literature in the field of causal inference that fo- cuses on the use of causal diagrams to determine when bias might arise in estimating a causal relationship of interest. Recent work by Daniel et al. (SMMR 21(3); 2011) used this approach to guide analyses with missing data. In the current talk, we adapt their approach to be specifically applicable to the RCT setting. Our goal is to provide a strategy to determine when a complete records analysis will be un- biased and therefore to determine when alternative methods (e.g. imputation or weighting) are required in order to avoid biased esti- mation of the intervention effect. Results and conclusion We show that in order to determine when a complete records ana- lysis is valid it is as important to focus on the form of the outcome data-generating model as well as the missing data mechanism. By providing an easily implementable strategy with simple rules, we seek to aid a broad audience to understand when analysis of only available RCT data will be sufficient to address the causal research question of interest without the need for the sometimes complex im- putation and weighting methods that are commonly used to address missing outcome data. Detection of incomplete data in surveillance and impact of missing data in assessment of effectiveness of financial incentives of viral load suppression in HPTN 065 pp Deborah Donnell1, Bernie Branson2, Wafaa El-Sadr3, Sahar Zangenah1 1Fred Hutchinson Cancer Research Center; 2Scientific Affairs LLC; 3 Columbia University Correspondence: Deborah Donnell Trials 2017, 18(Suppl 1):O99 y Correspondence: Deborah Donnell Trials 2017, 18(Suppl 1):O99 We present an enhancement to U&D, one that gradually limits dose allocations to within a tolerance interval around the MTD, using Sequential Probability Ratio Test (SPRT) decision rules. The interval boundaries can be rendered “soft” or “hard”, via SPRT decisions that are reversible or not, respectively. With some limitations, SPRT- enhanced U&D converges almost surely to a random walk confined inside the tolerance interval. An asymmetric “soft/hard” rule com- bination is possible, and is rather suitable for toxicity studies such as Phase I. Background Pragmatic trials often rely on pre-existing data systems to evaluate trial outcomes. HPTN 065 used US HIV Surveillance data to evaluate the outcomes of a clinic-randomized strategy trial to test the use of financial incentives to improve viral load suppression outcomes in the Bronx NY and Washington DC. p y Results Data triangulation throughout the trial revealed missing data in sur- veillance for some clinics in Washington DC. Evaluation of data in- consistencies and investigation into the causes of incompleteness, together with extensive collaboration with HIV surveillance staff, were largely successful in remediating the missing data for the trial evaluation period. Some baseline data could not be corrected due to lost access to data. During initial trial evaluation of effectiveness, ex- ploratory data analysis of time trends revealed a small number of clinics with more subtle data incompleteness issues, complicating the evaluation of the effectiveness of financial incentives. Increased imbalance on the restricted factors was observed using corrected data compared to the pre-trial data. Missing data in the baseline out- come assessment decreased the precision of efficacy estimates, with a 57% higher SE of the efficacy estimate in DC vs. NY. Trial efficacy results were sensitive to the effect of missing data, with the initial analysis of effectiveness of financial incentives showing an increase in viral load suppression of 3.9% (−3.4%, 11.1%; p = 0.27), changing to 3.7% (0.5, 6.9%) p = 0.022 after data completeness was addressed in Washington DC HIV surveillance. C l i Discussion Assaf Oron1, Nancy Flournoy2 1Seattle Children’s Hospital Research Institute; 2University of Missouri Correspondence: Assaf Oron Trials 2017, 18(Suppl 1):O100 In the future, we would like to test more scenarios and to evaluate more flexible models for dose-response relationship, providing the number of parameters to estimate remains in line with the sample size. Up-and-Down (U&D) is an established dose-finding approach used in many fields, yet all but forgotten by the Phase I design commu- nity. With respect to finding Phase I’s Maximum Tolerated Dose (MTD), when used with an appropriate estimation method U&D is on par with more complicated state-of-the-art designs, and its over- all behavior is more tractable and stable (Oron and Hoff, 2013). However, unlike some novel methods U&D cannot “sharpen” its dose-allocation random walk over time. Methods We present an adaptive dose-finding method, using a joint model- ling technique (Barrett J. et al., 2015) of a longitudinal outcome for continuous biomarker efficacy measurements and a probit time to first severe toxicity model, with shared random time slope. This method allows for exact likelihood inference, an important property, in the context of both missing at random due to low efficacy or in- creased toxicity and the case of small sample sizes. We combined this technique with the continual reassessment method, and used data collected over all treatment cycles. The selection of the dose at the entry of every new patient was based on the trade-off algorithm, proposed by Thall and Cook (2004), adapted to the situation of con- tinuous efficacy measures. For the evaluation of the method we run a set of simulations for two scenarios. Dose and time were included in both models, with the addition of dose-time interaction for the longitudinal outcome. We considered up to 9 dose levels, 3 treat- ment cycles, a maximum of 12 visits for the efficacy measurement and 66 patients. p Results In the first scenario the optimal dose was the first one and the MTD the second. The optimal dose was chosen at 85.6% of the cases, and another 12.8% was assigned to the MTD. For the second scenario, we assumed the fourth dose to be the optimal and the fifth to be the MTD. The optimal dose was suggested for 32.3% of the cases, while the MTD for 17.3% of them. At no point was the last dose level recommended. Our design performs very well when the optimal dose is at the edge of the dose range. This is because the desirability of certain doses around the optimal dose and MTD tends to be very close to one another. Even though this may be an issue when the optimal dose is located at the middle of the dose range, it is also the reason patients are very rarely allocated to doses far from the optimal dose. Finally, the method had interesting behavior regarding its ability to select the right dose in presence of missing data due to severe toxicity. Background Conventional dose-finding approaches in oncology of phase I trials aim to identify the maximum tolerated dose (MTD), based on toxicity events observed during the first treatment cycle. Even though this is relevant for cytotoxic agents, this may not be the case for molecularly targeted agents, usually administered in chronic schedules. Lately, continuous g Elizabeth Turner1, Melanie Prague2 1Duke University; 2INRIA, Bordeaux University Correspondence: Elizabeth Turner Trials 2017, 18(Suppl 1):O97 Page 227 of 235 Trials 2017, 18(Suppl 1):200 Page 227 of 235 biomarkers are used more and more to monitor efficacy. However, efficacy does not necessarily increase monotonically with dose and as corollary both toxicity and efficacy should be considered for the identification of the optimal dose. The optimal dose will be defined as the lowest dose that achieves high efficacy, while satisfying cer- tain toxicity requirements. of patients per clinic. During the trial, triangulation of data from the clinics, surveillance and the financial incentive delivery was used to as- sess data completeness. Sensitivity analysis and multiple imputation were subsequently used to evaluate 1) randomization balance based on incomplete pre-trial data in the restricted randomization; 2) the im- pact of incompleteness in baseline data on efficacy evaluation and 3) the sensitivity of the trial results to sites with missing data during the primary evaluation period. of patients per clinic. During the trial, triangulation of data from the clinics, surveillance and the financial incentive delivery was used to as- sess data completeness. Sensitivity analysis and multiple imputation were subsequently used to evaluate 1) randomization balance based on incomplete pre-trial data in the restricted randomization; 2) the im- pact of incompleteness in baseline data on efficacy evaluation and 3) the sensitivity of the trial results to sites with missing data during the primary evaluation period. g Conclusion Program assessments that utilize external data sources to evaluate outcomes need to conduct ongoing exploratory data analysis to understand and monitor data quality and completeness during the trial as trial results and study power will be affected by problems in the data source. Close collaboration with data source experts is crit- ical to assure quality and completeness of outcome data. O100 Up-and-down designs enhanced with SPRT rules for phase I cancer trials Assaf Oron1, Nancy Flournoy2 1Seattle Children’s Hospital Research Institute; 2University of Missouri Correspondence: Assaf Oron Trials 2017, 18(Suppl 1):O100 Methods SPRT-enhanced U&D generates treatment distributions more sharply peaked around the MTD than ordinary U&D, and with fewer toxicities on average. Despite the added complication, it is still simpler, more robust, and more tractable than most leading novel designs, thus presenting a highly attractive design choice for Phase I trials. The development and use of SPRT-enhanced U&D for several ongoing Phase I immunotherapy trials at Seattle Children’s Ben Towne Center is described. In a collaborative effort between study and surveillance staff, aggregate clinic outcomes were defined using variables available in surveillance data and aligned with the financial incentive intervention. Outcomes were centrally programmed by surveillance experts for evaluation using local surveillance databases. Pre-trial data on trial outcomes were used to conduct a restricted randomization of the 38 trial clinics, with the goal of achieving balance in pre-trial viral load suppression and number Page 228 of 235 Trials 2017, 18(Suppl 1):200 ( ) Conclusion We found that results for 33% of sampled clinical trials were available only on ClinicalTrials.gov, suggesting the database may be a useful resource to identify clinical trial results that would not otherwise be publically available for specific drug products. Although there were still gaps in the public availability of trial results, the existence of the trials is publicly known because of the registry entries. ClinicalTrials. gov data is increasingly being used to assess a broad range of research questions, many of which would have been difficult or impossible to address using published literature alone. Results Each new disease CDE working group (WG), consisting of 40–60 worldwide experts with varied fields of related expertise, met regu- larly to select from existing CDEs or refine and add from field- tested data elements from national registries and funded research studies. For most diseases, these WG members are divided into sub- groups applicable for each disease based on domains (e.g., Imaging, Demographics, Treatment/Interventions, Biomarkers). For Sport Concus- sion, subgroups are based on disease characteristics; Acute, Sub-Acute and Persistent/Chronic. All WGs began by reviewing CDEs previously developed for different diseases, conditions and symptoms to avoid unnecessary duplication. After the subgroup recommendations are completed, there is an internal review followed by public review in which time the recommendations are posted on the NINDS CDE Website for 1–2 months. For aim 1, our sample of 96 drug trial families included 329 trials, studying 86 drugs for 78 conditions by 45 companies. Of the 329 tri- als, 109 (33%) had results posted on ClinicalTrials.gov only, 42 (13%) in PubMed only, 81 (25%) in both, and 97 (29%) in neither. Of the 96 drug trial families, 45 families had results disclosed in at least one source for all trials. Of these 45 families, 15 families had results dis- closed only in ClinicalTrials.gov. For 18 drug trial families with a total of 48 trials, no results were available from any source. y For aim 2, we identified 404 research articles and 1,588 systematic re- views published between 2010–2015 that used data from the Clini- calTrials.gov registry, results database, or both. We categorized each research article into: characterization of clinical research for specific conditions (48%); research on ethics, adverse event reporting, and data mining (15%); quality of registered data and consistency with reporting policies (10%); characterization of the overall clinical re- search landscape (10%); evaluating publication bias or selective reporting (9%); and assessing specific research-related methods and issues (7%). O101 Impact of results reporting in clinicaltrials.gov Kevin Fain, Deborah A. Zarin, Rebecca J. Williams, Tony Tse, , , Thiyagu Rajakannan ClinicalTrials.gov Program Correspondence: Kevin Fain Trials 2017, 18(Suppl 1):O101 Disorders and Stroke Correspondence: Joy Esterlitz Trials 2017, 18(Suppl 1):O102 Correspondence: Joy Esterlitz Trials 2017, 18(Suppl 1):O102 Results ll All comments are collated and incorporated into the final CDE version release on the NINDS CDE website. The website provides uniform names and structures for each data element, as well as guidance docu- ments and template CRFs using the CDEs. WGs are also given the charge to classify the recommended CDEs as “Core, Supplemental- Highly Recommended, Supplemental or Exploratory” according to set definitions from the NINDS CDE project. Background j In 2016–2017, the National Institute of Neurological Disorders and Stroke (NINDS), NIH, and their partners have put forth new data stan- dards recommendations for the following diseases: (1) Cerebral Palsy; (2) Chiari I Malformation; (3) Headache, Version 2.0; (4) Sport Concus- sion; and (5) Subarachnoid Hemorrhage. The partners in develop- ment of these common data element (CDE) recommendations are the American Academy for Cerebral Palsy and Developmental Medi- cine, the Chiari & Syringomyelia Foundation, the Department of Defense, and the National Library of Medicine. Goals of the overall project include reducing study start-up time; increasing the efficiency and effectiveness of clinical research studies and treatment; and in- creasing data quality and data sharing across studies. Since 2008 U.S. federal law requires the submission of summary results to, and public posting on, ClinicalTrials.gov for certain clinical trials of FDA-regulated drugs, biologics and devices. We aimed to determine ways in which reporting to ClinicalTrials.gov has expanded the public availability and use of clinical trial information. Specifically, we assessed the extent to which ClinicalTrials.gov has improved the reporting of “full sets” of trials for specific drug products (aim 1). We also examined how researchers have used ClinicalTrials.gov data (aim 2). For aim 1, we examined ClinicalTrials.gov records for phase 2–4 clin- ical trials of drugs or biologicals with at least one U.S. study location, completed or terminated between 2007–2009, and funded by indus- try. We grouped trials into a “drug trial family” when the drug, condi- tion, and sponsor appeared to be the same across trials. We limited our analysis to a convenience sample of the first 96 drug trial families identified. For each trial, we assessed the presence of results posted on ClinicalTrials.gov and publications cited in PubMed as of June 2015. For aim 2, we searched PubMed to identify publications in Eng- lish between 2010–2015 that conducted original research using data retrieved from the ClinicalTrials.gov registry and/or results database. Authors manually reviewed eligible publications to categorize how ClinicalTrials.gov data were used in the study. O102 A common data language for clinical research studies: five (5) new disease recommendations from the national institute of neurological disorders and stroke, national institutes of health common data elements project Joy Esterlitz1, Sherita Ala’i1, Robin Feldman1, Kristen Joseph1, Muniza Sheikh1, Claudia S. Moy2, Sara S. Rue2, NINDS CDE Working Groups2 1The Emmes Corporation; 2The National Institute of Neurological Disorders and Stroke Correspondence: Joy Esterlitz Trials 2017, 18(Suppl 1):O102 A common data language for clinical research studies: five (5) new disease recommendations from the national institute of neurological disorders and stroke, national institutes of health common data elements project Oron AP, Hoff PD. Small-sample behavior of novel phase I cancer trial designs. Clinical Trials 2013; 10: 63–92. O101 Impact of results reporting in clinicaltrials.gov Kevin Fain, Deborah A. Zarin, Rebecca J. Williams, Tony Tse, Thiyagu Rajakannan ClinicalTrials.gov Program Correspondence: Kevin Fain Trials 2017, 18(Suppl 1):O101 O101 Impact of results reporting in clinicaltrials.gov Kevin Fain, Deborah A. Zarin, Rebecca J. Williams, Tony Tse, Thiyagu Rajakannan ClinicalTrials.gov Program Correspondence: Kevin Fain Trials 2017, 18(Suppl 1):O101 p j Joy Esterlitz1, Sherita Ala’i1, Robin Feldman1, Kristen Joseph1, Muniza Sheikh1, Claudia S. Moy2, Sara S. Rue2, NINDS CDE Working Groups2 1The Emmes Corporation; 2The National Institute of Neurological Disorders and Stroke Correspondence: Joy Esterlitz Trials 2017, 18(Suppl 1):O102 O101 O101 Impact of results reporting in clinicaltrials.gov Kevin Fain, Deborah A. Zarin, Rebecca J. Williams, Tony Tse, Thiyagu Rajakannan ClinicalTrials.gov Program Correspondence: Kevin Fain Trials 2017, 18(Suppl 1):O101 g Background CDEs were developed for 18 neurological disease areas prior to these five newly developed areas. There are over 11,000 CDEs in the NINDS library and over 600 validated instruments, Case Report Forms (CRFs), and study recommendations. The CDE initiatives strive to identify CDEs, template study forms, data dictionaries and guidelines to assist investigators who are initiating and conducting related clinical stud- ies. Use of standardized CDEs is critical to ensure that data housed within a database are of better quality; improve data sharing and meta-analyses; and help educate new clinical investigators. Methods g p Discussion ll d We will describe progress with accessing data and our approach to developing a data linkage pipeline. This includes: Mapping contact points where decisions are made about medicines management and identifying data providers at each contact; Mapping data fields from each provider with primary and key secondary endpoints; Identifying process to allow access to the data from the patient and from the data provider; Establishing robust processes to allow for the data to flow from the provider to the research team, including multiple data sharing agreements; Familiarisation with data sources through access to dummy data sets; Implementing governance requirements and submitting requests for data. O103 O103 Using electronic health records in clinical trials: rising to the challenge of developing a data linkage pipeline - experience from the ISCOMAT programme Suzanne Hartley1, Gerry Armitage2, Alison Blenkinsopp2, Chris Gale1, Beth Fylan Gwynn2, Ivana Holloway1, Claire Hulme1, Hanif Ismail2, Duncan Petty2, Mohamed Amin Mohammed2 1University of Leeds; 2University of Bradford Correspondence: Suzanne Hartley Trials 2017, 18(Suppl 1):O103 Using electronic health records in clinical trials: rising to the challenge of developing a data linkage pipeline - experience from the ISCOMAT programme Using electronic health records in clinical trials: rising to the challenge of developing a data linkage pipeline - experience from the ISCOMAT programme 1 2 2 1 Athanasia Gravani1, Marcus Jepson2, Caroline Wilson2, Athene Lane2, Chris Rogers3 g 1University of Bristol; 2MRC ConDuCT-II Hub for Trials Methodology Research, School of Social and Community Medicine, University of Bristol, Bristol, UK; 3Clinical Trials and Evaluation Unit, School of Clinical Sciences University of Bristol Bristol UK Suzanne Hartley1, Gerry Armitage2, Alison Blenkinsopp2, Chris Gale1, Beth Fylan Gwynn2, Ivana Holloway1, Claire Hulme1, Hanif Ismail2, Duncan Petty2, Mohamed Amin Mohammed2 1University of Leeds; 2University of Bradford Correspondence: Suzanne Hartley Trials 2017, 18(Suppl 1):O103 1University of Bristol; 2MRC ConDuCT-II Hub for Trials Methodology Research, School of Social and Community Medicine, University of Bristol, Bristol, UK; 3Clinical Trials and Evaluation Unit, School of Clinical Sciences, University of Bristol, Bristol, UK Correspondence: Athanasia Gravani Trials 2017, 18(Suppl 1):O104 p p Methods Six studies were purposefully selected to serve as case studies for the ATLAS study. Within each case study, trial managers (n = 6) fa- cilitating trial-specific training sessions as well as healthcare profes- sionals (principal investigators and research nurses, n = 13) from trial sites receiving the training, were interviewed between June 2015 and April 2016. Semi-structured face-to-face and telephone in- terviews were conducted using a topic guide. Interviews were audio-taped, transcribed verbatim and analysed thematically using NVivo. Non-participant observations of trial-specific training ses- sions (n = 13) as well as questionnaires completed by trial managers and site staff (n = 120) were also used to gain an overview of site staff and facilitators’ experience. y A data linkage project is in progress to determine the feasibility of the EHRs to obtain the data for the definitive trial. We will recruit 60 HF patients, who will provide written informed consent to access their EHR data from multiple data providers. The output will be (1) robust data linkage algorithm; (2) processes to access data for use in the definitive trial. Current progress We have recruited 43 patients and actively engaged with five data providers: National Institute for Cardiovascular Outcomes Research (NICOR) for national HF audit data; SystmOne & EMIS clinical systems for primary care data; NHS Digital for secondary care and mortality data and Community pharmacy/NHS Business Authority for dispens- ing and prescribing data. Results A variety of training modes (face-to-face, teleconference, online) were used to deliver training across the six case studies. However, face-to-face interaction was considered most beneficial in facilitating learning and enhancing personal relationship building and network- ing. Despite acknowledging the significance of the decision-making process in selecting the appropriate level and mode of training re- quired, this process was often overlooked at the early planning stages of trials and was usually poorly documented. Although evalu- ation of site initiation training to identify key areas where follow-up training might be required and improve future training sessions was considered best practice, it was not routinely conducted. The pur- pose of site initiations slightly differed between trial managers and site staff. Trial managers’ focus was to check that everything was in place at the site and that staff were aware of their roles and respon- sibilities in the trial, whereas, site staff viewed site initiations as the best time to meet and connect with the trial team. Additional train- ing and support provided to site staff over the course of the trial was considered of paramount importance in ensuring trial’s successful conduct. However, this was mainly provided retroactively on an ad- hoc basis. Conclusion Researchers who receive funding from NINDS are encouraged to use the CDEs in their CRFs and data management systems when- ever possible. To date, the feedback has included the CDE project’s impact on reducing study start-up time. Continued feedback is essential. The NINDS CDEs are a continually evolving resource, re- quiring updates as research advancements indicate. These newly Trials 2017, 18(Suppl 1):200 Page 229 of 235 Trials 2017, 18(Suppl 1):200 Page 229 of 235 The use of EHRs is becoming more established with trials, thus we provide recommendations for trialists seeking to access EHR across different health care providers. developed CDEs serve to be a valuable starting point or update for many neurological disease researchers and facilitate harmonization, streamlining and sharing of data. g g Support: This project was funded by HHSN271201200034C. O104 Analysis of trial-specific training during the site initiation phase: the ATLAS study 1 2 2 2 O 0 Analysis of trial-specific training during the site initiation phase: the ATLAS study Athanasia Gravani1, Marcus Jepson2, Caroline Wilson2, Athene Lane2, Chris Rogers3 1University of Bristol; 2MRC ConDuCT-II Hub for Trials Methodology Research, School of Social and Community Medicine, University of Bristol, Bristol, UK; 3Clinical Trials and Evaluation Unit, School of Clinical Sciences, University of Bristol, Bristol, UK Correspondence: Athanasia Gravani Trials 2017, 18(Suppl 1):O104 Background Electronic health records (EHRs), available for every patient who visits the UK NHS, are primarily used to inform the care of patients. They contain clinical information about primary and secondary care and treatments provided. Current literature suggests that EHRs can be used to inform the design and analysis of clinical trials, subject to ap- propriate data protection and governance, with potential to be more efficient in terms of costs and data return. Research funders are keen for trials to consider the use of routine data sources, where possible. Here we describe our progress with accessing EHRs and other elec- tronic data sources to support our research that crosses primary and secondary care. Methods: The National Institute for Health Research (NIHR) ISCOMAT Programme (Improving the safety and continuity of medicines management at care transitions) aims to design and evaluate an intervention to make best use of medicines and reduce harm through effective medicines management for heart failure (HF) patients following hospital discharge and across the primary care transition. It is a series of interlinked projects, culminating in a defini- tive cluster randomised controlled trial of 2,100 HF patients from 42 NHS Trusts in 3 UK regions. The primary endpoint is all-cause mortal- ity and HF related hospitalisations from hospital discharge. Key sec- ondary endpoints are medications prescribed post discharge. The majority of these data are held in the EHR. Trial-specific training provided during the start-up phase of studies has not been thoroughly investigated. In fact, little attention has been given to the role and structure of trial-specific training and how best to organise and implement it to ensure in-depth understanding of the trial protocol and trial-related procedures by trial staff. Aims To investigate the ways in which trial-specific training is provided during site initiation and explore trial managers; and site staff views on the importance of trial-specific training during study conduct. Moreover, ATLAS aimed to evaluate the process of trial-specific train- ing and make recommendations for improving the educational sup- port provided to site staff during the site start-up process. Methods O107 Continuous safety monitoring in large phase I cancer clinical trials with multiple expansion cohorts 1 2 Masha Kocherginsky1, Theodore Karrison2 1Northwestern University; 2University of Chicago Correspondence: Masha Kocherginsky Trials 2017, 18(Suppl 1):O107 Online interventions and data collection systems provide great prom- ise for efficient trial design however, there are challenges to ensure that systems are user friendly and intuitive to use, yet still allow the collection of data to validate the outcome of the study. Traditional phase I oncology clinical trial designs have morphed into multiple, parallel phase I trials incorporating concurrent expansion cohorts across multiple diseases or disease subtypes. These cohorts are used to provide additional safety data, as well as preliminary effi- cacy data. As a result, some of them have become quite large. For example, a recent study [Cancer Letter, Oct 7, 2016] looked at PD-1 drug development, and identified 35 phase I trials with 200 patients. In such trials, dose escalation is typically done using a standard phase I design, e.g. “3 + 3”, to determine the maximum tolerated dose (MTD), which is followed by the enrollment of multiple expan- sion cohorts in different disease types or biomarker-defined sub- groups. Safety is typically monitored only within each expansion cohort, and the results are not jointly evaluated in real time across the multiple expansion cohorts. y The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. O106 New cochrane risk of bias tool for cluster randomised tools Sandra Eldridge1, Marion Campbell2, Mike Campbell3, Amy Drahota4, Bruno Giraudeau5, Julian Higgins6, Barney Reeves6, Nandi Seigfried7 1Queen Mary University of London; 2Aberdeen University; 3Sheffield University; 4University of Porstmouth; 5University of Tours; 6Bristol University; 7South African Medical Research Council Correspondence: Sandra Eldridge Trials 2017, 18(Suppl 1):O106 New cochrane risk of bias tool for cluster randomised tools Sandra Eldridge1, Marion Campbell2, Mike Campbell3, Amy Drahota4, Bruno Giraudeau5, Julian Higgins6, Barney Reeves6, Nandi Seigfried7 1Queen Mary University of London; 2Aberdeen University; 3Sheffield University; 4University of Porstmouth; 5University of Tours; 6Bristol University; 7South African Medical Research Council Correspondence: Sandra Eldridge Trials 2017, 18(Suppl 1):O106 Here we propose to use a continuous safety monitoring approach based on the sequential generalized likelihood ratio test (SGLR) and discuss its application to adverse event (AE) monitoring for such large phase I trials with multiple concurrent expansion cohorts. Methods react trial Duncan Appelbe1, Heather Robinson2, Susie Dodd3, Andrew Walker2, Paula Williamson3, Fiona Lobban2 1The University of Liverpool; 2The Spectrum Centre for Mental Health Research, Lancaster University; 3NWHTMR, Department of Biostatistics, The University of Liverpool Correspondence: Duncan Appelbe Trials 2017, 18(Suppl 1):O105 Duncan Appelbe1, Heather Robinson2, Susie Dodd3, Andrew Walker2, Paula Williamson3, Fiona Lobban2 1The University of Liverpool; 2The Spectrum Centre for Mental Health Research, Lancaster University; 3NWHTMR, Department of Biostatistics, The University of Liverpool Duncan Appelbe1, Heather Robinson2, Susie Dodd3, Andrew Walker2, Paula Williamson3, Fiona Lobban2 A multi-disciplinary group of researchers including statisticians, other triallists, those leading the development of the new Cochrane risk of bias tool and experts in cluster randomised trials met over a period of a year to discuss the five different bias domains (bias arising from the randomization process, bias due to deviations from intended in- terventions, bias due to missing outcome data, bias in measurement of the outcome, bias in selection of the reported result) that are part of the new Cochrane risk of bias tool and how they relate to bias in cluster randomised trials. Paula Williamson , Fiona Lobban 1The University of Liverpool; 2The Spectrum Centre for Mental Health Research, Lancaster University; 3NWHTMR, Department of Biostatistics, The University of Liverpool Paula Williamson , Fiona Lobban 1The University of Liverpool; 2The Spectrum Centre for Mental Health Research, Lancaster University; 3NWHTMR, Department of Biostatistics, The University of Liverpool Correspondence: Duncan Appelbe Trials 2017, 18(Suppl 1):O105 Results The REACT study (https://www.reacttoolkit.co.uk/) is an online study to compare the effectiveness of a Relatives Education And Coping Toolkit (REACT) with an online Resource Directory. Half the people in the study receive the REACT toolkit, and half will receive the Re- source Directory. In this study the intervention and data collected from the participants is provided via the Internet, with little or no dir- ect interaction with the study team. This study opened in April 2016 and recruitment will close in early 2018. The REACT study comprises two disparate systems, the first being a bespoke data collection sys- tem that manages the eligibility/consent/registration/data collection processes, whilst the second system is a customised version of Word- Press that is used to deliver both arms of the intervention. Given the extent of the differences between assessing risk of bias in individually randomised trials and in cluster randomised trials, the group developed an adapted Cochrane risk of bias tool for clus- ter randomised trials. Differences occur in relation to assessing allocation concealment; appropriate assessment of bias in relation to blinding of participants and assessors; and ensuring missing clus- ters are considered in addition to missing values from participants. We also added an additional domain (bias arising from the timing of identification and recruitment of individual participants in rela- tion to timing of randomization) to cover the bias that may occur when individual participants in a cluster randomised trial are re- cruited after randomisation. Participants within this study do not attend regular clinical appoint- ments. As such retention at the two follow-up points and usage of the intervention requires automated reminders and more importantly the triggering of warnings/help to participants when they provide certain answers to specific questions. O107 Continuous safety monitoring in large phase I cancer clinical trials with multiple expansion cohorts 1 2 This approach is commonly used in vaccine studies [Shih et al.; Statistics in Medicine, 2009] to monitor rare events, and can be similarly used in phase I clinical trials to monitor the frequency of rarer high-grade or serious adverse events (SAEs), as well as to further evaluate whether the presumed MTD is near the targeted percentile. Trial Correspondence: Sandra Eldridge Trials 2017, 18(Suppl 1):O106 Conclusions Assessing bias in cluster randomised trials is not the same as asses- sing bias in individually randomised trials. Authors and peer re- viewers should be aware of key elements to include in trial reports to provide evidence that their trials are protected from bias. System- atic reviewers should use the Cochrane risk of bias tool adapted for cluster randomised trials to assess these trials for bias. p q The data required to determine the effectiveness of the intervention is taken from two sources: The response by the participants to out- come measures and data collected on the usage of the application (obtained from google analytics, server logs and custom reporting software). O107 O107 Continuous safety monitoring in large phase I cancer clinical trials with multiple expansion cohorts Masha Kocherginsky1, Theodore Karrison2 1Northwestern University; 2University of Chicago Correspondence: Masha Kocherginsky Trials 2017, 18(Suppl 1):O107 This presentation will discuss the electronic solutions required by the design of the study and the processes by which these systems were designed and implemented. A comparison of the data collected via google analytics and the server logs will be compared and commen- ted on, with a discussion about discrepancies being made. C l i Conclusions g We will describe challenges, variations in processes across data sources; the impact on establishing the data linkage pipeline and how this knowledge will streamline processes for the definitive trial. There is high variation and uncertainty on the ideal ways to provide trial-specific training to facilitate trial conduct. Based on the findings of the ATLAS study, a more standardised approach in the form of a Trials 2017, 18(Suppl 1):200 Page 230 of 235 randomised trials which takes account of the design of these trials and specific issues for assessing bias in these trials. Aim training toolkit was developed. The ATLAS training toolkit aims to bring together information from the current literature, regulations and various stakeholders’ views to advance the trial-specific training process. The aim of the tool is to support reviewers of trial reports to assess bias in cluster randomised trials. In this talk we describe the aspects of bias which are particular to cluster randomised trials or operate differently in these trials from the way they operate in individually randomised trials. How frequently should a disease be monitored? The interval length between observations in interval-censored data The National Health Service (NHS) has established procedures to sup- port research activities covered by the Research Governance Frame- work (RGF). These include Health Research Authority (HRA) approval to ensure regulatory compliance, and site-specific Research and De- velopment (R&D) agreement to support project delivery (capacity/ capability). In addition, the importance of research has been embed- ded in the NHS constitution (including Frameworks and Pledges) en- dorsed by the UK Government. The National Institute for Health Research (NIHR) Research Networks have also been established to embed Research infrastructure - primarily in the NHS setting -sup- porting research activities (trained research practitioners) and fund- ing via the Research Capability Funding (RCF). Research in non-NHS settings, such as Care Homes, is increasing however standardised procedures to support the delivery of high quality research have yet to be embedded in practice. Complexities arise in this setting as Care Homes are often independent businesses which vary in infrastructure with often a unique mix of funding streams including Local Author- ities, NHS (Continuing Healthcare), and self-funding - therefore rele- vant permissions will vary dependent upon the sites selected. Work has been done within the social care setting to bridge the gap, with the development of the “Implementation plan for research govern- ance in social care”, however the scope and applicability to Care Home research varies with no fixed format for formal research per- missions. Existing literature highlights the barriers to implementing the principles of the RGF in the Care Home setting, often referencing experiences from individual projects. However these experiences have yet to be consolidated into a robust summary of complexities with strategies to manage them in practice whilst still ensuring the delivery of quality research. We will present our experience from two randomised controlled trials within the care home setting alongside findings from a review of existing literature regarding trials imple- mentation in care homes. We will also outline an approach devel- oped to support the delivery of high quality research projects in this setting, summarising the key considerations for successful delivery in the absence of research infrastructure, the impact on timelines, re- source use, and trial implementation. How frequently should a disease be monitored? The interval length between observations in interval-censored data We will also discuss in more detail the key considerations during trial set-up, such as: i) Develop- ment and completion of formal permissions (Care Home Letter of Agreement) to document requirements for trial delivery at site, ii) Using quality/performance markers to inform site selection and on- going assessment of contextual factors impacting on trial delivery; iii) Selection of appropriate sites (eligibility/feasibility/recruitment) re- flective of the setting, iv) Developing processes to deliver the project in line with the principles of GCP (i.e. documented informed consent, data storage and transfer in accordance with DPA), and v) Incentives which sustain Care Home engagement, vi) Establishing informa- tion available within care records and development of appropriate data collection tools to support trial analysis. We have identified a series of challenges and proposed solutions which may help with future research in the Care Home setting and provides a useful re- source for researchers to deliver high-quality research in a research naïve setting. g Fang-Shu Ou, Martin Heller Mayo Clinic Correspondence: Fang-Shu Ou Trials 2017, 18(Suppl 1):O108 Background g Clinical trials in oncology use disease-free survival (DFS) or progression- free survival (PFS) as study endpoints. DFS and PFS are composite endpoints of disease status and overall survival. The tumor status is typ- ically measured by a computerized tomography (CT) scan which can be done every few months and the scan interval is typically determined by the treatment guideline. For example, DFS and PFS are common endpoints used in stage 3 colon cancer study and stage 4 colorectal cancer study, respectively. In stage 3 colon cancer, the CT scan interval used to be every 3 months and was extended to every 6 months. Re- cently, 12-month intervals between CT scans was proposed to reduce unnecessary radiation exposure. In stage 4 coloretal cancer, the typical CT scan interval is every 8–12 weeks due to the aggressive nature of the late stage disease. In clinical trials, if disease recurs or progresses, the event date is the date of the CT scan. As the interval length be- tween each assessment gets longer, we lose more precision regarding the actual event time and increase the potential bias as well. In this study, we examine how much information is lost due to interval- censoring, in particular, the number of events required to recover the loss of power. p Methods A numerical approximation of the exact distribution function for the hazard ratio (HR) sample statistic was used to assess the effect of interval censoring and determine the actual number of events needed to attain a given power. Monte-Carlo simulation was used to examine the extent of power loss using typical assumptions for stage 3 and 4 colorectal cancer trial design. Background g Bias has long been recognised as an issue that can seriously com- promise the validity of study trial results. Assessing bias from trial reports is often not easy. The Cochrane risk of bias tool is well estab- lished tool for facilitating this. This tool has recently been updated and as part of the new tool there is a specific adaptation for cluster Trials 2017, 18(Suppl 1):200 Page 231 of 235 Page 231 of 235 designs based on this approach will consist of a continuous safety monitoring boundary with a rejection number for each additional AE, while preserving the overall Type I and Type II error rates for the assumed AE rates. The boundaries can be calculated using existing software. can be quantified using a numerical approximation of the underlying distribution function. The effect of interval-censoring on accuracy of the time-to-event estimate is not the focus of this study but should be investigated to insure the point estimate is not horrendously biased when the scan interval lengthens. We consider two different implementations of the SGLR procedure: the first employs an overall monitoring rule based on pooled data across the multiple cohorts; the second monitors each cohort separ- ately, allowing for dose reduction in a given cohort if the boundary is exceeded. We simulate data using multiple expansion cohorts with different Poisson rates of AE’s, and evaluate the operating character- istics of these SGLR procedures. O109 Ensuring research quality in care home trials: learning from NHS governance procedures Amanda Lilley-Kelly, Rachael Kelley, Liz Graham, Vicki McLellan, Ian Wheeler, Suzanne Hartley Leeds Institute of Clinical Trials Research Correspondence: Amanda Lilley-Kelly Trials 2017, 18(Suppl 1):O109 Background Previous research published in the Lancet has estimated that as much as 85% of healthcare research might actually be wasted. A contributing factor to this may be interventions that are delivered with poor fidelity within clinical trials. Intervention fidelity is the extent to which an intervention is delivered as intended by its devel- opers, and assessing it is crucial as it increases confidence that changes in study outcomes are due to the effect of the intervention itself and not due to variability in implementation. Complex interven- tions involve several aspects with the potential to be implemented variably, therefore it is important to ensure a comprehensive evalu- ation of the fidelity of intervention delivery in such interventions. However, fidelity continues to be poorly assessed in clinical trials across multiple disciplines. At present there is a lack of pragmatic guidance and an insufficient focus on developing feasible ways of in- corporating fidelity assessment into clinical trials. Group-randomized trials (GRTs) are randomized studies that allocate intact groups of individuals to different comparison arms. A frequent practical limitation to adopting such research designs is that only a limited number of groups may be available, and therefore, simple randomization is unable to adequately balance multiple group-level covariates between arms. Therefore, covariate-based constrained randomization was proposed as an allocation technique to achieve this. Constrained randomization involves generating a very large number of possible allocation schemes (with a small number of groups to be randomized it is possible to generate all of them), cal- culating a pre-specified balance metric that assesses covariate imbal- ance, limiting the randomization space to a pre-specified percentage of those possible and randomly selecting one randomization to im- plement. However, related statistical issues on testing for interven- tion effect under such designs have not been thoroughly studied with binary outcomes. Motivated by two recent trials, we conduct a series of Monte Carlo simulations to evaluate the statistical properties of two model-based F-tests (linearization and likelihood F-test) and two randomization-based permutation tests (residual and likelihood P-test) under both simple and constrained randomization designs, with varying degrees of analysis-based covariate adjustment. Our re- sults indicate that constrained randomization improves the power of linearization F-test and two permutation tests when the prognostic group-level variables are controlled for in the analysis and the size of randomization space is reasonably small. We also demonstrate that constrained randomization reduces power loss from redundant analysis-based adjustment for non-prognostic covariates. Background g Phase II clinical trials aim to potentially screen out ineffective and iden- tify effective therapies to move forward to the randomized phase III set- ting. In phase II trials, the most common way of assessing tumor shrinkage is to dichotomize the patients by the response rate according to RECIST. Besides loss of statistical efficiency, studies have shown that designs using response rate require much more patients than those using continuous tumor size shrinkage. Further, drugs can be still active even if they do not lead to high levels of tumor regression, as could be observed with molecular targeted therapies and immunotherapies. These treatment strategies come often with lower toxicity profiles than traditional cytotoxic treatments, and have shifted the drug develop- ment paradigm into establishing evidence of biological activity, target modulation and pharmacodynamics effects of these therapies in early phase trials. As such, these trials need to address simultaneous Meng Liu, Emily V. Dressler Department of Biostatistics, College of Public Health, University of Kentucky T i l 2017 18(S l 1) O112 Meng Liu, Emily V. Dressler Department of Biostatistics, College of Public Health, University of Kentucky Correspondence: Meng Liu Trials 2017, 18(Suppl 1):O112 Correspondence: Meng Liu Trials 2017, 18(Suppl 1):O112 O110 Assessing implementation fidelity in clinical trials of behaviour change interventions 1 2 2 O111 An evaluation of constrained randomization for the design and analysis of group-randomized trials with binary outcomes Fan Li1, Elizabeth Turner1, William Vollmer2, David M. Murray3, Patrick J. Heagerty4, Elizabeth R. DeLong1 1Duke University; 2Kaiser Permanente; 3National Institutes of Health; 4University of Washington Correspondence: Fan Li Trials 2017, 18(Suppl 1):O111 Elaine Toomey1, James Matthews2, Deirdre Hurley2 1National University of Ireland Galway; 2University College Dublin Correspondence: Elaine Toomey Trials 2017, 18(Suppl 1):O110 Background Design con- siderations such as the choice of the balance metric and the size of randomization space are emphasized throughout. p g Objectives This research aimed to provide guidance on assessing fidelity of inter- vention delivery within clinical trials, i.e. to inform appropriate choice of fidelity assessment methods and how to achieve a balance between comprehensiveness and feasibility. Specifically, the study aimed to do this through establishing the fidelity of delivery of a behaviour-change physiotherapy-led intervention within the context of a clinical feasibility randomised controlled trial (RCT) and to explore the potential factors that may have influenced these fidelity results. Results For stage 3 colon cancer, assuming an exponential distribution with a 3-year DFS rate of 75% in the control arm and 84% in experimental arm (HR = 0.6), we found that 170 events are required to power the study with a one-sided alpha = 0.025 and beta = 0.1. If the patients received a scan every 3 months or 6 months, the study power is maintained. If scan frequency decreases to every 12 months, 2 add- itional events are required to maintain the study power. For stage 4 colorectal cancer, assuming the exponential distribution with median PFS equal to 2 months for the control arm and 4 months for the experimental arm (HR = 0.5), 90 events are required to provide 90% power with one-sided alpha = 0.25. For scan frequency of every 1 month, 6 additional events are required to maintain the study power. For scan frequency of every 2 and 3 months, which are stand- ard of care, 14 and 26 additional events are required to maintain the study power. y p Conclusions A sparse CT scan frequency can affect the study power requiring additional events to maintain the study power. The loss of power Trials 2017, 18(Suppl 1):200 Page 232 of 235 O110 Assessing implementation fidelity in clinical trials of behaviour change interventions Elaine Toomey1, James Matthews2, Deirdre Hurley2 1National University of Ireland Galway; 2University College Dublin Correspondence: Elaine Toomey Trials 2017, 18(Suppl 1):O110 y Methods This mixed methods study was a convergent triangulation design. 60 intervention sessions were delivered across seven sites by nine physio- therapists. Fidelity was assessed quantitatively using audio-recordings (n = 60), direct-observations (n = 24) and self-report checklists (n = 60) and qualitatively using individual semi-structured interviews with all physiotherapists (n = 9). Quantitative data were analysed using means and standard deviations. Qualitative data were analysed using thematic analysis. Integration of qualitative and quantitative data occurred at an interpretation level using a triangulation methodology. R l p Results Quantitatively, the fidelity scores were high for all assessment methods; with self-report (92.7%) consistently higher than direct-observations (82.7%), or audio-recordings (81.7%). There was significant variation be- tween physiotherapists’ fidelity scores but all scored above 50%. These findings were corroborated by the physiotherapist interviews. In terms of factors influencing fidelity, both qualitative and quantitative data found that physiotherapists’ knowledge and previous experience may have influenced their fidelity of the delivery. The physiotherapist quali- tative data additionally showed that participant-level and programme- level factors also influenced their fidelity. Meng Liu, Emily V. Dressler Department of Biostatistics, College of Public Health, University of Kentucky T i l 2017 18(S l 1) O112 A review of dose escalation methods in first-in-human small molecule oncology clinical trials 1 1 2 Bayesian approaches to phase II clinical trial designs usually base the inferences on the posterior distribution of the parameter of interest. If the posterior probability is computed and assessed in a sequential manner, the design may involve the problem of multipli- city, which, however, is often a neglected aspect in Bayesian trial designs. To effectively maintain the overall type I error rate, we propose solutions to the problem of multiplicity for Bayesian se- quential designs and, in particular, the determination of the cutoff boundaries for the posterior probabilities. We present both theoret- ical and numerical methods for finding the optimal posterior prob- ability boundaries with alpha-spending functions that mimic those of the frequentist group sequential designs. The theoretical ap- proach is based on the asymptotic properties of the posterior prob- ability, which establishes a connection between the Bayesian trial design and the frequentist group sequential method. The numerical approach uses a sandwich-type searching algorithm, which im- mensely reduces the computational burden. We apply least-square fitting to find the alpha-spending function closest to the target. We discuss the application of our method to single-arm and double- arm cases with binary and normal endpoints, respectively, and pro- vide a real trial example for each case. y ; y Correspondence: Daniel Greenwood Trials 2017, 18(Suppl 1):O115 Conclusions The research identified a number of key findings that can enable a feasible and comprehensive assessment of fidelity in clinical trials. Although conducted in the context of a feasibility RCT of a complex behavioural intervention, these findings can be generalised to other areas. Firstly, a ‘spectrum’ of quantitative assessment methods exists and choice may be guided by specific trial factors (e.g. size, resource availability). Secondly, a mixed methods approach provides a more comprehensive assessment of fidelity and can be feasibly done by utilising pre-existing trial qualitative data collection. Finally, assessing fidelity and its influencing factors can help understand how and why adaptations and deviations have occurred and can guide potential refinements. Trials 2017, 18(Suppl 1):200 Page 233 of 235 evaluation of safety as well as proof-of-concept biological marker activ- ity or changes in continuous tumor size instead of binary response rates. However, there is a lack of interim strategies developed to monitor futility and/or efficacy for these types of continuous outcomes, especially in the single-arm setting. p p Methods We aimed to examine stroke rehabilitation RCTs’ (i) recruitment effi- ciency (ii) trial features associated with recruitment efficiency and (iii) reporting of recruitment information. Lee and Liu (2008) developed a predictive probability design for binary outcomes in a single-arm phase II cancer clinical trial. We ex- tend their design into a two-stage setting for continuous endpoints assuming a normal distribution with known variance. We evaluate and present the design properties for both optimal and minimax designs. g Results Both simulation results and presented case study have demonstrated that the proposed design can incorporate an interim stop for futility that controls both type I and type II error rates. As expected, using continuous tumor size resulted in lower expected and maximum sample sizes. A limited exploration of the choice of prior was per- formed and the numeric results were shown to be robust. Conclusions The proposed two-stage design based on predictive probability is efficient with similar sample size reduction for continuous out- comes and possess desirable operating characteristics. As research rapidly moves to incorporate more immunotherapies and targeted therapies, it will accommodate new types of outcomes while allow- ing for flexible stopping rules for futility and/or efficacy to continue optimizing trial resources and prioritize agents with compelling early phase data. Background First-in-human clinical trials are a crucial stage in the development of novel oncology drug candidates. Despite their importance, the de- sign of these trials varies significantly. An improved understanding of these variations is needed in order to improve the impact of cancer research and make the process more economical. The aim of this re- search was to review, analyse and compare the dose escalation methods used in first-in-human small molecule oncology clinical trials to provide greater understanding of the variation of oncology clinical trial design. Background Efficient recruitment to randomised controlled trials (RCTs) is vital in high-quality, cost effective, clinical research. Under-recruitment, re- cruitment extensions and supplementary funding requests are com- mon. Under-recruitment leads to underpowered trials, inconclusive results and wasted research effort. Efficient recruitment to stroke re- habilitation RCTs is considered particularly problematic but has yet to be investigated. y Objective Objective We propose a two-stage single-arm design for continuous endpoints that allows for early futility stopping while maintaining desirable stat- istical properties. Aims and Objectives p g Methods We included all trials identified by the Cochrane Stroke Group’s trial register. This includes trials identified from 35 electronic databases (e.g. Medline, CINAHL, EMBASE); numerous clinical trial registers; and hand-searching other resources. We included publications between 2005–2015, grey literature, and had no language restrictions. Records that reported RCTs of stroke rehabilitation non-pharmacological interventions with patient populations were included. We extracted recruitment efficiency data (i) Rate: numbers randomised as a per- centage of those screened for eligibility (ii) Speed: average monthly recruitment numbers across sites (iii) overall Dropout rates. Data on recruitment sites, recruiters, setting, funding support, ethical review, intervention type, targeted impairment, control comparison, and coun- try of recruitment were extracted by two independent reviewers. Dis- crepancies were resolved by a third reviewer. p Results Two independent reviewers screened 12,939 titles, 1,270 abstracts and 788 full texts for eligibility. 515 trials were included. Only 39% of stroke survivors screened were subsequently randomised. Subgroup analysis revealed that recruitment efficiency was significantly affected by the intervention type, control condition, targeted impairment, re- cruitment time point and setting. C l i Conclusions Stroke rehabilitation trials experience notable recruitment inefficiencies. Control of type I error rates in Bayesian group sequential designs Haolun Shi, Guosheng Yin University of Hong Kong Correspondence: Haolun Shi Trials 2017, 18(Suppl 1):O113 O115 O115 A review of dose escalation methods in first-in-human smal molecule oncology clinical trials Daniel Greenwood1, Ian Stratford1, Steven Booth2 1University of Manchester; 2University of Hertfordshire Correspondence: Daniel Greenwood Trials 2017, 18(Suppl 1):O115 Method: The literature databases PubMED, Web of Science and Wiley Online Library were searched to identify published first-in-human trials of small molecule oncology candidates. Search terms used were: [1] “first-in-man” [title] OR [2] “first-in-human” [title] OR [3] “oncology OR cancer”. Further searches were completed which replaced terms [1] and [2] with [4] “dose escalation”. A total of 69 trial publications ap- plicable to the aim of this review were identified. Each publication was reviewed and relevant data extracted which included the type of dose escalation method and individual dose levels used. To enable comparative analysis of 3 + 3 and “accelerated trial design” (ATD) es- calation methods (the most popular methods with trials of suitable number for comparison), dose level data was translated to “Unique Dose Levels” (UDLs). The mean number of UDLs and dose increments of 3 + 3 and ATD trials were then compared. We hypothesised that, Efficiency of recruitment to stroke rehabilitation randomised controlled trials: secondary analysis of recruitment data ll1 d 1 d 1 h kl 2 y controlled trials: secondary analysis of recruitment data Kris McGill1, Marian Brady1, Jon Godwin1, Catherine Sackley2 1Glasgow Caledonian University; 2Kings College London Correspondence: Kris McGill Trials 2017, 18(Suppl 1):O114 Page 234 of 235 Trials 2017, 18(Suppl 1):200 Page 234 of 235 given its accelerated purpose, trials which employed the ATD escal- ation method would have fewer dose levels and larger increments between doses and thereby achieve the maximum tolerated dose (MTD) quicker. at random assumption. There may be lack of lack of awareness of the issue, or guidance on how to conduct such analyses. at random assumption. There may be lack of lack of awareness of the issue, or guidance on how to conduct such analyses. at random assumption. There may be lack of lack of awareness of the issue, or guidance on how to conduct such analyses. given its accelerated purpose, trials which employed the ATD escal- ation method would have fewer dose levels and larger increments between doses and thereby achieve the maximum tolerated dose (MTD) quicker. Chalmers Finalists Of the 69 trials identified for review, 64 were parallel multiple dose design and five were parallel single dose design. Of the parallel mul- tiple dose trials, 54/64 used a rule based dose escalation method with only 4/64 opting for a model based dose escalation method. Nonparametric overdose control for dose finding in drug-combination trials Nonparametric overdose control for dose finding in drug-combination trials Chi Kin Lam, Ruitao Lin, Guosheng Yin The University of Hong Kong Correspondence: Chi Kin Lam Trials 2017, 18(Suppl 1):O118 Methods We conducted a review of trial-based CEA published in the Health Technology Assessment journal between January 2013 and Decem- ber 2015. This journal is a key depository of full-scale economic eval- uations funded by the UK National Institute for Health Research Health Technology Assessment Programme (NIHR HTA), where each report includes details of the analysis methods and results. l Fifty-two eligible trials were identified. Nearly all of them had missing data, with a median proportion of participants with complete cost- effectiveness data of 63% (IQR 47% to 81%). Restricting analysis to complete-records remains the most common approach (43% of pri- mary analysis), followed by multiple imputation (30%). Only half of the studies conducted sensitivity analyses for missing data, and it was typically using a limited range of assumptions such as data being either “missing completely at random” or “missing at random”. Conclusions O119 Biomarker stratified design enriched by auxiliary variables Ting Wang1, Xiaofei Wang2, Haibo Zhou1, Jianwen Cai1, Stephen L. George2 1University of North Carolina at Chapel Hill; 2Duke University Correspondence: Ting Wang Trials 2017, 18(Suppl 1):O119 p g 1University of North Carolina at Chapel Hill; 2Duke University Correspondence: Ting Wang Trials 2017, 18(Suppl 1):O119 p g 1University of North Carolina at Chapel Hill; 2Duke University Correspondence: Ting Wang Trials 2017, 18(Suppl 1):O119 Missing data remain a major concern for trial-based CEA. In spite of its limitations, restricting analysis to the subset of complete records is the most common approach. Analysts typically do not follow guidelines in evaluating the robustness of inferences to departure from the missing g Conclusion The findings of this research suggest that the ATD dose escalation method may not achieve the MTD quicker than the traditional 3 + 3 dose escalation method. If ATD is in fact not accelerated, this finding has implications for drug development organisations where a pro- longed clinical trial process has large cost implications, and also for patients who may have to wait longer for drugs to reach the clinic. Future research will seek to compare the dose escalation methods used in first-in-human clinical trials of the same drug candidate (e.g. in Caucasian and Asian populations) to control the effect of drug variation on dose escalation whilst acknowledging and accounting for population differences. Missing data in trial-based cost-effectiveness analysis: a review of the current practice Baptiste Leurent, Manuel Gomes, James Carpenter London School of Hygiene & Tropical Medicine Correspondence: Baptiste Leurent Trials 2017, 18(Suppl 1):O116 Background C ff i drug combination trials Chi Kin Lam, Ruitao Lin, Guosheng Yin The University of Hong Kong Correspondence: Chi Kin Lam Trials 2017, 18(Suppl 1):O118 Cost effectiveness analyses (CEA) of randomised trials are an import- ant source of information for health commissioners to decide on how to best allocate limited resources. Missing data are a common issue in trials, and is particularly problematic in CEA which require complete information on both costs and effects. Recent guidelines addressing this issue encourage the use of suitable methods such as multiple imputation and recommend the conduct of sensitivity ana- lyses under varied missing data assumptions. It is however unclear to what extent these recommendations have affected practice. Aim: To review the extent of, and methods used to address, missing data in trial-based cost-effectiveness analysis. With the emergence of novel targeted anti-cancer agents, drug combinations have been recognized as cutting-edge development in oncology. However, limited attention has been paid to the overdose control in the existing drug-combination dose-finding trials. We develop the multi-agent nonparametric overdose control (MANOC) design for dose finding in phase I drug-combination trials. Based on a Bayesian decision-theoretic approach, we control the probability of overdosing in a local region at the current dose combination. Simula- tion studies are conducted to investigate the performance of the proposed design. While the MANOC can prevent patients from being allocated to overtoxic dose levels, its accuracy and efficiency are still competitive to the existing designs. As an illustration, the MANOC is applied to a phase I clinical trial for identifying the maximum toler- ated dose combination of buparlisib and trametinib. Method: The most popular rule based dose escalation methods were 3 + 3 (31/54) and ATD (16/54). The mean number of UDLs of 3 + 3 and ATD trials did not differ significantly (9.4 vs. 8.5; p-value = 0.31 for two-tailed t-test with 95% confidence intervals). Through use of a graphical heat map method of analysis, the dose increments of ATD trials do not appear to be greater than those of 3 + 3 trials. Given these findings, the research hypothesis was disproven. l O117 Adaptive prediction of event times in clinical trials Yu Lan1, Daniel F. Heitjan2 1Southern Methodist University; 2Southern Methodist University & UT Southwestern Medical Center Correspondence: Yu Lan Trials 2017, 18(Suppl 1):O117 In event-based clinical trials it is common to plan interim analyses to take place at planned event counts. Accurate prediction of these event times can support trial planning and the efficient allocation of re- sources. Available methods to create such predictions include paramet- ric cure and non-cure models and a nonparametric approach based on the Bayesian bootstrap. The parametric methods work well when their underlying assumptions are met, and the nonparametric method gives calibrated but inefficient predictions across a wide range of models. However, in the early stages of a trial, when predictions have the high- est marginal value, there is insufficient data to provide evidence about the form of underlying model, including whether a cure fraction exists. In this paper, we propose an adaptive method to address this defi- ciency. The method draws predictions from the model with the highest Bayesian posterior probability within a range of candidate models. To further capture the uncertainty in clinical trial prediction, we apply a simulation strategy using the Bayesian bootstrap. A simulation study demonstrates that the adaptive method produces prediction intervals that have good coverage and are slightly wider than non-adaptive in- tervals but narrower than nonparametric intervals. It leads to some im- provements in making predictions with data from the International Chronic Granulomatous Disease Study. Introduction In precision medicine, drugs are developed to target patients with certain genetic profiles. Targeted trials test treatment benefit only in Trials 2017, 18(Suppl 1):200 Page 235 of 235 that a patient with positive auxiliary variable also has a positive true biomarker, is large enough. that a patient with positive auxiliary variable also has a positive true biomarker, is large enough. the biomarker-positive patients. Trials with a biomarker-stratified design (BSD) allow a complete assessment of the effect of the new drug rela- tive to the standard drug overall as well as in various biomarker- defined subgroups. However, a BSD trial often requires enrolling a large number of patients, especially when the proportion of the biomarker positives is small and thus the conduct of a BSD trial is expensive when the cost of ascertaining the true biomarker status is high. Methods When employing the proposed design in a practical study, Gefitinib or Carboplatin-Paclitaxel in Pulmonary Adenocarcinoma in North America, for testing the treatment effect among EGFR mutants and the interaction effect, ABSD requires 155 randomized patients com- pared to the 930 randomized patients required by a BSD. In addition, ABSD reduces the total cost cost by 64.6%. We propose a special type of biomarker enrichment design, Biomarker Stratified Design Enriched by Auxiliary Variables (ABSD), in which a sub- group of patients, typically the biomarker-positive patients, are enriched based on the value of an inexpensive auxiliary variable that is positively correlated to the true biomarker. In such a design, all auxiliary-variable-positive patients and a proportion of the auxiliary- variable-negative patients are selected and included in the randomized trial. We compared the efficiency of ABSD with BSD in estimating vari- ous treatment parameters that are estimable in a BSD trial including the treatment effect in all patients and in specific biomarker subgroups and the interaction effect. We compared the efficiency of the two designs in term of the number of treated patients and the cost of the trial, assuming a range of prevalence of the true biomarker-positive patients in the overall population, the positive predictive value of the auxiliary variables for the true maker, and configurations of cost utilities of various items in conducting such trials. Another advantage of ABSD is that in most cases we can immedi- ately randomize patients selected in the screening process without waiting for the result of true biomarker test, which can substantially reduce reduce the waiting time. Introduction g Since PPV plays a very important role in the proposed design, a Bayesian adaptive ABSD is also proposed to deal with the mis- specified PPV. p Conclusion A biomarker stratified design enriched by an auxiliary variable can be more efficient than the standard BSD design. The efficiency gain can be particularly significant when the auxiliary variable has a high PPV, the prevalence rate of the biomarker-positive subgroup is small and the cost of ascertaining the true biomarker status is high relative to the auxiliary variable. Publisher’s Note S i N Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. The proposed ABSD always reduces the total cost of the trial relative to a BSD when the prevalence rate is small and the PPV, the probability Submit your next manuscript to BioMed Central and we will help you at every step: Results g Results Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step:
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DNA from extinct giant lemurs links archaeolemurids to extant indriids
BMC evolutionary biology
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Available at: https://works.bepress.com/laurie_godfrey/16/ April 28, 2008 April 28, 2008 University of Massachusetts Amherst University of Massachusetts Amherst From the SelectedWorks of Laurie R. Godfrey DNA from Extinct Giant Lemurs Links Archaeolemurids to Extant Indriids Laurie Godfrey, University of Massachusetts - Amherst Ludovic Orlando Sébastien Calvignac Céline Schnebelen Christoph J Douady, et al. BioMed Central Published: 28 April 2008 Published: 28 April 2008 BMC Evolutionary Biology 2008, 8:121 doi:10.1186/1471-2148-8-121 This article is available from: http://www.biomedcentral.com/1471-2148/8/121 This article is available from: http://www.biomedcentral.com/1 © 2008 Orlando et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creative which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cit Abstract Background: Although today 15% of living primates are endemic to Madagascar, their diversity was even greater in the recent past since dozens of extinct species have been recovered from Holocene excavation sites. Among them were the so-called "giant lemurs" some of which weighed up to 160 kg. Although extensively studied, the phylogenetic relationships between extinct and extant lemurs are still difficult to decipher, mainly due to morphological specializations that reflect ecology more than phylogeny, resulting in rampant homoplasy. Results: Ancient DNA recovered from subfossils recently supported a sister relationship between giant "sloth" lemurs and extant indriids and helped to revise the phylogenetic position of Megaladapis edwardsi among lemuriformes, but several taxa – such as the Archaeolemuridae – still await analysis. We therefore used ancient DNA technology to address the phylogenetic status of the two archaeolemurid genera (Archaeolemur and Hadropithecus). Despite poor DNA preservation conditions in subtropical environments, we managed to recover 94- to 539-bp sequences for two mitochondrial genes among 5 subfossil samples. Conclusion: This new sequence information provides evidence for the proximity of Archaeolemur and Hadropithecus to extant indriids, in agreement with earlier assessments of their taxonomic status (Primates, Indrioidea) and in contrast to recent suggestions of a closer relationship to the Lemuridae made on the basis of analyses of dental developmental and postcranial characters. These data provide new insights into the evolution of the locomotor apparatus among lemurids and indriids. than today. Pygmy hippos and the world's largest bird – the Great Elephant Bird Aepyornis maximus – are just two striking examples of those endemic species that began to Research article Address: 1Paléogénétique et Evolution Moléculaire, Université de Lyon, Institut de Génomique Fonctionnelle de Lyon, Institut Fédératif Biosciences Gerland Lyon Sud, Université Lyon 1, CNRS, INRA, Ecole Normale Supérieure de Lyon, 46 allée d'Italie, 69364 Lyon Cédex 07, France, 2CNRS UMR 5023, Laboratoire d'Ecologie des Hydrosystèmes Fluviaux, Université Claude Bernard Lyon 1, 6 rue R. Dubois, Bat. Darwin-C, F- 69622 Villeurbanne Cédex, France and 3Department of Anthropology, 240 Hicks Way, University of Massachusetts, Amherst, MA 01003, USA - ludovic.orlando@ens-lyon.fr; Sébastien Calvignac - sebastien.calvignac@ens-lyon.fr; Email: Ludovic Orlando - ludovic.orlando@ens-lyon.fr; Sébastien Calvignac - sebastien.calvignac@ens-lyon.fr; Céline Schnebelen - equipe.paleo@ens-lyon.fr; Christophe J Douady - douady@univ-lyon1.fr; Laurie R Godfrey - lgodfrey@anthro.umass.edu; Catherine Hänni* - catherine.hanni@ens-lyon.fr dovic Orlando - ludovic.orlando@ens-lyon.fr; Sébastien Calvignac - sebastien.calvignac@ens-lyon.fr; Email: Ludovic Orlando - ludovic.orlando@ens-lyon.fr; Sébastien Calvignac - sebastien.calvignac@ens-lyon.fr; Céline Schnebelen - equipe paleo@ens-lyon fr; Christophe J Douady - douady@univ-lyon1 fr; Laurie R Godfrey - Céline Schnebelen - equipe.paleo@ens-lyon.fr; Christophe J Douady - douady@univ-lyon1.fr; Laurie R Godfrey - lgodfrey@anthro.umass.edu; Catherine Hänni* - catherine.hanni@ens-lyon.fr Céline Schnebelen - equipe.paleo@ens-lyon.fr; Christophe J Douady - douady@univ-lyon1.fr; Laurie R Godfrey - Catherine Hänni* - catherine.hanni@ens-lyon.fr * Corresponding author Received: 11 December 2007 Accepted: 28 April 2008 Open Research article DNA from extinct giant lemurs links archaeolemurids to extant indriids Ludovic Orlando1, Sébastien Calvignac1, Céline Schnebelen1, Christophe J Douady2, Laurie R Godfrey3 and Catherine Hänni*1 Open Access Background g At the time of the first settlers over 2000 years ago [1,2], Madagascar harboured a greater faunal and floral diversity Page 1 of 9 (page number not for citation purposes) Page 1 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/8/121 BMC Evolutionary Biology 2008, 8:121 Other thorny phylogenetic questions still await a molecu- lar contribution. Such is the case for the least arboreal and most terrestrial lemurs, the Archaeolemuridae [19,20]. Among Lemuriformes, this family is presumed to belong to the superfamily Indrioidea and consists of three extinct species: Archaeolemur edwardsi, Archaeolemur majori, and Hadropithecus stenognathus [7,19]. Hadropithecus survived until the end of the first millennium A.D. whereas Archae- olemur experienced at least a half-a-millennium reprieve [1], possibly thanks to a greater plasticity of resources/ habitat exploitation [21]. All of the Archaeolemuridae were extremely powerfully built [19] and exhibited char- acters reminiscent of cercopithecoids [22]. On the basis of cranial anatomy and dental morphology, Archaeolemuri- dae have been considered the sister taxon of extant indri- ids and the palaeopropithecids (families Indriidae and Palaeopropithecidae) [23-25]. However, developmental features [26] as well as the postcranial characters of a newly excavated Hadropithecus subadult (Andrahomana Cave, southeastern Madagascar [27,28]) recently chal- lenged this consensual scenario and underscored striking similarities with lemurids (family Lemuridae). The debate is therefore still open [18]. decline and finally disappeared completely in the centu- ries following human colonization [1,3]. Albeit emblem- atic of Madagascar (>90 species among 15 genera currently alive in Madagascar, which represents about 15% of the whole diversity among Primates), lemurs do not stand as an exception [4-6]. Some 2,000 years ago, they inhabited a wide variety of wooded terrains, from forests to open woodlands and marshlands [7]. A spectac- ular array of life history traits derives from a single ances- tral primate that colonized Madagascar around 60MYA [8]. But human activities, such as overhunting and habitat modifications related to farming and pastoralism, led at least 17 species – belonging to nine different genera – to eventual extinction [9,10]. Several entire families – the Archaeolemuridae, Palaeopropithecidae, and Megaladap- idae – disappeared. Because all of the extinct species were larger than extant species, they are called 'giant' lemurs. Some of them dis- played particularly spectacular features (for example, enormous body size, elongated rostra and widely sepa- rated orbits, extremely rapid dental development). Background The Palaeopropithecidae, including the most specialized genus, Palaeopropithecus and its close relative Archaeoindris, as well as the smaller-bodied Babakotia and Mesoprop- ithecus, were so convergent on tree sloths that they have been dubbed the "sloth lemurs" [11]. However, Archaeoin- dris, despite its specializations for climbing, would have had to spend most of its time on the ground due to a body mass (ca. 160 kg) rivalling that of male gorillas [12]. The megaladapids, some of which rivalled female gorillas in body mass at ca. 88 kg [12], were slow climbers capable of suspension but not leaping [13]. They sported peculiar cranial specializations for consuming leaves and con- verged postcranially with koalas; they have thus been dubbed "koala lemurs." None of the 11 archaeolemurid specimens analysed so far by molecular biologists yielded amplifiable DNA [18]. In this study, we undertook an extensive analysis of 12 new Archaeolemur and Hadropithecus subfossil remains. We report for the first time the successful characterization of 94–539 bp along two mitochondrial genes (cytochrome b and 12S rRNA). This sequence data provides a good sup- port for a sistership between archaeolemurids and extant indriids, in agreement with the most generally accepted morphological phylogenetic scenario but in contrast to recent suggestions made on the basis of analyses of dental developmental and postcranial data. Extinct and extant lemurs exhibit an extraordinary range in body size and diversity of locomotor and dietary pat- terns. Ecologically driven convergences have considerably confounded interpretations of the phylogenetic relation- ships among lemurs. However, morphological data (cra- nial and postcranial characters, developmental features) have aided in the construction of a number of different phylogenetic hypotheses [14,15] some of which have been tested using molecular tools. For instance, Crovella et al. (1994) used hybridization features of highly repeated DNA probes to support the proximity of the extinct Pachylemur insignis to the ruffed lemurs (Varecia variegata) [16]. More recently, using PCR to recover short overlapping fragment over the cytochrome b gene, Yoder et al. (1999) [17] and Karanth et al. (2005) [18] were able to confirm Palaeopropithecidae as a sister group of extant indriids but challenged the long-standing proximity of Megaladapis to Lepilemur. Results f Histoire de la Terre, USM203/UMR5443 Paléobiodiversité, 8 rue Buffon, 75005 Paris. CNRS UP2147 = Dynamique de l'Évolution Humaine: Individus, Populations, Espèces, 44 rue de l'Amiral Mouchez, 75014 Paris Column 'Ancient DNA' summarizes the samples that gave authentic ancient DNA fragments. The specimen under collection number 1937–44 at the MNHN yielded authentic DNA fragments when a bone fragment (sample CH147) was sampled but no result when a molar root (sample CH190) was analyzed (Table 1 & Table S2, Additional file 1). All in all, these results sug- gest poor DNA preservation conditions in the subfossils, which is consistent with what is known about DNA decay in warm subtropical environments [29] and with a previ- ous ancient DNA survey of Malagasy subfossils [18,30]. ithecus stenognathus sample CH421 was found to exhibit a larger divergence from the Archaeolemur sequences (24 substitutions). Raw divergences were in good agreement with what was expected from morphological similarities between these species and genera [19,22]. Because our procedures respect the most stringent criteria of authentic- ity (independent extractions/amplifications, cloning and sequencing; see Materials and methods), we are confident that the ancient DNA sequences reported here are authen- tic and we used them in phylogenetic analyses (Figure 1ab). Despite such difficulties, we managed to recover authentic ancient DNA sequences from individuals belonging to each of the three genera sampled (Archaeolemur, Hadrop- ithecus and Megaladapis). Sample CH147 (Megaladapis edwardsi) allowed the recovery of a 190-bp cytochrome b sequence that exhibited one and three transitions with the two Megaladapis sequences reported in [18] (Accession numbers Genbank:AY894790 and AY894791, respec- tively). Notably, all these sequences were highly divergent (34 substitutions) from the Megaladapis haplotype described in [31] (Figure 1a, noted with a star). This hap- lotype (Accession number Genbank:AJ278142) was already criticized by Karanth et al. (2005) [18] and is now definitively confirmed as a probable PCR-contaminant. Archaeolemur majori samples (CH210 and CH146) deliv- ered respectively 269-bp and 335-bp in cytochrome b (Table 1). Both sequences were identical over the 269 shared nucleotides but markedly different from all availa- ble lemur sequences. Results f To further investigate the evolutionary history of giant lemurs, we performed an ancient DNA study on 12 sub- fossil remains representing 10 individuals belonging to three extinct giant lemur genera (Table 1). Short, overlap- ping segments of three mitochondrial genes (control region, cytochrome b and 12S rRNA) and one nuclear gene (IRBP) were targeted by PCR (Table S1, Additional file 1). The maximal amplicon length was about 160 bp (including primers). Among the 12 specimens analyzed, only 5 yielded amplifiable DNA although numerous extractions and PCR reactions were performed (Table S2, Additional file 1). It is noteworthy that none of the Con- trol Region fragments targeted gave positive results, sug- gesting poor efficiency of the PCR primers used here. Even for samples that delivered authentic mtDNA fragments, no nuclear DNA was recovered although fragments as short as 80 bp were targeted (Table S2, Additional file 1). Page 2 of 9 (page number not for citation purposes) BMC Evolutionary Biology 2008, 8:121 http://www.biomedcentral.com/1471-2148/8/121 Table 1: Subfossil samples examined. Species Sample DNA length (Nb. of overlapping fragments) Location Collection Reference Description Cyt b 12S rRNA Archaeolemur sp. CH70 - - Antsingiavo-A, Narinda CNRS UP2147 ref. ATA 2'01 Iliac CH71 - - Antsingiavo-A, Narinda CNRS UP2147 ref. ATA 2'01 Iliac CH125 - - Madagascar MNHN2 no ref. Right Femur Archaeolemur edwardsi CH126 190 (2) - Ménagerie MNHN2 ref. 1931–6 Left Canine sup. Archaeolemur majori CH127 - - Madagascar MNHN2 ref. MAD57-1906-16 2nd left Molar inf. CH145 - - Mitoho, Madagascar MNHN1 ref. 1938–537 Maxilla CH146 335 (4) 222 (2) Madagascar MNHN1 ref. 1935–419 Molar CH191 - - Madagascar MNHN1 ref. 1935–420 Tooth CH210 269 (3) - unknown MNHN1 ref. 1936–200 Tooth inf. Hadropithecus stenognathus CH421 94 (1) - Madagascar MNHN1 ref. 1935–408 Tooth Megaladapis edwardsi CH147 190 (2) - Madagascar MNHN1 ref. 1937–44 Bone CH190 - - Madagascar MNHN1 ref. 1937–44 Molar MNHN1 = Museum National d'Histoire Naturelle, Bâtiment d'Anatomie Comparée, CP 55 – 55, rue Buffon, 75005 Paris. MNHN2 = Museum National d'Histoire Naturelle, Dpt. Histoire de la Terre, USM203/UMR5443 Paléobiodiversité, 8 rue Buffon, 75005 Paris. CNRS UP2147 = Dynamique de l'Évolution Humaine: Individus, Populations, Espèces, 44 rue de l'Amiral Mouchez, 75014 Paris Column 'Ancient DNA' summarizes the samples that gave authentic ancient DNA fragments. MNHN1 = Museum National d'Histoire Naturelle, Bâtiment d'Anatomie Comparée, CP 55 – 55, rue Buffon, 75005 Paris. MNHN2 = Museum National d'Histoire Naturelle, Dpt. Page 3 of 9 (page number not for citation purposes) Phylogene Figure 1 Phylogenetic relationships among lemuriforms Figure 1 Phylogenetic relationships among lemuriforms. (A) Phylogenetic tree recovered after Bayesian analysis of Dataset #12 using different model parameters for 12S and Cytb genes. Numbers near the nodes refer to posterior probabilities. * Megala- dapis haplotype described in Montagnon et al. (2001) [31] (Accession number Genbank:AJ278142), criticized in Karanth et al. (2005) [18] and definitively confirmed here as a probable PCR-contaminant. (B) Summary Consensus of all the phylogenetic trees recovered from the analysis of our 12 datasets. y g p g g Phylogenetic relationships among lemuriforms. (A) Phylogenetic tree recovered after Bayesian analysis of Dataset #12 using different model parameters for 12S and Cytb genes. Numbers near the nodes refer to posterior probabilities. * Megala- dapis haplotype described in Montagnon et al. (2001) [31] (Accession number Genbank:AJ278142), criticized in Karanth et al. (2005) [18] and definitively confirmed here as a probable PCR-contaminant. (B) Summary Consensus of all the phylogenetic trees recovered from the analysis of our 12 datasets. three of them strongly support the classical view of a sis- tership of Propithecus and lemurids. Furthermore, the authors of this analysis note possible taxonomic bias in their analysis since except for Propithecus, no taxa from the family Indriidae (e.g. Indri and Avahi) have been consid- ered. This added to the limited amount of sequence infor- mation used in our study could explain these discrepancies. In any case, the sequences presented in this study sustain the sistership between Archaeolemur (and Hadropithecus) and indriids as the most likely phyloge- netic scenario. Consequently, we can now define a true series of synapomorphies for archaeolemurids, palaeo- propithecids, and indriids (collectively, the Indrioidea) at both the dental and postcranial levels (see Figure 2 and Additional data file: "Defining a series of synapomorphies for the Indrioidea clade" for a list of such synapomor- phies). Therefore, regardless of the phylogenetic method or the sequence dataset used, the ancient DNA sequences recov- ered in this study supported the existence of an Archaeole- muridae-Indriidae clade (Table 2; Figure 1b). Interestingly, despite significant variation among datasets (Table S3, Additional file 1), Approximately Unbiased (AU, [35]) and KH tests [36] come to similar conclusions by rejecting the 3 latter alternative topologies and by showing maximal p-values for the sistership between Archaeolemur (and Hadropithecus) and indriids. Results f Archaeolemur edwardsi sample CH126 instead yielded a 190-bp cytochrome b sequence (Table 1) that exhibited only a minimum number of 4 substitutions with Archaeolemur majori haplotypes while the 94-bp cytochrome b sequence obtained from Hadrop- Accordingly, we retrieved all strepsirrhine cytochrome b and 12S rRNA sequences available in Genbank and gener- ated 12 different datasets (Table 2 and Additional data file: for a complete list of the different sequences used). For each of these datasets, phylogenetic trees were built using both maximum likelihood and bayesian methods. Including the new Archaeolemur and Hadropithecus sequences in the dataset confirmed the indisputable monophyly of Malagasy Lemuriformes. This provided supplemental support to the scenario of a single origin for all Malagasy Primates [32] (reviewed in [33]) and there- fore to the authenticity of our sequences (the 'phyloge- netic sense' criterion; discussed for instance in [34]). Furthermore, most phylogenies showed the best boot- strap values and posterior probabilities for a sistership between Archaeolemur (and Hadropithecus) and indriids (up to 86.0% and 0.99, respectively; Table 2, summarized in Figure 1b). Alternative topologies relating Archaeolemur (and Hadropithecus) to either lemurids, cheirogaleids, lep- ilemurids or lorisiformes received no more than marginal bootstrap values or posterior probabilities (Table 2). Page 3 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/8/121 BMC Evolutionary Biology 2008, 8:121 Phylogenetic relationships among lemuriforms Figure 1 Phylogenetic relationships among lemuriforms. (A) Phylogenetic tree recovered after Bayesian analysis of Dataset #12 using different model parameters for 12S and Cytb genes. Numbers near the nodes refer to posterior probabilities. * Megala- dapis haplotype described in Montagnon et al. (2001) [31] (Accession number Genbank:AJ278142), criticized in Karanth et al. (2005) [18] and definitively confirmed here as a probable PCR-contaminant. (B) Summary Consensus of all the phylogenetic trees recovered from the analysis of our 12 datasets. Phylogene Figure 1 It is note- worthy, also, that the molecular topology presented here (Figure 1b) matches exactly that presented for extant taxa only by DelPero et al. (2004) [37] on page 440) as one of the "two remarkably similar topologies that were strongly supported at most of the internal nodes". However, a phy- logenomic toolkit using extensive nuclear and mitochon- drial sequence data came to different conclusions regarding the position of Propithecus (family Indriidae) and Lepilemur (family Lepilemuridae) [38]. Interestingly, the former position shows conflict between loci since Interestingly, if our molecular data unambiguously link archaeolemurids to indriids, they do not give insights into Page 4 of 9 (page number not for citation purposes) Page 4 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/8/121 BMC Evolutionary Biology 2008, 8:121 Table 2: Sequence datasets and phylogenetic supports for different phylogenetic hypotheses. Phylogene Figure 1 Method Dataset # Gene Root Length Taxa indriids lemurids cheirogaleids lepilemurids lorisiformes aye-aye Likelihood 1 Cytb Lorisiformes + Aye-aye 1140 125 21 0 0 0 0 2.5 2 Cytb Lorisiformes + Aye-aye 486 125 22.5 0 0.5 0 0 3 3 Cytb Aye-aye 486 99 18.5 0.5 0 0 NA 10.5 4 Cytb Aye-aye 486 99 0.7 0 0.7 0 NA 4 5 12S Lorisiformes + Aye-aye 985 124 72.5 0 3 0 0 1 6 12S Lorisiformes + Aye-aye 333 124 46.5 0.5 10.5 0 0 0 7 12S Aye-aye 333 89 42 5.5 9 0 NA 1 8 12S Aye-aye 197 89 36 4 5 0 NA 2 9 12S Aye-aye 372 89 86 0 0.5 0 NA 0.5 10 12S + Cytb Lorisiformes + Aye-aye 1934 49 39.5 0 0 0 0 0.5 11 12S + Cytb Lorisiformes + Aye-aye 819 49 33.5 0 0 0.5 0 1.5 12 12S + Cytb Aye-aye 819 36 33 0 1.5 1.5 NA 2 Bayesian 1 Cytb Lorisiformes + Aye-aye 1140 125 0.29 0 0 0 0 0.22 2 Cytb Lorisiformes + Aye-aye 486 125 0.52 0 0 0 0 0 3 Cytb Aye-aye 486 99 0.09 0 0 0 NA 0.64* 4 Cytb Aye-aye 486 99 0.44 0 0 0 NA 0.26 5 12S Lorisiformes + Aye-aye 985 124 0.84 0 0 0 0 0 6 12S Lorisiformes + Aye-aye 333 124 0.55 0 0.06 0 0 0 7 12S Aye-aye 333 89 0.54 0.06 0.09 0 NA 0 8 12S Aye-aye 197 89 0.53 0.08 0 0 NA 0 9 12S Aye-aye 372 89 0.99 0 0 0 NA 0 10 12S + Cytb Lorisiformes + Aye-aye 1934 49 0.64 0 0 0 0 0 11 12S + Cytb Lorisiformes + Aye-aye 819 49 0.68 0 0 0 0 0 12 12S + Cytb Aye-aye 819 36 0.88 0 0 0 NA 0 Bayesian partitioned 10 12S + Cytb Lorisiformes + Aye-aye 1934 49 0.63 0 0 0 0 0 11 12S + Cytb Lorisiformes + Aye-aye 819 49 0.68 0 0 0 0 0 12 12S + Cytb Aye-aye 819 36 0.86 0 0 0 NA 0 Phylogenetic supports for the nesting of Archaeolemur (and Hadropithecus) within different taxa (Indriidae, Lemuridae, Cheirogaleidae, Lepilemuridae, Lorisiformes or the Aye-aye, respectively) are given in the 6 last columns. Phylogene Figure 1 Bootstrap percentages or Posterior probabilities are given for Likelihood and Bayesian analyses, respectively. * In this topology, Archaeolemur actually appears as the sister taxon of the aye-aye but both are nested within paraphyletic indriids. This finding most likely results from a Long Branch Attraction artifact. Table 2: Sequence datasets and phylogenetic supports for different phylogenetic hypotheses. Phylogenetic supports for the nesting of Archaeolemur (and Hadropithecus) within different taxa (Indriidae, Lemuridae, Cheirogaleidae, Lepilemuridae, Lorisiformes or the Aye-aye, respectively) are given in the 6 last columns. Bootstrap percentages or Posterior probabilities are given for Likelihood and Bayesian analyses, respectively. * In this topology, Archaeolemur actually appears as the sister taxon of the aye-aye but both are nested within paraphyletic indriids. This finding most likely results from a Long Branch Attraction artifact. [41]. Since that time, craniodental studies have consist- ently recognized the phylogenetic affinity of Indrioidea and their separation from other lemurs. Often, the three families are treated as subfamilies within the family Indri- idae, which in turn is placed within the Lemuroidea. In their review of the craniodental evidence, Tattersall and Schwartz (1974) came to the same conclusion [25]. How- ever, they and other researchers since have recognized that the clade as a whole is not supported by a large number of morphological traits, and that different suites of morpho- logical traits might be used to defend very different phyl- ogenetic hypotheses. Moreover King et al. (2001) examined the sequence of fusion of postcranial epiphyses, dental eruption, and closure of cranial sutures in Archae- olemur and two living lemurs (Propithecus and Eulemur) in an effort to evaluate functional and phylogenetic implica- tions of developmental data [26]. These authors noted that the sequence data failed to demonstrate similarities of Archaeolemur to Propithecus, but rather showed greater likeness to Eulemur. In addition, studies of recently-found carpal elements of Palaeopropithecus, Archaeolemur, and Hadropithecus have demonstrated greater likenesses of the archaeolemurids to lemurids [22,27,28,42]. the phylogenetic relationships within the Indrioidea, as ingroup nodes do not receive conclusive bootstrap values and posterior probabilities (Figure 1A and Table S4, Addi- tional file 1). However, morphological and developmen- tal characters have recently provided unambiguous support for considering the palaeopropithecids as the sis- ter to the Indriidae (contra treating the Archaeolemuridae as the sister to the Indriidae) (Figure 2; see Additional data file: "Phylogenetics relationships within Indrioidea" for in-depth discussion). For this reason, this is our preferred phylogeny. Discussion The idea that the Archaeolemuridae, Palaeopropithecidae and Indriidae comprise a clade within the Lemuriformes is not new. Indeed, affinities of Archaeolemur to indriines were recognized by Lorenz von Liburnau in 1900 when he named "Protoindris globiceps" on the basis of a photograph of a skull (later synonymized with Archaeolemur majori) that had been collected by Franz Sikora in 1899 at Andra- homana Cave in southeastern Madagascar [39]. Standing (1908) treated Archaeolemur as an indriid [40], and G. E. Smith (1908) discussed the indriid character of its brain Page 5 of 9 (page number not for citation purposes) Page 5 of 9 (page number not for citation purposes) BMC Evolutionary Biology 2008, 8:121 http://www.biomedcentral.com/1471-2148/8/121 Model for the evolution indrioids, modified from Godfrey (1988) Figure 2 Model for the evolution indrioids, modified from Godfrey (1988).                                            ! " #$ $ ! " " " % &$ "  '  ( )*'+ ,-* . / '$"+$* $", ,(*'"$* $  &$" $* & $* 0$ " % "&$, #+", *&' "        ,+&,  ! " #$ $ ! "   &"$* "&  *$# !$" *$$* !&'1 2 ! "*3 4 !  +$, &+ , '*$ *$ (*3 *$# &$ +*+' $.*3 %*$ "# 1$&! $*  %*$"#  "&$, %* '1 +"3 $& (   5'*3 $&&*$, ,"$* ,(*'"  "&$, %* '1 +"3 $& (   5'*3 $&&*$, ,"$* ,(*'"   ,+&,  ! " #$ $ ! "   &"$* "&  *$# !$" *$$* !&'1 2 ! "*3 4 !  +$, &+ , '*$ *$ (*3 *$# &$ +*+' $.*3 %*$ "# 1$&! Page 6 of 9 (page number not for citation purposes) Discussion $*  %*$"#                                                     Model for the evolution indrioids, modified from Godfrey (1988) Figure 2 Model for the evolution indrioids, modified from Godfrey (1988). Given that the Archaeolemuridae-Lemuridae sistership is not supported by our molecular data, one interpretation is that the lemurid-likenesses of the Archaeolemuridae are primitive (symplesiomorphic) for indriids, palaeoprop- ithecids, archaeolemurids, and lemurids. Another is that they represent convergences of archaeolemurids and lemurids due to shared quadrupedalism, although this cannot account for developmental likenesses. Godfrey (1988) reconstructed the common ancestor of the Indrio- idea as a versatile and probably arboroterrestrial quadru- ped with limb proportions and a positional repertory not very different from those of the lemurids, Varecia or Pach- ylemur [14]. Such a scenario (Figure 2) would explain the manual similarities of the Archaeolemuridae and the Lemuridae. This author also reconstructed the common ancestor of the indriids and palaeopropithecids as a gen- eralized quadrupedal climber/hanger, with striking synapomorphies of the upper limb, hands, and feet. According to this interpretation, an initial split divided the Indrioidea into two clades, one of which (the Archae- olemuridae) specialized in terrestrial quadrupedalism while the other (the palaeopropithecid-indriid clade) spe- cialized in slow quadrupedal climbing and hanging. The latter subsequently split into two clades, one of which (the Palaeopropithecidae) sacrificed rapid locomotion of any sort to perfect deliberate hanging skills, the other of which (the Indriidae) sacrificed quadrupedalism to develop a new form of 'vertical clinging and leaping' while retaining sloth-like hanging skills. This unique combination of locomotor/postural features and dental adaptations in archaeolemurids is probably the reason why their phylogenetic status has been so diffi- cult to decipher. This study demonstrates the value of the ancient DNA approach in solving the phylogenetic rela- tionships among extinct and extant taxa, especially in sit- uations involving rampant morphological homoplasy, Page 6 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/8/121 http://www.biomedcentral.com/1471-2148/8/121 BMC Evolutionary Biology 2008, 8:121 morphological plasticity with rapid change in body size [43,44], or sexual dimorphism [45,46]. ries. PCR products were cloned using the Topo TA cloning kit (Invitrogen®) following the manufacturer instructions. Discussion Colonies positive for insertion were screened by PCR into a 12 μl reaction mix using universal M13 (5'-GTT TTC CCA GTC ACG ACG TTG) and REV (5'-TTT CAC ACA GGA AAC AGC TAT) primers and 35–45 cycles of dena- turation (94°C, 30s), annealing (55°C, 30s) and elonga- tion (72°C, 45s). PCR products were further sequenced by a service provider (Cogenics®). For each DNA fragment, the final sequence was deduced from the consensus of all clone sequences obtained from at least two independent PCR products. Such an approach is generally taken for dis- carding possible artifactual substitutions induced by DNA damage [51]. A total number of 75 PCR products and 399 clones were analyzed (Table S2, Additional file 1). For cytochrome b, no prematurate stop-codon is observed in the coding-phase of each of the final consensus. Finally, the sample CH146 (Archaeolemur majori) was independ- ently extracted, amplified and analysed in two different ancient DNA laboratories. It yielded identical consensus cytochrome b sequences. Conclusion We have been successful in amplifying and sequencing the first ancient DNA sequences of all the members of an enigmatic lemur family: the archaeolemurids (genera Archaeolemur and Hadropithecus). These 'giant lemurs' lived in Madagascar centuries ago but have been led to extinction by human activities. Our new sequences solve the phylogenetic position of archaeolemurids as close rel- atives of both the sloth lemurs and the indriids. This appears in sharp contrast with most recent ontogenetic studies as well as new discoveries of postcranial elements of the archaeolemurids that indicate striking similarities with lemurids. In light of our new phylogenetic frame- work, we were able to reinterpret the available cranial and postcranial data. Our data offer support for a particular scenario of the evolution of the Indrioidea locomotor apparatus (starting from arboroterrestrial ancestors that specialized in either terrestrial quadrupedalism or arbo- real skills). Methods A The new sequences reported in this manuscript were deposited in Genbank under Accession numbers EU441938–EU441943. All available sequences of extinct and extant Strepsirrhini were retrieved from Genbank and aligned using ClustalW. In order to investigate possible artifacts due to stochastic or systematic errors, 12 different datasets were constituted. Dataset composition is pro- vided in the Additional data file. Ancient DNA extraction, amplification and sequencing A total of 10 samples of subfossil lemurs belonging to the genera Archaeolemur and Hadropithecus were subjected to ancient DNA extraction (Table 1). These cover all the spe- cies currently allied to Archaeolemuridae. Furthermore, two subfossils of the extinct lemur Megaladapis edwardsi were also included in the analysis in order to compare the sequences retrieved with the sequences already reported by other laboratories [18,31]. DNA was extracted and amplified as previously described elsewhere [47,48], using appropriate ancient DNA techniques and respective of the most scrupulous ancient DNA authentication crite- ria [49]. Briefly, mock extractions and the three different amplification controls described in Loreille et al. (2001) [50] were included in each analysis to detect possible con- tamination. Only one lemur sample was extracted per extraction session to limit possible cross-contamination between specimens. All PCR reactions were conducted in a total volume of 25–100 μl using 2.5–10 units of Taq Gold (Perkin-Elmer®) together with 2 mM MgCl2, 1 mg/ ml BSA, 250 μM of each dNTP and 0,5–1 μM of the differ- ent primers listed in Table S1, Additional file 1. A 5–10 min activation step at 92–94°C was followed by 50–60 cycles of denaturation (92–94°C, 45–60s), annealing (44–50°C, 45–60s), extension (72°C, 45s) and a last extension step at 72°C (5–10 min). Primers (Table S1, Additional file 1) were designed to target overlapping DNA fragments of 80–200 bp among 3 different mtDNA genes (control region, cytochrome b and 12S rRNA) and one nuclear gene (IRBP). No specimen or DNA extract from modern lemurs was ever introduced in the laborato- References y 25. Tattersall I, Schwartz JH: Craniodental morphology and the sys- tematics of the Malagasy lemurs (Primates, Prosimii). Anthropol Pap Am Mus Nat Hist New York 1974, 52:139-192. 1. Burney DA, Burney LP, Godfrey LR, Jungers WL, Goodman SM, Wright HT, Jull AJ: A chronology for late prehistoric Madagas- car. J Hum Evol 2004, 47:25-63. 26. King SJ, Godfrey LR, Simons EL: Adaptive and phylogenetic sig- nificance of ontogenetic sequences in Archaeolemur, subfossil lemur from Madagascar. J Hum Evol 2001, 41:545-576. J , 2. Perez VR, Godfrey LR, Nowak-Kemp M, Burney DA, Ratsimbazafy J, Vasey N: Evidence of early butchery of giant lemurs in Mada- gascar. J Hum Evol 2005, 49:722-742. Vasey N: Evidence of early butchery of giant lemurs in Mada- gascar. J Hum Evol 2005, 49:722-742. 27. 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Karanth KP, Delefosse T, Rakotosamimanana B, Parsons TJ, Yoder AD: Ancient DNA from giant extinct lemurs confirms single origin of Malagasy primates. Proc Natl Acad Sci USA 2005, 102:5090-5095. [http://www.biomedcentral.com/content/supplementary/1471- 2148-8-121-S1.doc] [http://www.biomedcentral.com/content/supplementary/1471- 2148-8-121-S1.doc] 19. Godfrey LR, Jungers WL, Wunderlich RE, Richmond BG: Reap- praisal of the postcranium of Hadropithecus (Primates, Indri- oidea). Am J Phys Anthropol 1997, 103:529-556. ) J y p 20. Shapiro LJ, Seiffert CV, Godfrey LR, Jungers WL, Simons EL, Randria GF: Morphometric analysis of lumbar vertebrae in extinct Malagasy strepsirrhines. Am J Phys Anthropol 2005, 128:823-839. 21. Godfrey LR, Semprebon GM, Schwartz GT, Burney DA, Jungers WL, Flanagan EK, Cuozzo FP, King SJ: New insights into old lemurs: the trophic adaptations of the Archaeolemuridae. Int J Prima- tol 2005, 26:825-854. Authors' contributions 12. Jungers WL, Demes B, Godfrey LR: How big were the "giant" extinct lemurs of Madagascar? In Elwyn Simons: A search for origins Edited by: Fleagle JG, Gilbert CC. New-York: Springer; 2008:343-360. LO, SC and CS extracted, amplified and sequenced ancient DNA. LG provided palaeontological information. CD performed the phylogenetic analyses. CH initiated and coordinated the study. LO and LG wrote the paper. 13. Jungers WL: Adaptative diversity in subfossil Malagasy prosim- ians. Z für Morphol Anthropol 1980, 71:177-186. 14. Godfrey LR: Adaptive diversification of Malagasy strepsir- rhines. J Hum Evol 1988, 17:93-134. 15. Jungers WL, Godfrey LR, Simons EL, Chatrath PS, Rakotosamimanana B: Phylogenetic and functional affinities of Babakotia (Pri- mates), a fossil lemur from northern Madagascar. Proc Natl Acad Sci USA 1991, 88:9082-9086. test (AU, [35]) and unilateral KH test [36] using Consel [58]. test (AU, [35]) and unilateral KH test [36] using Consel [58]. 10. Godfrey LR, Jungers WL: Quaternary fossil lemurs. In The Primate fossil record Edited by: Hartwig W. New-York: Cambridge University Press; 2002:97-122. 11. Godfrey LR, Jungers WL: The extinct sloth lemurs of Madagas- car. Evol Anthropol 2003, 12:252-263. Additional material Additional file 1 Table S1. Fragment length, primer sequences and T annealing of the PCR fragments targeted under this study.Table S2. Number of independent PCR amplifications and clones sequenced per fragment and per sam- ple.Table S3. p-values of AU and KH tests for the clustering of Archae- olemur (and Hadropithecus) within different taxa (indriids, lemurids, cheirogaleids, lepilemurids, lorisiformes or aye-aye, respectively) or out- side indriids (no indriids).Table S4. Phylogenetic supports for three alter- native relationships among Indrioidea. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2148-8-121-S1.doc] Acknowledgements We thank four anonymous reviewers for fruitful comments, Marilyne Duf- fraisse for technical help and the following organizations for supporting this work: CNRS (APN), MENRT (ACI), ENS Lyon and UCB-Lyon1. We are especially indebted with Sandrine Hughes, Vincent Laudet, Marie Pagès and Michael Schubert for critical reading of the manuscript. We would also like to thank Drs. Christine Lefebvre, Marc Godinot and Pascal Tassy at the MNHN, and Dominique Gommery (CNRS UPR 2147) who kindly provided us the samples included in this study. 22. Jungers WL, Lemelin P, Godfrey LR, Wunderlich RE, Burney DA, Simons EL, Chatrath PS, James HF, Randrai GF: The hands and feet of Archaeolemur : metrical affinities and their functional sig- nificance. J Hum Evol 2005, 49:36-55. J 23. Tattersall I: Cranial anatomy of the Archaeolemurinae (Lemuroidea, Primates). Anthropol Pap Am Mus Nat Hist 1973, 52:1-110. 24. Tattersall I: The Primates of Madagascar New-York: Columbia Univer- sity Press; 1982. Phylogenetic analyses Bayesian Markov Chain Monte Carlo phylogenies were generated using MrBayes 3.12 [52] under a GTR model of evolution assuming a fraction of invariant sites and rate heterogeneity across sites. Two sets of four chains sampled every 100 generations were run until the average standard deviation of split frequencies between the two sets fell below the default critical value of 0.01 using a burn-in fraction of 25%. Bayesian posterior probabilities were finally recorded even if their significance, in term of robustness, remains an open question (e.g. [53]). For each dataset, the best-fitting model of substitution was then determined using Modeltest [54] following AIC criterion [55]. Maximum Likelihood (ML) trees were then built with Phyml [56]. The strength of the phylogenetic signal was assessed via non-parametric bootstrapping [57] among 200 pseudo replicates. For datasets #10–12, we analyzed either both genes under the same (Likelihood and Bayesian, respectively) or independent (Bayesian par- titioned) model parameters (Tables 2 & S4, Additional file 1). Statistical supports for different a priori selected hypotheses were assessed via the Approximately Unbiased Page 7 of 9 (page number not for citation purposes) Page 7 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/8/121 BMC Evolutionary Biology 2008, 8:121 BMC Evolutionary Biology 2008, 8:121 http://www.biomedcentral.com/1471-2148/8/121 32. Yoder AD, Cartmill M, Ruvolo M, Smith K, Vilgalys R: Ancient single origin for Malagasy primates. Proc Natl Acad Sci USA 1996, 93:5122-5126. 58. 33. Yoder A, Nowak M: Has vicariance or dispersal been the pre- dominant biogeographic force in Madagascar? Only time will tell. Ann Rev Ecol Evol Syst 2006, 37:405-431. y , 34. Shapiro B, Rambaut A, Gilbert TM: No proof that typhoid caused the Plague of Athens (a reply to Papagrigorakis et al ) Int J y 34. Shapiro B, Rambaut A, Gilbert TM: No proof that typhoid caused the Plague of Athens (a reply to Papagrigorakis et al.). Int J Infect Dis 2006, 10:334-335. f 35. Shimodaira H: An approximately unbiased test of phylogenetic tree selection. Syst Biol 2002, 51:492-508. y 36. Kishino H, Hasegawa M: Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in hominoidea. J Mol Evol 1989, 29:170-179. 37. DelPero M, Pozzi L, Masters JC: A composite molecular phylog- eny of living lemuroid primates. Folia Primatol 2006, 77:434-445. y g p 38. Horvath JE, Weisrock DW, Embry SL, Fiorentino I, Balhoff JP, Kappe- ler P, Wray GA, Willard HF, Yoder AD: Development and appli- cation of a phylogenomic toolkit: Resolving the evolutionary history of Madagascar's lemurs. Genome Res 2008 in press. 39. Lorenz von Liburnau L: über einige Reste ausgestorbener Pri- maten von Madagascar. Denkschriften der Kaiserlichen Akademie der Wissenschaften zu Wien 1901, 70:1-11. 40. Standing HF: On recently discovered subfossil Primates from Madagascar. Trans Zool Soc 1908, 18:69-162. g 41. Smith GE: On the form of the brain in the extinct lemurs of Madagascar, with some remarks on the affinities of the Indrisinae. Trans Zool Soc Lond 1908, 18:163-177. 42. Hamrick MW, Simons EL, Jungers WL: New wrist bones of the Malagasy giant subfossil lemurs. J Hum Evol 2000, 38:635-650. g y g J 43. Bunce M, Szulkin M, Lerner HR, Barnes I, Shapiro B, Cooper A, Hold- away RN: Ancient DNA provides new insights into the evolu- tionary history of New Zealand's extinct giant eagle. PLOS Biol 2005, 3:e9. 44. Lalueza-Fox C, Castersana J, Sampietro L, Marquès-Bonet T, Alcover JS, Bertranpetit J: Molecular dating of caprines using ancient DNA sequences of Myotragus balearicus, an extinct endemic Balearic mammal. BMC Evol Biol 2005, 5:70. 45. 57. Felsenstein J: Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985, 39:783-791. 58. Shimodaira H, Hasegawa M: CONSEL: for assessing the confi- dence of phylogenetic tree selection. Bioinformatics 2001, 17:1246-1247. g p 58. Shimodaira H, Hasegawa M: CONSEL: for assessing the confi- dence of phylogenetic tree selection. Bioinformatics 2001, 17:1246-1247. BMC Evolutionary Biology 2008, 8:121 Bunce M, Worthy TH, Ford T, Hoppitt W, Willerslev E, Drummond A, Cooper A: Extreme reversed sexual size dimorphism in the extinct New Zealand moa Dinornis. Nature 2003, 425:172-175. 46 H L Mill CD S fi ld RP L b t DM Nuclear DNA 45. Bunce M, Worthy TH, Ford T, Hoppitt W, Willerslev E, Drummond A, Cooper A: Extreme reversed sexual size dimorphism in the extinct New Zealand moa Dinornis. Nature 2003, 425:172-175. 46. Huynen L, Millar CD, Scofield RP, Lambert DM: Nuclear DNA sequences detect species limits in ancient moa. Nature 2003, 425:175 178 , 46. Huynen L, Millar CD, Scofield RP, Lambert DM: Nuclear DNA sequences detect species limits in ancient moa. Nature 2003, 425:175-178. 47. Hughes S, Hayden TJ, Douady CJ, Tougard C, Germonpré M, Stuart A, Lbova L, Carden RF, Hänni C, Say L: Molecular phylogeny of the extinct giant deer, Megaloceros giganteus. Mol Phylogenet Evol 2006, 40:285-291. 48. Orlando L, Mashkour M, Burke A, Douady CJ, Eisenmann V, Hänni C: Geographic distribution of an extinct equid (Equus hydrunti- nus : Mammalia, Equidae) revealed by morphological and genetical analyses of fossils. Mol Ecol 2006, 15:2083-2093. g y 49. Cooper A, Poinar HN: Ancient DNA: do it right or not at all. Science 2000, 289:1139. 50. Loreille O, Orlando L, Patou-Mathis M, Philippe M, Taberlet P, Hänni C: Ancient DNA analysis reveals divergence of the cave bear, Ursus spelaeus, and brown bear, Ursus arctos, lineages. Curr Biol 2001, 11:200-203. 51. Hofreiter M, Jaenicke V, Serre D, Haeseler Av A, Pääbo S: DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA. Nuc Acids Res 2001, 29:4793-4799. 57. Felsenstein J: Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985, 39:783-791. http://www.biomedcentral.com/1471-2148/8/121 BMC Evolutionary Biology 2008, 8:121 Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 9 of 9 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge 52. Huelsenbeck JP, Ronquist F: MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 2001, 17:754-755. p y g 53. Douady CJ, Delsuc F, Boucher Y, Doolittle WF, Douzery EJ: Com- parison of Bayesian and maximum likelihood bootstrap measures of phylogenetic reliability. Mol Biol Evol 2003, 20:248-254. 54. Posada D, Crandall KA: MODELTEST: testing the model of DNA substitution. Bioinformatics 1998, 14:817-818. 55. Akaike H: A new look at the statistical model identification. IEEE Transactions on Automatic Control 1974, 19:716-723. 56. Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 2003, 52:696-704. References In Natural change and human impact in Madagascar Edited by: Goodman SM, Patterson BD. Washington, DC: Smithsonian Institution Press; 1997:218-256. p 31. Montagnon D, Ravaoarimanana B, Rakotosamimanana B, Rumpler Y: Ancient DNA from Megaladapis edwardsi (Malagasy subfos- sil): preliminary results using partial cytochrome b sequence. Folia Primatol 2001, 72:30-32. 8. Yoder AD, Yang Z: Divergence dates for Malagasy lemurs esti- mated from multiple gene loci: geological and evolutionary context. Mol Ecol 2004, 13:757-773. 9. Simons EL: Lemurs: Old and New. In Natural change and human impact in Madagascar Edited by: Goodman SM, Patterson BD. Wash- ington, DC: Smithsonian Institution Press; 1997:218-256. 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English
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Isolated, Unilateral Inguinal Tuberculous Lymphadenitis
˜The œAmerican journal of tropical medicine and hygiene
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Isabel Ram´ırez1,2* 1Internal Medicine, Infectious Diseases, Hospital Pablo Tob ´on Uribe, Medell´ın, Colombia; 2Universidad de Antioquia, Medell´ın, Colombia Isabel Ram´ırez1,2* 1Internal Medicine, Infectious Diseases, Hospital Pablo Tob ´on Uribe, Medell´ın, Colombia; 2Universidad de Antio Isabel Ram´ırez1,2* 1Internal Medicine, Infectious Diseases, Hospital Pablo Tob ´on Uribe, Medell´ın, Colombia; 2Universidad de Antioquia, Medell´ın, Colombia Isabel Ram´ırez1,2* ctious Diseases, Hospital Pablo Tob ´on Uribe, Medell´ın, Colombia; 2Universidad de Antioquia, Medell´ın, Colombia A 17-year-old heterosexual man presented with a 3-month history of a painless, enlarging inguinal lymph node and mul- tiple discharging sinuses. There had been no clinical im- provement despite multiple empirical antibiotic treatments for other infections which share similar clinical presentations, such as lymphogranuloma venereum. Specific details in ref- erence to diagnosis and treatment could not be obtained (Figure 1). There was no history of recent unprotected sexual intercourse, previous tuberculosis (TB) exposure, symptoms suggestive of other systemic illnesses, fever, weight loss, cough, sore throat, urethral discharge, genital ulcer, or trauma to the lower extremities. On physical examination, an enlarged, 4 × 3-cm inguinal lymph node was noted; the remainder of the examination was unremarkable. The chest X-ray showed no abnormalitiesandatestfor humanimmunodeficiencyvirus was negative. The patient was evaluated for syphilis, but not for other sexually transmitted diseases, given that he had no his- tory of recent unprotected sexual exposure, no urethral dis- charge, or genital ulcer. Histologic examination of tissue obtained by an excisional biopsy showed granulomas with multinucleated giant cells (Figure 2) and acid-fast bacilli (Figure 3); culture grew Mycobacterium tuberculosis. Standard antituberculous treatment for 6 months was initiated. Clinical response was observed after 2 months of treatment. In the initial diagnosis of inguinal lymphadenitis, it is important to considerthepotentialetiologies,bothinfectiousandnoninfectious diseases. Painless lymphadenitis is usually due to toxoplasmosis, cat-scratch disease, syphilis, and TB; however, it can be painful at the beginning, and it can occur bilaterally. Painful unilateral lymphadenopathy can be caused by Chlamydia trachomatis and Haemophilus ducreyi; however, these are usually associated with urethral discharge or a genital ulcer. Other potential etiologies in- clude secondary bacterial infection of genital scabies or pedicu- losis pubis, plague, pyogenic infection of the leg, atypical mycobacterial infections, and persistent generalized lymphade- nopathy of AIDS. Noninfectious causes must also be considered, such as metastatic lymph node lesions and lymphoma. FIGURE 1. Isolated unilateral tuberculous lymphadenitis. This figure appears in color at www.ajtmh.org. The initial evaluation should include a history of recent sexual contact and testing for syphilis, Cytomegalovirus, Epstein–Barr virus, Toxoplasma gondii, hepatitis B and C vi- rus, herpes simplex virus, and HIV. * Address correspondence to Isabel Ram´ırez, Internal Medicine, Infectious Diseases. Hospital Pablo Tob ´on Uribe, calle 78B#69-240, Medell´ın, Colombia. E-mail iramirez@hptu.org.co Copyright © 2019 by The American Society of Tropical Medicine and Hygie Copyright © 2019 by The American Society of Tropical Medicine and Hygie Isabel Ram´ırez1,2* 1Internal Medicine, Infectious Diseases, Hospital Pablo Tob ´on Uribe, Medell´ın, Colombia; 2Universidad de Antioquia, Medell´ın, Colombia Cervical cultures for Neisseria gonorrhoeae and Ureaplasma urealyticum, poly- merase chain reaction (PCR) for Mycoplasma hominis, and nucleic acid amplification test for C. trachomatis should be obtained if the patient is sexually active. Empirical treat- ment is not recommended given that the causes can be FIGURE 2. Histologic examination of lymph node tissue. Hema- toxylin and eosine stain showing multiple granulomas with multinu- cleated giant cell (×100). This figure appears in color at www.ajtmh. org. FIGURE 1. Isolated unilateral tuberculous lymphadenitis. This figure appears in color at www.ajtmh.org. FIGURE 2. Histologic examination of lymph node tissue. Hema- toxylin and eosine stain showing multiple granulomas with multinu- cleated giant cell (×100). This figure appears in color at www.ajtmh. org. 770 INGUINAL LYMPHADENITIS 771 FIGURE 3. Ziehl-Neelsen stain showing asid-fast bacilli of Myco- bacterium tuberculosis (×100). This figure appears in color at www. ajtmh.org. of initial evaluation, and additional tests must be carried out based on clinical symptoms and findings. A fine needle aspi- ration is essential to make the diagnosis in tuberculous lymphadenopathy, as it can reveal granulomas in 61% of cases and a positive culture in 62%; for surgical specimens, the diagnostic yield is 88% and 71%, respectively.3,4 A 6- month TB treatment regimen is recommended as standard therapy. In countries with a high prevalence of mycobacterial resistance, a four-drug treatment regimen (isoniazid, rifampi- cin, pyrazinamide, and ethambutol) should be preferred within the first 2 months. Received March 10, 2018. Accepted for publication December 13, 2018. Received March 10, 2018. Accepted for publication December 13, 2018. Author’s addresses: Isabel Ram´ırez, Internal Medicine, Infectious Diseases, Hospital Pablo Tob ´on Uribe, Medell´ın, Colombia, and Uni- versidad de Antioquia, Medell´ın, Colombia, E-mail: iramirez@hptu. org.co. FIGURE 3. Ziehl-Neelsen stain showing asid-fast bacilli of Myco- bacterium tuberculosis (×100). This figure appears in color at www. ajtmh.org. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. multiple; therefore, it is always important to make the etio- logical diagnosis. REFERENCES Tuberculous lymphadenitis is the most common extrap- ulmonary manifestation of TB; it comprises 30–50% of these cases.1 Cervical lymph nodes are involved in 57% of cases, supraclavicular lymph nodes in 26%, submandibular lymph nodes in 3%, and axillary lymph nodes in 12%.2,3 Up to 17% of cases are bilateral and 78% of cases involve between one and three nodes. Isolated inguinal tuberculous lymphadenitis is infrequent, representing up to 8% of cases in all reported series.2,4 Active pulmonary TB occurs infrequently in immu- nocompetent patients with TB lymphadenitis; however, up to 15% of tuberculous lymphadenitis is associated with pulmo- nary TB.5 Therefore, a chest X-ray must be performed as part 1. Peto HM, Pratt RH, Harrington TA, LoBue PA, Armstrong LR, 2009. Epidemiology of extrapulmonary tuberculosis in the United States, 1993–2006. Clin Infect Dis 49: 1350–1357. 2. Thompson MM, Underwood MJ, Sayers RD, Dookeran KA, Bell PRF, 1992. Peripheral tuberculous lymphadenopathy: a review of 67 cases. Br J Surg 79: 763–764. g 3. Polesky A, Grove W, Bhatia G, 2005. Peripheral tuberculous lymphadenitis. Medicine 84: 350–362. 4. Dandapat MC, Mishra BM, Dash SP, Kar PK, 1990. Peripheral lymph node tuberculosis: a review of 80 cases. Br J Surg 77: 911–912. 5. Geldmacher H, Taube C, Kroeger C, Magnussen H, Kirsten DK, 2002. Assessment of lymph node tuberculosis in northern Germany a clinical review. Chest 121: 1177–1182.
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https://projecteuclid.org/journals/electronic-communications-in-probability/volume-6/issue-none/Kendalls-identity-for-the-first-crossing-time-revisited/10.1214/ECP.v6-1038.pdf
English
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Kendall's identity for the first crossing time revisited
Electronic communications in probability
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1Research supported by the Melbourne Research Development Scheme DOI: 10.1214/ECP.v6-1038 DOI: 10.1214/ECP.v6-1038 DOI: 10.1214/ECP.v6-1038 Elect. Comm. in Probab. 6 (2001) 91–94 ELECTRONIC COMMUNICATIONS in PROBABILITY 1Research supported by the Melbourne Research Development Scheme AMS 2000 Subject classification: 60G51 AMS 2000 Subject classification: 60G51 skip-free L´evy process, first crossing time, change of measure Abstract K. BOROVKOV1 Department of Mathematics and Statistics, University of Melbourne, Parkville 3010, Australia Department of Mathematics and Statistics, University of Melbourne, Parkville 3010, Australia email: K.Borovkov@ms.unimelb.edu.au email: K.Borovkov@ms.unimelb.edu.au Z. BURQ1 Department of Mathematics and Statistics, University of Melbourne, Parkville 3010, Australia Department of Mathematics and Statistics, University of Melbourne, Parkville 3010, Australia email: Z.Burq@ms.unimelb.edu.au submitted August 2, 2001 Final version accepted August 3, 2001 Abstract [1] and Section 21 in [10]. Some interesting extensions to the multidimensional case were given in [4]. Known proofs of the identities are either analytical (exploiting Laplace transforms in time and their multiplicative structure) or are based on a limiting procedure and a combinatorial argument (given, in particular in [5] and [10]) or factorization identities [3]. We present a new, rather short and elegant (from our point of view) proof of the above result which uses (a) martingale techniques to find the Laplace transform of the crossing time and then (b) change of measure to find the desired representation for the distribution of the time in terms of the distribution of Xt. To avoid a trivial situation, we assume that P (Xt > 0) > 0 for t > 0. Proof. (i) Since there are no positive jumps in the process {Xt}, the m.g.f. ϕ(λ) := E eλX1 < ∞for any λ ≥0 and is analytic on the positive half-line, and by independence of increments, Mt := eλXt/ϕt(λ), t ≥0, is a martingale. Denote by Pλ the probability corresponding to the respective Cram´er trans- form of the distribution of {Xt}, so that is a martingale. Denote by Pλ the probability corresponding to the respective Cram´er trans- form of the distribution of {Xt}, so that Pλ(Xt ∈dy) = eλy ϕt(λ)P (Xt ∈dy) (3) (3) and the process {Xt} still remains a L´evy process under Pλ. Next set λ0 := sup{λ ≥0 : ϕ(λ) = 1}. Clearly, 0 ≤λ0 < ∞always, and under Pλ the drift in {Xt} is linear with the coefficient Next set λ0 := sup{λ ≥0 : ϕ(λ) = 1}. Clearly, 0 ≤λ0 < ∞always, and under Pλ the drift in {Xt} is linear with the coefficient mλ := E λX1 = ϕ′(λ) ϕ(λ) = (ln ϕ(λ))′ > 0 for λ > λ0 (4) (4) (just recall the elementary fact that ln ϕ(λ) is convex and, when λ0 > 0, equal to 0 for both λ = 0 and λ = λ0). Note also that, for λ > λ0, one has ϕ(λ) > 1. (just recall the elementary fact that ln ϕ(λ) is convex and, when λ0 > 0, equal to 0 for both λ = 0 and λ = λ0). Note also that, for λ > λ0, one has ϕ(λ) > 1. Abstract We give a new relatively compact proof of the famous identity for the distribution of the first hitting time of a linear boundary by a skip-free process with stationary independent increments. The proof uses martingale identities and change of measure. Let {Xt}t≥0, X0 = 0, be a L´evy process which is skip-free in the positive direction. That is, Xt has no positive jumps and its increments are stationary independent. For an x > 0 set τ(x) := inf{t > 0 : Xt ≥x} τ(x) := inf{t > 0 : Xt ≥x} with τ(x) = ∞on the event {supt≥0 Xt < x}. The following result is well known. with τ(x) = ∞on the event {supt≥0 Xt < x}. The following result is well known. with τ(x) = ∞on the event {supt≥0 Xt < x}. The following result is well known. For any y, s > 0, Z ∞ y P (τ(x) ≤s)dx x = Z s 0 P (Xt > y)dt t . (1) (1) If Xt has a density pX(t, x) at x, then τ(x) also has a density pτ(t, x) at t and 1 xpτ(t, x) = 1 t pX(t, x). (2) (2) Relation (2) was first observed in a special case in [7]. Later the theorem was proved in [6], [9], [11] (under additional conditions) and [2]. Moreover, it was shown in [8] that (1) is a necessary condition for the process {Xt} to be skip-free. Relation (2) was first observed in a special case in [7]. Later the theorem was proved in [6], [9], [11] (under additional conditions) and [2]. Moreover, it was shown in [8] that (1) is a necessary condition for the process {Xt} to be skip-free. 91 92 Electronic Communications in Probability A discrete time (and, of course, space) analog of (2) is closely related to the classical ballot problem and was first given (in a special case) as early as in 1711 by A. de Moivre. For historical references and comments (and some generalizations) see e.g. [1] and Section 21 in [10]. Some interesting extensions to the multidimensional case were given in [4]. A discrete time (and, of course, space) analog of (2) is closely related to the classical ballot problem and was first given (in a special case) as early as in 1711 by A. de Moivre. For historical references and comments (and some generalizations) see e.g. KENDALL’S IDENTITY FOR THE FIRST CROSSING TIME REVISITED KENDALL’S IDENTITY FOR THE FIRST CROSSING TIME REVISITED 93 (iii) Next denote by TA = R ∞ 0 1A(Xt) dt the time spent by our process in the set A (i.e. the sojourn time of A). We will make use of the following fact: for any 0 < a < ∞and λ > λ0, E λT(0,a] = a mλ . (8) (8) To prove it, note that due to (4) and Wald’s identity one has for any a > 0 To prove it, note that due to (4) and Wald’s identity one has for any a > 0 a = E λXτ(a) = mλE λτ(a). (9) (9) Further, one can easily see that τ(a) −T(−∞,0] ≤T(0,a] ≤τ(a) + T (a) (−∞,0], (10) (10) where T (a) A denotes the time spent in A by the process {Xt+τ(a) −a}t≥0, which clearly has the same distribution as {Xt}t≥0, and hence T(−∞,0] and T (a) (−∞,0] are identically distributed, too. Moreover, for λ > λ0, they have a finite expectation under Pλ: where T (a) A denotes the time spent in A by the process {Xt+τ(a) −a}t≥0, which clearly has the same distribution as {Xt}t≥0, and hence T(−∞,0] and T (a) (−∞,0] are identically distributed, too. Moreover, for λ > λ0, they have a finite expectation under Pλ: Iλ := E λT(−∞,0] = E λ Z ∞ 0 1(−∞,0](Xt) dt ≤E λ Z ∞ 0 e−λXtdt = Z ∞ 0 E λe−λXtdt = Z ∞ 0 dt Z e−λy eλy ϕt(λ)P (Xt ∈dy) = Z ∞ 0 dt ϕt(λ) < ∞ since ϕ(λ) > 1. since ϕ(λ) > 1. Now this and the RHS of (10) imply that E λT(a,b] < ∞for any finite interval (a, b] [the last fact actually following from the well-known known recurrence-transience dichotomy for L´evy processes as well]. Together with the obvious observation that, for any 0 < a < b < c < ∞, Now this and the RHS of (10) imply that E λT(a,b] < ∞for any finite interval (a, b] [the last fact actually following from the well-known known recurrence-transience dichotomy for L´evy processes as well]. Abstract (ii) The last inequality, together with the obvious fact that Mt < eλx/ϕt(λ) on the event {τ(x) > t}, implies that, as t →∞, Mt →0 on {τ(x) = ∞} (so that we can formally set Mτ(x) = 0 on this event) and also E (Mt; τ(x) > t) →0. These relations ensure that the optional stopping theorem holds: 1 = M0 = E Mτ(x) = eλxE e−µτ(x) with µ := ln ϕ(λ) (5) (5) (this can be shown e.g. by applying the theorem to the bounded stopping time τ(x) ∧t and then letting t →∞). It is also clear that µ = µ(λ) is an increasing function mapping (λ0, ∞) onto (0, ∞) and hence has an inverse function λ = λ(µ), µ ∈(0, ∞), with (this can be shown e.g. by applying the theorem to the bounded stopping time τ(x) ∧t and then letting t →∞). It is also clear that µ = µ(λ) is an increasing function mapping (λ0, ∞) onto (0, ∞) and hence has an inverse function λ = λ(µ), µ ∈(0, ∞), with dλ dµ = 1 dµ/dλ = ϕ(λ) ϕ′(λ) = 1 mλ (6) (6) from (4). Now (5) is equivalent to E e−µτ(x) = e−λx, and differentiating this relation w.r.t. µ yields from (4). Now (5) is equivalent to E e−µτ(x) = e−λx, and differentiating this relation w.r.t. µ yields ( ) Now (5) is equivalent to E e−µτ(x) = e−λx, and differentiating this relation w.r.t. µ yiel Z ∞ 0 e−µtt P (τ(x) ∈dt) = xe−λx dλ dµ = x mλeλx . (7) (7) KENDALL’S IDENTITY FOR THE FIRST CROSSING TIME REVISITED And this completes the proof since the desired identity (1) just represents that fact in terms of the respective distribution functions. KENDALL’S IDENTITY FOR THE FIRST CROSSING TIME REVISITED Together with the obvious observation that, for any 0 < a < b < c < ∞, T(a,b] + T(b,c] = T(a,c] this means that a corresponding equality holds for the (finite) E λ-expectations of these vari- ables, and hence we have E λT(a,b] = γ(b −a) for some constant γ < ∞. But from (9) and (10) one gets |E λT(0,a] −a/mλ| ≤Iλ, and letting here a →∞we immediately see that this constant γ must be equal to 1/mλ which completes the proof of (8). (iv) Note that equality (8) can be re-written as Note that equality (8) can be re-written as a mλ = E λ Z ∞ 0 1(0,a](Xt) dt = Z ∞ 0 Pλ(Xt ∈(0, a]) dt = Z ∞ 0 dt Z (0,a] eλx ϕt(λ)P (Xt ∈dx) = Z (0,a]  eλx Z ∞ 0 e−µtP (Xt ∈dx) dt  . Since the above holds for any a > 0, the expression in the brackets is (for any λ > λ0) nothing else but a multiple of the Lebesgue measure: [· · ·] = m−1 λ dx for x ∈(0, ∞), so that Since the above holds for any a > 0, the expression in the brackets is (for any λ > λ0) nothing else but a multiple of the Lebesgue measure: [· · ·] = m−1 λ dx for x ∈(0, ∞), so that dx mλeλx = Z ∞ 0 e−µtP (Xt ∈dx) dt. 94 Electronic Communications in Probability Now dividing the LHS and RHS of (7) by x and integrating them w.r.t. dx over (y, ∞) we get, using the above formula, that Now dividing the LHS and RHS of (7) by x and integrating them w.r.t. dx over (y, ∞) we get, using the above formula, that Z ∞ y dx x Z ∞ 0 e−µtt P (τ(x) ∈dt) = Z ∞ 0 e−µtt Z ∞ y dx x P (τ(x) ∈dt) = Z ∞ y dx mλeλx = Z ∞ 0 e−µtP (Xt > y)dt. for any µ > 0. The equality of the Laplace transforms (in µ) implies that the measure tL(dt) := t Z ∞ y dx x P (τ(x) ∈dt) tL(dt) := t Z ∞ y dx x P (τ(x) ∈dt) has the density P (Xt > y), or, equivalently, that L(dt) has the density P (Xt > y)/t. References [1] Barton, D. E., and Mallows, C. L. (1965) Some aspects of the random sequence. Ann. Math. Statist. 36, 236–260. [2] Borovkov, A. A. (1965) On the first passage time for a class of processes with independent increments. Theor. Probab. Appl. 10, 360–364. [In Russian.] [3] Borovkov, A. A. (1976) Stochastic processes in queueing theory. Springer: New York. [4] Borovkov, K. A. (1995) On crossing times for multidimensional walks with skip-free com- ponents. J. Appl. Prob. 32, 991–1006. [5] Feller, W. (1971) An introduction to probability theory and its applications. Vol. 2. 2nd edn. Wiley: New York. [6] Keilson, J. (1963) The first passage time density for homogeneous skip-free walks on the continuum. Ann. Math. Statist. 34, 1003–1011. [7] Kendall, D. G. (1957) Some problems in the theory of dams. J. Royal Stat. Soc. B, 19, 207–212. [8] Rogozin, B. A. (1966) Distribution of certain functionals related to boundary value prob- lems for processes with independent increments. Theor. Probab. Appl. 11, 161–169. [In Russian.] [9] Skorohod, A. V. (1964) Random processes with independent increments. Nauka: Moscow [In Russian. English translation of the second Russian edition was published in 1991 (Kluwer: Dordrecht).] [10] Tak´acs, L. (1967) Combinatorial methods in the theory of stochastic processes. Wiley: New York. [11] Zolotarev, V. M. (1964) The first passage time of a level and the behaviour at infinity of a class of processes with independent increments. Theor. Probab. Appl. 9, 724–733. [In Russian.]
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RAS activation via CRLF2 signaling is a widespread mechanism in Down syndrome acute lymphoblastic leukemia regardless of RAS mutations
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5 Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia 6 National University Cancer Institute, Singapore, Singapore 7 University of Malaya Medical Centre, University of Malaya, Kuala Lumpur, Malaysia 8 Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA 9 Italian Institute for Genomic Medicine, Turin, Italy Abstract Leukemias are routinely sub-typed for risk/outcome prediction and therapy choice using acquired mutations and chromosomal rearrangements. Down syndrome acute lymphoblastic leukemia (DS‐ALL) is characterized by high frequency of CRLF2‐rearrangements, JAK2‐mutations, or RAS‐pathway mutations. Intriguingly, JAK2 and RAS-mutations are mutually exclusive in leukemic sub‐clones, causing dichotomy in therapeutic target choices. We prove in a cell model that elevated CRLF2 in combination with constitutionally active JAK2 is sufficient to activate wtRAS. On primary clinical DS‐ ALL samples, we show that wtRAS-activation is an obligatory consequence of mutated/hyperphosphorylated JAK2. We further prove that CRLF2-ligand TSLP boosts the direct binding of active PTPN11 to wtRAS, providing the molecular mechanism for the wtRAS activation. Pre‐inhibition of RAS or PTPN11, but not of PI3K or JAK‐signaling, prevented TSLP‐induced RAS‐GTP boost. Cytotoxicity assays on primary clinical DS‐ALL samples demonstrated that, regardless of mutation status, high-risk leukemic cells could only be killed using RAS‐inhibitor or PTPN11-inhibitor, but not PI3K/JAK‐ inhibitors, suggesting a unified treatment target for up to 80% of DS‐ALL. Importantly, protein activities-based principal- component-analysis multivariate clusters analyzed for independent outcome prediction using Cox proportional-hazards model showed that protein‐activity (but not mutation-status) was independently predictive of outcome, demanding a paradigm-shift in patient‐stratification strategy for precision therapy in high-risk ALL. David Koschut 1 ●Debleena Ray 1 ●Zhenhua Li2 ●Emanuela Giarin3 ●Jürgen Groet4 ●Ivan Alić 1,5 ● Shirley Kow-Yin Kham2 ●Wee Joo Chng6 ●Hany Ariffin 7 ●David M. Weinstock8 ●Allen Eng-Juh Yeoh2,6 ● Giuseppe Basso3,9 ●Dean Nižetić 1,4 Received: 9 June 2020 / Revised: 30 October 2020 / Accepted: 11 November 2020 / Published online: 27 November 202 © The Author(s) 2020. This article is published with open access * Dean Nižetić d.nizetic@qmul.ac.uk Oncogene (2021) 40:746–762 https://doi.org/10.1038/s41388-020-01567-7 Oncogene (2021) 40:746–762 https://doi.org/10.1038/s41388-020-01567-7 ARTICLE ARTICLE ARTICLE 1 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore RAS-protein activation but not mutation status is an outcome predictor and unifying therapeutic target for high-risk acute lymphoblastic leukemia David Koschut 1 ●Debleena Ray 1 ●Zhenhua Li2 ●Emanuela Giarin3 ●Jürgen Groet4 ●Ivan Alić 1,5 ● Shirley Kow-Yin Kham2 ●Wee Joo Chng6 ●Hany Ariffin 7 ●David M. Weinstock8 ●Allen Eng-Juh Yeoh2,6 ● Giuseppe Basso3,9 ●Dean Nižetić 1,4 David Koschut 1 ●Debleena Ray 1 ●Zhenhua Li2 ●Emanuela Giarin3 ●Jürgen Groet4 ●Ivan Alić 1,5 ● Shirley Kow-Yin Kham2 ●Wee Joo Chng6 ●Hany Ariffin 7 ●David M. Weinstock8 ●Allen Eng-Juh Yeoh2,6 ● Giuseppe Basso3,9 ●Dean Nižetić 1,4 5 Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia Patient samples Surplus clinical or archived clinical material for per- ipheral blood/bone marrow samples of DS-ALL and non-DS ALL patients was collected by the tissue bank of the Italian Association for Paediatric Haematology- Oncology (AIEOP). In accordance with the Declaration of Helsinki, informed written consent was obtained by the tissue bank for all subjects. Samples were processed and stored in the tissue bank at The Blizard Institute, which is licensed for tissue storage and monitored by UK-Human Tissue Authority. Detailed clinical descrip- tion of studied DS-ALLs and Non-DS B-ALLs is avail- able in Supplementary Table S1. Detailed cytogenetics was available in 12 cases. The acquired mutations landscape does not find a uni- fying profile that distinguishes HR childhood ALL from non-HR childhood ALL, suggesting the need for indivi- dualized therapy approach [16] preceded by individual patient sub-type assignment based on the mutational profile analysis. While Ph-like ALL has a high incidence (60%) of genomic rearrangements leading to an increased expression of the receptor to the cytokine TSLP, CRLF2 [17], and more than half of these have mutations in JAK and IL7R pathway - including constitutionally activating JAK2 mutations [11, 18, 19], less than 10% of Ph-like ALL also acquire RAS/MAPK pathway mutations. DS-ALL is dis- tinguished by the similarly high presence of both CRLF2- rearrangements (60%) (with JAK2 mutations at 32%), with a higher proportion of RAS-MAPK pathway mutations (36%) [20, 21]. Intriguingly, a near complete mutual exclusion between JAK2 and RAS mutations in diagnosis samples, or individual sub-clones of relapse samples of DS- ALL is repeatedly observable [20, 21]. MS2003/2010 cohort [22, 23] RNA-seq data was sub- mitted to the European Genome-phenome Archive (Accession# EGAS00001001858). RAS activity assays Cells were left uninduced or induced with human TSLP at 37 °C. Whenever indicated, DMSO or inhibitors were added for 3 h before TSLP-induction. Cells were lysed on ice at 1000 cells/µL lysis buffer according to the manu- facturer’s protocol of the active RAS detection kit (Cat. #8821; Cell Signaling Technology, Danvers, US). Total protein concentrations of samples were measured using a BCA protein-assay kit (Cat.#23225; ThermoFisher Scien- tific, Waltham, US). 50 µg total protein was loaded per column of the active RAS detection kit for Western blot (WB). In the RAS activation assay kit for ELISA (Cat.#17- 497; EMD Millipore, Burlington, US), 12 µg total protein was used at 100 ng/µL and the RAS-GTP pull-down was measured using a Synergy H1 plate reader (BioTek, Winooski, US) in luminescent mode. We hypothesized that the reason for this mutual exclu- sion is that increased CRLF2-levels in combination with JAK2 activation could be sufficient to activate wild-type (wt) RAS protein in the absence of RAS mutations. Introduction Acute lymphoblastic leukemia (ALL) is the most com- mon malignancy and cancer-related cause of death at pediatric age [1, 2]. Despite a considerable success rate of standard chemotherapy treatments, as many as Supplementary information The online version of this article (https:// doi.org/10.1038/s41388-020-01567-7) contains supplementary material, which is available to authorized users. 1 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore 2 Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore 3 Department of Women’s and Children’s Health (SDB), Hematology-Oncology Laboratory, University of Padua, Padua, Italy 9 Italian Institute for Genomic Medicine, Turin, Italy 4 The Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom RAS-protein activation but not mutation status is an outcome predictor and unifying therapeutic target. . . 747 10–15% of children with ALL have recurrent disease (relapses) [3, 4]. Patients with high-risk (HR) forms of ALL show increased incidence of relapses, poorer prognosis and lower overall 5-year survival rates fol- lowing relapse [5]. Recently, significant progress has been achieved in understanding the mechanistic con- sequences of individual pathways affected in HR-ALL, and the resulting selection of therapeutic targets leading to clinical trials using pathway-specific drugs, such as JAK/STAT inhibitors [6]. Recent detailed studies of the evolution of acquired genomic changes in ALL identified certain sub-types as being particularly HR forms [7, 8]. Among these are hypodiploid ALL [9], Philadelphia chromosome-like (Ph-like) type (defined as a type of ALL with the genomic profile similar to that of the Ph+ ALL) [8, 10, 11], ALL with an intrachromosomal amplification of chromosome 21 (iAMP21) [12, 13], and ALL in children with Down syndrome (DS-ALL) [14, 15]. DE); authenticated via multiplex PCR of minisatellite markers. Mycoplasma-free cells were routinely passaged (passage range for shown experiments: 15–35) according to the respective cell bank recommendations. Handling of primary patient samples is described in detail in Supple- mentary Material. Cell count and cell viability (percentage of acridine orange-positive cells not stained by 4′,6-diamidino-2-phe- nylindole (DAPI) were determined in an NC-250 automated cell-analyzer (ChemoMetec, Allerod, DK). CRLF2 and JAK2mut co-expression is sufficient to activate RAS in Ba/F3 cells We hypothesized that increased CRLF2-level in combi- nation with a mutation in JAK2 pathway genes could be sufficient to activate wtRAS protein in the absence of RAS mutations, as a mechanism to explain the mutual exclusion of JAK2 and RAS/MAPK mutations in DS-ALL. The level of RAS activity is generally assessed using a pull-down assay whereby the (activated) RAS-GTP is captured by virtue of its high affinity to RAS-binding-domain (RBD) of RAF proteins. In order to observe the effects of elevated CRLF2 signaling on the activation of RAS, we stably integrated a human CRLF2 overexpression construct [24] into the mouse pre-B-cell line Ba/F3. This alteration did not increase the level of pulled-down RAS-GTP (Fig. 1a) and neither did the stable overexpression of hJAK2R683G [24], the most prevalent specific activating JAK2 mutation in DS-ALL and Ph-like-ALL. However, when both of these alterations were combined, eight fold higher RAS- GTP level was measured, in the absence of cytokines (Fig. 1b, post-hoc Bonferroni p values are listed in Supple- mentary Table S2). An independent set of Ba/F3 lines, in which CRLF2 was transduced first, confirms that this increased RAS-activity is not due to variations in CRLF2 overexpression levels within the lines (Supplementary Fig. S1F). Growth of Ba/F3 cells depends on IL-3 (Supple- mentary Fig. S1A), which induces wtJAK2 phosphoryla- tion [25], and interestingly we found that it also activates RAS (Supplementary Fig. S1B). The cells with combined CRLF2 and JAK2R683G overexpression were the only ones in this series that grew in a cytokine-independent manner (Supplementary Fig. S1E), as also previously observed [24]. This proves that increased CRLF2-expression toge- ther with activated JAK2 is sufficient to activate wtRAS, and this coincides with the transition to cytokine- independent growth. Fig. 1 Combination of CRLF2 overexpression and constitutively active JAK2 is sufficient for wt RAS activation. WB analysis of the murine pro B cell line Ba/F3. Cells were stably transfected with human JAK2R683G and/or human CRLF2 (see Supplementary Figs. S1C and S1D) and cultured in IL-3-containing medium. All cells were then starved from IL-3 and cells were lysed. Each cell lysate was split up for analysis in RAS-GTP pull-down assay and for total proteins. An SDS-PAGE followed by WB was performed. a Left-hand side blot shows the RAS-GTP (activated RAS) pull-down while the right-hand side blots show whole cell lysates of the same samples. CRLF2 and JAK2mut co-expression is sufficient to activate RAS in Ba/F3 cells Antibody- targets are labeled on the right side of each image with black arrows marking the respective protein band; the antibody against HA-tag shows the expression of the human JAK2 construct. The experiment was repeated four times independently. b Quantification of a for active RAS (RAS-GTP) normalized to its level in untransfected cells. Error bars are SD and P values were determined in one-way ANOVA and post-hoc Bonferroni multiple comparison. Cell culture and cell viability For methods on proximity ligation assay (PLA), principal-component-analysis (PCA), statistical analysis, as well as lists of antibodies/chemicals, and standard protocols for the sequencing, SDS-PAGE/WB, phospho- protein antibody-microarray, and transduction please see “Supplementary Methods”. Ba/F3 (Cat.#RCB0805), a murine IL-3 dependent pro-B cell line, was obtained from RIKEN BioResource Center (Tsukuba, JP) and MUTZ-5 (Cat.#ACC490), a human B cell precursor leukemia cell line established at relapse, was obtained from the Leibniz Institute DSMZ (Braunschweig, D. Koschut et al. 748 Fig. 1 Combination of CRLF2 overexpression and constitutively active JAK2 is sufficient for wt RAS activation. WB analysis of the murine pro B cell line Ba/F3. Cells were stably transfected with human JAK2R683G and/or human CRLF2 (see Supplementary Figs. S1C and S1D) and cultured in IL-3-containing medium. All cells were then starved from IL-3 and cells were lysed. Each cell lysate was split up for analysis in RAS-GTP pull-down assay and for total proteins. An SDS-PAGE followed by WB was performed. a Left-hand side blot shows the RAS-GTP (activated RAS) pull-down while the right-hand side blots show whole cell lysates of the same samples. Antibody- targets are labeled on the right side of each image with black arrows marking the respective protein band; the antibody against HA-tag shows the expression of the human JAK2 construct. The experiment was repeated four times independently. b Quantification of a for active RAS (RAS-GTP) normalized to its level in untransfected cells. Error bars are SD and P values were determined in one-way ANOVA and TSLP-inducible RAS activity in absence of RAS mutations is a feature of human CRLF2 rearranged B-ALL However, neither the JAK inhibitor, nor the PI3K/mTOR inhibitor could block the wtRAS activation by TSLP as shown in WB (Fig. 3c, left- hand side blots) and ELISA (Fig. 3d). For the JAK2 inhibitor this might be explained by its failure to reduce the direct interaction between RAS and PTPN11 in PLA (Supplementary Fig. S2E). In contrast, the pan-wtRAS- inhibitor significantly blocked the TSLP-induced RAS- activity (Fig. 3c, left-hand side blot) and ELISA (Fig. 3d). Moreover, the pre-inhibition of RAS-direct interacting proteins (RAF and PTPN11) also reduced TSLP-induced wtRAS boost in human Ph-ALL cells (Fig. 3c). Combined, our data suggest that TSLP-activation of RAS in the absence of RAS mutations drives B-ALL cell growth, and represents an independent drug target, in addition to the PI3K/mTOR and JAK/STAT pathway targets. Fig. 2 Human Ph-like B-ALL (spontaneous CRLF2-rearrangment and JAK2R683G-mutation) cells activate wtRAS and RAS- interacting proteins upon TSLP-induction. MUTZ-5 cells were stimulated with 20 ng/mL hTSLP (maximal effective TSLP-con- centration, Supplementary Fig. S4B) for 10 min before lysis. Each lysate was split up for analysis in RAS-GTP pull-down assay and for WB. a An SDS-PAGE followed by WB was performed. To assess the total protein and phosphorylated protein amounts on the same PVDF- membrane, each membrane part was stripped and reprobed with new antibodies. RAS-GTP pull-down blots are on the left side while the right-hand side blots show whole cell lysates of the same samples. The gray arrow shows the unspecific signal of the GST-RAS binding domain (RBD) used in the active RAS pull-down assay acting as a loading control. The experiment was repeated five times independently and the graphs show the quantification for active RAS (RAS-GTP), phospho-MEK1/2, phospho-JAK2, and phospho-PTPN11. Beta-actin and total protein signals were used as a loading control to normalize samples. b A blot separate from a demonstrates the TSLP-inducibility of RAS-effector bRAF. c Quantification of five independent ELISA experiments in which RAS-GTP in MUTZ-5 cells was measured using a different, ELISA-specific active-RAS pull-down assay. d MUTZ-5 cells were probed for the activation of KRAS-GTP, HRAS-GTP, or NRAS-GTP isoforms (left side). The blots on the right show the total expression of the respective RAS proteins and the graphs show the average signal fold-change for KRAS-GTP, HRAS-GTP and NRAS- GTP (N = 4, means ± SD). P values were calculated using Student’s T test and adjusted with a Bonferroni-correction for sequential multiple-comparison. TSLP-inducible RAS activity in absence of RAS mutations is a feature of human CRLF2 rearranged B-ALL e Whole, non-denatured lysate from uninduced or TSLP-induced MUTZ-5 cells was subjected to an antibody- microarray. The graph shows relevant, most statistically significant changes in protein-phosphorylations, a heatmap-overview for all analyzed protein-phosphorylations can be found in Supplementary Fig. S4C. (N = 6, means ± SD). P values were calculated using Student’s T test (Bonferroni-correction for sequential multiple comparison can be found in Supplementary Table S2). induction (Fig. 2c). Interestingly, the genes for the same two isoforms (KRAS and NRAS), but not HRAS, acquire muta- tions in B-ALL [12, 20]. Therefore, we conclude that the RAS isoform activity pattern of TSLP-inducibility in wtRAS leukemia cells matches the isoforms that acquire mutations in RAS-mutated leukemia cases. Furthermore, we traced TSLP-signaling throughout cellular pathways in 68 indivi- dual protein-phosphorylation sites via an antibody-based phospho-array (Supplementary Fig. S4C). The phospho- array confirmed the increased phosphorylation observed for denatured proteins in WB for STAT5A, ERK1/2, MEK1, and JAK2 on their respective native epitopes (Supplemen- tary Fig. S4C) while the most statistically significant results demonstrate additional TSLP-effects by increasing activat- ing phosphorylations (AKT2, CDKN1A, ELK1) but also by downregulating pathway-inhibiting phosphorylations (cRAF (Ser296), GAB2, MYC, PTPN6) (Fig. 2e). TSLP-inducible RAS activity in absence of RAS mutations is a feature of human CRLF2 rearranged B-ALL 3a). Both of these compounds, at the concentrations used (tested to achieve high efficacy on the respective main pathway target in WB, Fig. 3c), showed a much stronger inhibitory effect on cell growth than the JAK inhibitor (Fig. 3a), despite the observation that this concentration of JAK-inhibitor, which blocks almost all STAT5-signaling (Fig. 3c, e), showed the strongest inhibition of TSLP-induced phos- phorylation of MEK1/2, PTPN11, ERK1/2 and rpS6 (Fig. 3c, right-hand side blots). However, neither the JAK inhibitor, nor the PI3K/mTOR inhibitor could block the wtRAS activation by TSLP as shown in WB (Fig. 3c, left- hand side blots) and ELISA (Fig. 3d). For the JAK2 inhibitor this might be explained by its failure to reduce the direct interaction between RAS and PTPN11 in PLA (Supplementary Fig. S2E). In contrast, the pan-wtRAS- inhibitor significantly blocked the TSLP-induced RAS- activity (Fig. 3c, left-hand side blot) and ELISA (Fig. 3d). Moreover, the pre-inhibition of RAS-direct interacting proteins (RAF and PTPN11) also reduced TSLP-induced wtRAS boost in human Ph-ALL cells (Fig. 3c). Combined, our data suggest that TSLP-activation of RAS in the absence of RAS mutations drives B-ALL cell growth, and represents an independent drug target, in addition to the PI3K/mTOR and JAK/STAT pathway targets. viability. We tracked the cell count and cell viability of MUTZ-5 cells after treatment with pan-wtRAS-inhibitor and in comparison to treatments with other compounds that have been previously reported to induce dose- dependent cytotoxicity in MUTZ-5 [27], some of which are currently in clinical trials for Ph-like ALL [6]. After 4 days treatment with pan-wtRAS inhibitor the growth and viability of MUTZ-5 cells were significantly reduced (Fig. 3a, b), and this was not affected by the presence of TSLP. In comparison, the dual PI3K/mTOR inhibitor also significantly reduced the growth and viability of MUTZ-5 cells, but this inhibitory effect could be partially counter- acted by the TSLP-induction (Fig. 3a). Both of these compounds, at the concentrations used (tested to achieve high efficacy on the respective main pathway target in WB, Fig. 3c), showed a much stronger inhibitory effect on cell growth than the JAK inhibitor (Fig. 3a), despite the observation that this concentration of JAK-inhibitor, which blocks almost all STAT5-signaling (Fig. 3c, e), showed the strongest inhibition of TSLP-induced phos- phorylation of MEK1/2, PTPN11, ERK1/2 and rpS6 (Fig. 3c, right-hand side blots). DS-ALL patients differ in the level of activity and inducibility of RAS, independently of RAS mutations The MUTZ-5 ALL cells used in the analysis so far share the increased CRLF2 expression and mutated JAK2 with approximately a third of DS-ALL patients [20], which also have no mutations in RAS genes. We therefore analyzed primary cells from presentation samples (at primary diag- nosis) of DS-ALL in RAS pull-down WB and ELISA assay measurements (+/- TSLP stimulation). The analyses were performed blinded to the mutation profile of the patient material and distinct DS-ALL patient profiles for RAS- activity and TSLP-inducibility of RAS were observed in WB (Fig. 4a) and confirmed by ELISA (Fig. 4b). As we see examples of RAS-mutated, wtRAS, or JAK2-mutated TSLP-inducible RAS activity in absence of RAS mutations is a feature of human CRLF2 rearranged B-ALL cultures was confirmed by performing standard Sanger DNA-sequencing of PCR-amplicons from genomic DNA, encompassing all exons of KRAS, NRAS and HRAS genes (Supplementary Table S3). We detected the presence of activated RAS in these cells by RAF-RBD pull-down of RAS-GTP (Fig. 2a), which was tripled upon a 10 min induction with the CRLF2-ligand TSLP. Similar results were reproduced using an ELISA-based RAS-pull-down (Fig. 2c). Both immediate upstream (PTPN11) and immediate down- stream (MEK1/2, bRAF) components of the RAS/MAPK pathway were also induced by TSLP induction (Fig. 2a, b). In order to prove the observations from Fig. 1 in human ALL cells, we selected a B-ALL cell line that harbors identical changes as our double-transfected model line in Fig. 1. The B cell precursor leukemia cell line MUTZ-5 from a relapsed Philadelphia-like B-ALL patient features a CRLF2-translocation leading to wt CRLF2 overexpression, as well as the JAK2R683G mutation, and the absence of mutations in any RAS-MAPK pathway genes [26]. The absence of RAS mutations in the MUTZ-5 cells grown in our RAS-protein activation but not mutation status is an outcome predictor and unifying therapeutic target. . . 749 The direct binding of activated RAS and bRAF proteins expressed in these cells (Supplementary Fig. S2A) was further validated via PLA (Supplementary Fig. S2B), as was RAS and phospho-PTPN11 interaction in the Ba/F3 model (Supplementary Fig. S3B). Both KRAS and NRAS, but not HRAS, isoforms showed increased activity after TSLP- The direct binding of activated RAS and bRAF proteins expressed in these cells (Supplementary Fig. S2A) was further validated via PLA (Supplementary Fig. S2B), as was RAS and phospho-PTPN11 interaction in the Ba/F3 model (Supplementary Fig. S3B). Both KRAS and NRAS, but not HRAS, isoforms showed increased activity after TSLP- 750 D. Koschut et al. viability. We tracked the cell count and cell viability of MUTZ-5 cells after treatment with pan-wtRAS-inhibitor and in comparison to treatments with other compounds that have been previously reported to induce dose- dependent cytotoxicity in MUTZ-5 [27], some of which are currently in clinical trials for Ph-like ALL [6]. After 4 days treatment with pan-wtRAS inhibitor the growth and viability of MUTZ-5 cells were significantly reduced (Fig. 3a, b), and this was not affected by the presence of TSLP. In comparison, the dual PI3K/mTOR inhibitor also significantly reduced the growth and viability of MUTZ-5 cells, but this inhibitory effect could be partially counter- acted by the TSLP-induction (Fig. RAS inhibitor can significantly block the growth of human B-ALL Ph-like wtRAS cells We next examined to what extent the direct RAS activa- tion in wtRAS leukemic cells affects the cell growth and RAS-protein activation but not mutation status is an outcome predictor and unifying therapeutic target. . . 751 p p y g p g DS-ALL in each of the profile types (Fig. 4c), with exception of low-RAS and non-inducible type, we can conclude that activity levels and TSLP-inducibility of RAS cannot be predicted on the basis of DNA-sequencing (acquired mutations) patterns. The most important conclusion of this analysis is th RAS is active/inducible in 14/20 (70%) of primary DS-AL samples analyzed, 8 of which had no RAS mutations, b 75% of those had either mutated or hyperphosphorylat JAK2 (Fig. 4c). This means that either the RAS mutation, DS-ALL in each of the profile types (Fig. 4c), with exception of low-RAS and non-inducible type, we can conclude that activity levels and TSLP-inducibility of RAS cannot be predicted on the basis of DNA-sequencing (acquired mutations) patterns. The most important conclusion of this analysis is that RAS is active/inducible in 14/20 (70%) of primary DS-ALL samples analyzed, 8 of which had no RAS mutations, but 75% of those had either mutated or hyperphosphorylated JAK2 (Fig. 4c). This means that either the RAS mutation, or The most important conclusion of this analysis is that RAS is active/inducible in 14/20 (70%) of primary DS-ALL samples analyzed, 8 of which had no RAS mutations, but 75% of those had either mutated or hyperphosphorylated JAK2 (Fig. 4c). This means that either the RAS mutation, or D. Koschut et al. 752 parallel to the same readouts from the MUTZ-5 Ph-like ALL reference cell line, and the PCA result was mapped onto a coordinate system (Fig. 5a) using the three principal components (PC1-3, Supplementary Fig. S6A). Unsu- pervised k-means clustering grouped ALL samples into Clusters 1–4 (Fig. 5a). This analysis grouped almost all presentation and remission samples of 9-year event-free survival patients (good outcome) together into Cluster 1 (green symbols). In contrast, out of 15 samples grouped into Cluster 2 (red symbols), 13 samples (87%) were from patients with death or subsequent relapse as outcome. Clusters 3 and 4, further along the PC1-axis, consisted of a small number of exclusively relapse samples. Using an independent mathematical approach, unsupervised hier- archical clustering of the 20 DS-ALL presentation samples (Supplementary Fig. RAS inhibitor can significantly block the growth of human B-ALL Ph-like wtRAS cells S6E) grouped 90% of the samples into the same groups as the PCA-mapping. The clustering revealed that presentation samples from Cluster 1 correlated with good outcome for DS-ALL patients while DS-ALL patients grouped into Cluster 2 showed a significantly increased risk of relapse (Fig. 5b, c). The PCA-derived protein activity score was independently predictive of outcome (P = 0.041) (Fig. 5c) when analyzed by a multi- variate Cox regression model together with CRLF2 protein- expression, NCI-risk and JAK2-mutation status (or RAS- mutation status, not shown). Cluster 2 contains a subgroup of DS-ALL presentation samples that clustered closer to the MUTZ-5 sample (Cluster 3). Like MUTZ-5, these patient samples had high CRLF2-expression, high JAK2-phoshor- ylation, and all featured the pattern of high basal RAS- activity that is TSLP-inducible. An event-free survival analysis that treats these MUTZ-5-like DS-ALL samples as a separate subcluster indicated a lower median survival (Supplementary Fig. S6D) but a higher number of samples is required to reach statistical significance for such subgrouping. Fig. 3 Inhibition of RAS stops wt-RAS Philadelphia-like ALL cell growth in the presence of TSLP. a MUTZ-5 cells were seeded at 6.5 × 105/mL density and cultured over 4 days with either 0.5% DMSO (vehicle control), 50 µM Salirasib (indirect Pan-RAS inh.), 10 µM PI- 103 (PI3K/mTOR dual inh.), or 5 µM Ruxolitinib (JAK inh.), each in absence or presence of 20 ng/mL human TSLP. Cell count and via- bility was determined in an NC-250 automated cell counter daily. The stacked-bar graph on the left side shows the growth rate after the 90 h timepoint, averaged from two independent experiments, each with triplicate wells. Red error bars are SD from the dead cell fraction while the black error bars show the SD of the viable cells. P values were calculated in one-way ANOVA from the total cell growth rate and adjusted in a post-hoc Bonferroni multiple comparison. Only relevant P values are shown in the graph, for a complete list see Supplementary Table S2. b The graph shows the cell viability of the experiment in a over time. c MUTZ-5 cells were pre-treated for 2 h with either 0.5% DMSO (vehicle control), 10 µM PI-103 (PI3K/mTOR dual inh.), 50 µM Salirasib (indirect pan-RAS inh.), 5 µM Ruxolitinib (JAK inh.), 50 µM Vemurafenib (Pan-Raf inh.), or 25 µM II-B08 (PTPN11 inh.), and then stimulated with 20 ng/mL human TSLP for 10 min followed by cell lysis. RAS inhibitor can significantly block the growth of human B-ALL Ph-like wtRAS cells Each lysate sample was split up for analysis in RAS-GTP pull-down assay and for total protein signal. RAS-GTP pull-down (left) and lysate samples (right) were loaded on separate gels. An SDS- PAGE followed by WB was performed. To assess the total protein and phosphorylated protein amounts on the same PVDF-membrane, membranes were stripped and reprobed with new antibodies. Antibody-targets are labeled on the right side of each image with black arrows indicating the respective protein band. d MUTZ-5 cells were treated with 50 µM Salirasib (pan-RAS-inhi- bitor), 10 µM PI-103 (PI3K/mTOR dual inhibitor), or 5 µM Rux- olitinib (JAK-inhibitor) like in c after which the RAS-GTP levels were measured in ELISA. N = 3 independent experiments, bar graph shows means ± SD. e MUTZ-5 cells were treated as in d and STAT5 activity was determined via Western blot. N = 3 independent experiments, bar graph shows means ± SD. P values for d and e were calculated in one- way ANOVA and post-hoc Bonferroni multiple comparison. the combination of high CRLF2 and hyperphosphorylated JAK2 (including mutated JAK2) can explain the mechan- ism for high RAS activity in 12/14 (86%) of DS-ALL with high RAS activity. Importantly, not a single wtRAS case with either mutated or hyperphosphorylated JAK2 was seen that lacked activated RAS protein (Fig. 4c), suggesting that RAS activation is an obligatory consequence in wtRAS DS- ALL cases with mutated or hyperphosphorylated JAK2. We restricted the further analysis only to DS-ALL pri- mary presentation samples, and quantitatively compared those that PCA grouped into Cluster 1 (PCA-predicted standard-risk (SR)) to those in Cluster 2 (PCA-predicted high-risk (HR)), for the basal activities (Fig. 5d top row) and TSLP-induced activities (Fig. 5d bottom row) of pan- RAS, JAK2, STAT5, MEK1/2, ERK1/2 and rpS6. We observed that basal and TSLP-induced activities of JAK2, ERK1/2 and rpS6 were significantly increased in HR-DS- ALL presentation samples compared to the SR group (within PCA-Cluster 1). For these proteins, correlation between risk and protein-activity/inducibility profile for our DS-ALL cohort, resembles previously reported findings for a different group of HR-ALL, the non-DS Ph-like ALL, grouped by the presence or absence of CRLF2 rearrange- ments [27]. Additionally, (and this has, to our knowledge, never been demonstrated for any ALL before), we also RAS activity and its TSLP inducibility correlate with outcome in DS-ALL patients Data from primary cells analysis from n = 20 presentation samples of DS-ALL for the RAS/MAPK, PI3K/mTOR, and JAK/STAT pathway activity profiles using WB (Supple- mentary Figs. S5A and S5B), as well as ELISA for acti- vated RAS-pull-down (Fig. 4c) were integrated with the similar data we obtained using n = 7 DS-ALL relapse and n = 4 DS-ALL remission samples, as well as n = 4 non-DS ALL presentation samples and n = 2 non-DS relapse sam- ples. We performed a PCA using all of these integrated data on N = 37 samples from n = 31 individual patients, in 753 753 4 70% f i DS ALL t ti l h ti i i hil i b l 10% RAS GTP i TSLP Fig. 4 70% of primary DS-ALL presentation samples show acti- vated and/or TSLP-inducible RAS, regardless of mutation-status. activity while an increase by at least 10% RAS-GTP in TSLP- stimulated samples over uninduced samples in ELISA was classed as TSLP-inducible RAS. For visualization, JAK2-phosphorylation levels measured in WB were categorized as –(negative) = 0.00–0.05; + = 0.05–0.50; ++ = 0.50–1.00; +++ = 1.00–2.00, and CRLF2 protein- levels were categorized as –(negative) = 0.00–0.05; + = 0.05–0.20; ++ = 0.20–0.50; +++ = 0.50–1.50. None of these arbitrary threshold-groupings were used in the clustering analysis (Fig. 5). Known CRLF2-rearrangements are marked (R). All values are nor- malized to those measured for uninduced MUTZ-5 cells processed in parallel to patient cells. Table boxes: Outcome of leukemia (white = good outcome, black = poor outcome), RAS mutations (=blue) or JAK2 mutations (=red) (gray = unsequenced). For patient/sample groups other than DS-ALL-diagnosis (Non-DS (NDS) at presentation, DS complete remission (CR), and DS/NDS at relapse) only averages are shown. For an overview of the WB data and analyzed protein expression/phosphorylation of all individual samples, see Supple- mentary Fig. S5. Fig. 4 70% of primary DS-ALL presentation samples show acti- vated and/or TSLP-inducible RAS, regardless of mutation-status. Primary presentation samples of DS-ALL patients were cultured for 2 days (detailed in Supplementary Fig. S5A-legend) and then induced for 10 min with 20 ng/mL TSLP (or uninduced) in serum-reduced medium. a Each lysate was split up for RAS-GTP pull-down assay (left blot) and for standard WB (right blot). Gray arrow shows the loading of the GST-RBD in the pull-down assay. b The RAS activity pattern in the patient samples from a was confirmed via ELISA measurement of RAS-activity in aliquots that were independently thawed and processed. c Overview of the ELISA-measured RAS activity for the DS-ALL cohort at diagnosis (not enough cell material was available for DS26, DS29, and DS30). The RAS-GTP pull-down ELISA was performed on lysates (100 ng/μL total-protein) from cells at minimum 75% viability. Brackets on top indicate the four RAS activity patterns presented in a and b. RAS-protein activation but not mutation status is an outcome predictor and unifying therapeutic target. . . Fig. 4 70% of primary DS-ALL presentation samples show acti- vated and/or TSLP-inducible RAS, regardless of mutation-status. Primary presentation samples of DS-ALL patients were cultured for 2 days (detailed in Supplementary Fig. S5A-legend) and then induced for 10 min with 20 ng/mL TSLP (or uninduced) in serum-reduced medium. a Each lysate was split up for RAS-GTP pull-down assay (left blot) and for standard WB (right blot). Gray arrow shows the loading of the GST-RBD in the pull-down assay. b The RAS activity pattern in the patient samples from a was confirmed via ELISA measurement of RAS-activity in aliquots that were independently thawed and processed. c Overview of the ELISA-measured RAS activity for the DS-ALL cohort at diagnosis (not enough cell material was available for DS26, DS29, and DS30). The RAS-GTP pull-down ELISA was performed on lysates (100 ng/μL total-protein) from cells at minimum 75% viability. Brackets on top indicate the four RAS activity patterns presented in a and b. For visualization purposes only in this graph, basal RAS-activity over 0.5 (median of all patient samples) MUTZ-5 basal RAS activity was grouped as high RAS activity while an increase by at least 10% RAS-GTP in TSLP- stimulated samples over uninduced samples in ELISA was classed as TSLP-inducible RAS. For visualization, JAK2-phosphorylation levels measured in WB were categorized as –(negative) = 0.00–0.05; + = 0.05–0.50; ++ = 0.50–1.00; +++ = 1.00–2.00, and CRLF2 protein- levels were categorized as –(negative) = 0.00–0.05; + = 0.05–0.20; ++ = 0.20–0.50; +++ = 0.50–1.50. None of these arbitrary threshold-groupings were used in the clustering analysis (Fig. 5). Known CRLF2-rearrangements are marked (R). All values are nor- malized to those measured for uninduced MUTZ-5 cells processed in parallel to patient cells. Table boxes: Outcome of leukemia (white = good outcome, black = poor outcome), RAS mutations (=blue) or JAK2 mutations (=red) (gray = unsequenced). For patient/sample groups other than DS-ALL-diagnosis (Non-DS (NDS) at presentation, DS complete remission (CR), and DS/NDS at relapse) only averages are shown. For an overview of the WB data and analyzed protein expression/phosphorylation of all individual samples, see Supple- mentary Fig. S5. RAS-protein activation but not mutation status is an outcome predictor and unifying therapeutic target. . . For visualization purposes only in this graph, basal RAS-activity over 0.5 (median of all patient samples) MUTZ-5 basal RAS activity was grouped as high RAS activity while an increase by at least 10% RAS-GTP in TSLP- stimulated samples over uninduced samples in ELISA was classed as TSLP-inducible RAS. For visualization, JAK2-phosphorylation levels measured in WB were categorized as –(negative) = 0.00–0.05; + = 0.05–0.50; ++ = 0.50–1.00; +++ = 1.00–2.00, and CRLF2 protein- levels were categorized as –(negative) = 0.00–0.05; + = 0.05–0.20; ++ = 0.20–0.50; +++ = 0.50–1.50. None of these arbitrary threshold-groupings were used in the clustering analysis (Fig. 5). Known CRLF2-rearrangements are marked (R). All values are nor- malized to those measured for uninduced MUTZ-5 cells processed in parallel to patient cells. Table boxes: Outcome of leukemia (white = good outcome, black = poor outcome), RAS mutations (=blue) or JAK2 mutations (=red) (gray = unsequenced). For patient/sample groups other than DS-ALL-diagnosis (Non-DS (NDS) at presentation, DS complete remission (CR), and DS/NDS at relapse) only averages are shown. For an overview of the WB data and analyzed protein expression/phosphorylation of all individual samples, see Supple- mentary Fig. S5. activity while an increase by at least 10% RAS-GTP in TSLP- stimulated samples over uninduced samples in ELISA was classed as TSLP-inducible RAS. For visualization, JAK2-phosphorylation levels measured in WB were categorized as –(negative) = 0.00–0.05; + = 0.05–0.50; ++ = 0.50–1.00; +++ = 1.00–2.00, and CRLF2 protein- levels were categorized as –(negative) = 0.00–0.05; + = 0.05–0.20; ++ = 0.20–0.50; +++ = 0.50–1.50. None of these arbitrary th h ld i d i th l t i l i (Fi 5) observed a significant increase in basal and TSLP-induced activity of both MEK1/2 and RAS in the HR-DS-ALL group, compared to the SR group. We also looked at protein expression levels and found RAS and rpS6 levels to correlate with the high-risk DS-ALL group (Supplementary Fig. S7A). This provided the rationale to look for differ- ences in a larger non-DS ALL cohort (see Supplementary Results, Supplementary Figs. S7B, S8A, S8B). observed a significant increase in basal and TSLP-induced activity of both MEK1/2 and RAS in the HR-DS-ALL group, compared to the SR group. We also looked at protein expression levels and found RAS and rpS6 levels to D. Koschut et al. 754 Fig 5 Sub stratification of DS ALL patients based on primary the two clusters (bar graph) c Kaplan Meier curves of cluster 1 (SR Fig. 5 Sub-stratification of DS-ALL patients based on primary cells: RAS-activation and downstream signaling in relation to standard-therapy outcomes. a A PCA was performed on the quan- tified data of Fig. 4 (data was given as continuous variables; no cutoffs nor pre-groupings were used) for the DS-ALL cohort at diagnosis, and (where available) at remission, and relapse, as well as presentation and relapse samples from Non-DS ALL patients. Top view of the PCA- mapping for all six analyzed protein-activities (basal and TSLP- induced) as well as CRLF2-protein expression of all samples along the calculated principal components (see also Supplementary Fig. S6A). K-means unsupervised clustering (with k set to 4 to achieve minimal class-class deviation, Supplementary Fig. S6B) grouped samples into clusters 1–4 (listed in Supplementary Fig. S6C). b PCA Clusters 1 and 2 contain all samples of the DS-ALL diagnosis cohort and were analyzed according to their outcome: A Fisher’s exact test determined the P value between the number of good and poor outcomes between the two clusters (bar graph). c Kaplan–Meier curves of cluster 1 (SR standard risk) and cluster 2 (HR high risk) DS-ALL patients. Table shows a Cox proportional-hazards model for protein activity score (PCA-derived principal component from all quantified protein activ- ities at basal and TSLP-induced level) together with CRLF2-protein expression level (for CRLF2+ samples), NCI risk groups (SR: age at diagnosis 1–10 years and WBC < 50.000/µL; HR = children age >10 years and/or WBC > 50.000/µL; or unknown), and presence of acti- vating JAK2-mutations. Reverse Kaplan–Meier median follow-up for N = 20 DS-ALL was 18.4 years. Patient numbers at risk for each year are given in the table. d The means of all analyzed basal or TSLP- induced protein activities are compared between the SR group (DS- ALL patients in PCA cluster 1) and the HR group (DS-ALL patients in PCA cluster 2). All error bars are SD; P values were calculated using Student’s T test and are adjusted with a Bonferroni-correction for sequential multiple comparison. Fig. 5 Sub-stratification of DS-ALL patients based on primary ll RAS i i d d i li i l i the two clusters (bar graph). c Kaplan–Meier curves of cluster 1 (SR d d i k) d l 2 ( hi h i k) S A i bl Fig. 5 Sub-stratification of DS-ALL patients based on primary cells: RAS-activation and downstream signaling in relation to standard-therapy outcomes. a A PCA was performed on the quan- tified data of Fig. 4 (data was given as continuous variables; no cutoffs nor pre-groupings were used) for the DS-ALL cohort at diagnosis, and (where available) at remission, and relapse, as well as presentation and relapse samples from Non-DS ALL patients. Top view of the PCA- mapping for all six analyzed protein-activities (basal and TSLP- induced) as well as CRLF2-protein expression of all samples along the calculated principal components (see also Supplementary Fig. S6A). K-means unsupervised clustering (with k set to 4 to achieve minimal class-class deviation, Supplementary Fig. S6B) grouped samples into clusters 1–4 (listed in Supplementary Fig. S6C). b PCA Clusters 1 and 2 contain all samples of the DS-ALL diagnosis cohort and were analyzed according to their outcome: A Fisher’s exact test determined the P value between the number of good and poor outcomes between the two clusters (bar graph). c Kaplan–Meier curves of cluster 1 (SR the two clusters (bar graph). c Kaplan–Meier curves of cluster 1 (SR standard risk) and cluster 2 (HR high risk) DS-ALL patients. Table shows a Cox proportional-hazards model for protein activity score (PCA-derived principal component from all quantified protein activ- ities at basal and TSLP-induced level) together with CRLF2-protein expression level (for CRLF2+ samples), NCI risk groups (SR: age at diagnosis 1–10 years and WBC < 50.000/µL; HR = children age >10 years and/or WBC > 50.000/µL; or unknown), and presence of acti- vating JAK2-mutations. Reverse Kaplan–Meier median follow-up for N = 20 DS-ALL was 18.4 years. Patient numbers at risk for each year are given in the table. d The means of all analyzed basal or TSLP- induced protein activities are compared between the SR group (DS- ALL patients in PCA cluster 1) and the HR group (DS-ALL patients in PCA cluster 2). All error bars are SD; P values were calculated using Student’s T test and are adjusted with a Bonferroni-correction for sequential multiple comparison. RAS-protein activation but not mutation status is an outcome predictor and unifying therapeutic target. . . 755 PI3K/mTOR pathway activation in TSLP-induced human ALL cells. PI3K/mTOR pathway activation in TSLP-induced human ALL cells. TSLP activates RAS directly and independently of PI3K/mTOR pathway activation 6b, c), at a concentration lower than required to block EIF4EBP1 activity via mTOR-complex destabilization [31]. PLA also detected a strong interaction between RAS and the RBD-containing PI3K-subunit p110α in these cells, which could be reduced using Rigosertib, a RAS-GTP mimetic that inhibits RAS by binding to the RBD of RAS-effectors (Supplementary Fig. S2D). O d t th f t l t th t di t tRAS Downstream effectors of the activated PI3K/mTOR pathway have been shown in some situations to cross- activate the downstream effectors of RAS-MAPK cascade, and vice versa [28, 29]. However, we observed that immediately upon addition of TSLP (0 min timepoint, Fig. 6a) the relative levels of activated pan-RAS, KRAS, NRAS, PTPN11, MEK1/2, and ERK1/2 were all higher than at any later timepoint, while in comparison, the activity onset of the PI3K/mTOR downstream target rpS6 was delayed (Fig. 6a). This makes it less likely, at least as the initial effect of TSLP, that the activation of MEK1/2 and ERK1/2 in such leukemic cells is caused by the cross-talk from the activated PI3K pathway. As PI3K can also be an effector of RAS [30], we used an alternative biochemical approach (PLA) by which we demonstrated the ability of a chemical inhibitor of RAS (Salisarib) to block the TSLP-induced rpS6-activating phosphorylation (Fig. 6b, c), at a concentration lower than required to block EIF4EBP1 activity via mTOR-complex destabilization [31]. PLA also detected a strong interaction between RAS and the RBD-containing PI3K-subunit p110α in these cells, which could be reduced using Rigosertib, a RAS-GTP mimetic that inhibits RAS by binding to the RBD of RAS-effectors (Supplementary Fig. S2D). Our data on primary patient material suggest the com- pulsory activation of RAS whenever elevated CRLF2 is present in combination with either mutated or otherwise activated JAK2. This would eliminate the selective advan- tage gained by a RAS mutation, explaining the mutual exclusion, however the underlying molecular mechanism remains to be explained. We therefore sought to further characterize the molecular mechanism behind the wtRAS activation in these leukemic cells. CRLF2-signaling increases direct interaction between active PTPN11 and RAS While the PTPN11-inhibitor reduced wtRAS activity in MUTZ5- cells (Fig. 3c), the connection between PTPN11 and RAS in ALL as well as in CRLF2-signaling is unknown. Active PTPN11 is thought to dephosphorylate RAS to prime it for activation [32], and we found PTPN11 phosphorylation to be increased by induced CRLF2- signaling (Fig. 2a). Furthermore, PTPN11 is published to be in complex with JAK2 upon cytokine-induction in tumor cells [33]. In order to confirm that the mechanism of acti- vating RAS in JAK2-mutated B-ALL cells is regulated via PTPN11, we designed a PLA assay that specifically detects the direct interaction between RAS and phosphorylated PTPN11 (Fig. 7a, c). Indeed, compared to the signal for two cytosolic proteins not expected to interact (PLA negative control (NC)), a strong PLA signal between RAS and p-PTPN11 was observed and this interaction almost dou- bled upon TSLP-induction (Fig. 7a). Of note, Ba/F3 cells cultured with IL-3, which activated RAS (Supplementary Fig. S1B) and JAK2-phosohorylation, also featured a higher level of RAS and p-PTPN11 interaction in PLA compared to unstimulated cells (Supplementary Fig. S3B). PLA assays also detected interactions between SOS1 and GRB2 in these leukemic cells, as well as other direct interactions involved in RAS activation, which also showed response to CRLF2-activation (RAS and SOS1; GRB2 and p-PTPN11) (Supplementary Fig. S2C). Remarkably, blocking PTPN11- activity via the PTPN11-inhibitor II-B08 reduced both endogenous, and TSLP-induced RAS activity in these cells (Fig. 7b). The PTPN11-inhibitor did not reduce the phos- phorylation marker on PTPN11 itself (Fig. 7b) but disrupted the direct interaction between RAS and p-PTPN11, low- ering it to levels below those in uninduced cells (Fig. 7c). Furthermore, a cytotoxic assay showed leukemic cell via- bility to be reduced by the PTPN11-inhibitor, similarly as with the RAS-inhibitor (Fig. 7d). Taken together, these results show that the mechanism of wtRAS activation by CRLF2 signaling depends on its direct interaction with catalytically active PTPN11. TSLP activates RAS directly and independently of PI3K/mTOR pathway activation The use of inhibitors on MUTZ-5 cells (Fig. 3a) suggested RAS activation to be independent from blocking of PI3K or JAK pathways. TSLP induction in high CRLF2- expressing and JAK2-mutated B-ALL is known to acti- vate STAT5 and PI3K/mTOR pathways [27], and this insight is exploited in innovative new therapeutic approaches that are currently clinically trialed [6]. We therefore first confirmed that our experimental system can reproduce these same results in WB (Supplementary Fig. S4A). In addition, we designed a quantitative method (PLA) to measure rpS6-phoshporylation in individual cells (Fig. 6b). Similar to the TSLP-induction in MUTZ-5 cells, the Ba/F3 CRLF2 + JAK2R683G cells also display an increased rpS6-phosphorylation in PLA compared to cells overexpressing only JAK2R683G (Supplementary Fig. S3A). The use of inhibitors on MUTZ-5 cells (Fig. 3a) suggested RAS activation to be independent from blocking of PI3K or JAK pathways. TSLP induction in high CRLF2- expressing and JAK2-mutated B-ALL is known to acti- vate STAT5 and PI3K/mTOR pathways [27], and this insight is exploited in innovative new therapeutic approaches that are currently clinically trialed [6]. We therefore first confirmed that our experimental system can reproduce these same results in WB (Supplementary Fig. S4A). In addition, we designed a quantitative method (PLA) to measure rpS6-phoshporylation in individual cells (Fig. 6b). Similar to the TSLP-induction in MUTZ-5 cells, the Ba/F3 CRLF2 + JAK2R683G cells also display an increased rpS6-phosphorylation in PLA compared to cells overexpressing only JAK2R683G (Supplementary Fig. S3A). Downstream effectors of the activated PI3K/mTOR pathway have been shown in some situations to cross- activate the downstream effectors of RAS-MAPK cascade, and vice versa [28, 29]. However, we observed that immediately upon addition of TSLP (0 min timepoint, Fig. 6a) the relative levels of activated pan-RAS, KRAS, NRAS, PTPN11, MEK1/2, and ERK1/2 were all higher than at any later timepoint, while in comparison, the activity onset of the PI3K/mTOR downstream target rpS6 was delayed (Fig. 6a). This makes it less likely, at least as the initial effect of TSLP, that the activation of MEK1/2 and ERK1/2 in such leukemic cells is caused by the cross-talk from the activated PI3K pathway. As PI3K can also be an effector of RAS [30], we used an alternative biochemical approach (PLA) by which we demonstrated the ability of a chemical inhibitor of RAS (Salisarib) to block the TSLP-induced rpS6-activating phosphorylation (Fig. Primary DS-ALL patient biopsies from high-risk sub- cohorts have a potent response to RAS-inhibition in vitro as a distinguishing feature We used primary surplus clinical material in Fig. 5 from n = 31 patients. Out of these, we had enough primary diagnosis material for 13 patients (before any therapy) to measure the effects of RAS, PI3K, or JAK inhibitors on Our data therefore strongly suggest that direct, wtRAS activation can precede, and to a certain extent promote, the D. Koschut et al. 756 6 Direct wtRAS-activation can precede PI3K/mTOR-pathway vation and resulting PI3K-downstream signaling activity was ked by RAS inhibitor. a Effect of TSLP induction over time. TZ-5 cells were incubated with 20 ng/mL human TSLP at 37 °C he indicated time points (0 min to 18 h) before cell lysis. Due to centrifugation step the TSLP can act for 5 min before lysis at point 0. Each cell lysate was split up for RAS-GTP pull-down y and WB. RAS-GTP pull-down elutions are on the left side while right-hand side blots show whole cell lysates of the same samples. body-targets are labeled on the right side of each image with black or JAK inhibitor. Cells were fixed and permeabilized in a 9 plate. After blocking, antibodies against phosphorylated rpS6 a rpS6 were used in conjunction with PLA rabbit and mouse pr allow specific readout of rpS6 activation in single cells in throughput manner. Histograms show the distribution for a experiment of the number of PLA spots in cells with at least spot (assay control is only shown in the bar graph). A minim 600 cells were analyzed per sample. Non-linear Gaussian fitting were plotted. Fluorescent microscope images show examples spots in MUTZ-5 cells for the respective treatment; white sca Fig. 6 Direct wtRAS-activation can precede PI3K/mTOR-pathway activation and resulting PI3K-downstream signaling activity was blocked by RAS inhibitor. a Effect of TSLP induction over time. MUTZ-5 cells were incubated with 20 ng/mL human TSLP at 37 °C for the indicated time points (0 min to 18 h) before cell lysis. Due to the centrifugation step the TSLP can act for 5 min before lysis at timepoint 0. Each cell lysate was split up for RAS-GTP pull-down assay and WB. RAS-GTP pull-down elutions are on the left side while the right-hand side blots show whole cell lysates of the same samples. Antibody-targets are labeled on the right side of each image with black arrows indicating the respective protein band. Primary DS-ALL patient biopsies from high-risk sub- cohorts have a potent response to RAS-inhibition in vitro as a distinguishing feature b Activation of PI3K/ mTOR downstream target rpS6 protein was monitored via PLA in high-throughput microscopy. MUTZ-5 cells were either not induced or induced with 20 ng/mL TSLP for 10 min. Where indicated, cells were pre-treated for 3 h with either DMSO (vehicle control), RAS inhibitor, or JAK inhibitor. Cells were fixed and permeabilized in a 96 well plate. After blocking, antibodies against phosphorylated rpS6 and total rpS6 were used in conjunction with PLA rabbit and mouse probes to allow specific readout of rpS6 activation in single cells in a high- throughput manner. Histograms show the distribution for a single experiment of the number of PLA spots in cells with at least 1 PLA spot (assay control is only shown in the bar graph). A minimum of 600 cells were analyzed per sample. Non-linear Gaussian fitting curves were plotted. Fluorescent microscope images show examples of PLA spots in MUTZ-5 cells for the respective treatment; white scale bars are 20 µm long. c The bar graph summarizes the average PLA spot counts of three independent experiments. Error bars are SD and P values were determined in one-way ANOVA and post-hoc Bon- ferroni multiple comparison. RAS-protein activation but not mutation status is an outcome predictor and unifying therapeutic target. . . 757 Fig. 7 CRLF2-signaling induces direct interaction between acti- vated PTPN11 and RAS, and PTPN11-activity is required for ALL cell growth. a Direct interaction between RAS and phos- phorylated PTPN11 was monitored via PLA using high-content microscopy. Serum-starved MUTZ-5 cells were induced (or not) with 20 ng/mL TSLP for 10 min. Cells were fixed and permeabilized in a 96 well plate. Antibodies against phosphorylated PTPN11 and pan-RAS were used in conjunction with PLA-probes to allow the amplification and staining of interaction-specific PLA-spots. The negative control (NC) are two cytosolic proteins not expected to interact. Fluorescent-microscopy images show examples of PLA-spots (scale bars = 20 µm). At least 250 cells per well were analyzed using Operetta-CLS automated high-content microscopy platform. The bar- graph shows the averages of three independent experiments (each performed in triplicates). Error bars are SD and P values were mined in one-way ANOVA and post-hoc Bonferroni multiple parison. b MUTZ-5 cells were pre-incubated with DMSO or 25 µ B08 (PTPN11 inhibitor) for 2 h and then stimulated or not with 2 mL TSLP for 10 min before cell lysis. Primary DS-ALL patient biopsies from high-risk sub- cohorts have a potent response to RAS-inhibition in vitro as a distinguishing feature Each cell lysate was split u RAS-GTP pull-down assay and for WB (whole-cell lysates). c M 5 cells were treated as in b before fixation. A PLA described in performed. d MUTZ-5 cells were seeded at 1.6 × 105/mL densit cultured for 7 days with either 0.5% DMSO (vehicle control), 5 Salirasib (indirect pan-RAS inhibitor), 25 µM II-B08, 50 μM V afenib (pan‐Raf inhibitor), 1 μM PD0325901 (MEK1/2-inhibito 5 µM Ruxolitinib (JAK-inhibitor), in presence of 20 ng/mL T Percentage of viable cells was determined in an NC-250 autom cell counter. Fig. 7 CRLF2-signaling induces direct interaction between acti- vated PTPN11 and RAS, and PTPN11-activity is required for ALL cell growth. a Direct interaction between RAS and phos- phorylated PTPN11 was monitored via PLA using high-content microscopy. Serum-starved MUTZ-5 cells were induced (or not) with 20 ng/mL TSLP for 10 min. Cells were fixed and permeabilized in a 96 well plate. Antibodies against phosphorylated PTPN11 and pan-RAS were used in conjunction with PLA-probes to allow the amplification and staining of interaction-specific PLA-spots. The negative control (NC) are two cytosolic proteins not expected to interact. Fluorescent-microscopy images show examples of PLA-spots (scale bars = 20 µm). At least 250 cells per well were analyzed using Operetta-CLS automated high-content microscopy platform. The bar- graph shows the averages of three independent experiments (each performed in triplicates). Error bars are SD and P values were deter- mined in one-way ANOVA and post-hoc Bonferroni multiple com- parison. b MUTZ-5 cells were pre-incubated with DMSO or 25 µM II- B08 (PTPN11 inhibitor) for 2 h and then stimulated or not with 20 ng/ mL TSLP for 10 min before cell lysis. Each cell lysate was split up for RAS-GTP pull-down assay and for WB (whole-cell lysates). c MUTZ- 5 cells were treated as in b before fixation. A PLA described in a was performed. d MUTZ-5 cells were seeded at 1.6 × 105/mL density and cultured for 7 days with either 0.5% DMSO (vehicle control), 50 µM Salirasib (indirect pan-RAS inhibitor), 25 µM II-B08, 50 μM Vemur- afenib (pan‐Raf inhibitor), 1 μM PD0325901 (MEK1/2-inhibitor), or 5 µM Ruxolitinib (JAK-inhibitor), in presence of 20 ng/mL TSLP. Percentage of viable cells was determined in an NC-250 automated cell counter. D. Koschut et al. 758 individual pathway activation status in the presence of TSLP. Primary DS-ALL patient biopsies from high-risk sub- cohorts have a potent response to RAS-inhibition in vitro as a distinguishing feature b Waterfall-plot shows the mean efficacies of 50 µM Salirasib, 10 µM PI-103 (PI3K/mTOR dual- inhibitor), or 5 µM Ruxolitinib (JAK-inhibitor) on pathway compo- nents (0% = no effect, 100% = full block of TSLP-induced protein- activation); tested on primary presentation samples from poor outcome DS-ALL patients in a. Error bars are SD; black P values were deter- mined in one-way ANOVA with post-hoc Bonferroni multiple- comparison. The red P values (Bonferroni-corrected for sequential multiple-comparison) indicate if each inhibitor on average significantly reduced the respective protein-activity in these samples (only P < 0.05 shown; Supplementary Table S2 lists all P values). c Cell-toxicity effect of inhibitors in DS-ALL. Samples from six patients were cul- tured for 2 days like in a before seeding 8 × 105 viable cells/mL in IMDM-complete medium (without IL-3/IL-7 but containing 20 ng/mL TSLP) together with 0.5% DMSO, 30 µM Rigosertib (non-ATP competitive RAS-GTP inhibitor), 10 µM PI-103, 5 µM Ruxolitinib, or Rigosertib&Ruxolitinib (DS23 cell count was insufficient). After 7 days, cell count and viability were measured (N = 3, means ± SD); vehicle-control cell numbers reduced to 1–4 × 105/mL, 70–90% via- bility. P values were determined in one-way ANOVA with post-hoc Dunnett’s multiple-comparison (all treatments compared to DMSO- control). e 8 × 105 viable cells/mL of patient-DS17 were handled like in c and treated with 0.5% DMSO, or 25 µM II-B08. After 7 days, cell count and viability were measured (N = 3, means ± SD); average vehicle-control viability: 74% (II-B08: 66%). on cell viability in six DS-ALL primary presentation sam- ples (Fig. 8c). The competitive RAS-inhibitor Rigosertib was chosen over Salisarib as Rigosertib shows more potential in current clinical trials and still can disrupt both wtRAS and mutant-RAS signaling activity. Remarkably, after 7 days the RAS-inhibitor treatment had significantly reduced the viable cell count (almost halved compared to vehicle-control) in almost all six samples, independent of RAS-mutation status (Fig. 8c). Only in one patient sample (DS17) the RAS inhibitor needed a combinatorial-treatment (adding JAK-inhibitor) to achieve a similar reduction of viable cells. Cells of this patient treated separately with PTPN11-inhibitor resulted in a significantly reduced viable cell count (Fig. 8d). The JAK-inhibitor alone showed no statistically sig- nificant effect in any of the DS-ALL samples, similar to what was observed for MUTZ-5 (Fig. 3a). Discussion Both DS-ALL and Ph-like ALL share CRLF2- rearrangements and various kinase-activating alterations as potential targets for individualized therapy using specific kinase-inhibitors [8, 34]. This lead to the use of phosphor- ylation patterns of individual kinase signaling cascades as informative biomarkers for combinatorial therapy design [6, 16, 35]. (Fig. 8b). As indicated by red P values, PI3K inhibitor significantly inhibited rpS6 phosphorylation, whereas JAK inhibitor significantly inhibited ERK, rpS6, and STAT5 phosphorylation. Notably, these inhibitors did not have any significant effect on RAS activity, reproducing the result obtained for the MUTZ-5 Ph-like ALL cell line (Fig. 3a). Only the RAS inhibitor was able to significantly block RAS activation in poor outcome DS-ALLs (Fig. 8b), in addition to blocking rpS6 phosphorylation, as likewise shown for the MUTZ-5 cells (Figs. 6b and 3b). This suggests that only RAS-inhibitor action is capable of efficiently blocking RAS activation in cells from both Ph-like/non-DS and DS-ALL poor outcome patient samples at the point of first clinical presentation, irrespective of the presence of RAS mutation. In contrast, JAK and PI3K inhibitor treatments alone did not significantly impact RAS activity in these samples (Fig. 8b). In order to better understand the physiological/ther- ti l d th ff t f RAS i hibiti (Fig. 8b). As indicated by red P values, PI3K inhibitor significantly inhibited rpS6 phosphorylation, whereas JAK inhibitor significantly inhibited ERK, rpS6, and STAT5 phosphorylation. Notably, these inhibitors did not have any significant effect on RAS activity, reproducing the result obtained for the MUTZ-5 Ph-like ALL cell line (Fig. 3a). Only the RAS inhibitor was able to significantly block RAS activation in poor outcome DS-ALLs (Fig. 8b), in addition to blocking rpS6 phosphorylation, as likewise shown for the MUTZ-5 cells (Figs. 6b and 3b). This suggests that only RAS-inhibitor action is capable of efficiently blocking RAS activation in cells from both Ph-like/non-DS and DS-ALL poor outcome patient samples at the point of first clinical presentation, irrespective of the presence of RAS mutation. In contrast, JAK and PI3K inhibitor treatments alone did not significantly impact RAS activity in these samples (Fig. 8b). In order to better understand the physiological/ther- In DS-ALL, recent studies of sub-clonal and single-cell evolution of changes in leukemic ALL blasts have iden- tified signaling activators (CRFL2-rearrangements, JAK2 mutations, RAS-MAPK mutations and iAMP21) as fre- quent events in primary and relapsed leukemic blasts [20, 21, 36]. Primary DS-ALL patient biopsies from high-risk sub- cohorts have a potent response to RAS-inhibition in vitro as a distinguishing feature The efficacy of RAS inhibition on intracellular protein activity (expressed as panRAS activity ratio between inhibitor and vehicle treated samples) for primary presentation samples showed a significant difference (P = .021 by Fisher’s exact test) between the good outcome (n = 7) and poor outcome DS-ALL groups (n = 6) (Fig. 8a). Also, samples in which RAS can be further activated by TSLP were more sensitive to RAS inhibitor treatment (Supplementary Fig. S9). For all poor outcome DS-ALL primary presentation samples, inhibitions of individual pathway effector activities via the RAS, PI3K, or JAK inhibitors were visualized as inverted bar graphs ranging from 0% (no inhibition) to 100% (complete inhibition) individual pathway activation status in the presence of TSLP. The efficacy of RAS inhibition on intracellular protein activity (expressed as panRAS activity ratio between inhibitor and vehicle treated samples) for primary presentation samples showed a significant difference (P = .021 by Fisher’s exact test) between the good outcome (n = 7) and poor outcome DS-ALL groups (n = 6) (Fig. 8a). Also, samples in which RAS can be further activated by TSLP were more sensitive to RAS inhibitor treatment (Supplementary Fig. S9). For all poor outcome DS-ALL primary presentation samples, inhibitions of individual pathway effector activities via the RAS, PI3K, or JAK inhibitors were visualized as inverted bar graphs ranging from 0% (no inhibition) to 100% (complete inhibition) RAS-protein activation but not mutation status is an outcome predictor and unifying therapeutic target. . . 759 Fig. 8 RAS-inhibitor blocks RAS-activity with greater efficiency in primary, poor outcome DS-ALL patient samples, prior to relapse. a Efficacy of RAS-inhibitor on ELISA-measured RAS-activity in DS- ALL, compared by outcome. Primary presentation DS-ALL samples were cultured for 2 days (detailed in Supplementary Fig. S5A-legend). Samples with sufficient cell count were treated with 0.5% DMSO (vehicle-control), or 50 µM Salirasib (indirect pan-RAS-inhibitor) for 3 h, and then induced for 10 min with 20 ng/mL TSLP in serum- reduced medium. Cells were lysed for RAS-GTP pull-down assay and whole-lysate WB (Supplementary Fig. S5A). Protein-activities of inhibitor-treated TSLP-induced samples were normalized to the activity-level of the respective vehicle-treated TSLP-induced samples. If inhibitor-treatment reduced the RAS-activity by over 10% compared to vehicle-control (dashed-line), the sample was tallied as successful RAS-blocking. A Fisher’s exact test was performed between the groups. Good outcome: N = 7 (3 RAS-mutations, 1 JAK2-mutation); poor outcome: N = 6 (1 JAK2-mutation). Primary DS-ALL patient biopsies from high-risk sub- cohorts have a potent response to RAS-inhibition in vitro as a distinguishing feature Interestingly, while the PI3K/mTOR-inhibitor and the RAS-inhibitor both showed effective reduction of viable cells in all three samples (DS16, DS20, DS27) that were clustered by PCA as SR (Fig. 5), only RAS-inhibition was able to reduce the viable cell count in the samples grouped by the PCA (Fig. 5) as HR (DS09, DS17, DS23) (Fig. 8c). These results suggest a paradigm shift in precision- therapy approach, by identifying HR sub-groups that are unlikely to respond to PI3K- or JAK-inhibitors alone and require direct RAS-inhibition. Importantly, the data confirm the notion that wtRAS-inhibitors could provide a uniform treatment for both mutated RAS and activated wtRAS cases (encompassing up to 80% of DS-ALL). Discussion It will be important to unravel the mechanisms behind the actions of these chromosome 21 genes, as their specific inhibition may be an additional component to consider in combinatorial therapy approaches [37, 49]. This is highlighted by very frequent observations of extra copies of chromosome 21 as acquired changes in DS and non-DS ALL, both at diagnosis, and at relapse [20, 50]. Taking RAS activity and inducibility, integrated with other protein activation patterns, we performed a multi- variate analysis clustering that identified SR and HR groups for DS-ALL and showed that protein activation pattern is independently predictive of outcome using multivariate Cox regression. In conclusion, our data show that activation of RAS protein is a common feature of up to 80% of DS-ALL, suggesting inhibition of overstimulated RAS pathway activity should be a unifying therapeutic strategy, even in the absence of RAS mutations. Importantly, our data indi- cate that patient pre-stratification for therapy optimization should assess RAS/MAPK protein activation status. Ultimately, patient-specific inhibitor combinations based on analyzed pathway activities should be part of future precision medicine approaches for HR-ALL groups. Ph-like ALL patients are already being studied for the combined effects of PI3K/mTOR and JAK/STAT inhibitor treatment [6]. “Supplementary Discussion” contains an expanded discussion on RAS-inhibitor strategies. Acknowledgements Singapore Ministry of Education Academic Research Funds Tier 2 grants (2015-T2-2-119 and 2015-T2-1-023) to DN; AIRC IG 19186 and Fondazione Cariparo 17/07 to GB. Acknowledgements Singapore Ministry of Education Academic Research Funds Tier 2 grants (2015-T2-2-119 and 2015-T2-1-023) to DN; AIRC IG 19186 and Fondazione Cariparo 17/07 to GB. p g Compared to RAS, mutations in PTPN11 are less pre- valent in DS-ALL but mutations in JAK2, RAS, and PTPN11 also appear to be mutually exclusive throughout different types of childhood ALL [20, 22, 38, 39]. Our data reveal that reducing RAS activity via inhibition of PTPN11 catalytical action may provide a functional alternative for ALL cells, while blocking the phosphor- ylation of PTPN11 via JAK inhibitors was not sufficient to prevent RAS activity, and concordantly with our mechanistic insight was also unable to block the direct interaction between PTPN11 and RAS. Our findings sug- gest that, depending on the patient’s protein activity pro- file, RAS inhibition (upstream, direct, or downstream) should be considered in combination with PI3K/mTOR and/or JAK/STAT inhibitors to further augment clinical treatment. Discussion In particular, JAK2 and RAS mutations were found to be both acquired and lost in relapse samples in a mutually exclusive manner [20, 21]. This emphasizes the need for individualized combined-therapy approaches that have a better chance of preventing the selection of sub- clones driven by a different signaling. Our data show that elevation in CRLF2 levels combined with JAK2 activation In order to better understand the physiological/ther- apeutic relevance, we measured the effect of RAS-inhibition D. Koschut et al. 760 are sufficient to activate wtRAS, and that TSLP has the potential to induce the wtRAS activity, independently of the PI3K/mTOR activity. This has implications on the choice of the combinatorial therapy design. Remarkably, our combined data from exome sequencing [20], and pri- mary ALL cell protein signaling (presented in this study), suggest that up to 65–82% of DS-ALL cases have highly activated RAS, either constitutively, or upon TSLP induction, regardless of their mutation profiles. 12 of 14 cases with high RAS-activity featured either RAS muta- tions or high CRLF2/JAK2 signaling (including JAK2- mutations). The only two samples featuring high wtRAS activity in absence of high JAK2 phosphorylation levels might activate RAS via a different pathway yet to be uncovered for DS-ALL. A very recent novel patient- derived xenograft models for DS-ALL found CBL-mutant (wtRAS) cells to have as high ERK1/2-phosphorylation as KRAS-mutant cells [37]. Interestingly in the same study, the leukemia burden in both wtRAS(JAK2-mutant) and mutant-RAS xenograft models was reduced via MEK- inhibitor, representing a strongly corroborating evidence to some of the conclusions of our study. seemed resistant to treatment with PI3K/mTOR or JAK inhibitors alone while only RAS-inhibition slashed the viable cell count in half. However, based on our data, the focus should not lie on targeting mutant-RAS alone but also the inhibition of overstimulated wtRAS pathway activity in absence of RAS mutations. Childhood leukemia in DS is distinguished by a rela- tively specific pattern of acquired mutation changes, for both AML [40–45] and ALL [20, 21, 46], and the reasons for this are not fully explained. More generally, people with DS have an unusual epidemiological pattern of malignancy: increased incidence and mortality for childhood leukemias of all types, but much decreased childhood and adult solid tumors [47, 48]. Functional consequences of an increased dose of some chromosome 21 genes may play important roles [48], and this is discussed in greater detail in “Sup- plementary Discussion”. Discussion In particular in DS-ALL, RAS/MAPK-inhibi- tion might be applicable to most HR patients, as we show that specifically samples stratified by our PCA as HR References 21. Schwartzman O, Savino AM, Gombert M, Palmi C, Cario G, Schrappe M, et al. Suppressors and activators of JAK-STAT signaling at diagnosis and relapse of acute lymphoblastic leukemia in Down syndrome. 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https://openalex.org/W3131066598
https://ejournal.unisayogya.ac.id/index.php/jkk/article/download/1901/pdf
Indonesian
null
Pengaruh Pola Asuh Orang Tua Terhadap Pernikahan Dini
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Abstrak Perkawinan remaja di Indonesia mengalami penurunan sejak tahun 2008-2018, namun sekarang terjadi peningkatan kembali. Termasuk di Desa Mergolangu dari tahun 2016 sampai 2019 tercatat sebanyak 50 pasangan suami istri dengan persentase 64% untuk pasangan wanita rentang usia <20. Pernikahan dini berpengaruh terhadap aspek kesehatan dan sosial, diantaranya adalah stunting, stress, dan perceraian. Tujuan penelitian ini untuk melihat pengaruh pola asuh terhadap pernikahan dini. Jenis penelitian ini adalah penelitian retrospektif desain case control dengan jumlah sampel 46 orang. Hasil penelitian menunjukan pola asuh permisif berpengaruh positif (T statistic 4,7540) terhadap pernikahan dini. Kejadian pernikahan dini 63,3% dan 36,7% disebabkan oleh karena faktor lain. Kata kunci: pola asuh; pernikahan dini; remaja Available online at https://ejournal.unisayogya.ac.id/ejournal/index.php/jkk Available online at https://ejournal.unisayogya.ac.id/ejournal/index.php/jkk 177 Available online at https://ejournal.unisayogya.ac.id/ejournal/index.php/jkk 1 Jurnal Kebidanan dan Keperawatan 'Aisyiyah, 16 (2), 2020, 177-185 Available online at https://ejournal.unisayogya.ac.id/ejournal/index.php/jkk Jurnal Kebidanan dan Keperawatan 'Aisyiyah, 16 (2), 2020, 177-185 Pengaruh Pola Asuh Orang Tua Terhadap Pernikahan Dini Mariah Ulfah1,*, Linda Yanti2, Prasanti Adriani3, Soliyah4 2,3,4Universitas Harapan Bangsa, Jalan Raden Patah No.100, Kedunglongsir, Ledug, Kec. Kembaran, Kab Banyumas, Jawa Tengah 53182 1mariahulfah@uhb.ac.id*; 2lindayanti@uhb.ac.id; 3Prasantiadriani@uhb.ac.id * corresponding author Tanggal Submisi: 2 Februari 2020, Tanggal Penerimaan: 6 Februari 2020 Mariah Ulfah1,*, Linda Yanti2, Prasanti Adriani3, Soliyah4 1,2,3,4Universitas Harapan Bangsa, Jalan Raden Patah No.100, Kedunglongsir, Ledug, Kec. Kemb Banyumas, Jawa Tengah 53182 1mariahulfah@uhb.ac.id*; 2lindayanti@uhb.ac.id; 3Prasantiadriani@uhb.ac.id * corresponding author Tanggal Submisi: 2 Februari 2020, Tanggal Penerimaan: 6 Februari 2020 Abstract Adolescent marriage in Indonesia has decreased from 2008-2018, but recently there has been an increase again. Including in Mergolangu Village from 2016 to 2019 recorded 50 married couples with a percentage of 64% for female partners aged <20 (32 people) and 36% (18 people) for female partners aged> 20 years. Early marriage affects health and social aspects, including stunting, stress, and divorce. This type of research is a retrospective case control design, with a total sample of 46 people using a retrospective. The purpose of this study was to see the effect of parenting on early marriage. The results showed that permissive parenting had a positive effect (T statistic 4.7540) on early marriage. The incidence of early marriage was 63.3% and 36.7% due to other factors. Key words: parenting, early marriage, adolescence PENDAHULUAN Pernikahan anak masih menjadi masalah kesehatan dan pemenuhan hak anak yang signifikan di negara dengan penghasilan rendah dan menengah (Rumble, 2018). Secara global, satu dari enam gadis remaja berusia antara 15 dan 19 tahun sudah menikah (Anjarwati, 2017) Tingkat pernikahan anak di dunia tertinggi adalah di sub Sahara Afrika yaitu 35% wanita muda menikah sebelum usia 18 tahun. Di Asia Selatan hampir 30% menikah sebelum usia 18 tahun. This is an open access article under the CC–BY-SA license. This is an open access article under the CC–BY-SA license. 10.31101/jkk.1901 10.31101/jkk.1901 10.31101/jkk.1901 This is an open access article under the CC–BY-SA license. Jurnal Kebidanan dan Keperawatan Aisyiyah, 16 (2), 2020 Mariah Ulfah, Linda Yanti, Prasanti Adriani, Soliyah 178 Tingkat pernikahan anak terendah ditemukan di Amerika Latin dan Karibia yaitu 24%, Timur Tengah dan Afrika Utara 17%, dan Eropa Timur dan Asia Tengah sekitar 12%. Diperkirakan anak perempuan yang menikah mencapai 12 juta per tahun, sedangkan 115 juta anak laki-laki menikah sebelum usia 18 tahun (Cossens & Jackson, 2020). Tingkat pernikahan anak terendah ditemukan di Amerika Latin dan Karibia yaitu 24%, Timur Tengah dan Afrika Utara 17%, dan Eropa Timur dan Asia Tengah sekitar 12%. Diperkirakan anak perempuan yang menikah mencapai 12 juta per tahun, sedangkan 115 juta anak laki-laki menikah sebelum usia 18 tahun (Cossens & Jackson, 2020). Di Indonesia sekitar 17% anak perempuan menikah sebelum usia 18 tahun (Badan Pusat Statistika, 2018). Tren perkawinan anak perempuan di Indonesia, menunjukkan penurunan di tahun 2008-2018. Prevalensi perkawinan anak adalah sebesar 14,67 persen, namun pada satu dekade kemudian (tahun 2018) hanya menurun sebesar 3,5 poin persen menjadi 11,21 persen (Badan Pusat Statistik, 2020) pernikahan anak dikaitkan dengan tempat tinggal pedesaan, perumahan yang lebih miskin kondisi dan rumah tangga dengan tingkat pengeluaran yang lebih rendah (Bidang Statistik Sosial, 2018). Faktor yang mempengaruhi pernikahan dini salah satunya adalah pola asuh orang tua. Pola asuh yang baik akan meningkatkan keterbukaan dengan anak (Kurniati, 2016), serta kegiatan pengasuhan orang tua diantaranya membimbing, mengarahkan termasuk terhadap pemilihan pernikahan dini. Wonosobo merupakan Kabupaten yang terdiri dari 15 Kecamatan dan 279 Desa. Pada tahun 2018 di Kabupaten Wonosobo persentase wanita menikah di umur <17 tahun sebanyak 29,99%, 17-18 tahun sebanyak 23,79%, umur 19-24 tahun sebanyak 37,24% dan umur lebih dari 25 tahun sebanyak 10,97%. PENDAHULUAN Dapat diketahui bahwa umur pertama pernikahan pada wanita di Wonosobo umur di bawah 19 tahun sebanyak 51,78% dan yang diatas 19 tahun sebanyak 48,21% (Badan Pusat Statistika, 2018). Dengan demikian angka pernikahan dibawah 20 tahun di Kabupaten Wonosobo Tinggi dibandingkan dengan yang menikah umur 20 tahun keatas, sebagaimana peraturan BKKBN bahawa usia ideal menikah adalah 20 tahun untuk wanita dan 25 tahun untuk pria. Berdasarkan data yang peneliti peroleh dari Kantor Urusan Agama Kecamatan Kalibawang di Desa Mergolangu dari tahun 2016 sampai 2019 tercatat sebanyak 50 pasangan suami istri dengan persentase 64 % untuk pasangan wanita rentang usia <20 (32 orang) dan 36 % (18 orang) untuk pasangan wanita rentang usia >20 tahun (Kalibawang, 2019). Pernikahan dini berisiko lebih tinggi mengakibatkan kematian pada ibu. Komplikasi kehamilan dan melahirkan merupakan salah satu penyebab utama kematian di antara gadis remaja (Gibbs, 2014). Pernikahan dini berdampak secara fisiologis, psikologis maupun sosial ekonomi (Indanah, Faridah, Sa’adah, &2020), termasuk berdampak terhadap Kesehatan reproduksi. ( Yohana, Beby, et al 2020). Dampak pernikahan dini memberikan kontribusi terhadap peningkatan kehamilan remaja yang diperkirakan mencapai lebih dari 500 kehamilan setiap tahunnya. Sedangkan Menurut Survei Demografi Kesehatan Indonesia tahun 2017, penyebabnya adalah penurunan penggunaan kontrasepsi modern pada segmen usia muda (15-29 tahun) secara segnifikan sekira 4 persen dari total populasi Indonesia. Sedangkan menurut data Susenas tahun 2017, hasil survei pada perempuan berumur 15-49 tahun diketahui bahwa 54,01% hamil pertama kali pada usia di atas 20 tahun (usia ideal kehamilan). Sisanya sebesar 23,79% hamil pertama kali pada usia 19-20 tahun, 15,99% pada usia 17-18 tahun, dan 6,21% pada usia 16 tahun ke bawah. Data terkait pernikahan usia dini masih sangat Copyright © 2020, Jurnal Kebidanan dan Keperawatan Aisyiyah ISSN 1858-0610 (print) | ISSN 2477-8184 Jurnal Kebidanan dan Keperawatan Aisyiyah, 16 (2), 2020 Mariah Ulfah, Linda Yanti, Prasanti Adriani, Soliyah 179 terbatas sehingga perlu dilakukan studi lebih lanjut untuk mendapatkan spesifikasi data terkait dengan pernikahan usia dini. Hal ini menunjukkan bahwa setengah dari perempuan yang pernah hamil di Indonesia mengalami kehamilan pertama pada usia muda atau remaja. Usia muda dibawah 20 tahun berisiko melahirkan bayi dengan BBLR dimana BBLR berisiko 20% terhadap stunting (Pramita & Kurniawati, 2011). Penelitian sebelumnya belum banyak yang membahas tentang pengaruh model pola asuh orang tua, akan tetapi sebagian besar penelitian terkait dengan pengakhiran pernikahan usia dini (Svanemyr, Chandra-Mouli, Raj, Travers, & Sundaram, 2015). Tujuan penelitian ini adalah untuk mengetahui pola asuh orang tua terhadap pernikahan dini. PENDAHULUAN Berdasarkan bahasan di atas, peneliti merasa penting untuk mengetahui pengaruh pola asuh terhadap keputusan remaja memilih menikah dini di Desa mergolangu kabupaten wonosobo? Mariah Ulfah et.al (Pengaruh Pola Asuh Orang Tua Terhadap Pernikahan Dini) Copyright © 2020, Jurnal Kebidanan dan Keperawatan Aisyiyah ISSN 1858-0610 (print) | ISSN 2477-8184 METODE Jenis penelitian ini adalah penelitian retrospektif desain case control. Populasi yang akan diambil dalam penelitian ini adalah seluruh remaja yang sudah menikah dari tahun 2016 sampai 2019 yang berjumlah 52 remaja yang di dapat dari Kantor Urusan Agama Kec. Kalibawang rentang umur 10 sampai 24 tahun di Desa Mergolangu Kecamatan Kalibawang Kabupaten Wonosobo yang terdiri dari 3 Dukuh yang terbagi menjadi sample remaja yang melakukan pernikahan dini dan tidak. Setelah dilakukan perhitungan dengan rumus lamessow Untuk menentukan besarnya sampel peneliti menggunakan rumus minimal sampel size menurut rumus Lameshow (Lamesh show, 1997), yaitu : n = Z21-α2P(1-P)N d2 (N-1) + Z21-α/2 P (1-P) Keterangan: n = besar minimal N = Jumlah populasi Z 1-α/2 =tingkat kepercayaan (95%=1,96) d = presisi(0,05)ketepatan 95% P = proporsi target populsi adalah 50% atau 0,5 1-P = 0,5 P hi l j d h ik h Perhitungan sampel remaja yang sudah menikah : Perhitungan sampel remaja yang sudah menikah : Perhitungan sampel remaja yang sudah menikah : p j y g 1,96² x 0,5 x 0,5 x 52 n = 1,96² x 0,5 x 0,5 x 52 0,05² x (52-1) + 1,96² x 0,5 x (1-0,5) 0,1275 = 49,9404 1, 0879 = 45,9 = dibulatkan menjadi 46 = dibulatkan menjadi 46 Teknik Pengumpulan data dalam penelitian ini adalah menggunakan angket yang berisi karakteristik responden, persepsi responden tentang pola asuh orang tua. Teknik analisis data menggunakan Partial Least Square (PLS) versi I (Ulum, Ghozali, & Chariri, 2008) dengan menggunakan analisis jalur untuk melihat pola asuh yang berpengaruh terhadap pemilihan menikah dini pada Copyright © 2020, Jurnal Kebidanan dan Keperawatan Aisyiyah ISSN 1858-0610 (print) | ISSN 2477-8184 Jurnal Kebidanan dan Keperawatan Aisyiyah, 16 (2), 2020 Mariah Ulfah, Linda Yanti, Prasanti Adriani, Soliyah 180 remaja. Analisis data yang digunakan dengan melihat uji T, uji T statistic diterima jika >1,96. (Garson, 2016). Etik penelitian dinyatakan laik etik melalui Komisi etik penelitian Kesehatan Universitas Harapan Bangsa Nomor: B.LPPM- UHB/115a/10/2019. Tabel 1. Jumlah Sampel dari Masing-Masing Dukuh Nama Dukuh Perhitungan Jumlah Sampel Yang menikah dini Dukuh Mergolangu 20/52 x 46 18 Dukuh Prigi 4/52 x 46 4 Dukuh Wonosari 8/52 x 46 8 Jumlah 30 Yang tidak menikah dini Dukuh Mergolangu Dukuh Prigi Dukuh Wonosari 11/52x46 5/52x46 2/52x46 10 4 2 Jumlah 16 Jumlah Total 46 Tabel 1. Jumlah Sampel dari Masing-Masing Dukuh Tabel 1. Jumlah Sampel dari Masing-Masing Dukuh HASIL DAN PEMBAHASAN Gambaran Umum Responden: p Responden dalam penelitian ini adalah remaja yang melakukan pernikahan dini dan tidak, dapat digambarkan sebagai berikut: Responden dalam penelitian ini adalah remaja yang melakukan pernikahan dini dan tidak, dapat digambarkan sebagai berikut: Tabel 2. Distribusi Frekuensi Karakteristik Responden yang Melakukan Pernikahan Dini dan Tidak Karakteristik Pernikahan Dini Ya Tidak Menikah dini 30 (65,2%) 16 (34,8%) Pendidikan ibu Dasar 29 (96,7%) 14 (87,5%) Menengah 1 (3,3%) 2 (12,5%) Pendidikan ayah Dasar 30 (100% 14 (87,5% Menengah 0 (0%) 2 (12,5%) Pendapatan Rendah 29 (96,7%) 1 (6,3%) Sedang 1 (3,3%) 14 (87,5% Tinggi 0 (0%) 1 (6,3%) Total 30 16 Tabel 2. Distribusi Frekuensi Karakteristik Responden yang Melakukan Pernikahan Dini dan Tidak Berdasarkan tabel 2 terlihat bahwa 65,2 % melakukan pernikahan dini, dengan latar belakang pendidikan ayah 100% dan ibu 96,7% pada kategori sekolah dasar , pendapatan orang tua 96,7% di kategorikan rendah pada remaja yang menikah dini. Analisis Bivariat: Sebelum melihat faktor pengaruh eksogen ke endogen, dilakukan uji nilai outer loading, uji validitas konvergen juga dievaluasi dengan melihat nilai Average Variance Extracted (AVE). Nilai AVE harus lebih besar dari 0,4. Mariah Ulfah et.al (Pengaruh Pola Asuh Orang Tua Terhadap Pernikahan Dini) Copyright © 2020, Jurnal Kebidanan dan Keperawatan Aisyiyah ISSN 1858-0610 (print) | ISSN 2477-8184 Jurnal Kebidanan dan Keperawatan Aisyiyah, 16 (2), 2020 Mariah Ulfah, Linda Yanti, Prasanti Adriani, Soliyah 181 Berdasarkan hasil uji statistik dengan menggunakan VPLS versi 1 ditunjukkan dalam gambar sebagai berikut: Berdasarkan hasil uji statistik dengan menggunakan VPLS versi 1 ditunjukkan dalam gambar sebagai berikut: Gambar 1. Analisis Jalur Peran Orang Tua dengan Pemilihan Menikah Dini pada Remaja Analisis tersebut dapat digambarkan dengan tabel sebagai berikut: Gambar 1. Analisis Jalur Peran Orang Tua dengan Pemilihan Menikah Dini pada Remaj Analisis tersebut dapat digambarkan dengan tabel sebagai berikut: Tabel 3. T Statistik Pengaruh Langsung Pola Asuh dengan Pernikahan Dini Structural Model-BootStrap Entire Sample Estimate Mean of Subsamples Standard Error T-Statistic Permisif->Nikah 0.695 0.6832 0.1462 4.754 Otoriter->Nikah -0.137 -0.1801 0.102 -1.3435 Demokratif->Nikah -0.076 -0.1134 0.0765 -0.9933 Tabel 3. T Statistik Pengaruh Langsung Pola Asuh dengan Pernikahan Dini Tabel 3. T Statistik Pengaruh Langsung Pola Asuh dengan Pernikahan Dini Structural Model-BootStrap Dari hasil uji T statistic terlihat bahwa: a. Pola asuh permisif berpengaruh secara signifikan terhadap pernikahan dini pada remaja dengan T statistic 4,7540>1,96. b. Pola asuh otoriter tidak berpengaruh secara signifikan terhadap pernikahan dini pada remaja dengan T statistic 1,3435<1,96. b. Pola asuh otoriter tidak berpengaruh secara signifikan terhadap pernikahan dini pada remaja dengan T statistic 1,3435<1,96. b. Pola asuh otoriter tidak berpengaruh secara signifikan terhadap pernikahan dini pada remaja dengan T statistic 1,3435<1,96. p j g c. Pola asuh sekuler tidak berpengaruh secara signifikan terhadap pernikahan dini pada remaja dengan T statistic 0.9933<1,96. p j g c. Pola asuh sekuler tidak berpengaruh secara signifikan terhadap pernikahan dini pada remaja dengan T statistic 0.9933<1,96. Pola asuh orang tua sangat berpengaruh terhadap karakter remaja (Hayyu, g g p g p j ( yy , 2018). Pola asuh orang tua terdiri dari permisif, demokratif dan otoriter. g g p g p j ( yy , 2018). Pola asuh orang tua terdiri dari permisif, demokratif dan otoriter. 2018). Pola asuh orang tua terdiri dari permisif, demokratif dan otoriter. 1. Pengaruh pola asuh permisif terhadap pernikahan dini. Dari hasil penelitian menyebutkan bahwa pola asuh permisif berpengaruh secara signifikan terhadap pernikahan dini pada remaja dengan T statistic 4,7540>1,96. Pola asuh permisif terbagi menjadi dua: neglectful parenting dan indulgent parenting. Model neglectful menghasilkan anak-anak yang kurang memiliki kompetensi social terutama karena adanya kecenderungan kontrol diri yang kurang. Berdasarkan hasil uji statistik dengan menggunakan VPLS versi 1 ditunjukkan dalam gambar sebagai berikut: Sedangkan pola asuh yang indulgent yaitu bila orang tua sangat terlibat dalam kehidupan anak, namun hanya memberikan kontrol dan tuntutan yang sangat sehingga mengakibatkan kompetensi sosial yang tidak adekuat dan kurang mampu untuk melakukan kontrol diri dan menggunakan kebebasannya Mariah Ulfah et.al (Pengaruh Pola Asuh Orang Tua Terhadap Pernikahan Dini) Copyright © 2020, Jurnal Kebidanan dan Keperawatan Aisyiyah ISSN 1858-0610 (print) | ISSN 2477-8184 Jurnal Kebidanan dan Keperawatan Aisyiyah, 16 (2), 2020 Mariah Ulfah, Linda Yanti, Prasanti Adriani, Soliyah 182 tanpa rasa tanggung jawab (Ambarwati, 2019). Hal ini sejalan dengan penelitian penelitian yang dilakukan Purwaningsih bahwa pernikahan usia dini lebih banyak terjadi pada anak dengan pola asuh permisif (Purwaningsih, 2015). Menurut penelitian rohayati responden dengan pola asuh permisif mengalami kejadian pernikahan dini, hal ini dikarenakan orang tua yang menerapkan kebebasan yang berlebihan pada anak tanpa kontrol dari orang tua (Rohayati, 2017), sehingga anak cenderung terjerumus pada hal – hal negatif seperti perilaku seksual pranikah, termasuk memilih untuk melakukan pernikahan dini (Sari, Winarni, & Dharminto, 2018) diperkuat juga dengan pendapat Nursal dalam (Hidayah dan Maryatun, 2013) remaja dengan pola asuh permisif dimana kedua orang tuanya tidak memberikan perhatian yang baik kepada mereka seperti membiarkan anak – anak tersebut berisiko melakukan perilaku menyimpang. Orang tua yang kurang memberikan rasa nyaman dan aman kepada anak ketika di rumah, menyebabkan anak memilih menghabiskan waktu lebih lama atau bahkan tinggal dengan sebayanya sehingga berisiko meniru perilaku dari orang-orang sekitar, budaya yang ada dimasyarakat, termasuk memilih melakukan pernikahan dini (Shabri & Raihana, 2019). 2. Pengaruh pola asuh demokratif terhadap pernikahan dini Hasil penelitian menunjukkan tidak ada pengaruh pola asuh demokratif terhadap pernikahan dini pada remaja dengan T statistic 0.9933<1,96. Tipe pola asuh demokratis memberikan kebebasan terhadap anaknya, namun tetap memberikan masukan dan bimibingan terhadap anak-anaknya. Termasuk setiap dalam mengambil keputusan musyawarah, mendukung anak dengan kesadaran, dan berkomunikasi dengan anak. Pola asuh ini mendorong anak untuk mandiri tetapi tetap di kontrol oleh orang tua, kehangatan yang diberikan orang menyebabkan membawa anak pada hal positif., termasuk Ketika memutuskan untuk menikah dini atau tidak (Sari et al., 2018). Hasil penelitian menyebutkan bahwa pola asuh demokratif tidak berpengaruh secara signifikan terhadap pernikahan dini pada remaja dengan T statistic 0.9933<1,96. Hal ini sejalan dengan penelitian Octiva sari 2016, dimana pola asuh demokratif tidak ada hubungan yang bermakna dengan pernikahan dini dengan p value 0,63 (Sari et al., 2018). Mariah Ulfah et.al (Pengaruh Pola Asuh Orang Tua Terhadap Pernikahan Dini) Copyright © 2020, Jurnal Kebidanan dan Keperawatan Aisyiyah ISSN 1858-0610 (print) | ISSN 2477-8184 Berdasarkan hasil uji statistik dengan menggunakan VPLS versi 1 ditunjukkan dalam gambar sebagai berikut: Menurut penelitian ini, pola asuh orang tua yang demokratis tidak berpengaruh terhadap pernikahan dini kemungkinan disebabkan karena budaya dan nilai-nilai yang dianut oleh masyarakat Desa mergolangu yang umumnya melakukan menikahkan secara dini anak-nak remajanya, hal ini sesuai dengan teori sosial cognitive (Manjarres-Posada, Onofre-Rodríguez, & Benavides-Torres, 2020), bahwa norma budaya, nilai atau keyakinan berpengaruh terhadap perilaku sehingga kemungkinan dari budaya dan nilai yang ada dimasyarakat tersebut berpengaruh terhadap perilaku remaja untuk memilih menikah secara dini. Pengaruh Pola asuh orang tua yang otoriter terhadap pernikahan dini. Pola asuh otoriter adalah pola asuh yang menuntut agar anak patuh dan tunduk terhadap semua perintah dan aturan yang dibuat oleh orangtua tanpa ada kebebasan untuk bertanya atau mengemukakan pendapat sendiri, sehingga dimungkinkan pola asuh ini memberikan kesempatan anak untuk memutuskan melakukan pernikahan dini ataupun tidak. Dalam penelitian ini pola asuh otoriter tidak berpengaruh secara signifikan terhadap pernikahan dini pada remaja dengan Jurnal Kebidanan dan Keperawatan Aisyiyah, 16 (2), 2020 Mariah Ulfah, Linda Yanti, Prasanti Adriani, Soliyah 183 T statistic 1,3435<1,96. Hal ini sejalan dengan Sari, 2018 yang mengatakan bahwa tidak ada hubungan antara pola asuh otoriter dengan usia menikah dini (Sari et al., 2018). Penelitian Hayu, 2018 menyebutkan bahwa orang tua dengan pola asuh gaya otoriter serta permissive, ternyata anaknya lebih banyak yang mengalami KTD dibandingkan pola asuh gaya demokratis hal ini dikarenakan anak tidak diberikan pengawasan sehingga merasa bebas melakukan perbuatan apapun sekalipun itu tidak baik. Pada remaja yang mempunyai orang tua dengan pola asuh permisif, mereka mempunyai orang tua yang terlalu sibuk bekerja,sehingga perhatian kepada anak kurang. Orang tua yang bisa mendidik anak dengan baik akan menghasilkan anak dengan perilaku baik pula, begitu juga sebaliknya..(Hayyu, 2018). Pola asuh ini banyak mengandung penerimaan orang tua responsive dan sangat memperhatikan kebutuhan anak dengan kontrol yang tepat sehingga anak tidak terlalu leluasa. Dalam penelitian ini tidak ada pengaruh kemungkinan disebabkan bahwa remaja yang menikah dini karena faktor budaya dimana di Desa Mergolangu yang umumnya melakukan pernikahan dini. Selain itu di dapatkan data dari observasi dan wawancara langsung oleh peneliti kepada pengurus pernikahan di Desa Mergolangu bahwa memang sudah menjadi tradisi di Desa Mergolangu melakukan pernikahan dini yaitu di umur <20 tahun yang di lakukan sudah sejak dari jaman dahulu dan sampai sekarang masih banyak yang melakukan pernikahan di usia <20 tahun SIMPULAN Simpulan dalam penelitian ini adalah pola asuh yang berpengaruh terhadap pemilihan pernikahan dini pada remaja yaitu pola asuh yang permisif. Disamping itu kejadian pernikahan dini dipengaruhi oleh pola asuh sebesar 63,3% dan 36,7% disebabkan oleh karena faktor lain. DAFTAR PUSTAKA Ambarwati, N. A. (2019). Determinan Pernikahan Dini Di Kecamatan Pakis Kabupaten Magelang. Retrieved from http://eprintslib.ummgl.ac.id/1200/ Ambarwati, N. A. (2019). Determinan Pernikahan Dini Di Kecamatan Pakis Kabupaten Magelang. Retrieved from http://eprintslib.ummgl.ac.id/1200/ Anjarwati. (2017). Increasing the minimum age of marriage program to improve maternal and child health in Indonesia. AIP Conference Proceedings, Vol. 1868. https://doi.org/10.1063/1.4995195 Badan Pusat Statistik. (2020). Pencegahan Perkawinan Anak percepatan yang tidak bisa ditunda. Badan Pusat Statistik, 0–44. Badan Pusat Statistika. (2018). Jumlah Penduduk di Indonesia. Https://Www.Bps.Go.Id/, (November), 30–31. Bidang Statistik Sosial. (2018). Statistik Sosial dan Kependudukan Provinsi Jawa Tengah. Retrieved from https://jateng.bps.go.id/publication/2019/05/08/c07e2688ac5b3b09c6e7c303/ statistik-sosial-dan-kependudukan-provinsi-jawa-tengah-hasil-susenas- 2018.html Cossens, S., & Jackson, S. (2020). White, classed and gendered: pre-teen girls negotiate successful young womanhood. Gender and Education. Retrieved from https://www.tandfonline.com/doi/abs/10.1080/09540253.2018.1482411 p Garson, G. D. (2016). Partial Least Squares: Regression & Structural Equation Garson, G. D. (2016). Partial Least Squares: Regression & Structural Equation Mariah Ulfah et.al (Pengaruh Pola Asuh Orang Tua Terhadap Pernikahan Dini) Copyright © 2020, Jurnal Kebidanan dan Keperawatan Aisyiyah ISSN 1858-0610 (print) | ISSN 2477-8184 Copyright © 2020, Jurnal Kebidanan dan Keperawatan Aisyiyah ISSN 1858-0610 (print) | ISSN 2477-8184 Jurnal Kebidanan dan Keperawatan Aisyiyah, 16 (2), 2020 Mariah Ulfah, Linda Yanti, Prasanti Adriani, Soliyah 184 Models. In G. David Garson and Statistical Associates Publishing. Models. In G. David Garson and Statistical Associates Publishing. Gibbs, S. (2014). Peer and community influences on the acceptance of premarital Gibbs, S. (2014). Peer and community influences on the acceptance of premarital sex among Vietnamese adolescents. Journal of Paediatrics and Child Health, 50(6), 438–443. https://doi.org/10.1111/jpc.12512 Hayyu, A. (2018). Gam mbaran fa ktor-fakt tor yang berhubun b ngan deng gan kejadi ian keham milan ti idak diing ginkan pa ada remaj ja Des cription of factors s related to the ev vent of un ndoired pregnanc p cy in youth. Journal of Health Science, 14(2), 124–132. Indanah, I., Faridah, U., Sa’adah, M., & ... (2020). Faktor Yang Berhubungan Dengan Pernikahan Dini. Jurnal Ilmu …. Retrieved from https://www.ejr.stikesmuhkudus.ac.id/index.php/jikk/article/view/796 Kalibawang, K. urusan agama K. (2019). Berita. 2019. Kurniati, A. (2016). Kontribusi Pola Asuh Orang Tua Dalam Pendidikan Karakter Anak (Studi Kasus di Dusun Tempurau Desa Batu Buil Kecamatan Belimbing). Jurnal Pendidikan Dasar Perkhasa. Retrieved from http://jurnal.stkippersada.ac.id/jurnal/index.php/JPDP/article/view/113 Lamesh show. (1997). Pemilihan Rumus dan Perhitungan Besar Sampel. Simple Size Determination for Health, 1–15. Manjarres-Posada, N. I., Onofre-Rodríguez, D. J., & Benavides-Torres, R. A. (2020). Social Cognitive Theory and Health Care: Analysis and Evaluation. Mariah Ulfah et.al (Pengaruh Pola Asuh Orang Tua Terhadap Pernikahan Dini) Copyright © 2020, Jurnal Kebidanan dan Keperawatan Aisyiyah ISSN 1858-0610 (print) | ISSN 2477-8184 Ulum, I., Ghozali, I., & Chariri, A. (2008). Intellectual Capital Dan Kinerja Keuangan Perusahaan ; Suatu Analisis Dengan Pendekatan Partial Least Squares. Simposium Nasional Akuntansi XI, 19(19), 23–24. Research priorities on ending child marriage and supporting married girls. Reproductive Health, 12(1), 10–13. https://doi.org/10.1186/s12978-015- 0060-5 DAFTAR PUSTAKA International Journal of Social Science Studies, 8(4), 132. https://doi.org/10.11114/ijsss.v8i4.4870 Pramita, D., & Kurniawati, T. (2011). Hubungan Antara Komunikasi Orang Tua Dan Anak Tentang Seks Dengan Perilaku Seks Remaja Di SMA N 1 Seyegan Sleman Yogyakarta. Retrieved from http://digilib.unisayogya.ac.id/id/eprint/1026 Purwaningsih, E., & ... (2015). Hubungan Pola Asuh Orang Tua Dengan Kejadian Pernikahan Usia Dini Di Desa Jambu Kidul, Ceper, Klaten. INVOLUSI Jurnal Ilmu. Retrieved from http://jurnal.stikesmukla.ac.id/index.php/involusi/article/view/43/0 Rohayati, D. A. (2017). Hubungan Pola Asuh Orangtua Dengan Perilaku Moral Tidak Baik Siswa Smp Negeri 14 Muaro Jambi. Retrieved from https://repository.unja.ac.id/1728/ Rumble, L. (2018). An empirical exploration of female child marriage determinants in Indonesia. BMC Public Health, 18(1). https://doi.org/10.1186/s12889-018-5313-0 Sari, O., Winarni, S., & Dharminto, D. (2018). Hubungan Adat Setempat, Pola Asuh, dan Persepsi Orang Tua dengan Umur Menikah Wanita PUS pada Pernikahan Dini di Kecamatan Karangrayung, Kesehatan Masyarakat (eRetrieved from https://ejournal3.undip.ac.id/index.php/jkm/article/view/19852 Shabri, M. R., & Raihana, P. A. (2019). Hubungan Pola Asuh Permisif Dan Kontrol Diri Dengan Perilaku Seksual Pranikah Remaja Di Lembaga Pembinaan Khusus Anak Kelas 1 Kutoarjo. Retrieved from http://eprints.ums.ac.id/id/eprint/75122 Svanemyr, J., Chandra-Mouli, V., Raj, A., Travers, E., & Sundaram, L. (2015). Copyright © 2020, Jurnal Kebidanan dan Keperawatan Aisyiyah ISSN 1858-0610 (print) | ISSN 2477-8184 Jurnal Kebidanan dan Keperawatan Aisyiyah, 16 (2), 2020 Mariah Ulfah, Linda Yanti, Prasanti Adriani, Soliyah Jurnal Kebidanan dan Keperawatan Aisyiyah, 16 (2), 2020 Mariah Ulfah, Linda Yanti, Prasanti Adriani, Soliyah 185 Research priorities on ending child marriage and supporting married girls. Reproductive Health, 12(1), 10–13. https://doi.org/10.1186/s12978-015- 0060-5 Ulum, I., Ghozali, I., & Chariri, A. (2008). Intellectual Capital Dan Kinerja Keuangan Perusahaan ; Suatu Analisis Dengan Pendekatan Partial Least Squares. Simposium Nasional Akuntansi XI, 19(19), 23–24. q Yohana, B., Sunarsih, T., & Rokhanawati, D. (2020). Parenting Styles and Resilience towards Reproductive Health in Married Female Adolescents in Gunungkidul, Yogyakarta, Indonesia. Journal of Health Promotion and Behavior, 5(2), 96–103. https://doi.org/10.26911/thejhpb.2020.05.02.04 Mariah Ulfah et.al (Pengaruh Pola Asuh Orang Tua Terhadap Pernikahan Dini) Copyright © 2020, Jurnal Kebidanan dan Keperawatan Aisyiyah ISSN 1858-0610 (print) | ISSN 2477-8184
https://openalex.org/W4361844611
https://figshare.com/articles/journal_contribution/Supplementary_Table_5_from_Biomarkers_of_HPV_in_Head_and_Neck_Squamous_Cell_Carcinoma/22392222/1/files/39837798.pdf
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Supplementary Table 1 from Biomarkers of HPV in Head and Neck Squamous Cell Carcinoma
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Death/Cases Not Adjusted for Tumor Stage (n=62) Adjusted for Tumor Stage (n=62) without adjustment for tumor stages, Eastern Massachusetts, 1999-2003,. Death/Cases (n=62) (n=62) HR(95% CI) HR(95% CI) 5/22 ref. ref. 16/40 2 0 (0 7-5 9) 2 0 (0 7-5 7) 16/40 2.0 (0.7 5.9) 2.0 (0.7 5.7) 16/42 ref. ref. 5/20 0.7 (0.2-2.1) 0.6 (0.2-1.8) 16/44 ref. ref. 5/18 1.2 (0.4-4.1) 0.9 (0.3-3.2) 14/39 ref ref 14/39 ref. ref. 7/23 0.6 (0.2-1.6) 0.4 (0.1-1.3) 17/40 ref. ref. 4/22 0 2 (0 1 0 8) 0 1 (0 01 0 3) 4/22 0.2 (0.1-0.8) 0.1 (0.01-0.3) 17/43 ref. ref. 4/19 0.3 (0.1-1.0) 0.1 (0.03-0.6) 12/36 9/26 0.9 (0.3-2.5) 0.6 (0.2-2.4)
https://openalex.org/W2952547656
https://europepmc.org/articles/pmc6630642?pdf=render
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Study on ASJ Cutting of TC18, Based upon Multivariate Nonlinear Regression and SA-BP-AGA
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Received: 19 May 2019; Accepted: 12 June 2019; Published: 13 June 2019 Received: 19 May 2019; Accepted: 12 June 2019; Published: 13 June 2019 Abstract: TC18 titanium alloy has been widely applied, but is considered as a difficult machining material. Taking the kerf angle as the quality criterion, this paper studied the cutting performance of TC18 by the use of an abrasive slurry jet (ASJ), based upon multivariate nonlinear regression and SA-BP-AGA. Cutting experiments were carried out according to the Taguchi orthogonal method. The experimental factors included traverse speed, standoffdistance, pressure and slurry concentration, with five levels set, respectively. Meanwhile, a characterization method of the major influencing factors was proposed. A multiple nonlinear regression model and a back propagation artificial neural network (BP) prediction model, based on adaptive genetic algorithm (AGA), were established. The reliability was verified by statistics equations for the 22 groups of the fitting or training model and the three groups of experimental results. The BP-AGA and Simulated annealing algorithm (SA) were used to form a set of prediction optimization systems, called integrated SA-BP-AGA. Finally, the results showed that the main factor influencing the kerf angle is the slurry concentration. BP-AGA is easier to model, offers better robustness and is more accurate than a multivariate nonlinear regression model. The best kerf angle can be predicted by the integration system. The study results can improve the performance for the machining of TC18 by ASJ. Keywords: ASJ cutting; retardation coefficient; SA-BP-AGA; TC18 materials materials materials Keywords: ASJ cutting; retardation coefficient; SA-BP-AGA; TC18 Study on ASJ Cutting of TC18, Based upon Multivariate Nonlinear Regression and SA-BP-AGA 1 School of Electrical and Power Engineering, China University of Mining and Technology, Xuzhou 221116, China; TS17130003A3@cumt.edu.cn (J.L.); jianniang_zhouxin@163.com (X.Z.); cumthui@126.com (H.Z.); wfc0317@163.com (F.W.); xqw0703@163.com (Q.X.) 2 Xuhai College, China University of Mining and Technology, Xuzhou 221116, China 2 Xuhai College, China University of Mining and Technology, Xuzhou 221116, China * C d @ d 2 Xuhai College, China University of Mining and Technology, Xuzhou 221116, China * Correspondence: cwguo@cumt.edu.cn * Correspondence: cwguo@cumt.edu.cn 1. Introduction Titanium alloy TC18 (Ti-5Al-5Mo-5V-1Cr-1Fe) possesses the common excellent performance of both alpha phase and beta phase titanium alloy, such as high strength to weight ratio, high toughness, high hardness, high corrosion resistance, being non-magnetic and so on, with a new style alloy (alpha + beta). It has been widely used in aviation, biomedical, automotive fields, etc. [1,2]. However, there exist enormous challenges for traditional machining approaches due to its properties, including poor heat transfer performance, work-hardening and unstable chemical reaction, and deformation under high temperature conditions, which tends to cause serious tool wear, even much lower durability and shorter life than expectancy [3–5]. Moreover, some researches showed that the quality of the machined surface of the work piece has significant influence on its mechanical properties, especially upon fatigue properties [6–8]. To address these issues, some researchers used an improved genetic algorithm to optimize the milling parameters of TC18, and studied the forging process parameters of TC18 based upon the BP neural network, but it did not overcome the negative effect of the poor property of TC18 on the quality of the traditional processing technology, such as the heat affected zone, etc. [9,10]. Therefore, there is an urgent need for an advanced processing method to solve various problems that arise in the processing of TC18. www.mdpi.com/journal/materials www.mdpi.com/journal/materials Materials 2019, 12, 1902; doi:10.3390/ma12121902 2 of 14 Materials 2019, 12, 1902 ASJ technology is one of the fastest growing and most advanced non-traditional processing technologies. It has the advantages of no thermal effects, no residual stress, good incision quality, high applicability of materials, being environment friendly and highly competitive in material processing [11]. Wang studied the mechanism of kerf width and kerf angle formation during abrasive water jet machining [12]. Azmir used the Taguchi experimental method and variance analysis to study the influence of processing parameters on the kerf angle upon the cutting of glass/epoxy composite laminate, and concluded that the type of abrasive is the most important controlling factor [13]. Alberdi established a mathematical model based on pressure, the mass flow of the abrasive, target distance and transverse velocity processing parameters, which is used to predict the profile produced by AWJ cutting 1075-T651 [14]. Feng used numerical simulations and experiments, concluding that the jet with the added polymer has better stability in air [15]. 1. Introduction Wang found that adding high polymer to the abrasive slurry to cut stainless steel would have better processing performance [16]. Just as traditional machining relies on computer optimization control and an optimization of processing efficiency [17–19], advanced computer algorithms can also be used to optimize the processing parameters of an abrasive water jet in order to obtain high-quality products. Azlan used an integrated system of SA and GA algorithms to optimize the parameters of the abrasive processing process [20]. However, it depends on a great multivariate nonlinear regression model which is difficult to obtain. By considering the diameter of the focused nozzle and controllable process parameters such as work pressure, traverse speed and abrasive flow rate, Srinivasu modeled the artificial neural network to predict the depth of cut in the AWJ process, and also used a genetic algorithm to find out the optimal parameters combination [21]. However, the accurate ANN (artificial neural networks) prediction modeling was constructed directly with enormous work and difficulty. y y In order to study the effect of processing parameters on TC18, we used Taguchi’s orthogonal method to carry out the experiments. To optimize the machining process by ASJ for TC18, and at the same time taking into account the stability of the jet pressure in the experiment, we provided some new measures to analyze the experimental data. Firstly, a multivariate nonlinear regression model was established, and the reliability of the prediction model was verified by using a mathematical statistics formulae (MAPE, MSE and R2) and some specific experimental data. Based on the verification results, the model was only used to determine the main influencing factors of the experiment, which indicated that Azlan’s method [20] was not applicable here. We comprehensively utilized the good methods proposed by Azlan [20] and Srinivasu [21], meanwhile avoiding the restrictive conditions in [20] that it must rely on a great nonlinear prediction model, and solved the difficult problem of directly establishing the neural network prediction model in [21]. A back propagation artificial neural network (BP) prediction model, based on adaptive genetic algorithm (AGA) was established, and of which (BP-AGA) the validation was checked by using the same method as above. Then this study compared the multiple nonlinear regression method with the neural network method optimized by the adaptive genetic algorithm. It is found that BP-AGA is easier to model, offers better robustness and is more accurate. 1. Introduction Finally, the BP-AGA and simulated annealing algorithm (SA) optimization technology were used to form a set of prediction systems, called integrated SA-BP-AGA. Through this integrated system, the best kerf angle and the parameters affecting the kerf angle were obtained. The study results can improve the performance for TC18 machining by ASJ. 2.1. Experimental System The experiment was carried out on the DWJ1313-FC abrasive jet cutting system (DARDI, Nanjing, China) at the Water Jet Research Center of the China University of Mining and Technology, and the set of equipment is shown in Figure 1. 3 of 14 3 f 14 Materials 2019, 12, 1902 M t i l 2019 12 FOR Figure 1. DWJ1313-FC Abrasive Jet Machining system. Figure 1. DWJ1313-FC Abrasive Jet Machining system. Figure 1. DWJ1313-FC Abrasive Jet Machining system. ure 1, the experimental equipment consists of a ical control) cutting table, booster pump, the wate Figure 1. DWJ1313-FC Abrasive Jet Machining system. Figure 1. DWJ1313-FC Abrasive Jet Machining system. re 1, the experimental equipment consists of cal control) cutting table booster pump the wate As shown in Figure 1, the experimental equipment consists of a control cabinet, CNC (computerized numerical control) cutting table, booster pump, the water tank, an abrasive tank, etc. Firstly, the working pressure is set by the control cabinet, and then the booster pump is started to force the polyacrylamide (PAM) slurry in the water tank flow into the abrasive tank at high speed, and then mix with the abrasive particles to form the abrasive slurry high-speed fluid. Then the steady jet is polymerized by the jet nozzle on the numerical control cutting platform. Finally, the cutting platform can be activated, and the nozzle will carry out the cutting experiment according to As shown in Figure 1, the experimental equipment consists of a control cabinet, CNC (computerized numerical control) cutting table, booster pump, the water tank, an abrasive tank, etc. Firstly, the working pressure is set by the control cabinet, and then the booster pump is started to force the polyacrylamide (PAM) slurry in the water tank flow into the abrasive tank at high speed, and then mix with the abrasive particles to form the abrasive slurry high-speed fluid. Then the steady jet is polymerized by the jet nozzle on the numerical control cutting platform. Finally, the cutting platform can be activated, and the nozzle will carry out the cutting experiment according to the planning road path. etc. Firstly, the working pressure is set by the control cabinet, and then the booster pump is started to force the polyacrylamide (PAM) slurry in the water tank flow into the abrasive tank at high speed, and then mix with the abrasive particles to form the abrasive slurry high-speed fluid. 2.1. Experimental System Then the steady jet is polymerized by the jet nozzle on the numerical control cutting platform. Finally, the cutting platform can be activated, and the nozzle will carry out the cutting experiment according to the planning road path. 2 2 Experimental Scheme the planning road path. 2.2. Experimental Scheme As shown in Figur 2.2. Experimental Scheme As shown in Figure 2, there are many processing parameters to be set in the ASJ cutting experiment. In this experiment, the effects of traverse speed, standoff distance, system pressure and slurry concentration on the cutting quality characteristics (the kerf angle), are studied. The kerf angle indicates the inclination of the cutting wall. To solve the problems of expensive costs and inefficiency caused by the full-factor experimental method, we adopted the Taguchi orthogonal theory to carry out experiments [22]. Five levels were set for each variable, as shown in Table 1, and other processing parameters were kept constant for all cuts, as shown in Table 2. Based on the same As shown in Figure 2, there are many processing parameters to be set in the ASJ cutting experiment. In this experiment, the effects of traverse speed, standoffdistance, system pressure and slurry concentration on the cutting quality characteristics (the kerf angle), are studied. The kerf angle indicates the inclination of the cutting wall. To solve the problems of expensive costs and inefficiency caused by the full-factor experimental method, we adopted the Taguchi orthogonal theory to carry out experiments [22]. Five levels were set for each variable, as shown in Table 1, and other processing parameters were kept constant for all cuts, as shown in Table 2. Based on the same experimental system, Wang [12] provided a reference for the experimental processing parameter design of this paper. experiment. In this experiment, the effects of traverse speed, standoff distance, system pressure and slurry concentration on the cutting quality characteristics (the kerf angle), are studied. The kerf angle indicates the inclination of the cutting wall. To solve the problems of expensive costs and inefficiency caused by the full-factor experimental method, we adopted the Taguchi orthogonal theory to carry out experiments [22]. Five levels were set for each variable, as shown in Table 1, and other processing parameters were kept constant for all cuts, as shown in Table 2. Based on the same experimental system, Wang [12] provided a reference for the experimental processing parameter design of this paper. xperimental system, Wang [12] provided a reference for the experimental processing paramet design of this paper. Figure 2. Processing principle schematic. Figure 2. Processing principle schematic. Figure 2. Processing principle schematic. Figure 2. Processing principle schematic. Table 1 Variable parameters and their levels Table 1. Variable parameters and their levels. 2.3. Experimental Results 2.3. Experimental Results This experiment followed the L25 (54) orthogonal experiment table designed by MINITAB 17 software. The actual processed data and the measured characterization data were shown in Table 3. The kerf width was measured with an OLYMPUS DSX510 Microscope (Olympus, Tokyo, Japan). For high-quality image acquisition, we used an external 10× objective lens and an internal default 50× eyepiece combination. Then, through the DSX software system, we set the focal length to 1×, then set the acquisition mode to a 3D bright field, and set the image stitching overlap ratio to 20%, and finally set the acquisition area to 10 mm × 3 mm. After the automatic acquisition and splicing were completed, the geometric measurement mode in the DSX510 software measurement module was selected to measure the kerf width in the high quality image. In order to reduce the error caused by the measurement, the top kerf width and the bottom kerf width corresponding areas of the same cut sample, were respectively measured 25 times, and the average values were taken as their final measurement results. Image measurement is shown in Figure 3. This experiment followed the L25 (54) orthogonal experiment table designed by MINITAB 17 software. The actual processed data and the measured characterization data were shown in Table 3. The kerf width was measured with an OLYMPUS DSX510 Microscope (Olympus, Tokyo, Japan). For high-quality image acquisition, we used an external 10× objective lens and an internal default 50× eyepiece combination. Then, through the DSX software system, we set the focal length to 1×, then set the acquisition mode to a 3D bright field, and set the image stitching overlap ratio to 20%, and finally set the acquisition area to 10 mm × 3 mm. After the automatic acquisition and splicing were completed, the geometric measurement mode in the DSX510 software measurement module was selected to measure the kerf width in the high quality image. In order to reduce the error caused by the measurement, the top kerf width and the bottom kerf width corresponding areas of the same cut sample, were respectively measured 25 times, and the average values were taken as their final measurement results. Image measurement is shown in Figure 3. (a) (b) Figure 3. Measure mode diagram. (a) Top kerf width; (b) bottom kerf width. Figure 3. Measure mode diagram. (a) Top kerf width; (b) bottom kerf width. (b) (a) (b) (a) Figure 3. the planning road path. 2.2. Experimental Scheme As shown in Figur Table 1 Variable parameters and their levels Table 1. Variable parameters and their levels. Figure 2. Processing principle schematic. Table 1. Variable parameters and their levels. Number Variables L1 L2 L3 L4 L5 1 Traverse speed V (mm/min) 30 40 50 60 70 2 Standoff distance H (mm) 0.5 1 1.5 2 2.5 3 Slurry concentration C (%) 0 0.05 0.1 0.15 0.2 Table 1. Variable parameters and their levels. Number Variables L1 L2 L3 L4 L5 1 Traverse speed V (mm/min) 30 40 50 60 70 2 Standoff distance H (mm) 0.5 1 1.5 2 2.5 3 Slurry concentration C (%) 0 0.05 0.1 0.15 0.2 4 System pressure P (MPa) 18 22 26 30 34 Table 1. Variable parameters and their levels. Number Variables L1 L2 L3 L4 L5 1 Traverse speed V (mm/min) 30 40 50 60 70 2 Standoffdistance H (mm) 0.5 1 1.5 2 2.5 3 Slurry concentration C (%) 0 0.05 0.1 0.15 0.2 4 System pressure P (MPa) 18 22 26 30 34 4 of 14 4 of 14 Materials 2019, 12, 1902 Materials 2019, 12, x FOR Table 2. Constant Parameters. Table 2. Constant Parameters. Table 2. Constant Parameters. Table 2. Constant Parameters. Invariables Values Material size 200 × 30 × 5 (mm3) Nozzle diameter 1.0 (mm) Volume fraction of abrasive 20% High Polymer PAM Average diameter of abrasive 0.27 (mm) or 80 (mesh) Abrasive material type garnet Angle of influence 0 (degree) Invariables Values Material size 200 × 30 × 5 (mm3) Nozzle diameter 1.0 (mm） Volume fraction of abrasive 20% High Polymer PAM Average diameter of abrasive 0.27 (mm) or 80 (mesh) Abrasive material type garnet Angle of influence 0 (degree) Material size Nozzle diameter Volume fraction of abrasive High Polymer Average diameter of abrasive Abrasive material type Angle of influence Material size Nozzle diameter Volume fraction of abrasive High Polymer Average diameter of abrasive Abrasive material type Angle of influence 3. Predictive Model of Kerf Angle based on Multivariate Nonlinear Regression Modeling 3.1. Methodology 2.3. Experimental Results 2.3. Experimental Results Measure mode diagram. (a) Top kerf width; (b) bottom kerf width. Figure 3. Measure mode diagram. (a) Top kerf width; (b) bottom kerf width. 5 of 14 Materials 2019, 12, 1902 Table 3. The experimental data for model constructions. NO. Operating Variables Result V H C P Actual P Top Kerf Width (mm) Bottom Kerf Width (mm) Kerf Angle (◦) 1 1 1 1 1 18.1 0.880234 0.51703 4.1547 2 2 2 4 18.4 0.901138 0.752207 1.7061 3 3 3 2 18.3 0.940592 0.820456 1.3764 4 4 4 5 17.8 0.896659 0.708886 2.1507 5 5 5 3 17.9 1.013852 0.867836 1.6727 6 1 4 3 2 22.1 1.027128 0.852151 2.0043 7 2 5 1 22.2 1.003986 0.516018 5.574 8 3 1 4 21.7 0.913358 0.732508 2.0715 9 4 2 2 22.8 0.909006 0.77915 1.4877 10 5 3 5 22.4 0.908018 0.767484 1.61 11 1 2 5 3 25.9 0.901708 0.785861 1.3273 12 2 3 3 25.9 0.940733 0.59944 3.9049 13 3 4 1 26.1 0.958826 0.514941 5.0732 14 4 5 4 26.7 0.999784 0.786822 2.4389 15 5 1 2 26.8 0.925032 0.767567 1.8038 16 1 5 2 4 30.5 1.048323 0.919693 1.4737 17 2 1 5 30 0.923354 0.768629 1.7725 18 3 2 3 30.6 0.979514 0.822616 1.7973 19 4 3 1 30.4 0.946768 0.532342 4.7381 20 5 4 4 31.6 0.996658 0.798935 2.2646 21 1 3 4 5 34.8 1.027287 0.864021 1.8702 22 2 4 2 33.5 0.965137 0.852903 1.2859 23 3 5 5 34.1 0.991862 0.782701 2.3954 24 4 1 3 35.4 0.927152 0.855971 0.8156 25 5 2 1 33.9 0.960782 0.54892 4.709 The inclination of the kerf is defined as: q = tan−1 Wtop −Wbottom 2h ! (1) (1) where, θ, Wtop, Wbottom, and h are the kerf angle, the thickness of the cutting specimen, the top kerf width, and the bottom kerf width, respectively, as shown in Figure 2. where, θ, Wtop, Wbottom, and h are the kerf angle, the thickness of the cutting specimen, the top kerf width, and the bottom kerf width, respectively, as shown in Figure 2. where, θ, Wtop, Wbottom, and h are the kerf angle, the thickness of the cutting specimen, the top kerf width, and the bottom kerf width, respectively, as shown in Figure 2. 3. Predictive Model of Kerf Angle based on Multivariate Nonlinear Regression Modeling 3.1. Methodology Due to the slight fluctuation of the instability of the jet pressure during the real processing, the fluctuation error can be quantized by the mean relative level error function (MRLE). MRLE = 1 d X i (|ti −oi| n ) × 100% (2) (2) where, d is the tolerance among the adjacent levels, ti is the actual experiment value of the group i, oi is the designed value or the predictive value, n is the number of designs at the same level. 6 of 14 Materials 2019, 12, 1902 The Mean relative pressure level error calculated by the above Equation (2) is shown in Figure 4. In Figure 4, the corresponding value of Y is the decimal form of MRLE. It can be seen from the diagram that the maximum average relative error is 15.5%. Therefore, the commonly used analysis method-single factor multivariate variance based upon the Taguchi orthogonal method cannot be applied in this article. Materials 2019, 12, x FOR PEER REVIEW 6 of 14 method-single factor multivariate variance based upon the Taguchi orthogonal method cannot be applied in this article. Figure 4. Mean relative pressure level error. Figure 4. Mean relative pressure level error. Figure 4. Mean relative pressure level error. Figure 4. Mean relative pressure level error. Figure 4. Mean relative pressure level error Figure 4. Mean relative pressure level error. Therefore, we proposed a new idea to deal with this kind of situation. First of all, through multivariate nonlinear regression methods, the fitting was performed on the actual jet pressure values in all the odd groups and all the even numbers except (18, 22, 24) in Table 3. Then, the fitting degree of the regression equation was determined based on the multivariate correlation coefficient R, after which the reliability of the prediction model was verified by mathematical statistics formulas with the 22 groups’ modeling data and the remaining three groups’ data of experiments. These formulae included the determination coefficient (R2), mean squared error (MSE) and the mean absolution percentage error (MAPE). Therefore, we proposed a new idea to deal with this kind of situation. First of all, through multivariate nonlinear regression methods, the fitting was performed on the actual jet pressure values in all the odd groups and all the even numbers except (18, 22, 24) in Table 3. 3.1. Methodology Materials 2019, 12, 1902 7 of 14 These comprehensive values of the slope of each variable can be figured out to characterize the sensitivity of the kerf angle to each processing variable, which are shown as the Equations (8)–(11). These comprehensive values of the slope of each variable can be figured out to characterize the sensitivity of the kerf angle to each processing variable, which are shown as the Equations (8)–(11). Mean(Sk) = 1 n n X i Slope(k)i (8) (8) Ct = ∆θ/ ∆k kmax −kmin ! = ∆θ ∆k × (kmax −kmin) (9) (9) In which, In which, ∆θ ∆k = Mean(Sk) (10) (10) where, Mean (Sk) is the mean of each variable’s slope; Ct means that when the other variables are unchanged, the change of k relative to its own interval will theoretically cause the maximum change of θ; and k represents one of the variables selected in the experiments. where, Mean (Sk) is the mean of each variable’s slope; Ct means that when the other variables are unchanged, the change of k relative to its own interval will theoretically cause the maximum change of θ; and k represents one of the variables selected in the experiments. Cr = θmax −θmin Ct (11) (11) where, Cr is the interval length of the actual kerf angle change divided by the Ct (in theory, the maximum change length of the k variable can cause the maximum change in the kerf angle). This also means that this k factor hinders the change of inclination caused by the change of the other factors through its own changes, which is called the retardation coefficient of k. where, Cr is the interval length of the actual kerf angle change divided by the Ct (in theory, the maximum change length of the k variable can cause the maximum change in the kerf angle). This also means that this k factor hinders the change of inclination caused by the change of the other factors through its own changes, which is called the retardation coefficient of k. The smaller the retardation coefficient is, the more powerful the influence of k is. The method was compared with the results of multiple variances, and it was found that when the Cr corresponding to the factor was less than 10, the factor had an important influence. 3.1. Methodology Through this method, the influence of factors can be sorted, and the main influencing factors are identified. The Regression Model of Kerf Angle 3.1. Methodology Then, the fitting degree of the regression equation was determined based on the multivariate correlation coefficient R, after which the reliability of the prediction model was verified by mathematical statistics formulas with the 22 groups’ modeling data and the remaining three groups’ data of experiments. These formulae included the determination coefficient (R2), mean squared error (MSE) and the mean absolution percentage error (MAPE). APE). i i 2 2 i i (t t) (o t) R= (t t) (o t) − − − −     (3) ( ) 2 i i 2 i 2 i i t o R =1 (o )   −   −      (4) 1 R = P (ti −t)P (oi −t) qP (ti −t)2P (oi −t)2 (3) R2 = 1 −   P i(ti −oi)2 P i (oi)2   (4) MSE = ( 1 N X i|ti −oi| 2) (5) MAPE = ( 1 N X i ti −oi ti × 100) (6) (3) (4) (4) (5) (6) 2 i i i 1 MSE = ( t o ) N −  (5) where t is the average of the actual experiment value; N is the total amount of participating in the calculation of runs. i i i i t o 1 MAPE = ( 100) N t − ×  (6) Finally, partial derivatives of each processing variable of the established regression equation were calculated. Substituting the values of the processing variables in each group into the partial derivative equation, the slope of the aimed partial variable of the regression equation in each group coordinates was obtained, as described in Equation (7). actual experiment value; N is the total amount of participating in the f h i i bl f h bli h d i i Slope(k) = f′ k(V, H, C, P) (Vi,Hi,Ci,Pi) (7) n the (7) Finally, partial derivatives of each processing variable of the established regression equation were calculated. Substituting the values of the processing variables in each group into the partial derivative equation, the slope of the aimed partial variable of the regression equation in each group where, Slope(k) is the slope of the equation about the k variable, k can be V, H, C or P. In addition, Vi, Hi, Ci and Pi are the values of group i in Table 3, respectively. 3.2. The Regression Model of Kerf Angle What is given here mainly showed the transformation of the original data by us to complete the regression fitting. The mathematical model established in this paper is expressed as Equation (12), which is one of the commonly used fitting basic equations in the field of waterjet machining [20,23,24]. (12) θ = aVqHs(C + C0)zPue′ (12) re, ε’ is the experimental error, and a, q, s, C0, z and u are pending parameters to be estimated by experimental data. Equation (12) can be linearized by performing a logarithmic transformation as follows: ln θ = lnc + qlnV + slnH + tln(C + C0) + ulnP + lne′ (13) (13) The final Equation (13) can be written as: The final Equation (13) can be written as: The final Equation (13) can be written as: ˆθ = aVqHs(C + C0)tPu (14) (14) The regression model for the kerf angle that has been determined, is as follows: ˆθ = 2.038V0.0094H−0.0088(C + 1 × 10−5) −0.0971P−0.0896 (15) (15) Materials 2019, 12, 1902 Materials 2019, 12, x FOR 8 of 14 8 of 14 The multivariate correlation coefficient R is calculated as the linearized regression Equation (13), and R = 0.8316, greater than 0.8, which can be accepted. The rest of the mathematical statistics test is calculated according to Equation (15). Using the remaining 3 groups of experimental parameters to check the prediction accuracy of the equation, and quantified by statistical formulae R2, MSE, and MAPE. The results listed into the table 5 are 0.8383, 0.6415, and 69.4384%, respectively. Although the mean absolute error percentage exceeds 20%, indicating that the regression model prediction accuracy is not high, the coefficient of determination and the average error are both within acceptable limits, and the statistical result of training groups are 0.9558, 0.3647, and 19.5027, which are listed into the Table 4. So the training groups can be used to assess the influence of the four factors. g q (13), and R = 0.8316, greater than 0.8, which can be accepted. The rest of the mathematical statistics test is calculated according to Equation (15). Using the remaining 3 groups of experimental parameters to check the prediction accuracy of the equation, and quantified by statistical formulae R2, MSE, and MAPE. The results listed into the table 5 are 0.8383, 0.6415, and 69.4384%, respectively. 3.3. Analysis of Main Influencing Factors 3.3. Analysis of Main Influencing Factors By Equations (7) and (8), the slope of each variable is calculated and expressed in Figure 5. By Equations (7) and (8), the slope of each variable is calculated and expressed in Figure 5. By Equations (7) and (8), the slope of each variable is calculated and expressed in Figure 5. By Equations (7) and (8), the slope of each variable is calculated and expressed in Figure 5. By Equations (7) and (8), the slope of each variable is calculated and expressed in Figure 5. By Equations (7) and (8), the slope of each variable is calculated and expressed in Figure 5. Figure 5. Slope (V, H, C, P). As shown in Figure 5, it can be obviously observed that the slope fluctuation of the slurry concentration is particularly intense, and its absolute mean value is also large, which is several orders of magnitude larger than the other three variables. So a simple estimate can be obtained that the main influencing factor is C. By Equation (11), the Cr values of V, H, C and P are 228.77, 116.5388, 0.0025, and 32.61, respectively. It can be clearly known that C has a significant influence, followed by P, H, and V, which has little influence. Figure 5. Slope (V, H, C, P). As shown in Figure 5, it can be obviously observed that the slope fluctuation of the slurry concentration is particularly intense, and its absolute mean value is also large, which is several orders of magnitude larger than the other three variables. So a simple estimate can be obtained that the main influencing factor is C. By Equation (11), the Cr values of V, H, C and P are 228.77, 116.5388, 0.0025, and 32.61, respectively. It can be clearly known that C has a significant influence, followed by P, H, and V, which has little influence. y quat o s ( ) a d (8), t e s ope o eac va ab e s ca cu ated a d e p essed gu e 5 By Equations (7) and (8), the slope of each variable is calculated and expressed in Figure 5. Figure 5. Slope (V, H, C, P). Figure 5. Slope (V, H, C, P). Figure 5. Slope (V, H, C, P). Figure 5. Slope (V, H, C, P). 3.3. Analysis of Main Influencing Factors 3.3. Analysis of Main Influencing Factors As shown in Figure 5, it can be obviously observed that the slope fluctuation of the slurry concentration is particularly intense, and its absolute mean value is also large, which is several orders of magnitude larger than the other three variables. So a simple estimate can be obtained that the main influencing factor is C. By Equation (11), the Cr values of V, H, C and P are 228.77, 116.5388, 0.0025, and 32.61, respectively. It can be clearly known that C has a significant influence, followed by P H and V which has little influence As shown in Figure 5, it can be obviously observed that the slope fluctuation of the slurry concentration is particularly intense, and its absolute mean value is also large, which is several orders of magnitude larger than the other three variables. So a simple estimate can be obtained that the main influencing factor is C. By Equation (11), the Cr values of V, H, C and P are 228.77, 116.5388, 0.0025, and 32.61, respectively. It can be clearly known that C has a significant influence, followed by P, H, and V, which has little influence. 4 Predictive Model of Kerf Angle Based on ANN AGA 4. Predictive Model of Kerf Angle Based on ANN-AGA 4. Predictive Model of Kerf Angle Based on ANN-AGA It can be seen from the above calculation results that the multivariate nonlinear regression is not good at prediction Therefore a new prediction model is established It can be seen from the above calculation results that the multivariate nonlinear regression is not good at prediction. Therefore, a new prediction model is established. 3.2. The Regression Model of Kerf Angle Although the mean absolute error percentage exceeds 20%, indicating that the regression model prediction accuracy is not high, the coefficient of determination and the average error are both within acceptable limits, and the statistical result of training groups are 0.9558, 0.3647, and 19.5027, which are listed into the Table 4. So the training groups can be used to assess the influence of the four factors. 3.3. Analysis of Main Influencing Factors 3.3. Analysis of Main Influencing Factors 4 1 M th d l 4.1. Methodology 4.1. Methodology During neural network training, network structure parameters and initial thresholds and weights determine the training duration and network quality of the network to a large extent. Due to During neural network training, network structure parameters and initial thresholds and weights determine the training duration and network quality of the network to a large extent. Due to the nature of the “black box” of neural networks, it leads to blindness in debugging and low training efficiency. the nature of the “black box” of neural networks, it leads to blindness in debugging and low training efficiency. However, by using the AGA method to find the optimal initial training thresholds and weights of the neural network, the blindness of debugging is reduced to a certain extent, the efficiency of However, by using the AGA method to find the optimal initial training thresholds and weights of the neural network, the blindness of debugging is reduced to a certain extent, the efficiency of network training is greatly improved, and the quality of the network is indirectly improved. Finally, the test results are analyzed by the same statistical formulae (R2, MSE and MAPE). network training is greatly improved, and the quality of the network is indirectly improved. Finally the test results are analyzed by the same statistical formulae (R2 MSE and MAPE) An adaptive genetic algorithm optimizes the BP neural network flow as shown in Figure 6. 9 of 14 9 of 14 Materials 2019, 12, 1902 Materials 2019 12 x FOR Figure 6. Back propagation-adaptive genetic algorithm (BP-AGA) structure schematic diagram. Figure 6. Back propagation-adaptive genetic algorithm (BP-AGA) structure schematic diagram. Figure 6. Back propagation-adaptive genetic algorithm (BP-AGA) structure schematic diagram. Figure 6. Back propagation-adaptive genetic algorithm (BP-AGA) structure schematic diagram. After debugging the structure of neural network and random initial weights and thresholds, it is supplemented by the adaptive genetic algorithm to improve efficiency and quality. This genetic algorithm mainly includes chromosome coding, selection operation, mutation operation, crossover operation and fitness operation. Among them, the crossover ratio (pc) and the mutation ratio (pm) in the parameters of the genetic algorithm play very important roles in the performance of the algorithm. If the fixed pc and pm values are adopted, it is difficult to adapt to the change of population, and sometimes leads to the evolution of the past. In this paper, an adaptive algorithm based on Srinvivas is proposed. 4 1 M th d l 4.1. Methodology The pc and pm in the algorithm can change automatically with fitness values, which can maintain group diversity and ensure convergence, as shown in Equation After debugging the structure of neural network and random initial weights and thresholds, it is supplemented by the adaptive genetic algorithm to improve efficiency and quality. This genetic algorithm mainly includes chromosome coding, selection operation, mutation operation, crossover operation and fitness operation. Among them, the crossover ratio (pc) and the mutation ratio (pm) in the parameters of the genetic algorithm play very important roles in the performance of the algorithm. If the fixed pc and pm values are adopted, it is difficult to adapt to the change of population, and sometimes leads to the evolution of the past. In this paper, an adaptive algorithm based on Srinvivas is proposed. The pc and pm in the algorithm can change automatically with fitness values, which can maintain group diversity and ensure convergence, as shown in Equation (16) below. max min max max max max (pc pc )(f f ') pc ,f ' f f f pc pc ,f ' f − −  − ≥  − =   <  avg avg avg (16) pc =  pcmax −(pcmax−pcmin)(fmax−f′) fmax−favg , f′ ≥favg pcmax, f′ < favg pm =  pmmax −(pmmax−pmmin)(fmax−f) fmax−favg , f ≥favg pmmax, f < favg (16) (16) max min max max avg max avg max avg (pm pm )(f f) pm ,f f f f pm pm ,f f − −  − ≥  − =   <  (16) where, pcmax, pcmin, pmmax and pmmin are the maximum, minimum crossover rate and mutation rate, respectively. fmax, favg are the maximum fitness value and the average fitness for each generation of population. f’ is the larger fitness value of the two individuals to cross, and f is the fitness value of the variant individuals. where, pcmax, pcmin, pmmax and pmmin are the maximum, minimum crossover rate and mutation rate, respectively. fmax, favg are the maximum fitness value and the average fitness for each generation of population. 4 1 M th d l 4.1. Methodology f’ is the larger fitness value of the two individuals to cross, and f is the fitness value of the variant individuals Firstly, the chromosome was constructed by a binary encoding of the initial threshold and weight of the neural network, and the prediction error of the neural network was used as our fitness value. Then the individual difference was produced by chromosomal variation and cross, and the selection of the wheel was executed by the principle of survival of the fittest. the variant individuals. Firstly, the chromosome was constructed by a binary encoding of the initial threshold and weight of the neural network, and the prediction error of the neural network was used as our fitness The optimal threshold and weight of the neural network were found. Finally, based on these parameters, the neural network was trained to get the best network model. value. Then the individual difference was produced by chromosomal variatio selection of the wheel was executed by the principle of survival of the fittest 4.2. Neural Network Optimized by Adaptive Genetic Algorithm Based on Kerf Angle selection of the wheel was executed by the principle of survival of the fittest. The optimal threshold and weight of the neural network were found. Finally, based on these parameters, the neural network was trained to get the best network model. 4.2. Neural Network Optimized by Adaptive Genetic Algorithm Based on Kerf Angle It was finally determined that the structure of ANN is 4-11-1 that is with 4 input nodes 11 It was finally determined that the structure of ANN is 4-11-1, that is, with 4 input nodes, 11 hidden nodes and 1 output nodes. The adaptive genetic algorithm parameters included, the population size is 24, the maximum cross rate is 0.7, the minimum cross rate is 0.1, the maximum mutation rate is 0.05, and the minimum mutation rate is 0.01. Similarly, the 22 sets of data selected above were used for training. A neural network prediction model with high accuracy was established by MATLAB 2016. 10 of 14 l t d elected 10 of 14 l t d elected Materials 2019, 12, 1902 mutation rate is 0.0 The structure of the neural network is shown in Figure 7. The regression performance of the neural network is shown in Figure 8. above were used for training. A neural network prediction model with high accuracy was established by MATLAB 2016. The structure of the neural network is shown in Figure 7. The regression performance of the neural network is shown in Figure 8. established by MATLAB 2016. The structure of the neural network is shown in Figure 7. The regression performance of the neural network is shown in Figure 8. Figure 7. ANN’s (BP-AGA) structure diagram. Figure 7. ANN’s (BP-AGA) structure diagram. Figure 7. ANN’s (BP-AGA) structure diagram. Figure 7. ANN’s (BP-AGA) structure diagram. Figure 8 ANN’S (BP AGA) regression performance diagram Figure 7. ANN’s (BP-AGA) structure diagram. Figure 7. ANN’s (BP-AGA) structure diagram. Figure 8. ANN’S (BP-AGA) regression performance diagram. Figure 8. ANN’S (BP-AGA) regression performance diagram. Figure 7. ANN’s (BP-AGA) structure diagram. Figure 7. ANN’s (BP-AGA) structure diagram. Figure 7. ANN’s (BP-AGA) structure diagram. Figure 8 ANN’S (BP-AGA) regression performance diagram Figure 8. ANN’S (BP-AGA) regression performance diagram. Figure 8. ANN’S (BP-AGA) regression performance diagram. Figure 8 ANN’S (BP AGA) eg essio pe fo a ce diag a Figure 8. ANN’S (BP-AGA) regression performance diagram. Figure 8. ANN’S (BP-AGA) regression performance diagram. o u ae , E a A E e e u a e i 5 C i f th T A l i M th d 5. Comparison of the Two Analysis Methods 5. Comparison of the Two Analysis Methods 5. Comparison of the Two Analysis Methods Currently, the main common methods for multivariate regression analysis are Forward, Forward, Backward and Stepwise. But in most cases, they are difficult to fit and are prone to multiple collinear troubles [24]. Therefore, a large amount of time has to be used to perform various p y Currently, the main common methods for multivariate regression analysis are Forward, Forward, Backward and Stepwise. But in most cases, they are difficult to fit and are prone to multiple collinear troubles [24]. Therefore, a large amount of time has to be used to perform various transformations on the data to obtain a higher coefficient of multiple correlation and determination. Currently, the main common methods for multivariate regression analysis are Forward, Forward, Backward and Stepwise. But in most cases, they are difficult to fit and are prone to multiple collinear troubles [24]. Therefore, a large amount of time has to be used to perform various transformations on the data to obtain a higher coefficient of multiple correlation and determination. transformations on the data to obtain a higher coefficient of multiple correlation and determination. However, using the artificial neural network to learn experimental data, and then predicting, only the optimization algorithm is needed to optimize the initial threshold and weight of the transformations on the data to obtain a higher coefficient of multiple correlation and determination. However, using the artificial neural network to learn experimental data, and then predicting, only the optimization algorithm is needed to optimize the initial threshold and weight of the network, and then the simple network structure parameter adjustment can achieve the purpose. However, using the artificial neural network to learn experimental data, and then predicting, only the optimization algorithm is needed to optimize the initial threshold and weight of the network, and then the simple network structure parameter adjustment can achieve the purpose. network, and then the simple network structure parameter adjustment can achieve the purpose. From Tables 4 and 5, the fitting quality and prediction performance of the regression model are not as good as the training quality and prediction performance of neural network model. Moreover, compared with the statistical analysis results of training quality and prediction performance, it can b h h l k h b b d f l l h h fi i i network, and then the simple network structure parameter adjustment can achieve the purpose. value. Then the individual difference was produced by chromosomal variatio selection of the wheel was executed by the principle of survival of the fittest 4.2. Neural Network Optimized by Adaptive Genetic Algorithm Based on Kerf Angle g ( ) g p g From Figure 8, it can be found that the multivariate correlation coefficient R of training and testing is 0.97172 and 0.99783, respectively, with a high goodness of fit, listed in the Table 4. Then, the remaining three groups are still used as checking groups, which are calculated by the statistical formulae R2 MSE and MAPE The results are listed into the Table 5 From Figure 8, it can be found that the multivariate correlation coefficient R of training and testing is 0.97172 and 0.99783, respectively, with a high goodness of fit, listed in the Table 4. Then, the remaining three groups are still used as checking groups, which are calculated by the statistical formulae R2, MSE and MAPE. The results are listed into the Table 5. From Figure 8, it can be found that the multivariate correlation coefficient R of training and testing is 0.97172 and 0.99783, respectively, with a high goodness of fit, listed in the Table 4. Then, the remaining three groups are still used as checking groups, which are calculated by the statistical formulae R2, MSE and MAPE. The results are listed into the Table 5. 6. The Integrated SA-BP-AGA Optimization 6. The Integrated SA-BP-AGA Optimization Based on the trained neural network prediction model, a simulated annealing algorithm was used to find the optimal parameter combination of the minimum processing kerf angle. The integrated SA-BP-AGA of the above description is shown in Figure 9. Based on the trained neural network prediction model, a simulated annealing algorithm was used to find the optimal parameter combination of the minimum processing kerf angle. The integrated SA-BP-AGA of the above description is shown in Figure 9. Figure 9. SA-BP-AGA structure schematic diagram. Figure 9. SA-BP-AGA structure schematic diagram. Figure 9. SA-BP-AGA structure schematic diagram. Figure 9. SA-BP-AGA structure schematic diagram. Figure 9. SA-BP-AGA structure schematic diagram Figure 9. SA-BP-AGA structure schematic diagram. The simulated annealing algorithm is a random search technique that is able to escape local optima using a probability function [25]. SA is a relatively mature algorithm, widely used in VLSI (Very Large Scale Integration) design, image recognition and neural network computer research. It can be decomposed into three parts: Solution space, objective function and initial solution. Here, the solution space is composed of the ranges of various processing variables. The objective function is the AGA-optimized neural network, and the initial solution is set as the best parameter group that appears in the experiment, which is the 24th in the Table 3. The optimal solutions of the MATLAB Optimization Toolbox is given in Figure 10 on the base of these criteria as listed in Table 6 The simulated annealing algorithm is a random search technique that is able to escape local optima using a probability function [25]. SA is a relatively mature algorithm, widely used in VLSI (Very Large Scale Integration) design, image recognition and neural network computer research. It can be decomposed into three parts: Solution space, objective function and initial solution. Here, the solution space is composed of the ranges of various processing variables. The objective function is the AGA-optimized neural network, and the initial solution is set as the best parameter group that appears in the experiment, which is the 24th in the Table 3. The optimal solutions of the MATLAB Optimization Toolbox is given in Figure 10 on the base of these criteria as listed in Table 6. Table 6. Combination of simulated annealing (SA) parameter rates. o u ae , E a A E e e u a e i 5 C i f th T A l i M th d 5. Comparison of the Two Analysis Methods 5. Comparison of the Two Analysis Methods From Tables 4 and 5, the fitting quality and prediction performance of the regression model are not as good as the training quality and prediction performance of neural network model. Moreover, compared with the statistical analysis results of training quality and prediction performance, it can be seen that the neural network has better robustness and fault tolerance than the fitting regression From Tables 4 and 5, the fitting quality and prediction performance of the regression model are not as good as the training quality and prediction performance of neural network model. Moreover, compared with the statistical analysis results of training quality and prediction performance, it can be seen that the neural network has better robustness and fault tolerance than the fitting regression method. 11 of 14 11 of 14 Materials 2019, 12, 1902 Materials 2019, 12, x FOR Table 4. Fitting or Training quality performance. Table 4. Fitting or Training quality performance. Table 4. Fitting or Training quality performance. Table 4. Fitting or Training quality performance. Table 4. Fitting or Training quality performance. Model Fitting or Training Quality MSE MAPE (%) R2 Regression 0.3647 19.5027 0.9558 network 0.0987 6.0343 0.9881 Table 5. Prediction quality. Model Number Prediction Quality 18 22 24 MSE MAPE (%) R2 Regression 1.9460 2.0897 1.9359 0.6415 69.4384 0.8383 network 1.8161 1.2147 0.8946 0.0039 5.4244 0.9979 experiment 1.7973 1.2859 0.8156 Table 4. Fitting or Training quality performance. Model Fitting or Training Quality MSE MAPE (%) R2 Regression 0.3647 19.5027 0.9558 network 0.0987 6.0343 0.9881 Table 5. Prediction quality. Model Number Prediction Quality 18 22 24 MSE MAPE (%) R2 Regression 1.9460 2.0897 1.9359 0.6415 69.4384 0.8383 network 1.8161 1.2147 0.8946 0.0039 5.4244 0.9979 experiment 1.7973 1.2859 0.8156 6 Th I t t d SA BP AGA O ti i ti 7. Conclusions 7. Conclusions The paper carried out experiments according to Taguchi's orthogonal method, and established two predictive models for kerf taper based on a multivariate nonlinear model and the BP-AGA model. Meanwhile, statistical formulae (MAPE, MSE and R2) were used to verify and compare the reliability of the two prediction models. The BP-AGA prediction model is more reliable than the multivariate nonlinear regression prediction model. Moreover, neural network prediction shows better robustness and fault tolerance. The main influencing factors can be determined by the retardation coefficient proposed in this paper. This coefficient proposes to broaden the application of the Taguchi orthogonal test method in the field of the non-precision control industry, and provides a solution for the study of fluctuation factors by the Taguchi orthogonal horizontal experiment method. It was found that the main factor affecting the kerf angle is slurry concentration, and then the system pressure and standoff distance, while the traverse speed has barely influence. The optimal processing parameter combination under the condition of the minimal kerf angle are obtained by SA based on the prediction model of BP-AGA. The study The paper carried out experiments according to Taguchi’s orthogonal method, and established two predictive models for kerf taper based on a multivariate nonlinear model and the BP-AGA model. Meanwhile, statistical formulae (MAPE, MSE and R2) were used to verify and compare the reliability of the two prediction models. The BP-AGA prediction model is more reliable than the multivariate nonlinear regression prediction model. Moreover, neural network prediction shows better robustness and fault tolerance. The main influencing factors can be determined by the retardation coefficient proposed in this paper. This coefficient proposes to broaden the application of the Taguchi orthogonal test method in the field of the non-precision control industry, and provides a solution for the study of fluctuation factors by the Taguchi orthogonal horizontal experiment method. It was found that the main factor affecting the kerf angle is slurry concentration, and then the system pressure and standoffdistance, while the traverse speed has barely influence. The optimal processing parameter combination under the condition of the minimal kerf angle are obtained by SA based on the prediction model of BP-AGA. The study results can improve the performance for TC18 machining by ASJ and guide actual production. p p g y J g p Author Contributions: conceptualization, J.L. and C.G.; methodology, J.L.; software, J.L.; validation, J.L., X.Z. 6. The Integrated SA-BP-AGA Optimization 6. The Integrated SA-BP-AGA Optimization Parameters Setting Value/Function Type Objective limit 1 × 10−4 Annealing function Boltzmann annealing Reannealing interval 100 Temperature update function Exponential temperature Initial temperature 100 Acceptance probability function Simulated annealing acceptance Table 6. Combination of simulated annealing (SA) parameter rates. Parameters Setting Value/Function Type Objective limit 1 × 10−4 Annealing function Boltzmann annealing Reannealing interval 100 Temperature update function Exponential temperature Initial temperature 100 Acceptance probability function Simulated annealing acceptance Data type Double Table 6 Combination of simulated annealing (SA) parameter rates Table 6. Combination of simulated annealing (SA) parameter rates. Table 6 Combination of simulated annealing (SA) parameter rates Table 6. Combination of simulated annealing (SA) parameter rates. g ( ) p Setting Value/Function Type 1 × 10−4 Boltzmann annealing 100 Exponential temperature 100 imulated annealing acceptance Setting Value/Function Type 1 × 10−4 Boltzmann annealing 100 Exponential temperature 100 Simulated annealing acceptance Double a a ete s Objective limit Annealing function Reannealing interval Temperature update function Initial temperature Acceptance probability function Objective limit Annealing function Reannealing interval Temperature update function Initial temperature Acceptance probability function Data type Materials 2019, 12, 1902 12 of 14 As shown in Figure 10, the theoretically optimal solution was observed that the minimum kerf angle is 6.9425 × 10−5. The set value of process parameters that lead to the minimum θ value are 31.5 mm/min for traverse speed, 0.7806 mm for standoffdistance, 0.07841% for slurry concentration and 33.73 MPa for jet pressure. Materials 2019, 12, x FOR PEER REVIEW 12 of 14 As shown in Figure 10, the theoretically optimal solution was observed that the minimum kerf angle is 6.9425 × 10−5. The set value of process parameters that lead to the minimum θ value are 31.5 mm/min for traverse speed, 0.7806 mm for standoff distance, 0.07841% for slurry concentration and 33 73 MP f j t j p Figure 10. Fitness function plot of SA-BP-AGA. Figure 10. Fitness function plot of SA-BP-AGA. Figure 10. Fitness function plot of SA-BP-AGA. Figure 10. Fitness function plot of SA-BP-AGA. preparation, J.L.; writing review and editing, J.L. and C.G; visualization, J.L.; supervision, C.G.; project administration, C.G and J.L.; funding acquisition, C.G. Funding: This work was funded by the Fundamental Research Funds for the Central Universities Funding: This work was funded by the Fundamental Research Funds for the Central Universities (2017XKZD02). Conflicts of Interest: The authors declare no conflict of interest. results can improve the performance for TC18 machining by ASJ and guide actual production. Author Contributions: conceptualization, J.L. and C.G.; methodology, J.L.; software, J.L.; validation, J.L., X.Z. and F.W.; formal analysis, J.L.; investigation, J.L.; resources, C.G.; data curation, Q.X.; writing—original draft e a atio J L iti e ie a d editi J L a d C G i uali atio J L u e i io C G oje t Author Contributions: Conceptualization, J.L. and C.G.; methodology, J.L.; software, J.L.; validation, J.L., X.Z. and F.W.; formal analysis, J.L.; investigation, J.L.; resources, C.G.; data curation, Q.X.; writing—original draft preparation, J.L.; writing—review and editing, J.L. and C.G; visualization, J.L.; supervision, C.G.; project administration, C.G and J.L.; funding acquisition, C.G. aration, J.L.; writing review and editing, J.L. and C.G; visualization, J.L.; supervision, C.G.; projec inistration, C.G and J.L.; funding acquisition, C.G. ding: This work was funded by the Fundamental Research Funds for the Central Universities (2017XKZD02) Funding: This work was funded by the Fundamental Re Conflicts of Interest: The authors declare no conflict of interest. 7. Conclusions 7. Conclusions and F.W.; formal analysis, J.L.; investigation, J.L.; resources, C.G.; data curation, Q.X.; writing—original draft ti J L iti i d diti J L d C G i li ti J L i i C G j t Author Contributions: Conceptualization, J.L. and C.G.; methodology, J.L.; software, J.L.; validation, J.L., X.Z. and F.W.; formal analysis, J.L.; investigation, J.L.; resources, C.G.; data curation, Q.X.; writing—original draft preparation, J.L.; writing—review and editing, J.L. and C.G; visualization, J.L.; supervision, C.G.; project administration, C.G and J.L.; funding acquisition, C.G. preparation, J.L.; writing review and editing, J.L. and C.G; visualization, J.L.; supervision, C.G.; administration, C.G and J.L.; funding acquisition, C.G. 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Preparation of novel β-CD/P(AA-<i>co</i>-AM) hydrogels by frontal polymerization
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Preparation of novel β-CD/P(AA-co-AM) hydrogels by frontal polymerization Bin Li  (  libin_027@126.com ) Wuhan Polytechnic University Haibo Qin  Wuhan Polytechnic University Ming Ma  Wuhan Polytechnic University Xiaojia Xu  Wuhan Polytechnic University Mengjing Zhou  Wuhan Polytechnic University Wenrui Hao  Wuhan Polytechnic University Zhigang Hu  Wuhan Polytechnic University Research Article Keywords: Frontal polymerization, Hydrogel, β-cyclodextrin, Drug release, Deep eutectic solvent Posted Date: November 9th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2243374/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. d ll License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/20 Page 1/20 Page 1/20 Page 1/20 Abstract In this paper, betaine (Bet) was used as a hydrogen bond acceptor (HBA), acrylic acid (AA) and acrylamide (AM) were used as hydrogen bond donors (HBD) and mixed to form a deep eutectic solvent (DES). Different concentrations of β-cyclodextrin (β-CD) were dispersed in DES, and a novel β-CD/P(AA- Co-AM) hydrogel was prepared by frontal polymerization (FP). Characteristic structure and morphology of the hydrogels were analyzed using Fourier infrared spectroscopy (FTIR) and scanning electron microscopy (SEM), and the properties of the hydrogels were investigated. The results show that the mechanical properties of the hydrogel were improved by β-CD acting as a second cross-linking agent in the polymerization process, thus increasing the cross-link density of the hydrogel. Because the carboxyl groups contained in acrylic acid dissociate under alkaline conditions, the composite hydrogel shows excellent pH responsiveness under alkaline conditions. Tetracycline hydrochloride was used as a drug model to test the drug loading and drug release performance of hydrogels. With the increase of β-CD content, the loading capacity of hydrogels to tetracycline hydrochloride gradually increased. The data of drug release indicated that the hydrogel has good drug delivery performance and has promising applications in drug delivery systems and other areas. 1. Preface Hydrogel is a cross-linked polymer with a three-dimensional network structure that can absorb a large number of water molecules without being soluble in water, and can have both solid and liquid properties. It has mechanical strength and a wide range of stimulus responsiveness1–3 and has physicochemical properties suitable for use in the human body4. Its unique network structure and properties can be used in many applications, such as in biomedicine5, drug delivery6–8, tissue engineering9,10 and other fields. Typically, hydrogel networks are constructed from hydrophilic polymers, giving them a good ability to bind hydrophilic drugs11–13. However, the inability of the polymer network of hydrogels to bind drugs into the hydrogel leads to the inability of the hydrogel to control the drug release behavior, limiting the application of hydrogels for drug delivery. β-cyclodextrin (β-CD) is a cyclic oligosaccharide with a hydrophobic cavity inside, allowing organic or inorganic molecules can be trapped14. The non-polar hydrophobic cavity of β-CD can absorb drug molecules into the hydrophobic cavity, forming an inclusion complex known as host-guest15.β-CD can be grafted through polymerization of β-CD to macromolecular chains, thus allowing the unique ability to form inclusion complexes to be transferred to the hydrogel. Moreover, the hydrophilic network of the hydrogel enhances the biocompatibility of β-CD and improves the stability of the inclusion complex, while β-CD enhances the mechanical of the hydrogel, and changes the release behavior of the drug, which corresponds to an increase in the water solubility of the drug molecules and an increase in the drug loading capacity. Thus, β-CD is suitable for overcoming the limited absorption of hydrophobic drugs and the control of hydrophilic drug release16, 17. The introduction of β-CD into polymeric matrices as polymeric fillers offers the possibility of preparing hydrogels with more excellent properties. Page 2/20 Page 2/20 Hydrogels have various polymerization methods, such as suspension polymerization18 and emulsion polymerization19, However, these polymerization methods have disadvantages such as difficult process control and impact on polymer performance. Frontal polymerization (FP) is primarily the conversion of monomers into polymers by the formation and propagation of thermal polymerization fronts using in situ self-propagation techniques and the ability to self-sustain and propagate in the region of the monomer mixture20. Compared with other polymerization methods, FP has process advantages such as shorter time, lower energy consumption, and no waste emission21. 2.1 Materials acrylamide (AM), Betaine (Bet), β-cyclodextrin (β-CD) and acrylic acid (AA) were bought from Shanghai Aladdin Biochemical Technology Co. N,N-methylene bisacrylamide (MBA) was obtained from Tianjin Comio Chemical Reagent Co. The potassium persulfate (KPS) was obtained from Sinopharm Chemical Reagent Co. All the reagents were of analytical grade and did not need further purification; the water used in this experiment was ultrapure water. 1. Preface In recent years, frontal polymerization has synthesized numerous polymer hydrogels, such as poly (itaconic acid-acrylic acid-acrylamide) hydrogels22 and poly (acrylic acid-acrylamide) / activated carbon23. Deep eutectic solvent (DES) is a novel ionic liquid formed by mixing hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD)24. Its preparation method is relatively simple, only quaternary ammonium salts and HBD compounds are stirred at a certain temperature to form a eutectic mixture to obtain high purity DES. DES has low volatility, thermal stability, high electrical conductivity and good biocompatibility, which greatly facilitates the preparation of polymer hydrogels with excellent properties25–27. Based on previous research, we prepared a novel β-CD/P(AA-co-AM) hydrogel with good drug release properties by using frontal polymerization in DES, and investigated the effect of β-CD content on the properties associated with the novel hydrogel. We first prepared a ternary DES composed of acrylic acid- betaine-acrylamide, and then prepared β-CD/P(AA-AM) novel hydrogels by dispersing β-CD in the DES, Fourier Transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) were used to characterize its structure, and further analyzed the effect law of β-CD on the mechanical properties and drug release properties of the hydrogels. 2.5 Characterization and testing of hydrogels Before characterization, the hydrogels after complete soaked were dried in a freeze dryer at -60°C for 48 h to obtain dry gel samples. The spectral characteristics were analyzed using Fourier infrared spectroscopy between wave number 500 and 4000 cm− 1. The cross-sections of the dried hydrogels were sprayed with gold using a high vacuum ion sputterer, and the microstructure of the hydrogels after gold spraying was observed by scanning electron microscopy (SEM). 2.3 Hydrogel preparation by frontal polymerization The crosslinker and initiator were included in the mixture of DES and β-CD, mixed well and moved to a test tube (10 mm in diameter and 100 mm in length) and left for some time to remove the air bubbles generated during the stirring process. The heated electric soldering iron was leaned against the upper face of the solution for thermal triggering, and the upper end of the reactor was kept under atmospheric pressure, and the soldering iron was removed when the frontal appeared and the polymerization reaction started. After the reaction is completed, the prepared hydrogel is removed, cut into uniform discs and soaked in deionized water for one week to dissolve the unreacted monomers. The obtained novel hydrogels were dried in a freeze-dryer until the quality was constant and stored for the next test. 2.4 Frontal speed and temperature measurement In the FP reaction, the front end of the reaction moves toward the unreacted region at a constant rate, and the rate of frontal movement can be derived by recording the change in front end position at different times. To monitor the front-end temperature in relation to time, a test tube was held in a gripper at room temperature and a K-type thermocouple with a digital thermometer was dipped into the liquid to record the temperature of the front-end during the reaction. 2.2 Synthesis of DES Bet was used as HBA and AM and AA as HBD. the three raw materials were mixed in a molar ratio of 1:2:2 in a collector-type thermostatic heating magnetic stirrer with constant stirring until a uniform and transparent DES was formed. the synthesized DES was allowed to stand until there were no air bubbles, and then β-CD was added to the DES according to the ratio in Table 1 with thorough stirring. Page 3/20 Page 3/20 Table 1 Composition and proportioning of hydrogels Samples AA/AM/Bet (molar ratio) β-CD(wt%) MBA(wt%) KPS(wt%) FP0 2:2:1 0 1 0.5 FP1 2:2:1 0.25 1 0.5 FP2 2:2:1 0.50 1 0.5 FP3 2:2:1 1.00 1 0.5 2.5.1 Mechanical performance testing A universal testing machine was used to test the stress-strain properties of the composite hydrogel, and the prepared hydrogel samples were stretched at a speed of 100 mm/min until the hydrogel fractured, Page 4/20 Page 4/20 and the test was repeated several times. The hydrogel was tested for compression resistance using TA.XTC-18 texture analyzer. Before the test, the hydrogel was cut into a cylindrical shape of 10mm in diameter and 10 mm in length, then immersed in deionized water for a period of time and compressed at a compression rate of 0.2 mm/s until the deformation of the hydrogel reached 80%, and the test was repeated several times. The formula for calculating the compressive strength of hydrogel to Eq. (1). he formula for calculating the compressive strength of hydrogel to Eq. (1). P = F S P = F S 1 F is the applied force and S is the cross-sectional area of the hydrogel. F is the applied force and S is the cross-sectional area of the hydrogel. 2.5.2 PH responsiveness performance test of hydrogel Buffer solutions with pH 2.4, 4.5 (sodium citrate/citric acid) and pH 9.4, 10.7 (sodium carbonate/sodium bicarbonate) were prepared, and the pH of the buffer solutions was accurately tested with a digital pen acidity meter (PH-208, accuracy 0.01). The dry hydrogel with a mass of about 30mg was put into the buffer solution until the swelling equilibrium was reached, and the water on the surface of the hydrogel was drained and weighed, and the equation for the swelling equilibrium (SR) was calculated to Eq. (2): SR = mt −m0 m0 m0 2 mt is the swell weight of the hydrogel at different pH, m0 is the dry weight of the hydrogel 2.5.3 Drug loading The dry gel was immersed in 40 ml (10 mg/ml) of tetracycline hydrochloride solution at 37°C until the weight of the hydrogel did not change, the hydrogel was removed from the solution and the solution on the surface of the hydrogel was absorbed using filter paper, and the absorbance of the drug solution before and after the immersion of the hydrogel was measured to calculate the amount of drug absorbed. 3.1 Measurement of frontal fronts The frontal polymerization reaction creates a frontal front, which is an interface between the polymer produced by the reaction and the unreacted monomer. As illustrated in Fig. 3(a), the frontal position versus time curve shows that the frontal front moves toward the monomer region at a constant rate and completes the polymerization reaction rapidly in less than 6 min. As can be observed in Fig.(b), the frontal temperature change curve has a nearly horizontal section at the beginning, indicating that spontaneous polymerization has not occurred in FP28. As the β-CD content in the hydrogel increases, the movement of the frontal front decreases gradually, and Vf decreases from 2.82 to 1.41 cm/min when the β-CD content increases from 0 to 1wt%. The FP maximum temperature decreased from 168.7°C to 142.3°C. The increase of β-CD content decreased the Vf value because β-CD as an inert substance in the polymerization reaction would lead to thermal dispersion, which decreased the polymerization reaction temperature, thus slowing down the reaction rate, and the Tmax value also decreased29. Table 2 Parameters of frontal polymerization Sample β-CD (wt%) Tmax (℃) Vf (cm/min) FP0 0 168.7 2.82 FP1 0.25 160.6 2.18 FP2 0.50 150.3 1.92 FP3 1.00 142.3 1.41 Table 2 Parameters of frontal polymerization Sample β-CD (wt%) Tmax (℃) Vf (cm/min) FP0 0 168.7 2.82 FP1 0.25 160.6 2.18 FP2 0.50 150.3 1.92 FP3 1.00 142.3 1.41 2.5.4 Drug release performance testing The fully drug loaded hydrogels were placed into 60 ml of deionized water at 37°C. After a certain interval, 5 ml of drug release solution was taken out. The absorbance of the solution was measured at 357 nm with a UV-5900PC UV-visible spectrophotometer, and the solution was poured back into the container immediately after measurement. Linear regression was used to establish a pharmacokinetic model to study the drug release performance of hydrogels, and to calculate the cumulative release of drugs loaded on hydrogels. Page 5/20 The drug release is calculated by the following equation is Eq. (3). The drug release is calculated by the following equation is Eq. (3). The drug release is calculated by the following equation is Eq. (3). Cumulative Release(%) = × 100% Wdrug Wgel 3 In Eq. (3), Wdrug is the drug release at different times; Wgel is the total drug loading of the hydrogel. In Eq. (3), Wdrug is the drug release at different times; Wgel is the total drug loading of the hydrogel. 3.3 Microscopic morphological of composite hydrogels (SEM) To study the effect of β-CD on the internal structure of P(AA-AM) hydrogels, SEM observations were performed on the hydrolyzed composite hydrogels. Before performing SEM on the hydrogels, the hydrogels were pre-frozen after one week of immersion and subsequently freeze-dried at -60°C for 48 h. After the freeze-drying treatment, the SEM scans of the four sets of hydrogels, the morphology is shown in Fig. 5. The cross section of FP0 appears to have folds, which may be caused by the collapse of the polymer network and the contraction of the structure during freezing. When the ice sublimates from the hydrogel, the flexible polymer chains in the hydrogel come into contact with each other, resulting in a collapsed hydrogel network35, 36. The addition of β-CD makes the cross section of the hydrogel dense and smooth, which is due to the increased cross-link density of β-CD during polymerization, which contributes to the formation of a denser polymer network in the hydrogel, resulting in the contact between polymer chains becoming more frequent and the structure of the hydrogel becoming more dense. 3.2 Fourier Infrared Spectroscopy (FTIR) To further analyze the hydrogel condition, the hydrogel was analyzed by infrared spectroscopy, and the results are shown in Fig. 4. Figure 4(a) shows the FTIR spectral profile of AM, the absorption peak at 3338 cm− 1 is the stretching vibration peak of -NH2 on the amide group (-CONH2), and the absorption peak at 1664 cm− 1 is the stretching vibration peak of C = O and the C = C vibration peak30, 31. From Fig. 4(b), it Page 6/20 Page 6/20 can be seen that there is a strong absorption peak of P(AA-AM) at 3438 cm− 1, which corresponds to the stretching vibration peak of -NH2 stretching vibration peak, and the absorption peak at 1647 cm− 1 corresponds to the stretching vibration peak of C = O and C = C vibration peak. And the absorption peak at 2927 cm− 1 is an asymmetric vibration of the C-H band, which is caused by the cleavage of C = = C in acrylamide32. The absorption peak present at 1449 cm− 1 is a symmetric stretching peak formed by the dissociation of the hydroxyl group of AM into COO− during the polymerization process33. The IR spectral curve of β-CD shows that the absorption peak at 3388 cm− 1 is the O-H stretching vibration peak, and the absorption peak at 1368 cm− 1 is the bending vibration peak of O-H34. From the FTIR spectral profile of P(AA-AM)/β-CD, it can be seen that there are a large number of absorption peaks identical to those of P(AA-AM), but a stronger absorption peak appears at 1374 cm− 1, which corresponds to the bending vibration peak generated by O-H in β-CD. The above results indicate that β-CD enters in the polymer network of P(AA-AM) hydrogel. 3.4 PH responsive The effect of pH buffer solution on the dissolution equilibrium behavior of hydrogels is presented in Fig. 7. As can be observed in Fig. 7, the trend of hydrogel changes remained basically the same in solutions with different pH values. At pH 2.4, the carboxylic acid groups exist in the form of -COOH, which reduces the electrostatic repulsive force of the polymer chains and causes the polymer chains of the whole hydrogel network to be intertwined and contracted, so the swelling capacity of the hydrogel is low at low this environment40. When the pH is at 4.8 environment, the functional groups start to dissociate, the hydrogen bonding between -COOH groups is weakened, and the osmotic pressure inside the hydrogel gradually increases, leading to an increase in ESR33. The reason for the reduced swelling behavior of the hydrogel near pH = 7 may be due to the hydrogen bonding present within the molecules of -CONH2 groups, which interacts with the hydrogen bonding between the molecules of carboxylic acid groups leading to an increase in cross-link density, resulting in the contraction of the polymer network31. When the pH is greater than 7, the carboxylic acid groups ionize, -COOH dissociates into -COO−, and the ions between generates strong electrostatic repulsion, which expands the polymer network and thus leads to an increase in osmotic pressure inside the hydrogel, resulting in an increase in the swelling rate of the hydrogel. As can be show in Fig. 7, the swelling rate of the hydrogel gradually decreases with the increase of β-CD content, which is because the increase of β-CD content increases the cross-link density of the hydrogel, while the hydrophobic cavity of β-CD can prevent water molecules from penetrating into the hydrogel, thus limiting the swelling performance of the hydrogel41. 3.4 Mechanical properties In order to test the mechanical properties of the hydrogels, tensile and compression experiments were performed on the hydrogels, and the experimental results are shown in Fig. 6. From Fig. 6(a), we can see that the tensile strength of the hydrogel gradually increases with the increase of β-CD in the hydrogel, and the highest tensile strength of FP3 hydrogel can reach 2.0 MPa, which is about two times of that of FP0 hydrogel. Figure 6(b) shows the compression curve of the hydrogel, and the compressive strength of the hydrogel increases with the increase of β-CD. The above results show that the introduction of β-CD enhances the mechanical properties of the composite hydrogels, which is due to the role of β-CD as a chemical cross-linker in the polymerization process. The increase of β-CD content in the hydrogel network increases the cross-link density of the hydrogel. The higher the crosslinking density of the hydrogel, the Page 7/20 Page 7/20 denser the gel network formed, the more hydrogen bonds formed within and between molecules, and the stresses were dispersed at the fractures, resulting in the enhancement of the mechanical strength of the hydrogel37, 38. The mechanical properties of hydrogels can usually be enhanced by increasing the concentration of hydroxyl groups of the constituent groups of polymeric materials, so the mechanical properties of hydrogels were also improved with the addition of β-CD39. 3.5 Drug loading of hydrogels We chose tetracycline hydrochloride as the model drug and loaded it into the hydrogel. Figure 8 shows the drug loading data of the hydrogel, the drug loadings of FP0, FP1, FP2 and FP3 were 63.51, 75.61, 115.63 and 154.67 mg/g, with the increase of β-CD content, the loading of the hydrogel to the drug increased. Figure 9 shows a schematic diagram of the uptake of drug molecules by the hydrogel. β- CD/P(AA-co-AM) composite hydrogel is loaded with drug mainly by two ways. One is, the hydrogel swelling leads to the drug-containing solution into the hydrogel network; the other is, the hydrophobic cavity of β-CD can bind to the hydrophobic end of tetracycline hydrochloride, so that β-CD can form host- guest inclusion complexes with drug molecules, increasing the affinity of the polymer network for drug molecules and increasing the number of drug molecules captured by the hydrogel, thereby enhancing the drug adsorption capacity of the hydrogel42, 43. The increase in β-CD content in the hydrogels allowed more drugs to be loaded into the polymer network, improving the drug loading of the hydrogels. Page 8/20 Page 8/20 3.6 Drug release of hydrogels The cumulative drug release rate of the hydrogel is shown in Fig. 10 by performing drug release tests on the hydrogel, thereby investigating the effect produced by β-CD on drug release. In drug release studies, when deionized water penetrates into the polymer matrix, the trapped drug molecules are released from the hydrogel due to diffusion that occurs due to osmotic pressure. As can be shown in Fig. 10, the drug release efficiency of the hydrogels showed a significant difference with the increase of β-CD content in the hydrogels, and the drug release rate gradually decreased. At the beginning, the greater osmotic pressure between the drug molecules in the hydrogel network and the external environment resulted in a greater drug release velocity from the hydrogel within 600 min, while after 600 min, the drug release velocity from the hydrogel started to decrease. β-CD content increases the cross-link density of the hydrogel and makes the polymeric network of the polymer more compact, resulting in a restricted rate of water entry into the hydrogel, thus making the drug molecule's slow release time becomes longer44–46. 4. Conclusion In this paper, β-CD was mixed with Bet-AA-AM ternary DES to prepare new β-CD/P(AA-co-AM) hydrogels by front-end polymerization, and the structure and properties of the hydrogels were investigated, and the results showed that: (1) Compared with the conventional polymerization method, FP prepared composite hydrogels with faster polymerization rate and greener raw materials. A new hydrogel of β-CD/P(AA-AM) was prepared by frontal polymerization, and after the addition of β-CD, the polymerization rate of the hydrogel gradually decreased due to the thermal diffusion of β-CD. (1) Compared with the conventional polymerization method, FP prepared composite hydrogels with faster polymerization rate and greener raw materials. A new hydrogel of β-CD/P(AA-AM) was prepared by frontal polymerization, and after the addition of β-CD, the polymerization rate of the hydrogel gradually decreased due to the thermal diffusion of β-CD. (2) The mechanical properties of the hydrogels were gradually improved with the addition of β-CD. The addition of β-CD increased the cross-link density of the hydrogels and increased the number of hydrogen bonds in the hydrogels, which led to the improvement of the mechanical properties of the hydrogels. (2) The mechanical properties of the hydrogels were gradually improved with the addition of β-CD. The addition of β-CD increased the cross-link density of the hydrogels and increased the number of hydrogen bonds in the hydrogels, which led to the improvement of the mechanical properties of the hydrogels. (3) β-CD controls the release behavior of drug molecules by forming host-guest inclusion complexes. When β-CD is added to the hydrogel it can make the hydrogel have drug retardation, and after loading the composite hydrogel with drug, it can achieve prolonged release in deionized water, making the hydrogel potentially useful for applications such as drug delivery systems. Acknowledgements The work is supported by 2022 Knowledge Innovation Dawn Special Plan Project (2022010801020393), Marine Defense Technology Innovation Center Innovation Fund (JJ-2020-719-01), Natural Science Foundation of Hubei Province (2021CFB292) and Research and Innovation Initiatives of WHPU (2022J04). We are grateful for the technical support from the Wuhan Polytechnic University Conflict of interest The authors declare that they have no relevant financial or non-financial interests to disclose. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by BL, HQ, MM, XX, MZ, WH and ZH. The first draft of the manuscript was written by HQ and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Page 9/20 References 1. 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Figure 1 Molecular formula for the formation of DES Molecular formula for the formation of DES Page 13/20 Figure 2 Schematic diagram of the synthesis of novel hydrogels Figure 3 (a) Speed of hydrogel polymerization frontal; (b) temperature of hydrogel polymerization frontal Figure 2 Schematic diagram of the synthesis of novel hydrogels Figure 2 Figure 2 Schematic diagram of the synthesis of novel hydrogels Figure 3 (a) Speed of hydrogel polymerization frontal; (b) temperature of hydrogel polymerization frontal Figure 3 (a) Speed of hydrogel polymerization frontal; (b) temperature of hydrogel polymerization frontal (a) Speed of hydrogel polymerization frontal; (b) temperature of hydrogel polymerization frontal Page 14/20 Page 14/20 Figure 4 (a) FTIR plots of acrylamide (AM); (b) FTIR plots of β-CD, P(AA-AM) and P(AA-AM)/β-CD Figure 4 Figure 4 (a) FTIR plots of acrylamide (AM); (b) FTIR plots of β-CD, P(AA-AM) and P(AA-AM)/β-CD (a) FTIR plots of acrylamide (AM); (b) FTIR plots of β-CD, P(AA-AM) and P(AA-AM)/β-CD (a) Page 15/20 Figure 5 Figure 5 SEM images of FP0 (a), FP1 (b), FP2 (c) and FP3 (d) for the freeze-dried hydrogels SEM images of FP0 (a), FP1 (b), FP2 (c) and FP3 (d) for the freeze-dried hydrogels Page 16/20 Figure 6 (a) Hydrogel tensile property test curve; (b) Hydrogel compression property test curve (a) Hydrogel tensile property test curve; (b) Hydrogel compression property test curve (a) Hydrogel tensile property test curve; (b) Hydrogel compression property test curve (a) Page 17/20 Figure 7 pH responsiveness curve of hydrogels Figure 7 Figure 7 pH responsiveness curve of hydrogels pH responsiveness curve of hydrogels Page 18/20 Page 18/20 Page 19/20 Figure 8 Drug loading of hydrogels Figure 9 Figure 8 Figure 8 Page 19/20 Drug loading of hydrogels Figure 9 Page 19/20 Drug loading of hydrogels Figure 9 Drug loading of hydrogels Figure 9 Schematic diagram of the absorption of drug molecules by hydrogels Schematic diagram of the absorption of drug molecules by hydrogels Schematic diagram of the absorption of drug molecules by hydrogels Figure 10 Drug release profile of hydrogels Figure 10 Drug release profile of hydrogels Page 20/20
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Facies distribution and depositional cycles in lacustrine and palustrine carbonates: The Lutetian–Aquitanian record in the Paris Basin
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To cite this version: Kévin Moreau, Simon Andrieu, Justine Briais, Benjamin Brigaud, Magali Ader. Facies distribution and depositional cycles in lacustrine and palustrine carbonates: The Lutetian–Aquitanian record in the Paris Basin. Depositional Record, 2024, 10 (1), pp.124-158. ￿10.1002/dep2.264￿. ￿hal-04372428￿ Distributed under a Creative Commons Attribution 4.0 International License O R I G I N A L R E S E A R C H A R T I C L E O R I G I N A L R E S E A R C H A R T I C L E Correspondence Kévin Moreau, CNRS, GEOPS, Université Paris-Saclay, Orsay Cedex, France. Email: kevin.moreau.arnage@gmail. com Kévin Moreau1   | Simon Andrieu2,3,4  | Justine Briais2  | Benjamin Brigaud1,5  | Magali Ader6 1Université Paris-Saclay, CNRS, GEOPS, Orsay Cedex, France 2BRGM, Orléans, France 3Department of Geoscience, Aarhus University, Aarhus C, Denmark 4Laboratoire de Géologie de Lyon: Terre, Planètes et Environnement, CNRS, Ecole Normale Supérieure de Lyon, Université de Lyon 1, Villeurbanne, France 5Institut universitaire de France (IUF), Paris, France 6Institut de physique du globe de Paris, CNRS, Université Paris Cité, Paris, France 1Université Paris-Saclay, CNRS, GEOPS, Orsay Cedex, France 2BRGM, Orléans, France 3Department of Geoscience, Aarhus University, Aarhus C, Denmark 4Laboratoire de Géologie de Lyon: Terre, Planètes et Environnement, CNRS, Ecole Normale Supérieure de Lyon, Université de Lyon 1, Villeurbanne, France 5Institut universitaire de France (IUF), Paris, France 6Institut de physique du globe de Paris, CNRS, Université Paris Cité, Paris, France Correspondence Kévin Moreau, CNRS, GEOPS, Université Paris-Saclay, Orsay Cedex, France. Email: kevin.moreau.arnage@gmail. com Funding information 1Université Paris-Saclay, CNRS, GEOPS, Orsay Cedex, France 2BRGM, Orléans, France 3Department of Geoscience, Aarhus University, Aarhus C, Denmark 4Laboratoire de Géologie de Lyon: Terre, Planètes et Environnement, CNRS, Ecole Normale Supérieure de Lyon, Université de Lyon 1, Villeurbanne, France This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2023 The Authors. The Depositional Record published by John Wiley & Sons Ltd on behalf of International Association of Sedimentologists. |  1 wileyonlinelibrary.com/journal/dep2 Abstract Abstract The difficulty of correlating continental deposits hinders predicting lacustrine and palustrine carbonate facies variations in time and space. This study aims to understand better the factors governing these facies heterogeneities by measur- ing carbonate isotopes and conducting facies, petrographic and sequence strati- graphic analyses of the Lutetian–Aquitanian deposits of the Paris Basin, that record the transition from marine to lacustrine environments. Large-scale cor- relations enabled the definition of two lacustrine–palustrine carbonate facies models. (1) The coastal lacustrine system (Bartonian to Rupelian), consists of fine-grained brackish carbonate exhibiting episodic marine inputs during short- term relative sea-level maxima and evaporite sedimentation during relative sea- level minima. Lacustrine sediments differ notably from marine ones with more negative δ13C and δ18O compositions that co-vary and a biota adapted to low salinity conditions. In the associated palustrine environment, depositional se- quences evolve upwards from micritic lacustrine deposits to nodular and then laminar calcretes. Microbial-coated grains and rhizoliths indicate biological pro- cesses during repeated subaerial exposure phases in sub-tropical to arid climates. (2) The inland lacustrine system (Rupelian and Aquitanian) was disconnected from the marine domain and showed evidence of microbial activity with micro- bial crusts and oncoidal rudstones. Facies rich in micritic intraclasts composed of palustrine and lacustrine facies indicate the reworking of already lithified sedi- ments along the margins. In the palustrine domain, the calcrete facies are less abundant than breccias formed in-situ by desiccation, limestones with root traces, or organic-rich wackestones and marls. This system reflects a more temperate cli- mate with more developed microbial structures and less exposed carbonates than the coastal lacustrine system. The southward migration of the depocentre and the transition from marine environments to (1) coastal and then (2) inland sys- tems are controlled by uplift phases induced by Pyrenean and Alpine orogenesis. Depositional Rec. 2023;00:1–35. HAL Id: hal-04372428 https://hal.science/hal-04372428v1 Submitted on 4 Jan 2024 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Received: 10 July 2023  |  Revised: 15 November 2023  |  Accepted: 18 November 2023 DOI: 10.1002/dep2.264 Received: 10 July 2023  |  Revised: 15 November 2023  |  Accepted: 18 November 2023 DOI: 10.1002/dep2.264 Received: 10 July 2023  |  Revised: 15 November 2023  |  Accepted: 18 November 2023 Facies distribution and depositional cycles in lacustrine and palustrine carbonates: The Lutetian–Aquitanian record in the Paris Basin u1   | Simon Andrieu2,3,4  | Justine Briais2  | Benjamin Brigaud1,5  | Kévin Moreau1   | Simon Andrieu2,3,4  | Justine Briais2  | Benjamin Brigaud1,5  | Magali Ader6 K E Y W O R D S Cenozoic, facies model, lacustrine, non-marine carbonate, palustrine, Paris Basin, stratigraphy K E Y W O R D S Cenozoic, facies model, lacustrine, non-marine carbonate, palustrine, Paris Basin, stratigraphy Funding information Funding information Bureau de Recherches Géologiques et Minières, Grant/Award Number: 2019-157; French Ministry of Research and Higher Education, Grant/Award Number: 2019-105 g Bureau de Recherches Géologiques et Minières, Grant/Award Number: 2019-157; French Ministry of Research and Higher Education, Grant/Award Number: 2019-105 2  | 2 MOREAU et al. 1  |  INTRODUCTION Kendall (1985) suggest that the sulphate supply leading to gypsum precipitation in coastal salinas/playas comes from marine groundwater. The impact of the marine do- main on coastal lacustrine and palustrine sedimentation can therefore play a significant role in sedimentation pro- cesses and requires further investigation. In continental systems, facies vary significantly, both spa- tially and temporally, making it difficult to predict and model them. This heterogeneity is partly explained by the fact that continental environments are more sensitive than marine environments to changes in accommoda- tion space (Allen & Collinson, 1986; Bohacs et al., 2000). Variation in accommodation space is directly controlled by (1) vertical tectonic movements induced by subsidence and uplifts and (2) climate, which alters lake levels and sedimentation rates by varying inflow/evaporation ratios (Allen & Collinson,  1986; Bohacs et  al.,  2000; Alonso- Zarza,  2003). The respective influences of climate and tectonics on accommodation space and sedimentary fa- cies variations are still difficult to disentangle, not least because of the incomparably more significant carbonate facies heterogeneity than that found on marine platforms (Della Porta,  2015; De Boever et  al.,  2017; Capezzuoli et al., 2022). Despite their significant variability, it is dif- ficult to discriminate among deposits from nearby envi- ronments, such as palustrine and pedogenic facies due to similar sedimentary features (roots, desiccation cracks, etc.; Freytet & Plaziat,  1982; Wright & Tucker,  1991; Tandon & Andrews, 2001; Alonso-Zarza, 2003; MacNeil & Jones, 2006; Brasier, 2011). Another question related to the impact of the marine domain on continental environments concerns the abil- ity or otherwise to correlate marine and non-marine sed- imentary series. Indeed, the sedimentation processes on continental systems are largely controlled by autocycli- cal parameters (river inflow and discharge, local climate, aquifer-level evolution, glacial storage, tides for very large lakes…) all depending on the evolution of continental systems controlled by allocyclical parameters (tectonics and climate; Allen & Collinson, 1986). Eustatic variations should not affect lakes or marshes. Nonetheless, MacNeil and Jones (2006) show that palustrine carbonates devel- oped during sea-level fall in ancient coastal areas. In the same way, Platt and Wright (2023) suggest that palustrine deposits occurring in inland basins may have been influ- enced by sea level due to ephemeral connections with the marine domain. The coastal realm, even if predominantly non-marine, could therefore exhibit cyclicity similar to that of the marine realm. 2 The Paris Basin is an intracratonic sedimentary basin filled by Triassic to Quaternary deposits above a Cadomian to Variscan basement (Guillocheau et al., 2000). During the Cenozoic, a low subsidence phase occurred along E–W axes of subsidence until the late Eocene while NE–SW subsidence continued until the Miocene (Guillocheau et al., 2000; Briais, 2015). A number of compressive struc- tures such as NW–SE-orientated folds and faults were active during these periods related to the Pyrenean col- lision, while NE–SW flexure is interpreted as a response to the Alpine collision (Robin et  al.,  1998; Guillocheau et al., 2000; Bourgeois et al., 2007; Briais, 2015). The Cenozoic carbonate deposits of the Paris Basin formed in marine and non-marine settings from the Lutetian to the Aquitanian (Eocene, Oligocene, Miocene). Their bio- stratigraphic fauna and flora have been closely studied, pro- viding an excellent stratigraphic framework for answering some of the questions raised above (Abrard, 1925; Blondeau et  al.,  1965; Cavelier,  1969; Turland,  1974; Pomerol & Riveline, 1975; Gitton et al., 1986). However, most previous facies and stratigraphic works on the Cenozoic sedimen- tary rocks of the Paris Basin have focussed on the coastal and marine domains (Gély & Lorenz, 1991; Briais, 2015; Briais et al., 2016). Only a few (albeit detailed) studies have concentrated on interpreting non-marine carbonate facies (Ménillet, 1974; Guillemin, 1976; Freytet & Plaziat, 1982), where no magnetostratigraphy or chemostratigraphy is available. This study has the objective to determine fa- cies and the depositional environments of both marine and non-marine Lutetian-Aquitanian carbonates of the Paris Basin. The relative controls of climate, tectonics and proximity of the coastline to the variety and distribu- tion of non-marine carbonate facies are then discussed. For this purpose, a field and petrographic study was con- ducted to define facies and depositional environments. Based on pre-existing biostratigraphic data and by extend- ing the pre-existing sequence stratigraphic framework to new outcrops and boreholes (Delhaye-Prat et  al.,  2005; Briais,  2015), large-scale cross-sections are proposed to reconstruct the geometries from the marine to lacustrine or palustrine domains. To constrain the hydrology and the type of water (marine vs. meteoric) where carbonate facies formed, oxygen and carbon stable isotopic data on carbon- ates were acquired. The main contribution of this study is to propose two facies models for lacustrine–palustrine environments corresponding to the newly defined coastal lake and inland lake systems. 1  |  INTRODUCTION ey.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Furthermore, there is difficulty in dating non-marine deposits due to (1) the poor preservation of fossils, (2) the scarcity of good stratigraphic fossils, and (3) the abun- dance of sedimentary hiatuses. This creates difficulty in establishing correlations in non-marine carbonates that prevent the precise characterisation of sedimentary archi- tectures, further reinforcing the difficulty in understand- ing and predicting their facies continuity in the continental domain. Numerous studies have focussed on spatially constraining non-marine facies in extensional contexts, such as in lacustrine rift basins, where the difference in subsidence rates leads to profiles with steep slopes (Platt & Wright, 1991; Thompson et al., 2015). In these contexts, shallow high-energy platforms can develop on topographic highs, displaying shell or oncoid accumulations, ooid shoals, mudflats, or wave-dominated sedimentary fea- tures (Platt & Wright, 1991; Mercedes-Martín et al., 2014; Thompson et al., 2015; Deschamps et al., 2020; Lettéron et  al.,  2022). There is less literature on basins in more stable tectonic contexts, such as intracratonic basins. In Non-marine carbonates represent, by definition, “car- bonate sediments that form and may be syn-deposition- ally transformed (“diagenetically altered”) under the strong influence of meteoric waters, including situations with various degrees of mixing with seawater, evaporative or basinal fluids” (De Boever et al., 2017). This definition, although generic, has limitations in ancient coastal envi- ronments since it lacks criteria for determining in which environments carbonates were deposited: non-marine (lacustrine, palustrine) or marine (lagoons). The coasts of the Yucatán, Florida and Bahamas platforms are a perfect example of the close spatial relationship that can exist between non-marine (mainly palustrine, brackish to freshwater environments) and marine (lagoon, beach bar- riers, reefs …) carbonates (Platt & Wright, 2023). Moreover, foraminifera have even been found in Cenozoic and Quaternary lake deposits, attesting a marine water influ- ence (Dye & Barros, 2005; Strotz, 2015; Lettéron et al., 2017; Pint et  al.,  2017; Fritz et  al.,  2018), while  Warren and |  3 MOREAU et al. 3 these cases, depositional profiles are flatter and smoother, and the lake margins are more subject to variation in the lake water level that strongly depends on climate (Platt & Wright, 1991; Bohacs et al., 2000; Alonso-Zarza, 2003). depocentre to the palustrine setting. Moreover, evidence is produced that both climate and geographic location con- trol facies type and heterogeneity. 1  |  INTRODUCTION While depositional models have already been pro- posed for lacustrine systems, mostly on extensive contexts (Mercedes-Martín et  al.,  2014; Thompson et  al.,  2015; Deschamps et  al.,  2020; Lettéron et  al.,  2022), several questions remain. How do climate and tectonics influence lacustrine and palustrine carbonate facies in intracratonic basins? How does the relative position of the basin in rela- tion to the coastline influence non-marine carbonate sedi- mentation and facies? What are the criteria for identifying lacustrine carbonates from lagoon carbonates? Do eustatic variations affect coastal lake systems? If so, do they allow us to correlate stratigraphic surfaces from the marine to the continental domain? 2 For each of these models, the study assesses the evolution of facies from the lake From the Lutetian to the Aquitanian, the Paris Basin was dominated by carbonate and clastic sedimentation fluctuating from shallow marine to lacustrine and pa- lustrine environments (Ménillet, 1974; Mégnien, 1980; Ziegler,  1990; Meulenkamp et  al.,  2000; Copestake et  al.,  2003; Londeix et  al.,  2014). Coastal environ- ments, including estuaries, restricted marine domains, intertidal zones, supratidal zones, or lakes, were im- portant depositional environments during this inter- val (Blondeau et  al.,  1965; Delhaye-Prat et  al.,  2005; Figure  1). This variety of depositional environments depends on the connection of the northern part of the basin with the marine domain via a NW–SE oriented channel (Figure  1A,B). This channel is recorded by littoral shelly sands between the Bray and Vigny anti- clines (Morellet & Morellet, 1948; Pomerol et al., 1965; Mathelin & Bignot, 1989; Gély & Lorenz, 1991). The con- nection became less and less active from the Lutetian (Figure 1A,B) and was severed by the late Rupelian with the transition from estuarine and marine environments to lacustrine systems from the Bartonian (Figure  1C; Mégnien,  1980). The sedimentary material consists of carbonates, sands, marls, clays and gypsum evaporites deposited in shallow marine to lacustrine environments. Today, these deposits outcrop in the vicinity of Paris (Figure  1D). Brackish foraminifera, charophytes and palynomorphs were attributed to calcareous nannofos- sil assemblages (Calcareous Nannofossil Palaeogene NP and Neogene NN zones) in an effort to correlate non-ma- rine and marine deposits by comparing palaeontologi- cal records with the neighbouring basins of Hampshire in the United Kingdom and Belgium (Blondeau et al., 1965; Le Calvez, 1970; Pomerol & Riveline, 1975; Aubry, 1985; Riveline et al., 1996). Five major episodes ley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01 4 4 MOREAU et al. IGURE 1  (A–C) Location of the study area on a palaeogeographical map of Western Europe during (A) the Lutetian, (B) the Rupelian, C) the Aquitanian (Londeix et al., 2014; Copestake et al., 2003; Meulenkamp et al., 2000). The study area is highlighted by the red rectangle. D) Location of the study outcrops and boreholes, outcrop and borehole data from the literature and databases, cross-sections in this study, nd major faults intersecting the Cenozoic deposits in the Paris Basin. 2 wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 1  (A–C) Location of the study area on a palaeogeographical map of Western Europe during (A) the Lutetian, (B) the Rupelian, (C) the Aquitanian (Londeix et al., 2014; Copestake et al., 2003; Meulenkamp et al., 2000). The study area is highlighted by the red rectangle. (D) Location of the study outcrops and boreholes, outcrop and borehole data from the literature and databases, cross-sections in this study, and major faults intersecting the Cenozoic deposits in the Paris Basin. 5 MOREAU et al. of carbonate production are identified for the Lutetian– Aquitanian period, with N–S migration of the deposi- tional environment (Figure 2): first, from the Lutetian to early Bartonian, marine bioclastic grainstones (Calcaire grossier Formation; equivalent to NP14b to NP15b zones) gave way to muddy marine facies (top of the Calcaire grossier and Marnes et Caillasses formations; equivalent to NP14b-NP16 zones; Figure 2) in the northern part of the basin (Abrard, 1925; Blondeau et al., 1965; Pomerol & Riveline, 1975; Toulemont, 1982). In the southern and eastern parts, planorbid limestones formed in freshwater environments (Planorbis pseudoammonius; Blondeau et al., 1965; Calcaire de Morancez, Calcaire de Darvault and Calcaire de Provins formations). Second, during the late Bartonian (equivalent to NP17 zone), deposition of a charophyte-rich lagoon–lacustrine limestone domi- nated (Chara friteli, Gyrogona tuberosa and Raskyella vadaszi zones; Calcaire de Saint-Ouen Formation) between short marine sandy episodes (Sables de Mortefontaine and Sables de Cresnes-Monceau mem- bers). Third and fourth during the Priabonian to early Rupelian (equivalent to NP18, 19, 20, and 21 zones), lacustrine limestones poor in fossils were deposited in the southern and eastern parts of the basin (Calcaire de Champigny and Calcaire de Brie formations; Figure 2). Gypsum evaporites or marls accumulated in the central part of the Paris Basin during the Priabonian (Marnes et Masses de gypses Formation and Marnes blanches de Pantin Member; Mégnien, 1974; Turland, 1974), giving way to limestones or marls with marine species during the early Rupelian (Caillasses d'Orgemont Formation). 2 Fifth, during the late Rupelian and Aquitanian, la- custrine carbonates were deposited in the southern part of the basin, between Chartres, Etampes and Montargis (Figure 1) with the Calcaire d'Etampes (late Rupelian, Chara microcera, equivalent to NP24 zone, Riveline, 1983) and the Calcaire de Beauce formations (Aquitanian, Stephanochara bertotensis zone; Figure 2) .1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley On FIGURE 2  Schematic lithostratigraphic illustration following a NE–SW cross-section across the study area (modified after Gély, 2016). Members are written in italics. Palaeoenvironmental interpretations are from Mégnien (1980), Briais (2015), and this study. The names of the localities are given in Figure 1D. NP-NN: Calcareous Nannofossil zones for the Palaeogene (NP) and Neogene (NN) epochs. The following abbreviations are used for short-term sequence stratigraphic surfaces and systems tracts: CC: Correlative conformity; HST: Highstand Systems Tract; MFS: Maximum Flooding Surface; TST: Transgressive Systems Tract; SU: Subaerial Unconformity; U: Unit. wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 2  Schematic lithostratigraphic illustration following a NE–SW cross-section across the study area (modified after Gély, 2016). Members are written in italics. Palaeoenvironmental interpretations are from Mégnien (1980), Briais (2015), and this study. The names of the localities are given in Figure 1D. NP-NN: Calcareous Nannofossil zones for the Palaeogene (NP) and Neogene (NN) epochs. The following abbreviations are used for short-term sequence stratigraphic surfaces and systems tracts: CC: Correlative conformity; HST: Highstand Systems Tract; MFS: Maximum Flooding Surface; TST: Transgressive Systems Tract; SU: Subaerial Unconformity; U: Unit. 6 MOREAU et al. due to the separation of the basin from the marine domain to the north (Denizot,  1927; Cavelier,  1969; Ménillet, 1974; Guillemin, 1976; Lozouet, 2012). A sed- imentary hiatus is recognised during the Chattian, be- tween these two units (Pomerol, 1989). et al., 2011; Roche, 2020). Classification of microbial-rich build-ups after Vennin et al. (2021) is used for the descrip- tion of microbial features in sedimentary facies. Facies and microfacies have been characterised based on their lithology, texture, sedimentary structures and compo- nent grains observed on macro samples and on 205 thin sections. 2 These five major episodes of carbonate production are separated by four siliciclastic episodes (Figure  2): coastal sands of the Auvers-Beauchamp group (early Bartonian; Mégnien, 1980; Briais, 2015) and the Sables de Cresnes-Monceau Member (late Bartonian); marl deposits with brackish to marine fauna of the Marnes à Pholadomya ludensis Member (early Priabonian; Pomerol et  al.,  1965); the Marnes bleues d'Argenteuil Member (late Priabonian) and the Argiles vertes de Romainville (early Rupelian) Member; and finally a sandy siliciclastic marine episode topped by aeolian dunes with the Sables de Fontainebleau Formation (Rupelian; Alimen,  1936; Gitton et  al.,  1986; Delhaye- Prat et al., 2005). 3.2  |  Sequence stratigraphy and cross-sections To understand the depositional geometries and to es- tablish stratigraphic cross-sections, outcrops and bore- holes were interpreted in terms of sequence stratigraphy. Three types of stratigraphic surfaces used in marine areas can be applied to continental deposits (Hanneman & Wideman,  2010): subaerial unconformities, correla- tive conformities and maximum flooding surfaces. Units are bounded by sequence boundaries (subaerial uncon- formities and their correlative conformities), which rep- resent the shallowest environment recorded within a sequence and coincide with shifts in stacking patterns be- tween shallowing-upward and deepening-upward trends (Strecker et  al.,  1999; Changsong et  al.,  2001; Keighley et al., 2003; Hanneman & Wideman, 2010; Pérez-Rivaréz et al., 2018; Deschamps et al., 2020; Guan et al., 2021; Melo et al., 2021; Lettéron et al., 2022). Subaerial unconformi- ties were identified by surfaces characterised by features such as erosion karstification or palaeosoils occurrence— whereas correlative conformities are surfaces that were not (Hanneman & Wideman, 2010). Maximum flooding surfaces (MFS) represent the deepest deposits encoun- tered within a sequence and mark the shift between deep- ening-upward and shallowing-upward trends (Hamilton & Tadros, 1994; Keighley et al., 2003). In the succession of palustrine deposits encountered in this study, the beds with the least evidence of pedogenic modifications were interpreted as MFS, recording the periods when the water level was highest. Subaerial unconformities, correlative conformities and maximum flooding surfaces separate transgressive systems tracts, characterised by deepening- upward facies, and highstand systems tracts, character- ised by shallowing-upwards facies (Strecker et al., 1999; Bohacs et  al.,  2000; Changsong et  al.,  2001; Keighley et al., 2003; Bohacs et al., 2007; Deschamps et al., 2020; Guan et al., 2021). The nomenclature of the short-term and long-term surfaces used in this work for the Cenozoic of the Paris Basin was initially defined by Briais (2015). h d Four long-term cycles and 22 short-term depositional cycles were established by previous work in the Lutetian– Aquitanian interval of the Paris Basin (Delhaye-Prat et al., 2005; Briais, 2015). The major stratigraphic sur- faces that can be recognised throughout the basin and define these cycles are detailed in the supplementary material. 4.1 The Cenozoic carbonates of the Paris Basin were only slightly buried (a hundred metres at most) implying lim- ited burial diagenesis of the micrite. Carbon and oxygen isotope analyses were conducted on 43 micritic carbon- ate samples collected in the Maisse well [location 8 on Figure 1D] in deposits dated from the Lutetian (top of the Calcaire grossier Formation) to the end of the Rupelian (Calcaire d'Etampes Formation; Supplementary data). The micrites all have a low-magnesium calcite mineral- ogy (>1% MgO). They are autochthonous micrites ex- cept for the palustrine facies where pseudomicrites are present (Flügel & Munnecke, 2010). Carbonates (2.5 mg) were sampled by micro-drilling on newly sawn faces in homogeneous micritic areas, with the powder placed in sealed tubes and dissolved using H3PO4 to produce CO2. Carbon and oxygen isotopic compositions of the evolved CO2 were measured using a gas chromatograph coupled to a GVInstruments Analytical Precision 2003 mass spec- trometer at the Université Paris Cité (Institut de Physique du Globe de Paris, Paris, France) (for method details see Assayag et  al.,  2006). Three internal standards (Rennes 1; Merck and Accros) were used to convert raw isotope values into δ18O/PDB and δ13C/PDB values and evaluate the reproducibility of the analyses. The isotopic values repre- sent the mean of four analyses made for each sample. The This facies association contains granular bioclastic lime- stones with two distinctive facies: a siliciclastic and foraminifera rudstone (facies F1a) and a bioclastic grain- stone or rudstone (facies F1b) (Figure  3A through F). The biota is composed of gastropods, bivalves, annelids (serpulids and ditrupes), echinoderms, and a wide vari- ety of benthic foraminifera (nummulitids, orbitolinids, miliolids, etc.). Based on previous studies, bryozoans, sponges, brachiopods, fishes or turtles have also been found in these facies in the Paris Basin (Merle,  2008). Siliciclastic and foraminifera rudstones (facies F1a) pre- sent Thalassinoides burrows while bioclastic grainstones display Ophiomorpha bioturbations (facies F1b). Facies F1a, which is coarser than facies F1b, presents nummu- lites (often oriented), erosion gullies and erosive surfaces. Facies F1b shows cross bedding in dunes ranging from 10 cm to 1 m in height (types II to IV of Allen, 1980), pla- nar bedding (Figure  3A) or wave ripples. The smallest dunes more frequently have reactivation surfaces. 3.1  |  Core description, facies and petrography For that, the east–west cross-section provided here inter- sects those of Briais (2015) between locations 71 to 78 (Figure 1D). 3.3  |  Stable isotope analyses (δ13C and δ18O) 3.3  |  δ18O) 3.1  |  Core description, facies and petrography iley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License This study is based on the detailed examination of 17 out- crops in the eastern and southern part of the basin and 18 boreholes located in Paris [locations 3–9, 12, 25 and 28 in Figure 1D]. The data set is completed with gamma-ray well-logs (http://​infot​erre.​brgm.​fr/​ and http://​www.​miner​ gies.​fr/​fr) from 51 boreholes available online from the French Geological Survey databases, supplemented by 11 outcrop or borehole sedimentary logs from 1:50000 scale geological maps (Marchand,  1968; Labourguigne,  1971; Labourguigne & Turland, 1974; Gigot, 1973, 1980, 1984) and other studies (Cavelier,  1968; Aubry et  al.,  1977; Delhaye-Prat et  al.,  2005; Le Callonnec et  al.,  2018). Outcrops and boreholes have been described in detail using the classifications of Dunham  (1962) and Embry and Klovan  (1971) for texture, and the commonly used terminology for non-marine facies based on the structure of specific facies such as calcretes or microbial carbon- ates (Platt,  1989; Platt & Wright,  1991; Alonso-Zarza & Wright, 2010a; Gierlowski-Kordesch, 2010; Alonso-Zarza Two stratigraphic cross-sections were constructed using the outcrops and boreholes listed in Figure  1D. The two cross-sections intersect at the Maisse borehole [location 8] for which detailed sedimentary description |  7 |  7 MOREAU et al. external reproducibilities (1σ) for δ13C and δ18O values are 0.1% and 0.2% respectively. external reproducibilities (1σ) for δ13C and δ18O values are 0.1% and 0.2% respectively. and gamma-ray well-log interpretation were under- taken. This allows sedimentary facies and stratigraphic sequences to be matched with gamma-ray logs. This matching is used as a reference for interpreting other gamma-ray logs in terms of sedimentary facies/facies associations and then to establish correlations between sedimentary logs and well logs. The first cross-section is oriented north–south from the Mont-Pagnotte bore- hole [location 29 in Figure 1D] to Orléans [12] by way of Paris. The second one is oriented east–west, extend- ing from Baronnie quarry [18] to Villermain quarry [13]. The age model of the depositional sequences relies on (1) the biostratigraphic fauna (foraminifera, charophytes, palynomorphs, dinocysts, malacofauna and mammals) identified in previous studies in sections from Creil to Etampes (N–S cross-section, Cavelier, 1968; Pomerol & Riveline,  1975; Aubry et  al.,  1977) and (2) correlation of these sequences with the ones defined in the Briais cross-sections (2015), which relate to biostratigraphi- cally well-anchored zones in the north of the basin. 4  |  RESULTS: DEPOSITIONAL ENVIRONMENTS AND SEDIMENTARY FACIES Thirty-two sedimentary facies were grouped into six depo- sitional environments: (1) the open marine inner platform for facies deposited in a high hydraulic energy marine set- ting above the fair-weather wave base; (2) the restricted marine domain for facies deposited in calm and shallow marine environments with varying salinity; (3) the coastal lake for facies deposited under fresh to brackish waters in the coastal environment with rare sporadic connections with the marine domain; (4) the floodplain; (5) the palus- trine environment, recording the deposition of carbonates under fresh or brackish water which were then subjected to subaerial exposure and modification of the sediment; and (6) the inland lake, never connected to a marine set- ting. Observations, descriptions and isotopic data are pre- sented in detail below. A summary table is given in the supplementary material. 4.1 (B) Sandy rudstone with coarse quartz and shell fragments; Facies F1a; Calcaire grossier Formation, Lutetian [5]. (C and D) Rudstone and grainstone with marine bivalves, gastropods, echinoderms and foraminifera, see the bioturbation (Ophiomorpha) in picture D; Facies F1b; Calcaire grossier Formation, Lutetian [3 and 6]. (E) Bioclastic grainstone in thin sections with quartz (Qz), benthic foraminifera (B.F.), miliolids (Mil.) and echinoderms (Ech.); Facies F1b; Calcaire grossier Formation, Lutetian [6]. (F) Same facies as picture D with bivalves (Bi.); base of the Sables de Fontainebleau Formation, Rupelian [8]. alternating periods of flooding and then exposure during dry episodes (Kendall & Warren, 1987). This facies association is abundant in the Calcaire grossier Formation (Lutetian) and at the base of the Sables de Fontainebleau Formation (Rupelian). Oxygen and carbon isotopes—Isotopic values range from −4.6 to 1.1‰ for δ13C and from −4.3 to 1.9‰ for the δ18O (mean value of −2.2 and −1.2‰, respectively), i.e., close to the range of non-recrystallised marine fos- sils obtained from marine molluscs from the Paris Basin (Huyghe, 2010) and from ostracods from the Hampshire Basin (Marchegiano & John, 2022), which, for a similar age, range from −2.5 to 1.5‰ for the δ13C and from −5 to −0.5‰ for the δ18O (Figure 5). This is consistent with the marine origin mentioned before. Some micrites (es- pecially in facies F2e and g) exhibit variable oxygen iso- topic compositions with some positive values, showing fluctuations in water chemistry during phases of evapora- tion. This association is exclusive to the upper part of the Calcaire grossier and the Marnes et Caillasses formations (Lutetian to early Bartonian). 4.2  |  Facies association FA2 Restricted marine inner platform Petrography and sedimentary structures—Seven facies compose this association: (1) grainstone/packstone with miliolids (facies F2a) (Figure  4A,B,C), (2) alternations of foraminifera-rich wackestone and grainstones (fa- cies F2b) (Figure 4A,D), (3) bioturbated floastone with a wackestone matrix (facies F2c) (Figure 4E,F), (4) alterna- tions of dolomite and wackestone with foraminifera and storm wash-over (facies F2d) (Figure 4G,H), (5) alterna- tions of miliolid grainstone and green marls (facies F2e) (Figure  4I), (6) alternations of gypsum and mudstone/ wackestone levels (facies F2f) (Figure 4J), and (7) biotur- bated mudstone (facies F2g) (Figure 4K). They correspond to centimetre-sized to decimetre-sized muddy limestone alternations with low faunal diversity (benthic foraminif- era, dasyclads, echinoderms, gastropods, bivalves) and less detrital input than facies association FA1. Beds are planar or present metre-scale undulations (Figure  4L). Marine foraminifera families identified in these facies are, among others, Calcarinidae, Miliolidae, Fabulariidae, Polymorphinidae or Valvulinidae. Marine-brackish fami- lies are also present, such as Bolivinidae, Buliminidae, Discorbidae, Elphidiidae or Rosalinidae. 4.1 iley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License The marine biota, the sedimentary structures, the com- mon bioturbations (Figure 3D) and the large quantity of detrital quartz, all suggest a well-oxygenated open marine domain with a high energy wave-dominated or tide-domi- nated environment located above the fair-weather wave base 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/ 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules o 8 MOREAU et al. 8 (Briais, 2015). Dunes in facies F1b indicate an open marine environment between the subtidal and the intertidal do- mains while cross-bedding alternating with planar bedding marks high energy tidal flat environments. Facies F1a in- dicates a shoreface environment further to the shoreline where storm events are recorded (Pemberton et al., 1992). //onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License (Briais, 2015). Dunes in facies F1b indicate an open marine environment between the subtidal and the intertidal do- mains while cross-bedding alternating with planar bedding marks high energy tidal flat environments. Facies F1a in- dicates a shoreface environment further to the shoreline where storm events are recorded (Pemberton et al., 1992). ce, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms and conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Lic wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License marks high energy tidal flat environments. Facies F1a in- dicates a shoreface environment further to the shoreline where storm events are recorded (Pemberton et al., 1992). (Briais, 2015). Dunes in facies F1b indicate an open marine environment between the subtidal and the intertidal do- mains while cross-bedding alternating with planar bedding |  9 9 MOREAU et al. FIGURE 3  Open marine inner platform facies association FA1. The numbers indicated [#] correspond to the location of the boreholes and sedimentary sections in Figure 1. (A) Quarry face revealing cross-bedding in dunes and planar bedding; Facies F1b; Calcaire grossier Formation, Lutetian [27]. 4.3 Petrography and sedimentary structures—This facies as- sociation consists of five facies showing rare sporadic connections of the basin with the marine domain: (1) alternating gypsum limestones and marls (facies F3a) (Figure 6A,B,C), (2) euryhaline foraminifera wackestone to mudstone (facies F3b) (Figure  6D,E), (3) shell rich packstone (facies F3c) (Figure 6F), (4) varves (facies F3d), and (5) gypsiferous marls (facies F3e). Facies F3a is locally composed of alternating gypsum, millimetre-scale planar microbial laminae, or calcite dendrites in a micritic matrix (Figure 6A,B,C). The gypsum crystallised as saccharoidal, lenticular or dendritic forms and is sometimes recrystal- lised in calcite or in silica. Facies F3b, F3c and F3d present a biota of ostracods, euryhaline foraminifera (families: Bolivinidae, Buliminidae, Discorbidae, Nonionidae, and Rosalinidae; genus: Elphidiella and Caucasina), gastro- pods, bivalves and charophytes (Figure 6D,E,F). The varve facies F3d corresponds to alternating limestone beds sev- eral tens of centimetres thick and thin clayey interbeds. The fossil assemblage in the gypsiferous marls F3e consists of brackish to marine species (Corbulidae, Turritellidae, Potamididae families) associated with charophytes in the northern part of the basin. However, they show an affinity ley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License All these facies show few detrital grains, sometimes abundant bioturbations, laminations and alternation be- tween muddy and granular lamina and/or beds, indicating fluctuating hydrodynamic energy from low to moderate energy environments, typical of restricted marine envi- ronments (Wilson, 1974; Arribas et al., 2004). The fora- minifera assemblage characterises a marine environment with salinity fluctuations. The low biota diversity is dom- inated by miliolids and dasyclad algae, which suggests a protected marine environment such as an inner lagoon (Wilson, 1974; Langer & Lipps, 2003; Amao et al., 2016). Alternating muddy and shelly beds suggests deposition during storm wash-overs. In facies F2f, alternating gypsum and mudstone/wackestone with tepee structures indicates a supratidal environment along the margin, subjected to 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Cr 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/ 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of 10 10 MOREAU et al. 4.3 (I) Alternating cross-bedded and bioturbated green marls and grainstone with foraminifera; Facies F2e; Calcaire grossier Formation, Lutetian [26]. (J) Alternating mudstone to wackestone layers with fluid escape and tepees; Facies F2f; sparite has precipitated between the laminae; Marnes et Caillasses Formation, Lutetian–Bartonian [1]. (K) Bioturbared mudstone; Facies F2g; green clay fragments are present; Calcaire grossier Formation, Lutetian [8]. (L) Alternating marls and mudstones with undulating bedding; Facies F2g; Marnes et Caillasses Formation, Lutetian–Bartonian [27]. tps://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com .1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley On FIGURE 5  Cross-plot of stable carbonate isotopic composition (δ13C and δ18O values vs PDB) of micrite for marine, lacustrine and palustrine facies of the Cenozoic of the Paris Basin. Although some of the facies analysed show patches of sparitic cements or grains, the analyses were systematically carried out on micrite to reduce the impact of diagenesis on isotopic signals. The marine isotopic domain is given by the two blue rectangles based on the isotopic composition of ostracods from the Hampshire Basin (Marchegiano & John, 2022) and marine molluscs from the Paris Basin (Huyghe, 2010). et al., 2017; Fritz et al., 2018; Lettéron et al., 2018). These salinity variations are supported by the presence of gyp- sum levels in the depositional record. Oxygen and carbon isotopes—Isotopic values range from −7.0 to −2.3‰ for the δ13C and from −6.2 to 1.7‰ for the δ18O (means of −5.6 and −3.5‰, respectively; Figure 5). The isotopic composition of the micrite clearly distinguishes this environment from a restricted marine setting since its δ13C and δ18O values are more negative and within the range of open lakes (Talbot, 1990). This is best interpreted here as reflecting a higher contribution of meteoric waters (Allan & Matthews, 1982; Talbot, 1990). Synthesis—Facies association 3 therefore presents mud-dominated carbonate with a low detrital content, a dominance of brackish species, and records meteoric isotope values. These observations are interpreted as in- dicating a coastal lake environment. The varve facies F3d represents the deeper and/or calmer part of the coastal lake whereas the shell packstone of facies F3c indicates moder- ate hydrodynamic energy with local accumulation of shells along the lake margin (Gierlowski-Kordesch, 2010). 4.3 Facies F3a with gypsum levels and microbial mats indicates saline water deposits in a playa-like environment (Schreiber, 1988; Rouchy et  al.,  2001; Flügel & Munnecke,  2010). These gypseferous marls (F3e) are most influenced by marine in- cursions, as shown by the common occurrence of marine species. Some levels composed by this facies even show a marine environment like lagoons. This facies association made up the limestone and marl formations in the centre and the northern part of the basin during the Bartonian, Priabonian and early Rupelian. FIGURE 5  Cross-plot of stable carbonate isotopic composition (δ13C and δ18O values vs PDB) of micrite for marine, lacustrine and palustrine facies of the Cenozoic of the Paris Basin. Although some of the facies analysed show patches of sparitic cements or grains, the analyses were systematically carried out on micrite to reduce the impact of diagenesis on isotopic signals. The marine isotopic domain is given by the two blue rectangles based on the isotopic composition of ostracods from the Hampshire Basin (Marchegiano & John, 2022) and marine molluscs from the Paris Basin (Huyghe, 2010). of bioturbated, brownish sandy clays to marls with ter- restrial gastropods and sinuous vertical millimetre-wide bioturbations with a fine, organic-rich centre interpreted as ancient roots. This facies is present above erosional surfaces. 4.3 for brackish to freshwater environments (Lymnaeidae, Planorbidae, Truncatellidae families with genus Nystia) in the southern part. These mud-rich facies with few detrital grains (>10%) i di t t i t d d iti l i t ith l terrestrial input. The fossil assemblage is characteristic of a brackish to freshwater environment with fluctuating salinity (mostly mesohaline to oligohaline conditions) in coastal environments like estuaries, lagoons and coastal l k (Pl i t 1993 D & B 2005 St t 2015 Pi t , 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative C wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License for brackish to freshwater environments (Lymnaeidae, Planorbidae, Truncatellidae families with genus Nystia) in the southern part. for brackish to freshwater environments (Lymnaeidae, Planorbidae, Truncatellidae families with genus Nystia) in the southern part. terrestrial input. The fossil assemblage is characteristic of a brackish to freshwater environment with fluctuating salinity (mostly mesohaline to oligohaline conditions) in coastal environments like estuaries, lagoons and coastal lakes (Plaziat, 1993; Dye & Barros, 2005; Strotz, 2015; Pint These mud-rich facies with few detrital grains (>10%) indicate a restricted depositional environment with low 11 MOREAU et al. FIGURE 4  Restricted marine facies association FA2. The numbers indicated [#] correspond to the location of the boreholes and sedimentary sections in Figure 1. (A) Horizontal alternations of bivalve-rich grainstone (facies F2a) and wackestone with foraminifera (facies F2b); Calcaire grossier Formation, Lutetian [6]. (B) Grainstone with miliolids; Facies F2a: miliolidae (mil.), dasycladian algae (da.), echinoderms (ech.), glauconite and quartz; Calcaire grossier Formation, Lutetian [6]. (C) Same facies as picture B; Calcaire grossier Formation, Lutetian [3]. (D) Wackestone with foraminifera; Facies F2b: benth. Foram: benthic foraminifera; Calcaire grossier Formation, Lutetian [6]. (E and F) Floatstone with large gastropods (gast.) and bivalves (bi.) in a wackestone matrix; Facies F2c with foraminifera, bivalves, ostracods and ditrupes, miliolids (Mil.) and benthic foraminifera (benth. Foram.). Bivalves are marine species (Veneridae); Calcaire grossier Formation, Lutetian [8]. (G and H) Alternating dolomite (dol.) and wackestone with foraminifera subjected to turbation by a tempestite with crushed gastropods (gast.). Note the desiccation cracks (DsC) at the bottom of the sample; Facies F2d; Marnes et Caillasses Formation, Lutetian–Bartonian [1]. 4.5  |  Facies association FA5 Palustrine environment beds. Sometimes, the presence of laminated structures lying horizontally within the intraclasts confirms that the clasts have not been remobilised in in-situ brecci- ated limestones (facies F5f; Figure  7J). In Figure  7J, they also highlight that the two intraclasts were for- merly continuous. The surface of the intraclasts ranges from rough to smooth and may be locally covered by micritic laminar calcretes (Figure  7K). Between in- traclasts, the filling consists either of gastropods and reworked polygenetic carbonate intraclasts within a micritic matrix that is rarely desiccated or by sparite cements (Figure 7J,K,L). The alternation of mudstone and wackestone with root traces (facies F5h) exhibits a palustrine carbonate in the upper part of the beds, while the bottom is unaffected by subaerial exposure (Figure  7N). Peloidal grainstone F5e fills desiccation cracks, microcavities or karstic channels within previ- ously described facies. Petrography and sedimentary structures—The palus- trine facies association is composed of eight facies dis- playing abundant subaerial exposure markers such as desiccation cracks or pedogenetic processes: (1) tubular limestones (facies F5a; Figure 7A,B), (2) nodular brec- ciated limestones (facies F5b, Figure 7C,D,E), (3) lami- nar brecciated limestones (facies F5c, Figure 7F), and chalky altered limestones (facies F5d, Figure 7G), (5) peloidal grainstones (facies F5e, Figure  7H,I), (6) in- situ brecciated limestones (facies F5f, Figure  7J,K,L), (7) organic-rich wackestone and marls with reworked intraclasts (facies F5g, Figure 7M), and (8) alternating mudstone and wackestone with root traces (facies F5h, Figure 7M,N). Facies F5a to F5d present intense suba- erial exposure leading to extensive transformation of the primary sediment, which is difficult to recognise in some cases. Fauna is either absent or limited to rare ostracods. In facies F5b and F5c, intraclasts are mainly desiccated, displaying a jigsaw-puzzle structure. They are locally silicified by opal A, microcrystalline quartz or chalcedony. In thin section, facies F5a to F5c show alveolar-septal structures (Figure  7F), coated grains with micritic agglutinated fabrics or concentric micro- bial laminations (Figure 7C,D), rhizoliths and clotted micrite. Tubular voids interpreted as rootlet moulds and rarely preserved polygonal white or brownish prisms (Microcodium) extend throughout the matrix and the intraclasts (Figure 7B,C,F). Micrite laminae with desic- cation cracks, fenestral fabrics and rootlet moulds mak- ing up the laminar brecciated limestones without any significant change in thickness (Figure 7F). A micritic matrix or a sparitic calcitic cement separate the clasts, while vadose calcitic cements develop in the porosity (Figure  7E). 4.4  |  Facies F4 Floodplain The floodplain is represented by only one facies in the Paris Basin: coarse to fine sandy clays. This facies is present at the base of the Lutetian deposits. It consists This facies is interpreted as floodplain associated with fluvial systems, marking the transgression of the Lutetian deposits above the Ypresian sedimentary formations. 12 MOREAU et al. FIGURE 6  Coastal lake facies association FA3. The numbers indicated [#] correspond to the location of the boreholes and sedimentary sections in Figure 1. (A) Cores presenting alternating gypsum, limestone and organic matter; Facies F3a; Masses de gypses Formation, Priabonian [28]. (B) Gypsum developing in limestone; Facies F3a; Marnes et Caillasses Formation, Lutetian–Bartonian [1]. (C) Limestone with calcite dendrites and recrystallised gastropods (some are shown with black arrows); Facies F3a; Calcaire de Saint-Ouen Formation, Bartonian [5]. (D) Wackestone with microfossils; Facies F3b; Calcaire de Saint-Ouen Formation, Bartonian [2]. (E) Wackestone with pelloids (Pel.), ostracods and small foraminifera (benth. foram.); Facies F3b; Calcaire de Saint-Ouen Formation, Bartonian [7]. (F) Shell- rich packstone with ostracods, charophytes, gastropods (gastr.) and euryhaline benthic foraminifera (benth. foram.); Facies F3c; Calcaire de Saint-Ouen Formation, Bartonian [6]. 4.5  |  Facies association FA5 Palustrine environment Facies F5e to F5h also present markers of subaerial exposure, although these are notably less important than in facies F5a–d. The top of the beds are often irregular but well recognisable in outcrops. The primary sediment is partly preserved and can be identified and described. Gastropods and ostracods are common, charophytes are locally present but rare, and former roots are abundant in the upper part of some Oxygen and carbon isotopes—The isotope composi- tions are depleted in 13C and 18O with values ranging from −8.8 to −4.7‰ for the δ13C and from −6.2 to −2.5‰ for the δ18O (mean values of −7.1 and −4.6‰, respectively; Figure 5). The highly negative δ13C and δ18O values indi- cate the influence of meteoric fluids (Talbot, 1990). Note however that the micrite of these facies has been modified early by biological activity. Synthesis—All facies of this facies association FA5 present characteristics of subaerial exposure suggest- ing the ephemeral presence of water in lacustrine sys- tems or seasonal wetlands, i.e., typical of a palustrine environment (Freytet & Plaziat, 1982; Platt, 1989; Platt & Wright,  1991; Freytet & Verrecchia,  2002; Alonso- Zarza & Wright,  2010a). The muddy limestone rep- resents the highest water level while the subaerial exposure features (peloids, roots, desiccation, …) record the depositional system with the lowest water level (Freytet & Plaziat, 1982; Platt & Wright, 1991; Bohacs et al., 2000; Freytet & Verrecchia, 2002; Alonso-Zarza & Wright, 2010a). Facies F5a to F5d are interpreted as calcretes composed mainly of beta microfabrics which were formed during long or intense episodes of sub- aerial exposure (Wright,  1994; Kosir,  2004; Kabanov et al., 2008; Alonso-Zarza & Wright, 2010a). The pres- ence of alveolar-septal structures, rootlet moulds and 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/ |  13 13 MOREAU et al. | Microcodium in these facies, plus fenestral fabrics in Alonso-Zarza & Wright,  2010). These facies record 877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Cr Microcodium in these facies, plus fenestral fabrics in some layers in facies F5c suggest a rhizogenic origin (Wright et al., 1988; Wright, 1989; Alonso-Zarza, 1999; Alonso-Zarza & Wright,  2010). These facies record the strong weathering of the primary carbonate of the Lutetian, Priabonian, Rupelian and Aquitanian. 4.5  |  Facies association FA5 Palustrine environment They display often mic- ritic and rarely hybrid laminated microfabrics (Vennin et al., 2021). Finally, the mudstone-marl alternation (fa- cies F6g) is composed of beds of mudstones or wacke- stones several centimetres thick with reworked intraclasts and gastropods alternating with marls (Figure  8I). No subaerial exposure features were identified in this facies. Oxygen and carbon isotopes—The isotopic values of covariation is identified (mean values of −6.8 and −3.2‰, respectively; Figure  5). These values correspond to the range found in modern temperate lakes and confirm the formation of the micrite in meteoric water (Talbot, 1990). Synthesis—The biota and isotope composition of the micrite of this association indicates deposition in a fresh to brackish-water lake, isolated from any marine influence. “Pure” lacustrine limestones are rare, with most showing slight signs of subaerial exposure towards the top of the beds (desiccation cracks, tubular voids interpreted as rootlet moulds, fenestral porosity). The polyphasic breccia indicate multiple reworking of the sediment (Figure 8B). Black litho- clasts present in these breccia (facies F6a), known as “black pebbles” (Platt, 1989), attest to a short transport distance from the palustrine system (Platt, 1989; Miller et al., 2013). Because such breccia generally occur above subaerial expo- sure surfaces, they are interpreted as resulting from rework- ing of primary sediment in shallow water. The facies F6b is interpreted as resulting from deposition of the smallest reworked intraclasts transported over longer distances than that seen in facies F6a formed. In the mud–wackestone and clayey marl alternations (facies F6g) (Figure  8I), the ab- sence of subaerial features and organic matter-rich or clayey levels suggests a deposit far from the lake margins but still above the hypolimnion (Platt & Wright, 1991; Gierlowski- Kordesch, 2010). This facies association is encountered in the last episodes of carbonate sedimentation in the Paris Basin in the Calcaire d'Etampes (late Rupelian) and the Calcaire de Beauce (Aquitanian) formations. 4.5  |  Facies association FA5 Palustrine environment (G) Chalky altered limestone with recrystallised micrite in microsparite and silica (Si.); Facies F5d; Calcaire de Champigny Formation, Priabonian [8]. (H) Peloidal grainstone (large arrows) filling a karstic cavity (small arrows); Facies F5e; Calcaire d'Etampes Formation, Rupelian [8]. (I) Peloidal grainstone (facies F5e) filling spaces between two in-situ intraclasts with gastropods (gast.); Facies F5e and F5f; Calcaire de Beauce Formation, Aquitanian [16]. (J) In-situ brecciated limestones with stromatolitic-like structures inside the intraclasts; Facies F5f; Calcaire de Beauce Formation, Aquitanian [14]. (K) In-situ brecciated limestones with a thin layer of laminar calcrete visible around the in-situ intraclasts; Facies F5f; Calcaire de Beauce Formation, Aquitanian [16]. (L) In-situ brecciated limestones showing jigsaw- puzzle like centimetre-sized intraclasts marking desiccation of the former carbonate rock. The intergranular space is cemented with vadose and phreatic cements; Facies F5f; Calcaire de Provins Formation, Lutetian [18]. (M) Alternation of palustrine facies F5g and F5h affected by the development of a metre-sized karstic cavity; Calcaire de Beauce Formation; Aquitanian [11]. (N) Wackestone with root traces and peloid development; Facies F5h; Calcaire d'Etampes Formation, Rupelian [8]. F6f), and (7) alternations of mudstone and marls (facies F6g; Figure 8). Gastropods are the main fossil found in these facies; ostracods and charophytes are present in lesser amounts. Fenestral porosity is present and filled by dogtooth or drusy sparite cements. Polygenetic brec- cia facies (F6a) display reworked carbonate intraclasts of a few centimetres to a few tens of centimetres in size with different morphologies (rounded to sub-angular) and origins (black pebbles, microbial crusts, palustrine carbonate) without preferential orientation, and locally attesting to a polyphase process of breccia formation (Figure 8A,B). Lithoclastic wackestones (facies F6b) ex- hibit similar reworked intraclasts but with smaller mil- limetre-sized fragments and lower proportions of grains. The clasts are mostly rounded and sometimes concen- trated along horizontal levels a few millimetres thick (Figure 8C). Within centimetre-thick to decimetre-thick grey to bluish indurated beds, floatstones and rudstones with gastropods (F6d) or with oncoids (F6e) are present (Figure 8E,F,G). Desiccation is often identified at the top of the beds. Alternation of clear and dark millimetre- thick micritic laminae, with the dark ones occasionally containing some fragments of organic matter can form columnar or planar structures covering exposure surfaces or filling the intergranular space (Figure 8H). Encrusted shells (Lymnaeidae) are unaltered and intact when in contact with the laminae. These laminae are interpreted as microbial crusts (facies F6f). 4.5  |  Facies association FA5 Palustrine environment ownloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Common m/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Alonso-Zarza & Wright,  2010). These facies record the strong weathering of the primary carbonate of the Lutetian, Priabonian, Rupelian and Aquitanian. Microcodium in these facies, plus fenestral fabrics in some layers in facies F5c suggest a rhizogenic origin (Wright et al., 1988; Wright, 1989; Alonso-Zarza, 1999; 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/ 14 14 MOREAU et al. 4.6  |  Facies association FA6 Inland lake Petrography and sedimentary structures—This association is composed of seven facies: (1) polygenetic breccia (facies F6a), (2) wackestone with lithoclasts (facies F6b), (3) grainstone with peloids and shell fragments (facies F6c), (4) shell-rich floatstone (facies F6d), (5) oncoidal wacke- stone to packstone (facies F6e), (6) microbial crust (facies , Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons iley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License F6a), (2) wackestone with lithoclasts (facies F6b), (3) grainstone with peloids and shell fragments (facies F6c), (4) shell-rich floatstone (facies F6d), (5) oncoidal wacke- stone to packstone (facies F6e), (6) microbial crust (facies 4.6  |  Facies association FA6 Inland lake 4.6 Petrography and sedimentary structures—This association is composed of seven facies: (1) polygenetic breccia (facies 15 MOREAU et al. FIGURE 7  Palustrine carbonate facies association FA5. The numbers indicated [#] correspond to the location of the boreholes and sedimentary sections in Figure 1. (A and B) Rhizoliths. Root cells are still identifiable (black arrows). Rootlet moulds are filled by calcedony (in white); Facies F5a; Calcaire de Beauce Formation, Aquitanian [12]. (C and D) Peloids coated by micritic envelopes (mi.env); Facies F5b; Calcaire de Champigny Formation, Priabonian [18 and 21]. (E) Geopetal cement in nodular brecciated limestone; Facies F5b; Calcaire de Champigny Formation, Priabonian [8]. (F) Laminar brecciated limestone displaying a recrystallised millimetre-thick root level with alveolar septal structures (Al. Sep. St.) around which desiccation cracks (Dess.C.) develop; Facies F5c; Calcaire de Brie Formation, Rupelian [8]. 5  |  STRATIGRAPHIC SEQUENCES, FACIES DISTRIBUTION AND PALAE​ OEN​VIR​ONM​ENTAL EVOLUTION (H) Columnar laminated microbial crusts overlying an eroded surface; Facies F6f; Calcaire de Beauce Formation; Aquitanian [16]. (I) Alternating mudstones and gastropod marls; Facies F6g; Calcaire d'Etampes Formation, Rupelian [10]. FIGURE 8  Inland lake facies association FA6. The numbers indicated [#] correspond to the location of the boreholes and sedimentary sections in Figure 1. (A) Polygenetic breccia with black angular limestone pebbles and reworked carbonate intraclasts; Facies F6a; Calcaire de Beauce Formation, Aquitanian [16]. (B) Polyphase and polygenetic limestone breccia; Facies F6a; Calcaire de Beauce Formation, Aquitanian [14]. (D) Grainstone with peloids and shell fragments (red arrows); Facies F6c; Calcaire de Beauce Formation, Aquitanian [12]. (E and F) Gastropod (gast.)-rich rudstone beds. An erosional surface separates facies F6d from facies F6e in picture F; Facies F6d; Calcaire de Beauce Formation, Aquitanian [13]. (G) Beds of oncoidal rudstone; Facies F6e; Calcaire de Beauce Formation, Aquitanian [13]. (H) Columnar laminated microbial crusts overlying an eroded surface; Facies F6f; Calcaire de Beauce Formation; Aquitanian [16]. (I) Alternating mudstones and gastropod marls; Facies F6g; Calcaire d'Etampes Formation, Rupelian [10]. have been identified from the Lutetian to the Rupelian (47–28 Ma) using the facies associations described above. The average duration of a cycle is approximately 1.25 Myr, which matches the range of duration of third-order cycles (Vail et al., 1991; Hardenbol et al., 1999). All these cycles are included in four lower-order cycles (longer than 4 Myrs long), considered by Briais (2015) as second-order cycles. The first one ranges from the Lutetian to the early Bartonian (LB cycle, duration ≈7 Myr) and is delimited by the L1 and B2 subaerial unconformity. It is followed by the early Bartonian to middle Priabonian (BP cycle, ≈7 Myr) and the middle Priabonian to late Rupelian (PO cycle, ≈7–8 Myr) cycles topped, respectively, by the P3 and A1 subaerial unconformity. The final cycle ranges from the Aquitanian to the Burdigalian (A cycle, ≈4–5 Myr). These cycles are detailed below. Biota suggest that, during the LB1 and LB2 cycles, the deepest bathymetries were located in the northern part of the basin, decreasing southwards and along the Bray and Remarde anticlines (Merle,  2008). The upper regressive systems tract of the LB2 cycle re- cords the shallowing of the basin with the appearance of restricted marine limestones (FA2), which persists throughout the LB3 cycle with several evaporitic epi- sodes around Paris (Figures 9 and 11B). 5.1  |  Lutetian– early Bartonian LB cycle: Isolation of the marine carbonate platform 5.2  |  Early Bartonian to middle Priabonian BP cycle: from an estuarine to a carbonate-evaporitic lacustrine system 5  |  STRATIGRAPHIC SEQUENCES, FACIES DISTRIBUTION AND PALAE​ OEN​VIR​ONM​ENTAL EVOLUTION Restricted ma- rine deposits pass laterally into a palustrine domain extending westwards to Provins (Figures 10 and 11B) with in-situ brecciated limestones (facies F5f, Calcaire de Provins Formation) and southwards from Pithiviers to Orléans with several facies displaying the freshwa- ter gastropod Biomphalaria pseudoammonia (facies F5f, F5g, F5h; Turland,  1974; Mégnien,  1980; Gély & Lorenz, 1991; Merle, 2008). 5  |  STRATIGRAPHIC SEQUENCES, FACIES DISTRIBUTION AND PALAE​ OEN​VIR​ONM​ENTAL EVOLUTION Oxygen and carbon isotopes—The isotopic values of these facies are negative, ranging from −7.8 to −4.3‰ for the δ13C and from −4.8 to −2.3‰ for the δ18O, and a The two cross-sections are constructed above and below the major subaerial unconformity R2, which is taken as 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules 16 16 MOREAU et al. | a horizontal surface (Figures  9 and 10). This preceeds the marine transgression of the Sables de Fontainebleau Formation above palustrine or lacustrine formations The stratigraphic surfaces defined by Briais  (2015) were correlated further south and east in the lacustrine and palustrine facies and help to define cycles. Fifteen 877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative C wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License The stratigraphic surfaces defined by Briais  (2015) were correlated further south and east in the lacustrine and palustrine facies and help to define cycles. Fifteen short-term stratigraphic cycles (shorter than 4 Myrs long) a horizontal surface (Figures  9 and 10). This preceeds the marine transgression of the Sables de Fontainebleau Formation above palustrine or lacustrine formations (Calcaire de Brie and Caillasses d'Orgemont formations). 17 MOREAU et al. FIGURE 8  Inland lake facies association FA6. The numbers indicated [#] correspond to the location of the boreholes and sedimentary sections in Figure 1. (A) Polygenetic breccia with black angular limestone pebbles and reworked carbonate intraclasts; Facies F6a; Calcaire de Beauce Formation, Aquitanian [16]. (B) Polyphase and polygenetic limestone breccia; Facies F6a; Calcaire de Beauce Formation, Aquitanian [14]. (C) Reworked limestone intraclasts forming beds in a micritic matrix; Facies F6b; Calcaire de Beauce Formation, Aquitanian [14]. (D) Grainstone with peloids and shell fragments (red arrows); Facies F6c; Calcaire de Beauce Formation, Aquitanian [12]. (E and F) Gastropod (gast.)-rich rudstone beds. An erosional surface separates facies F6d from facies F6e in picture F; Facies F6d; Calcaire de Beauce Formation, Aquitanian [13]. (G) Beds of oncoidal rudstone; Facies F6e; Calcaire de Beauce Formation, Aquitanian [13]. 5.2  |  Early Bartonian to middle Priabonian BP cycle: from an estuarine to a carbonate-evaporitic lacustrine system The Lutetian to early Bartonian LB cycle contains three short-term cycles (LB1, LB2 and LB3), each one mainly composed of marine carbonate rocks from the FA1 and FA2 facies associations. This cycle extends from the ero- sional subaerial unconformity L1, recording a 2 Myrs long hiatus (Pomerol,  1989; Briais et  al.,  2016), to the erosional subaerial unconformity B2 at the top of the Marnes et Caillasses Formation. The deposits thin out from north to south (Figure  9). Biostratigraphic data and sequence stratigraphic correlations suggest that the maximum flooding surfaces of each cycle, L2, L3 and B1, are onlapping southwards and westwards (Figures 9 and 10). The Lutetian marks a long-term regression from open marine (facies association FA1) to restricted ma- rine environments (facies association FA2). Nonetheless, the maximum flooding surfaces L2 and L3 seem to not extend beyond the Remarde Anticline (Figure  9) and Melun (Figure 10), where they merge with the L1 sur- face, while the B1 maximum flooding surface stretches further southwards and westwards to Pithiviers and Etampes (Figures 9 and 10). The long-term BP cycle records six short-term cycles (BP1 to BP6), marking a marine transgression above the Marnes et Caillasses Formation (subaerial unconformity B2) dur- ing the early Bartonian with estuarine and tidal siliciclas- tic sands (BP1 to BP3; Briais, 2015), and then isolation of the basin during the late Bartonian to middle Priabonian with lacustrine and evaporitic deposits (facies association F3, BP4 to BP6) (Figures 9 and 10). The cycle ends with the subaerial unconformity P3 at the top of the Marnes et Masses de gypses and the Calcaire de Champigny formations. The first three short-term cycles (BP1 to BP3) present the same geometries as the Lutetian cycles LB1 and LB2, i.e., a maximum thickness and depth in the northern part of the basin and thinning southwards where the maximum flooding surfaces are interpreted to onlap and facies are shallower (Figure 9). A sedimentary hiatus is identified in the eastern part during these cycles (Figure 10). During the last three short-term cycles of the BP cycle (BP4 to 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA a 18 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. 5.2  |  Early Bartonian to middle Priabonian BP cycle: from an estuarine to a carbonate-evaporitic lacustrine system See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the a 18 MOREAU et al. al. ; ; y ; ; 54877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Comm FIGURE 9  Correlation diagram of Lutetian to Miocene sections along a north–south transect between the Mont-Pagnotte borehole and Orléans. This diagram incorporates 12 outcrop sections examined in this study supplemented by two sections from the literature and 28 boreholes. Correlations are based on biostratigraphy (foraminifera, mammals, dinocysts, charophytes, malacofauna and palynomorphs; Cavelier, 1968; Le Calvez, 1970; Pomerol & Riveline, 1975; Aubry et al., 1977; Riveline, 1983) and the definition of 16 stratigraphic cycles delimited by subaerial unconformities and their correlative conformities. The names of the localities associated with the numbers indicated [#] are in Figure 1D. The facies architecture is not described for the Sables de Fontainebleau Formation. 1: Sables de Mortefontaines; 2: Sables de Cresnes-Monceau; 3: Marnes à Phalodomya ludensis; 4: Marnes à lucines; 5: Marnes d'entre-deux-Masses. Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Lice FIGURE 9  Correlation diagram of Lutetian to Miocene sections along a north–south transect between the Mont-Pagnotte borehole and Orléans. This diagram incorporates 12 outcrop ections examined in this study supplemented by two sections from the literature and 28 boreholes. Correlations are based on biostratigraphy (foraminifera, mammals, dinocysts, charophytes, malacofauna and palynomorphs; Cavelier, 1968; Le Calvez, 1970; Pomerol & Riveline, 1975; Aubry et al., 1977; Riveline, 1983) and the definition of 16 stratigraphic cycles delimited by ubaerial unconformities and their correlative conformities. The names of the localities associated with the numbers indicated [#] are in Figure 1D. The facies architecture is not described for he Sables de Fontainebleau Formation. 1: Sables de Mortefontaines; 2: Sables de Cresnes-Monceau; 3: Marnes à Phalodomya ludensis; 4: Marnes à lucines; 5: Marnes d'entre-deux-Masses. Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. 5.2  |  Early Bartonian to middle Priabonian BP cycle: from an estuarine to a carbonate-evaporitic lacustrine system See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Co FIGURE 9  Correlation diagram of Lutetian to Miocene sections along a north–south transect between the Mont-Pagnotte borehole and Orléans. This diagram incorporates 12 outcrop sections examined in this study supplemented by two sections from the literature and 28 boreholes. Correlations are based on biostratigraphy (foraminifera, mammals, dinocysts, charophyte malacofauna and palynomorphs; Cavelier, 1968; Le Calvez, 1970; Pomerol & Riveline, 1975; Aubry et al., 1977; Riveline, 1983) and the definition of 16 stratigraphic cycles delimited by subaerial unconformities and their correlative conformities. The names of the localities associated with the numbers indicated [#] are in Figure 1D. The facies architecture is not described fo the Sables de Fontainebleau Formation. 1: Sables de Mortefontaines; 2: Sables de Cresnes-Monceau; 3: Marnes à Phalodomya ludensis; 4: Marnes à lucines; 5: Marnes d'entre-deux-Masses. |  19 RE 10  Correlation diagram of Lutetian to Miocene sections along an east west transect between La Baronnie quarry and Les Grands Réages quarry. This diagram incorporates eight p sections examined in this study supplemented by three sections from the literature and 29 boreholes. Correlations are based on the definition of nine stratigraphic cycles delimited by ial unconformities and their correlative conformities at Maisse. The names of the localities associated with the numbers indicated [#] are in Figure 1D. 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable C |  19 relative conformities at Maisse. The names of the localities associated with the numbers indicated [#] are in Figure 1D. 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules |  19 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Lib 19 MOREAU et al. | FIGURE 10  Correlation diagram of Lutetian to Miocene sections along an east–west transect between La Baronnie quarry and Les Grands Réages quarry. This diagram incorporates eight utcrop sections examined in this study supplemented by three sections from the literature and 29 boreholes. 5.2  |  Early Bartonian to middle Priabonian BP cycle: from an estuarine to a carbonate-evaporitic lacustrine system Correlations are based on the definition of nine stratigraphic cycles delimited by ubaerial unconformities and their correlative conformities at Maisse. The names of the localities associated with the numbers indicated [#] are in Figure 1D. 4877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Co Lutetian to Miocene sections along an east–west transect between La Baronnie quarry and Les Grands Réages quarry. This diagram incorporates eight supplemented by three sections from the literature and 29 boreholes. Correlations are based on the definition of nine stratigraphic cycles delimited by lative conformities at Maisse. The names of the localities associated with the numbers indicated [#] are in Figure 1D. 77, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rul FIGURE 10  Correlation diagram of Lutetian to Miocene sections along an east–west transect between La Baronnie quarry and Les Grands Réages quarry. This diagram incorporates eight utcrop sections examined in this study supplemented by three sections from the literature and 29 boreholes. Correlations are based on the definition of nine stratigraphic cycles delimited by ubaerial unconformities and their correlative conformities at Maisse. The names of the localities associated with the numbers indicated [#] are in Figure 1D. ownloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Co 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01 20 MOREAU et al. 11  Palaeoenvironmental evolution during five periods of carbonate sedimentation. These reconstructions are based ns of this study (Figures 9 and 10) and on pre-existing palaeoenvironmental maps (Mégnien, 1980; Merle, 2008; Briais, nthic Zone (SBZ) and the Calcareous Nannofossils (NP) are based on the global chronostratigraphic chart (Speijer et al 0.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley On ibrary on [04/01/2024]. 5.4  |  Miocene A cycle, a lake on a low energy microbial asymmetrical lacustrine– palustrine margin This last long-term cycle corresponds to the Neogene de- posits in the Paris Basin and contains the carbonate la- custrine Aquitanian Molasse du Gatinais and Calcaire de Beauce formations and the siliciclastic fluvial Sables et Marnes de l'Orléanais Formation. They occur after a 5 Myr long hiatus covering the Chattian (Pomerol, 1989) throughout the basin (Figure 11E). The transgressive sys- tems tract of this long-term cycle is composed of lacustrine and palustrine limestones (commonly facies F5f, F5g, F5h, 5.2  |  Early Bartonian to middle Priabonian BP cycle: from an estuarine to a carbonate-evaporitic lacustrine system From the BP4 to BP6 cycles, a palustrine domain dominated by calcretes (F5a to F5d) migrates from Pithiviers, Melun and Provins towards Paris (Figures 9, 10 and 11D). Along the margins of the basin, palustrine sequences presenting coastal la- custrine facies FA3, in-situ brecciated limestones F5f or chalky altered limestones F5d overlayed by other calcretes (facies F5a-c) develop in several locations [8, 17 and 20 in Figures  1D, 10 and 12]. Maximum flooding surfaces of each cycle are located inside the less modified beds by pedogenetic processes, mainly in limestone and marl beds contaning ostracods, charophytes and foraminifera (facies F3b, F5f or F5g; Figure 12). During the PO1 and PO2 cycles, gypsiferous marls (fa- cies F3e, Argiles bleues d'Argenteuil, Marnes de Pantin, Glaises à cyrènes and Argiles vertes de Romainville mem- bers) displaying brackish to marine fauna develop in a coastal lake environment in the northern part of the basin from Creil to Pithiviers (Figure 9). They pass southwards and eastwards into palustrine deposits with calcrete fa- cies (facies association FA5, Calcaire de Brie Formation). Palustrine environments migrate strongly northwards during the deposition of the highstand systems tracts of the PO2 sequence (Figure  9). These cycles exhibit the same facies and sequences as the BP5 and BP6 cycles previously described. The maxium thickness of the PO3, PO4 and PO5 cycles is seen around Paris and Etampes and thins southwards and eastwards with the Sables de Fontainebleau Formation (Figures 9 and 10). This forma- tion marks a sandy marine transgression from the north- west to the south-east of the basin (Figure 9). Cycle PO6, however, displays a maximum thickness in the south and east of the study area with alternating lacustrine and pa- lustrine facies passing to calcrete facies from Orléans to Etampes (facies F5b, F5c, F5e, F5g, F5h, F6b, F6d, F6f and F6g; Calcaire d'Etampes Formation; Figure  9). It is correlated with a sedimentary hiatus from Paris to Creil (Figures 9 and 10). The descriptions in the west and north-west of the basin of the same palustrine limestones as in the south and south-east (Mégnien, 1980) and the thinning of sed- iments in the north of the basin (Figure 9) suggest that the lake was periodically closed to the marine domain and surrounded by a frequently exposed palustrine area (Figure 11D). 5.2  |  Early Bartonian to middle Priabonian BP cycle: from an estuarine to a carbonate-evaporitic lacustrine system See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed //onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 11  Palaeoenvironmental evolution during five periods of carbonate sedimentation. These reconstructions are based on the cross-sections of this study (Figures 9 and 10) and on pre-existing palaeoenvironmental maps (Mégnien, 1980; Merle, 2008; Briais, 2015). The Shallow Benthic Zone (SBZ) and the Calcareous Nannofossils (NP) are based on the global chronostratigraphic chart (Speijer et al., 2020). 21 MOREAU et al. BP6), lacustrine limestones, marls and evaporites develop from Creil to Etampes (FA3, Calcaire de Saint-Ouen to the top of Marnes et Masses de gypses formations), pass- ing southwards and eastwards into palustrine limestones (FA5, Calcaire de Champigny Formation, Figures 9 and 11C,D). The maximum thickness of deposits occurs in Paris and decreases northwards. Deposits also thin out around the Meudon and Remarde anticlines (Figure 9). At Paris, the transgressive systems tract of the BP4 cycle is composed of lacustrine marl and limestone facies mixed with thin evaporite layers while it thins out com- pletely eastwards (facies F3a-d; Calcaire de Saint-Ouen Formation; Figures  9 and 10). The maximum flooding surfaces of cycles BP4 to BP6 are identified as gypsiferous marls (facies F3e) with marine or brackish fauna (respec- tively inside the Marnes à Pholadomya ludensis Member for BP4, Marnes à lucines Member for BP5, and Marnes d'entre-deux-Masses Member for BP6). Episodes of con- nection with marine water are marked by the occurrence of marine bivalves or foraminifera along the maximum flooding surfaces (Pomerol et al., 1965; Mégnien, 1980), which record salinity close to but lower than that of nor- mal seawater. The highstand systems tracts of cycles BP4, BP5 and BP6 correspond to gypsum deposits around Paris, and spread as far as the Meudon Anticline as noted by Gély & Lorenz (1991) and Mégnien (1974). From the BP4 to BP6 cycles, a palustrine domain dominated by calcretes (F5a to F5d) migrates from Pithiviers, Melun and Provins towards Paris (Figures 9, 10 and 11D). Along the margins of the basin, palustrine sequences presenting coastal la- custrine facies FA3, in-situ brecciated limestones F5f or chalky altered limestones F5d overlayed by other calcretes (facies F5a-c) develop in several locations [8, 17 and 20 in Figures  1D, 10 and 12]. 5.2  |  Early Bartonian to middle Priabonian BP cycle: from an estuarine to a carbonate-evaporitic lacustrine system Maximum flooding surfaces of each cycle are located inside the less modified beds by pedogenetic processes, mainly in limestone and marl beds contaning ostracods, charophytes and foraminifera (facies F3b, F5f or F5g; Figure 12). The descriptions in the west and north-west of the basin of the same palustrine limestones as in the south domain (facies association FA3) into a sandy marine en- vironment. The PO cycle ends with the establishment of lacustrine and palustrine domains (F5 and F6) in the southern part of the basin. It ends with the subaerial un- conformity A1, corresponding to a major sedimentary hiatus beginning at the end of the Rupelian and extend- ing throughout the Chattian (Figure 9). In some localities between Etampes and Orléans, this hiatus is located be- tween the Calcaire d'Etampes (Rupelian) and the Molasse du Gatinais (Aquitanian) formations with sandy or silty marl levels directly overlying lacustrine carbonates. Depocentres are located around Paris until the last short- term cycle PO6, when the depocentre migrates southward. BP6), lacustrine limestones, marls and evaporites develop from Creil to Etampes (FA3, Calcaire de Saint-Ouen to the top of Marnes et Masses de gypses formations), pass- ing southwards and eastwards into palustrine limestones (FA5, Calcaire de Champigny Formation, Figures 9 and 11C,D). The maximum thickness of deposits occurs in Paris and decreases northwards. Deposits also thin out around the Meudon and Remarde anticlines (Figure 9). At Paris, the transgressive systems tract of the BP4 cycle is composed of lacustrine marl and limestone facies mixed with thin evaporite layers while it thins out com- pletely eastwards (facies F3a-d; Calcaire de Saint-Ouen Formation; Figures  9 and 10). The maximum flooding surfaces of cycles BP4 to BP6 are identified as gypsiferous marls (facies F3e) with marine or brackish fauna (respec- tively inside the Marnes à Pholadomya ludensis Member for BP4, Marnes à lucines Member for BP5, and Marnes d'entre-deux-Masses Member for BP6). Episodes of con- nection with marine water are marked by the occurrence of marine bivalves or foraminifera along the maximum flooding surfaces (Pomerol et al., 1965; Mégnien, 1980), which record salinity close to but lower than that of nor- mal seawater. The highstand systems tracts of cycles BP4, BP5 and BP6 correspond to gypsum deposits around Paris, and spread as far as the Meudon Anticline as noted by Gély & Lorenz (1991) and Mégnien (1974). 5.3  |  Middle Priabonian to early Rupelian PO cycle: from coastal lakes to inland lakes The long-term PO cycle is composed of six short-term cy- cles (PO1 to PO6) showing the evolution of a lacustrine 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01 22 MOREAU et al. MOREAU et al. y.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 23 MOREAU et al. FIGURE 12  Carbonate section of the Bartonian-Priabonian in the Maisse borehole (location 8 in Figure 1) with the associated gamma- ray log, the vertical facies succession, the interpretation of sediment exposure time to subaerial processes, and the sequence stratigraphic interpretations. Refer to Section 4 for the facies and facies association nomenclature and explanations of their interpretation. Note that the gamma-ray log effectively distinguishes between different facies associations (FA). Abrupt facies changes and erosive surfaces characterise subaerial unconformities. F6a, F6b, F6d, F6e and F6f) in the north of the Miocene basin (locations 11, 13, 14, 15, 16 and 24 in Figures 1D, 9, 10 and 11E) and more clayey facies near the depocentre at Orléans (facies F6b, F6c and F6g) (Figures 9, 10 and 11E). Multiple shorter depositional sequences are topped by desiccation cracks and indicate successive lake-level vari- ations. The last highstand systems tract recorded by a suc- cession of desiccated limestones (facies F6d, F5f and F5h) covered by cryoturbated white marls, with the A2 subae- rial unconformity at its top, is identified at the top of the Calcaire de Beauce Formation in several localities in the basin (locations [11], [13], [14], [15] and [16] in Figures 10 and 13). with the marine realm, these cycles are compared to the global sea-level curve and European marine cycles (Figure  14; Hardenbol et  al.,  1999; Miller et  al.,  2020; Speijer et al., 2020). The maximum flooding surfaces of the BP4, BP6 and PO1 cycles in the central and northern parts of the study area can be correlated to those in other European basins (Figure  14). This could indicate that these cycles are being controlled by large-scale sea-level variations. However, it was not possible to identify these cyclesin the eastern part of the basin. Only the major regressive trend is recorded (Figure 14). This indicates that the control of relative sea-level variations over short-order cycles decreased or even disappeared east- wards, where environments lay further from the marine domain. 5.3  |  Middle Priabonian to early Rupelian PO cycle: from coastal lakes to inland lakes However, the Bartonian-Priabonian short-term cycles of the Paris Basin and of European basins are not consistent with global sea-level variations, which means that these sea-level variations are not principally a re- sult of eustacy. For example, the major unconformity P3 coincides well with a sequence boundary in European cycles but corresponds to a global sea-level maximum (Figure 14). Moreover, the short-term cycles identified in the Paris Basin during the Lutetian-Bartonian differ somewhat from third-order cycles in other European basins, both in number and age while the study area was mostly occupied by estuarine, restricted or even open marine platform environments during this period (Figure 14; Hardenbol et al., 1999; Speijer et al., 2020). These differences may be explained by local and/or re- gional tectonic movements (Briais,  2015). When the tectonic constraints relaxed, the palaeotopography flat- tened, enabling the deposition of marine sediments over long distances, which then represent maximum flood- ing surfaces. There is no discernible link between the PO6 and A cycles and the eustatic cycle. Differences in facies on either side of the basin are recognised and record differences in water inflow. Facies F6b is associated with in-situ brecciated limestones (fa- cies F5f) along the western margin [locations 13, 14, 15, 16 and 24 in Figure 1D], and with limestones with root traces and wackestones or marls with lithoclasts (respec- tively facies F5h and F5g) on the eastern margin [location 11] (Figure  13). As the asymmetrical lacustrine system model from Arenas and Pardo  (1999) suggests, poorly drained margins are more brecciated and subject to the erosion of pre-existing material, while heavily vegetated fringes develop along well-drained margins. Therefore, the Aquitanian lake shows an asymmetrical profile with a poorly drained western margin and a well-drained eastern margin. This scheme is consistent with the presence of a fluvial system arriving from the south-east at the begin- ning of the A cycle. ley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licens FIGURE 12  Carbonate section of the Bartonian-Priabonian in the Maisse borehole (location 8 in Figure 1) with the associated gamma- ray log, the vertical facies succession, the interpretation of sediment exposure time to subaerial processes, and the sequence stratigraphic interpretations. Refer to Section 4 for the facies and facies association nomenclature and explanations of their interpretation. Note that the gamma-ray log effectively distinguishes between different facies associations (FA). Abrupt facies changes and erosive surfaces characterise subaerial unconformities. 6.1  |  Marine and tectonic influences on palaeoenvironments and sedimentary geometries The long-term cycles (5–10 Ma) of the Paris Basin do not correlate with those of the European basins. It likely indicates that long-term cycles are mainly controlled by tectonics (Figure 14). The cross-sections highlight three different basin configurations (Figures 9, 10 and 14): (1) during the Lutetian and Bartonian, the depocentre was located north of the Bray Fault and deposits progressively onlapped southwards; (2) from the late Bartonian to early Rupelian, the depocentre migrated to Paris and to the east- ern part of the basin while the northern part of the basin and the Meudon Anticline underwent uplift; (3) during Each short-term and long-term cycle was readjusted in time thanks to the biostratigraphic framework de- tailed in Sections 2 and 3 in three locations: in the cen- tre and the north (locations around Paris [1 to 7] in Figure 1D), in the south (locations around Maisse [8 to 16], and to the east (Provins [17 to 21]). Some uncertain- ties remain, however, about the exact age of surfaces. Because coastal lacustrine facies show connections 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are gove 24 24 MOREAU et al. iley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms and conditions) on W line Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are gov wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 13  Schematic logs of the Viabon (A) and Crambes (B) quarries (respectively locations [16] and [11] on Figure 1D) and their stratigraphic sequences. FIGURE 13  Schematic logs of the Viabon (A) and Crambes (B) quarries (respectively locations [16] and [11] on Figure 1D) and their stratigraphic sequences FIGURE 13  Schematic logs of the Viabon (A) and Crambes (B) quarries (respectively locations [16] and [11] on Figure 1D) and their stratigraphic sequences. Pyrenean deformations whereas the last configuration is linked to Alpine deformations (Guillocheau et al., 2000; Bourgeois et  al.,  2007; Briais,  2015). 6.1  |  Marine and tectonic influences on palaeoenvironments and sedimentary geometries The depocentre the late Rupelian, the depocentre moved further south before the large uplift of the basin during the Chattian. The first and second configurations are associated with the late Rupelian, the depocentre moved further south before the large uplift of the basin during the Chattian. The first and second configurations are associated with 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rul 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01 |  25 MOREAU et al. 25 FIGURE 14  Synthetic chronostratigraphic diagram from the Lutetian to the Aquitanian showing stratigraphic cycles in Europe and in the Paris Basin (1: Speijer et al., 2020), global sea-level variations (2: Miller et al., 2020), the evolution of the Paris Basin configuration resulting from tectonic activity and the evolution of depositional systems/environments, and climate (3: Châteauneuf, 1980; 4: Mégnien, 1980; 5: Huyghe et al., 2015; 6: Cramer et al., 2009). The time of each cycle is based on the biostratigraphic fauna discovered in the basin correlated to the global chronostratigraphic chart (see Section 2 for details; Gradstein et al., 2020). y Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for ru FIGURE 14  Synthetic chronostratigraphic diagram from the Lutetian to the Aquitanian showing stratigraphic cycles in Europe and in the Paris Basin (1: Speijer et al., 2020), global sea-level variations (2: Miller et al., 2020), the evolution of the Paris Basin configuration resulting from tectonic activity and the evolution of depositional systems/environments, and climate (3: Châteauneuf, 1980; 4: Mégnien, 1980; 5: Huyghe et al., 2015; 6: Cramer et al., 2009). The time of each cycle is based on the biostratigraphic fauna discovered in the basin correlated to the global chronostratigraphic chart (see Section 2 for details; Gradstein et al., 2020). migration and major palaeogeographical changes related to these deformations resulted in different sedimentary profiles. The first configuration favours the northward connection of the basin to the marine domain, and then the deposition of open and restricted marine facies (as- sociations FA1 and FA2; Calcaire grossier and Marnes et Caillasses formations). 6.2.1  |  The coastal lacustrine system The coastal lacustrine system (facies association FA3 and FA5, Figure 15) was dominated by freshwater, re- corded in the lacustrine sediments by the abundance of ostracods, charophytes and gastropods, but had ephem- eral connections with the marine domain (Figure 15A). Carbon and oxygen isotopes show a large range of val- ues but are lower than those from the restricted marine carbonates (Figure 15A). This indicates variation in sa- linity due to (1) the mixing between meteoric and ma- rine waters and (2) a decrease in the inflow-evaporation ratio (Figures 5 and 15; Talbot, 1990). Carbonate facies are micritic with some bioclastic levels (both marine and lacustrine fauna). The faunal content and its diver- sity are generally greater in marine influenced deposits (Fürsich,  1993), which present packstone to rudstone textures (facies F3b and F3c) or marly facies (F3e). Freshwater deposits often present mudstone to pack- stone textures. Ephemeral connections with the marine domain, probably during storms as attested by the pres- ence of tempestites, favour the development of euryha- line foraminifera and marine fauna in brackish to saline waters (Pint et al., 2017; Fritz et al., 2018). It is a challenge to classify and relate facies between marine and non-marine domains in many ancient sedi- mentary systems especially where outcrops are rare (e.g., the Issirac Basin, France) or with subsurface data (co- quina formations in the Campos, Santos or Sergipe ba- sins, Brazil; Thompson et al., 2015; Lettéron et al., 2018; Favoreto et al., 2021). In some previous works focussing on the Cenozoic of the Paris Basin, palaeontologists based their palaeoenvironmental definitions on salinity crite- ria (Abrard, 1925; Denizot, 1927; Blondeau et al., 1965; Pomerol et  al.,  1965; Cavelier,  1968; Mégnien,  1974; Turland,  1974; Pomerol & Riveline,  1975; Aubry et al., 1977; Mégnien, 1980). Indeed, deposits with a pal- aeontological record suggesting normal marine salinity were defined as open to restricted marine, deposits with a brackish water fauna were classified as coastal brackish lagoonal, and deposits with freshwater to brackish fossils were interpreted as lacustrine. The large-scale cross-sec- tions traced in this study indicate that levels with marine or brackish fauna are correlated with those with brackish to freshwater fauna without any thickness variations and without “barrier” facies (sand bars, palaeosols, marshes etc.). This suggests a salinity gradient within a single waterbody rather than separate lakes or lagoons. 6.2.1  |  The coastal lacustrine system These salinity gradients must therefore have resulted from the mixing of seawater and meteoric water from the drain- age basin, and/or from a variable evaporation rate. Three distinct settings (restricted marine domain, coastal lake and inland lake) can be defined based on facies, salinity, sedimentary geometries, palaeogeography and carbon and oxygen isotope data of micrites. This system developed under two different climates in the Paris Basin: a subtropical climate (wet with contrasted seasons) during the Bartonian and the Rupelian, and a semi-arid climate (dry with strong seasonality) during the Priabonian (Figure 14; Châteauneuf, 1980). Under a sub- tropical climate, gypsum precipitation was reduced and micritic facies (facies F3b and F3c) and varves (facies F3d) formed on the margins and in the depocentre of a strat- ified lake (Figure 15). Under semi-arid conditions, high gypsum-content deposits formed in the depocentre of the lake (facies F3a and F3e) (Priabonian, Figure 15). Planar microbial laminae formed on the lake margins, mostly with gypsum deposits (facies F3a). The same facies dis- tribution was identified depending on the climate in the oligohaline to mesohaline and hypersaline lake models (Eugster & Hardie, 1978; Allen & Collisson, 1986; Bohacs et al., 2000; Lettéron et al., 2022) or in sabkha and non-ma- rine evaporitic environments (Evans et al., 1969; Warren & Kendall, 1985; Shaw et al., 1990; Cooke et al., 1993; Rouchy et al., 1993; Arenas & Pardo, 1999; Bouton et al., 2016). However, it differs with respect to the presence of marine fauna due to marine connections. First, the restricted marine platform (facies associa- tion FA2) was attached to the marine domain, experi- encing variable salinity (marine, brackish or hypersaline water) and deposition of marine influenced sediments (diversified marine fauna, slightly negative δ13C val- ues). This setting developed in the Paris Basin during Lutetian– early Bartonian times (Figure 11A,B). Second, during Bartonian–Rupelian times the coastal lake (FA3) in the Paris Basin was dominated by meteoric and flu- vial waters although ephemeral connections occurred with the marine domain (δ13C and δ18O values covary and are negative; Figure 11C,D). Finally, the inland lake setting (FA6) is disconnected from any marine influ- ences and freshwater sediments formed (late Rupelian and Aquitanian stages in the Paris Basin; Figure 11E). The following parts of the discussion presents detailed facies models and differences for the newly defined coastal lake and inland lake settings. 6.1  |  Marine and tectonic influences on palaeoenvironments and sedimentary geometries The progressive southward migra- tion of the depocentre during the Bartonian–Priabonian implies a progressive disconnect of the basin from the marine domain and the deposition of coastal lake facies (association FA3, Calcaire de Saint-Ouen and Marnes et Masses de gypses formations). During the Rupelian, the migration of the depocentre even further south implies a complete disconnect with the marine domain, and thus the formation of an inland lake facies (association FA6, Calcaire d'Etampes and Calcaire de Beauce formations). 20554877, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/dep2.264 by Cochrane France, Wiley Online Library on [04/01 26 26 MOREAU et al. 15  Depositional and facies models of lacustrine–palustrine carbonate systems of the Paris Basin during the Cenozoi lacustrine system model: mixed carbonate-evaporitic brackish to hypersaline lacustrine to palustrine environments, w y Cochrane France, Wiley Online Library on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for ru ibrary on [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed 15  Depositional and facies models of lacustrine–palustrine carbonate systems of the Paris Basin during the Cenozoic lacustrine system model: mixed carbonate-evaporitic brackish to hypersaline lacustrine to palustrine environments, wit nnections with the marine domain; semi-arid, subtropical climate; Bartonian to Rupelian. (B) Inland lacustrine system: water microbial lacustrine margin isolated from the marine domain; temperate climate; Rupelian and Aquitanian. e Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Co wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 15  Depositional and facies models of lacustrine–palustrine carbonate systems of the Paris Basin during the Cenozoic. (A) Coastal lacustrine system model: mixed carbonate-evaporitic brackish to hypersaline lacustrine to palustrine environments, with episodic connections with the marine domain; semi-arid, subtropical climate; Bartonian to Rupelian. (B) Inland lacustrine system: low energy freshwater microbial lacustrine margin isolated from the marine domain; temperate climate; Rupelian and Aquitanian. FIGURE 15  Depositional and facies models of lacustrine–palustrine carbonate systems of the Paris Basin during the Cenozoic. (A) Coastal lacustrine system model: mixed carbonate-evaporitic brackish to hypersaline lacustrine to palustrine environments, with episodic connections with the marine domain; semi-arid, subtropical climate; Bartonian to Rupelian. (B) Inland lacustrine system: low energy freshwater microbial lacustrine margin isolated from the marine domain; temperate climate; Rupelian and Aquitanian. 27 27 MOREAU et al. 6.2.1  |  The coastal lacustrine system Laterally to the lake (facies association FA3), a pa- lustrine domain (FA5) developed, strongly affected by subaerial exposure (Figure 15A). The negative δ13C and δ18O values indicate the influence of meteoric fluids (Figures 5 and 15). In this palustrine environment, the ini- tial carbonate formed in low saline meteoric water judg- ing from the biota and the negative carbon and oxygen 28 28 MOREAU et al. Vera & Jiménez de Cisneros, 1993; Miller et al., 2013). In the deepest part of the lake, alternations of marls and mudstones (facies F6g) form. As mentioned by Platt and Wright (1992), the highly extended palustrine domain indicates that the lacustrine–palustrine system was mainly shallow, as attested by the abundance of desic- cated lacustrine facies (facies F6a, b, d and e) and the rarity/absence of deep-water facies. The palustrine do- main that developed laterally to the inland lake is differ- ent to the palustrine domain associated with the coastal lake. Fewer calcretes formed and they are localised in the areas furthest from the lake (Figure 15). Instead, or- ganic-rich marls and alternating mudstones and wacke- stones with root traces (facies F5g and F5h) were more frequent along the lake margins. Breccia facies formed in-situ by desiccation or by recycling of the pre-existing and early cemented carbonate are also abundant (facies F5f, F6a). This model is similar to those of Freytet and Plaziat (1982), Allen and Collinson (1986), Platt (1989) or Bohacs et  al. (2000) when open or balanced-filled lakes have low-energy margins. Nonetheless, it differs by showing a lower detrital input, abundant microbial crusts and reworked intraclasts, and a large variety of palustrine facies. The differences in facies distribution between the coastal and inland lacustrine systems can be explained by the wetter climate during the Rupelian– Aquitanian than during the Priabonian (Figure  15B; Châteauneuf, 1980). isotope values of the micrites (Arenas et al., 1997; Alonso- Zarza & Arenas, 2004; Leng & Marshall, 2004; Huerta & Armenteros,  2005; Fischer-Femal & Bowen,  2021). The palustrine domain was greatly extended in a semi-arid climate, which is consistent with the current view of pa- lustrine carbonate formation (Freytet & Plaziat,  1982; Wright & Tucker,  1991; Platt & Wright,  1992; Alonso- Zarza, 2003; Huerta & Armenteros, 2005; Alonso-Zarza & Wright, 2010; Azerêdo et al., 2015). 6.2.1  |  The coastal lacustrine system Palustrine facies show varying degrees of modification by subaerial exposure processes, ranging from simple desiccation of the primary mud (facies F5e and f) to total pedogenic overprinting (fa- cies F5a to d). These varied features (desiccation cracks, microkarsts, root traces, calcretes, rhizoconcretions) re- veal different durations of exposure to surface processes, directly dependent on the climate. Under subtropical con- ditions, these features are less developed and the calcrete facies are thinner in metre-thick depositional sequences. The depositional sequences begin at the base with in-situ brecciated limestones (facies F5f) and chalky altered lime- stones (facies F5d) marking high sedimentation rates and moderate pedogenetic processes, i.e., a high lake-water level during a relatively wet period (Figure  12; Alonso- Zarza & Wright, 2010). The common presence of lacus- trine facies below palustrine facies within depositional sequences indicates a lacustrine origin for the micrite. Facies F5f and F5d pass upwards into nodular brecciated limestones (facies F5b) and then tubular and laminar brecciated limestones (facies F5a and F5c), which indi- cate a lower sedimentation rate and formation by intense pedogenesis during longer periods of subaerial exposure (Figure 12). Subaerial exposure increasingly modified pal- ustrine facies with distance from the lake (Figure 15). In the inland lake, micrite is still dominant but mi- crobial activity is clearly more important than in coastal lakes, with the abundance of laminated microbial crusts and microbial oncoids (facies F6e and f). These differences in microbial structures between coastal and inland lakes probably result from climate changes. Indeed, microbial structures are more developed during periods of temperate climate (Rupelian and Aquitanian) (Châteauneuf,  1972; Utescher et al., 2000; Scherler et al., 2013). The enhanced development of microbial structures in wet climates during the Aquitanian is already known from other lo- cations in Western Europe (Roche et  al.,  2018; Arenas- Abad, 2021; Vennin et al., 2021). High precipitation rates may favour microbial activity by bringing nutrients into the lakes. iley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6.2.2  |  The inland lacustrine system The inland lacustrine system (facies associations FA6 and FA5) presents no marine influence but instead indi- cate sedimentation in freshwater (δ13C and δ18O values covary and are negative during the late Rupelian and Aquitanian; Figure 15B). Fossils such as gastropods, os- tracods and charophytes are present but only abundant in specific locations on the lake margins, where they may form coquinas (facies F6d; Gierlowski-Kordesch, 2010). These fossil-rich beds alternate with thick micro- bial crusts (facies F6f) and oncoidal beds (facies F6e). Desiccation in the lacustrine facies is frequent and marks subaerial exposure. The abundance of reworked intra- clast facies (F6a and F6b) and grainstones with peloids and shell fragments (facies F6c) highlights the rework- ing by waves of desiccated carbonates from the palus- trine and lacustrine margins (Strasser, 1984; Platt, 1989; Palustrine carbonates formed therefore periodically from the late Lutetian to the Aquitanian in the Paris Basin and pass laterally to restricted marine and lacustrine en- vironments (facies associations FA2, FA3 and FA6). In the fossil record, palustrine carbonates have been identi- fied in numerous settings associated with deposits from internal ramps, lagoons, rivers and lakes (Alonso-Zarza & Wright,  2010a). These carbonates are often linked to significant underlying karstic aquifer systems (Platt & Wright,  2023). These aquifers can supply the necessary 29 MOREAU et al. CaCO3 for carbonate formation in marshes through ris- ing groundwaters (“Groundwater Dependent Carbonate Factories” by Platt & Wright,  2023). Therefore, the ap- pearance and development of palustrine carbonates in the fossil record can be interpreted in response to the aquifer level rising due to marine transgression in coastal settings or regional watershed responses to inland basin filling. fauna adapted to low salinity conditions (mesohaline to oligohaline), a strong negative carbon and oxygen anom- aly of the micrite indicating a major contribution from continental waters and sediment thicknesses reducing towards the marine domain. Facies correspond to varves or evaporites in the depocentre while micritic carbonates with a brackish fauna were deposited on the lake mar- gins. The associated palustrine domain is mainly marked by the formation of calcrete facies presenting different textures (chalky, nodular, laminar and rhizoliths) and showing successive metre-sized shallowing upwards se- quences, recording subaerial exposure. In-situ brecciated limestones and chalky calcretes are the palustrine facies located closest to the lake margins. Moving away from the lake, they pass into nodular and then into laminated calcretes and rhizoliths. 6.2.2  |  The inland lacustrine system The latter are the most devel- oped palustrine facies in the coastal lake system. In the case studied here, the palustrine and lacustrine carbonates are currently karstified and overlay the karst- ified Cretaceous chalk aquifer in the eastern and south- ern parts of the basin. Although no information about the exact age of these karstifications is known, the presence of Burdigalian mammals in the karsts within the Aquitanian Calcaire de Beauce Formation suggests that some of these karsts formed very early after sedimentation, no later than a few million years. Consequently, the chalk aquifer and possibly underlying karstic aquifers within non-marine carbonates may have controlled the water supply in these lacustrine-palustrine environments. While brief marine connections have been identified in the coastal lacustrine system, they are not recorded in the palustrine domain; their contributions may be relatively minor compared to the inputs from the watershed and underlying aquifers. Moreover, the drier climate of Priabonian-early Rupelian times likely contributed to the extensive development of palustrine facies compared to the late Rupelian and the Aquitanian, affecting the water inflow from the watershed and the evaporation rate. The preservation of these palus- trine deposits, which can reach thicknesses of 40–50 m in the Calcaire de Champigny Formation, is likely favoured by local subsidence occurring to the east and south of the basin during the Lutetian-Aquitanian. Inland lacustrine systems developed during the Rupelian and the Aquitanian in the Paris Basin. Lakes became disconnected from the marine domain and were home to freshwater fauna. As a result, oxygen and carbon isotope values are highly negative and less variable than in the coastal lake. Microbial crusts with micritic or hy- brid laminated microfabrics and oncoids are frequent and attest to intense microbial activity. In the palustrine do- main, organic-rich layers, limestones with root traces and peloidal grainstones formed close to the lake margins. They present subaerial exposure features such as rootlet moulds, desiccation cracks and microkarstification, and they are frequently interbedded with lake facies. In-situ brecciated limestones and rare calcrete facies reflect a longer exposure time and are located further from the lake margin. y.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licen In the intracratonic Paris Basin, large-scale and long- term tectonic processes were the main control of long-term cycles and led to the southward migration of the dep- ocentre. 6.2.2  |  The inland lacustrine system Tectonism was responsible for the changes from dominant marine environments in the Lutetian to coastal lacustrine systems during the Bartonian and then inland lacustrine systems during the Rupelian and Aquitanian by modifying the connection of the basin with the marine do- main. Third-order relative sea-level variations recorded in other European basins seem to control short-term cycles in the coastal lacustrine systems (BP4, BP6 and PO1 cycles), but not in the inland systems. As a result, short and long- term sequence stratigraphic surfaces (maximum regressive and maximum flooding surfaces) could be extended from the marine domain to the coastal lacustrine systems, allow- ing correlation between these two depositional settings. This reinforces the importance of differentiating between coastal and inland lacustrine environments. The impact of climate on non-marine carbonate production was a major feature in the Paris intracratonic basin. In more temperate Kévin Moreau  https://orcid.org/0000-0001-5165-8740 Kévin Moreau  https://orcid.org/0000-0001-5165-8740 7  |  CONCLUSIONS The petrographic and facies studies of 35 new sedimen- tary sections coupled with data from the literature for the coastal and continental carbonate formations of the Cenozoic of the Paris Basin allow us to define 32 facies formed in depositional environments ranging from open marine to lacustrine and palustrine. Key short-term and long-term sequence stratigraphic surfaces (maximum flooding and maximum regressive surfaces) were corre- lated along two large-scale cross-sections to distinguish spatial and temporal facies evolution and depositional ge- ometries. Two facies models are proposed for coastal and inland lacustrine systems, encompassing both the lacus- trine and palustrine environments. Coastal lacustrine systems developed during the Bartonian and Rupelian in the Paris Basin. These lakes had episodic connections with the marine domain. This environment differs from lagoon environments by a 30 30 MOREAU et al. Alonso-Zarza, A.M. (1999) Initial stages of laminar calcrete forma- tion by roots: examples from the Neogene of central Spain. Sedimentary Geology, 126, 177–191. to humid climates, lacustrine carbonate production was in- duced by microbial activity. Finally, it is highly likely that the presence of aquifers in the Cretaceous chalk and in the lacustrine-palustrine carbonate formations played a significant role in the for- mation of the palustrine deposits on the surface, by supply- ing the calcium carbonate necessary for their formation. However, the existence of such aquifers when palustrine carbonates were formed remains to be proven. Alonso-Zarza, A.M. (2003) Palaeoenvironmental significance of pal- ustrine carbonates and calcretes in the geological record. Earth Science Reviews, 60, 261–298. https://​doi.​org/​10.​1016/​S0012​-​ 8252(02)​00106​-​X Alonso-Zarza, A.M. & Arenas, C. (2004) Cenozoic calcretes from the Teruel Graben, Spain: microstructure, stable isotope geochem- istry and environmental significance. Sedimentary Geology, 167, 91–108. https://​doi.​org/​10.​1016/j.​sedgeo.​2004.​02.​001 Alonso-Zarza, A.M. & Wright, V.P. (2010a) Chapter 2: palustrine carbonates. In: Alonso-Zarza, A.M. & Tanner, L.H. (Eds.) Developments in Sedimentology Carbonates in continen- tal settings: facies, environments, and processes. Amsterdam: Elsevier 61, pp. 103–131. https://​doi.​org/​10.​1016/​S0070​-​4571​ (09)​06102​-​0 ORCID Arribas, M.E., Bustillo, A. & Tsige, M. (2004) Lacustrine chalky car- bonates: origin, physical properties and diagenesis (Palaeogene of the Madrid Basin, Spain). Sedimentary Geology, 166, 335– 351. https://​doi.​org/​10.​1016/j.​sedgeo.​2004.​01.​012 n [04/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by Amao, A.O., Kaminski, M.A. & Setoyama, E. (2016) Diversity of foraminifera in a shallow restricted lagoon in Bahrain. Micropaleontology, 62, 197–211. https://​doi.​org/​10.​47894/​mpal.​ 62.3.​01 Arenas, C., Casanova, J. & Pardo, G. (1997) Stable-isotope char- acterization of the Miocene lacustrine systems of Los Monegros (Ebro Basin, Spain): palaeogeographic and palae- oclimatic implications. Palaeogeography Palaeoclimatology Palaeoecology, 128, 133–155. https://​doi.​org/​10.​1016/​S0031​ -​0182(96)​00052​-​1 Arenas, C. & Pardo, G. (1999) Latest Oligocene–Late Miocene la- custrine systems of the north-central part of the Ebro Basin (Spain): sedimentary facies model and palaeogeographic syn- thesis. Palaeogeography Palaeoclimatology Palaeoecology, 151, 127–148. https://​doi.​org/​10.​1016/​S0031​-​0182(99)​00025​-​5 DATA AVAILABILITY STATEMENT Data used in this research are available in the Supplementary Information associated with the article as well as upon reasonable request from the corresponding author. Arenas-Abad, C. (2021) A multi-scale approach to laminated micro- bial deposits in non-marine carbonate environments through examples of the Cenozoic, north-east Iberian Peninsula, Spain. Depositional Record, 8, 67–101. ACKNO​WLE​DGE​MENTS This study was conducted within the framework of the research collaboration agreement 2019-157 between the BRGM (Bureau de Recherches Géologiques et Minières, French Geological Survey) and Université Paris-Saclay. This project was funded by the BRGM through the “Référentiel Géologique de la France” programme and by a doctoral PhD from the French Ministry of Research and Higher Education (2019-105). We thank all the companies that gave us access to their quarries, with- out which some of this work would not have been pos- sible: a2c-materiaux, Cemex, Eurovia, Fulchiron, Imerys Ceramic France, Lafarge Holcim, SMBP, and Roland and Dadonville quarries. We are also grateful to the SIARCE for allowing us to describe the Maisse borehole. Thanks to Chantal Bourdillon (Stratigraphie et biochronologie) for the identification of foraminifera species and to Phillipe Blanc (Lithologie Bourgogne) for the high-quality of thin sections. 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(2021) The Limagne Basin: a journey through modern and P., Wattinne, A., Kolodka, C., Gaucher, E., Virgone, A. & Visscher, P.T. 00102​C1865D (1989) Lacustrine carbonates and pedogenesis: Sedimentology and origin of palustrine deposits from the Early Cretaceous Rupelo Formation, W Cameros Basin, N Spain. 34 34 MOREAU et al. Rouchy, J.M., Taberner, C. & Peryt, T.M. (2001) Sedimentary and diagenetic transitions between carbonates and evaporites. Sedimentary Geology, 140, 1–8. https://​doi.​org/​10.​1016/​S0037​-​ Sedimentology, 36, 665–684. https://​doi.​org/​10.​1111/j.​1365-​ 3091.​1989.​tb020​92.​x Platt, N.H. & Wright, V.P. (1991) Lacustrine carbonates: facies models, facies distributions and hydrocarbon aspects. Special Publications of the International Association, 13, 57–74. https://​ doi.​org/​10.​1002/​97814​44303​919.​ch3 0738(00)​00169​-​X Scherler, L., Mennecart, B., Hiard, F. & Becker, D. (2013) Evolutionary his- tory of hoofed mammals during the Oligocene–Miocene transition in Western Europe. Swiss Journal of Geosciences, 106, 349–369. doi.​org/​10.​1002/​97814​44303​919.​ch3 (2021) The Limagne Basin: a journey through modern and 35 MOREAU et al. 35 fossil microbial deposits. Bulletin de la Société géologique de France, 192, 41. https://​doi.​org/​10.​1051/​bsgf/​2021030 Wright, V.P. (1994) Paleosols in shallow marine carbonate se- quences. Earth Science Reviews, 35, 367–395. https://​doi.​org/​10.​ 1016/​0012-​8252(94)​90002​-​7 Vera, J.A. & Jiménez de Cisneros, C. (1993) Palaeogeographic signif- icance of black pebbles (Lower Cretaceous, Prebetic, southern Spain). Palaeogeography Palaeoclimatology Palaeoecology, 102, 89–102. https://​doi.​org/​10.​1016/​0031-​0182(93)​90007​-​6 Ziegler, P.A. (1990) Geological atlas of Western and Central Europe. London: Shell International Petroleum Mij. B.V. and Geological Society, Bath, p. 239. Warren, J.K. & Kendall, C.G.S.C. (1985) Comparison of sequences formed in marine Sabkha (Subaerial) and salina (subaqueous) settings—modern and ancient. AAPG Bulletin, 69, 1013–1023. https://​doi.​org/​10.​1306/​AD462​B46-​16F7-​11D7-​86450​00102​ C1865D Warren, J.K. & Kendall, C.G.S.C. (1985) Comparison of sequences formed in marine Sabkha (Subaerial) and salina (subaqueous) settings—modern and ancient. AAPG Bulletin, 69, 1013–1023. SUPPORTING INFORMATION Additional supporting information can be found online in the Supporting Information section at the end of this article. Wilson, J.L. (1974) Characteristics of carbonate-platform margins. AAPG Bulletin, 58, 810–824. https://​doi.​org/​10.​1306/​83D91​ 49D-​16C7-​11D7-​86450​00102​C1865D 49D-​16C7-​11D7-​86450​00102​C1865D How to cite this article: Moreau, K., Andrieu, S., Briais, J., Brigaud, B. & Ader, M. (2023) Facies distribution and depositional cycles in lacustrine and palustrine carbonates: The Lutetian– Aquitanian record in the Paris Basin. The Depositional Record, 00, 1–35. Available from: https://doi.org/10.1002/dep2.264 Wright, V.P., Platt, N.H. & Wimbledon, W.A. (1988) Biogenic laminar calcretes: evidence of calcified root-mat horizons in paleosols. Sedimentology, 35, 603–620. Wright, V.P. (1989) Terrestrial stromatolites and laminar calcretes: a review. Sedimentary Geology, 65, 1–13. Wright, V.P. & Tucker, M.E. (1991) Calcretes: an Introduction. In: Wright, V.P. & Tucker, M.E. (Eds.) Calcretes. Oxford: Blackwell Publishing Ltd, pp. 1–22. https://​doi.​org/​10.​1002/​97814​44304​ 497.​ch
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DPP4 in diabetes
Frontiers in immunology
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Edited by: Heidi Noels, Institute for Molecular Cardiovascular Research, Germany Reviewed by: Yasuo Terauchi, Yokohama City University, Japan Noriyasu Hirasawa, Tohoku University, Japan *Correspondence: Juergen Eckel, Paul-Langerhans-Group for Integrative Physiology, German Diabetes Center, Auf’m Hennekamp 65, Düsseldorf D-40225, Germany eckel@uni-duesseldorf.de Edited by: Heidi Noels, Institute for Molecular Cardiovascular Research, Germany Reviewed by: Yasuo Terauchi, Yokohama City University, Japan Noriyasu Hirasawa, Tohoku University, Japan Keywords: CD26/DPP4, soluble DPP4, type 2 diabetes mellitus, incretins, DPP4 inhibitors/gliptins, multifunctional enzyme Diana Röhrborn, Nina Wronkowitz and Juergen Eckel* l-Langerhans-Group for Integrative Physiology, German Diabetes Center, Düsseldorf, Germany Paul-Langerhans-Group for Integrative Physiology, German Diabetes Center, Düsseldorf, Germany Dipeptidyl-peptidase 4 (DPP4) is a glycoprotein of 110 kDa, which is ubiquitously expressed on the surface of a variety of cells. This exopeptidase selectively cleaves N- terminal dipeptides from a variety of substrates, including cytokines, growth factors, neu- ropeptides, and the incretin hormones. Expression of DPP4 is substantially dysregulated in a variety of disease states including inflammation, cancer, obesity, and diabetes. Since the incretin hormones, glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide (GIP), are major regulators of post-prandial insulin secretion, inhibition of DPP4 by the gliptin family of drugs has gained considerable interest for the therapy of type 2 diabetic patients. In this review, we summarize the current knowledge on the DPP4–incretin axis and evaluate most recent findings on DPP4 inhibitors. Furthermore, DPP4 as a type II transmembrane protein is also known to be cleaved from the cell membrane involving different metalloproteases in a cell-type-specific manner. Circulating, soluble DPP4 has been identified as a new adipokine, which exerts both para- and endocrine effects. Recently, a novel receptor for soluble DPP4 has been identified, and data are accumulating that the adipokine-related effects of DPP4 may play an important role in the pathogenesis of cardiovascular disease. Importantly, circulating DPP4 is augmented in obese and type 2 diabetic subjects, and it may represent a molecular link between obesity and vascular dysfunction. A critical evaluation of the impact of circulating DPP4 is presented, and the potential role of DPP4 inhibition at this level is also discussed Introduction Dipeptidyl-peptidase (DPP) 4, which is also known as CD26, is a ubiquitously expressed glycopro- tein of 110 kDa, which was first characterized by Hopsu-Havu and Glenner (1). DPP4 is a type II transmembrane protein, which is also cleaved off the membrane and released into the circulation by a process called shedding (2, 3). The importance of DPP4 for the scientific and medical community raised substantially since the approval of DPP4 inhibitors for the treatment of type 2 diabetes mellitus (T2DM). These so-called gliptins increase the incretin levels and therefore prolong the post-prandial insulin action. Since soluble DPP4 is characterized as an adipokine (4) and also correlates with parameters of the metabolic syndrome (5), it might also be an important molecular biomarker. DPP4 is a multifunctional enzyme, which serves as a binding partner for numerous peptides, among which are adenosine deaminase (ADA) and extracellular matrix proteins (2, 6, 7). Moreover, as a serine protease, DPP4 cleaves numerous substrates, which further amplifies its complexity of action. Thus, DPP4 is involved in signaling processes, immune cell activation, and its dysregulated expression and release is associated with numerous diseases. Specialty section: This article was submitted to Chemoattractants, a section of the journal Frontiers in Immunology Received: 06 May 2015 Accepted: 13 July 2015 Published: 27 July 2015 REVIEW published: 27 July 2015 doi: 10.3389/fimmu.2015.00386 Keywords: CD26/DPP4, soluble DPP4, type 2 diabetes mellitus, incretins, DPP4 inhibitors/gliptins, multifunctional enzyme Citation: July 2015 | Volume 6 | Article 386 1 Frontiers in Immunology | www.frontiersin.org Röhrborn et al. DPP4 in diabetes classically secreted proteins, the signal peptide is not cleaved off, but serves as a membrane anchor. We were able to show that the circulating form of DPP4 (sDPP4), which lacks the cytoplasmic domain and the transmembrane region, is cleaved off the mem- brane of human adipocytes and smooth muscle cells in a process called shedding by the involvement of matrix metalloproteases (MMPs) (3). In the present review, we wanted to emphasize the complex function of DPP4 with special focus on its association to T2DM. Furthermore, we wanted to offer a different perspective of the current view of DPP4 beyond the inhibition of its protease activity (8–10). The first part of the present review is dealing with general information about DPP4 and its numerous biological functions in regard to T2DM and its treatment. The last section collects the current knowledge about how DPP4 with its pleiotropic func- tions, as described before, affects several organs, thereby playing a pivotal role in the development of T2DM and its comorbidities. Within the TMD, it could be shown that proline residues play an important role for the translocation of membrane-anchored proteins, such as DPP4. Chung and colleagues (11) studied sin- gle proline substitution throughout the TMD of DPP4. They were able to show that translocation and integration into the membrane are determined by the hydrophobicity, conforma- tion, and also the location of proline within the TMD. Further- more, the position of proline relative to other prolines and the location of highly hydrophobic residues within the TMD are important for correct translocation and membrane integration of DPP4. Biology of DPP4 The following part will deal with the domain architecture and respective relevance of these domains for the functionality of DPP4. Dipeptidyl-peptidase 4 (EC3.4.14.5) is a type II transmembrane protein, which groups together with fibroblast-activation protein α (FAP), the resident cytoplasmic proteins, DPP8 and DPP9, and the non-enzymatic members, DPP6 and DPP10, to the serine peptidase subfamily S9B. All of these proteins share a typical α/β hydrolase fold (2, 6). The DPP4 protein consists mainly of 4 domains: a short cytoplasmic domain (1–6), a transmembrane domain (TMD) (7–28), a flexible stalk segment (29–39), and the extracellular domain (40–766), which can be further separated by a highly glycosylated region, the cysteine-rich region, and the catalytic region (Figure 1). In addition to the TMD, the glycosylation of DPP4 is also important for the correct trafficking of DPP4. Carbohydrates account for approximately 20% of the total molecular mass of DPP4 and cause heterogeneity of this protein depending on the location on different cell types. Two highly conserved gluta- mate residues (205 and 206) within the glycosylated region are essential for the activity of DPP4 (12). Interestingly, six of the nine N-glycosylation sites are located within the glycosylated region. These glycosylation sites are mostly conserved among species. They are necessary for folding, stability, and intracellu- lar trafficking (13). Other modifications like sialylation and/or O-glycosylation have an impact on targeting DPP4 to the cell membrane. Sialyation of DPP4 increases significantly with age, and hypersialyation occurs in patients with HIV infection (14). As a member of the type II transmembrane proteins, DPP4 contains a typical signal peptide, which is necessary for the targeting to the endoplasmatic reticulum and the initiation of the translocation across the cell membrane. In contrast to the FIGURE 1 | Domain structure of DPP4 [adapted from Ref. (2)]. Schematic representation of the membrane-bound DPP4 monomer. The extent of the circulating and soluble form of DPP4 is illustrated on the left in blue. The shedding of DPP4 from the membrane by indicated matrix metalloproteinases is shown by a scissor symbol in red. The vertical black bar on the right represents the primary structure with the delineation of the different regions. In green are interactions collected, which occur in the indicated region of the DPP4 structure. MMP, matrix metalloproteinase; M6P/IGFII, mannose-6 phosphate/insulin-like growth factor 2. FIGURE 1 | Domain structure of DPP4 [adapted from Ref. (2)]. Schematic representation of the membrane-bound DPP4 monomer. DPP4 Expression and Its Regulation Dipeptidyl-peptidase 4 is ubiquitously expressed on numerous different cell types among which are epithelial cells, fibroblasts, and leukocyte subsets. Mechanisms that regulate DPP4 gene tran- scription and enzymatic activity are not fully understood so far and may be dependent on the studied cell type. y p yp The human DPP4 gene is located on chromosome 2, spans 70 kb, and consists of 26 exons (2). The DPP4 promoter region contains consensus sites for different transcription factors like NFκB, SP-1, EGFR, and AP-1 factor NF-1 (18). At least in chronic b lymphocytic leukemia cells, it could be shown that there is a consensus interferon γ-activated sequence (GAS), which is a binding motif for STAT1. The interferons, α, β, and γ, stimulate STAT1α binding to this region and thus lead to an increased DPP4 expression and activity (19). Interleukin (IL) 12, which is a key factor in differentiation of naïve T-cells into the Th1 subtype, is also able to upregulate DPP4 expression. Therefore, DPP4 is important in immune cell activation (20, 21). Our group was able to show that release of soluble DPP4 is increased upon TNFα stimulation and insulin in vitro (4). However, IL-12 and TNFα also seem to play a regulatory role in translation and translocation of DPP4. In activated lymphocytes, IL-12 upregulates DPP4 trans- lation whereas TNFα decreases cell surface expression, which might be due to elevated sDPP4 release (22). Also transcription factors, such as HIF-1α and HNFs, target DPP4 expression (23), which fits to the observation of our group that hypoxia induces DPP4 release in human smooth muscle cells, which might be mediated by MMPs (3). Beside its role in inflammation, adenosine is also an important player in glucose homeostasis. Already in 1988 it was shown that, by lowering endogenous adenosine levels, ADA contributes to a reduced insulin sensitivity of glucose transport stimulation (30). Additionally, adenosine seems to facilitate insulin action in adipocytes (31). Another study could show a correlation of increased ADA activity in T2DM with fasting plasma glucose, HbA1c, aspartate, and alanine aminotransferase (ALT). DPP4 inhibitors exert no additional effects on ADA activity despite glycemic control or HbA1c-dependent effects (32). All these studies emphasize that the effects of ADA/DPP4-interaction are independent of DPP4 enzymatic activity. Another known interaction partner of DPP4 is Caveolin-1, which is present on antigen-presenting cells (APCs) and binds to residues 630 and 201–211 of DPP4 expressed on T-cells. Binding Partners of Membrane-Bound DPP4 g The best-studied interaction in this regard is certainly the binding of DPP4 and ADA. It was already identified in 1993 by Morrison and colleagues (24). Importantly, the interaction of DPP4 and ADA preserves the enzymatic function of both binding partners. It has been shown that residues 340–343 of DPP4 are essential for the interaction with ADA. Regulation of the DPP4/ADA inter- action occurs, e.g., via tetramerization of DPP4 or glycosylation at Asn281, which interferes with ADA binding (25). Also, the HIV envelope glycoprotein, gp120, which interacts with DPP4 on lymphocytes via its C3 region, is able to inhibit the association with ADA (2). Upon ADA binding, activation of plasminogen-2 occurs, which raises plasmin levels. This leads to a degradation of matrix proteins and an activation of MMP, thereby indicating that the interaction of DPP4 and ADA might be involved in tissue remodeling (26). The serine in the active site of DPP4 is located in the sequence Gly–Trp–Ser–Tyr–Gly and is part of the catalytic triad (Ser 630, Asp 708, His 740) within the catalytic region of DPP4. DPP4 is an exopeptidase, which cleaves dipeptides from the penultimate N- terminal position of its substrates and thereby either inactivates these peptides and/or generates new bioactive compounds (7). There are numerous different DPP4 substrates known to date and they will be addressed in a separate section within this review. Furthermore, ADA catalyzes the irreversible deamination of adenosine and 2′-deoxyadenosine and is therefore a crucial player in the cellular and humoral immunity. Via interaction with CD45, the complex of ADA and DPP4 enhances T-cell activation. Inter- estingly, DPP4 is also able to promote T-cell proliferation inde- pendent from ADA binding or even its enzymatic activity (27). Zhong et al. were able to show that the interaction of DPP4 and ADA on dendritic cells might potentiate inflammation in obe- sity upon activation and proliferation of T-cells, which could be competitively inhibited by exogenous sDPP4, but not by inhibit- ing DPP4 enzymatic function (28). Furthermore, ADA activity is elevated in T2DM patients and may serve as a marker of inflammation and obesity (29). DPP4 Expression and Its Regulation Thereby, an upregulation of CD68 occurs and initiates a signaling cas- cade, which might be implicated in the pathogenesis of arthritis, and may be relevant for other inflammatory diseases as well (33). Intracellular signaling is also initiated by DPP4 via inter- action with Caspase recruitment domain containing protein 11 (CARMA-1) (6). Another well-known interaction of DPP4 is with extracellular matrix proteins like collagen and fibronectin (34, 35). The inter- action of DPP4 with fibronectin was revealed via nitrocellulose binding assays in rat hepatocytes and seems to play a role in the interaction of these cells with the ECM and with matrix assembly (36). Interaction of DPP4 with FAPα leads to a local degradation of ECM and thus migration and invasion of endothelial cells (37). Biology of DPP4 The extent of the circulating and soluble form of DPP4 is illustrated on the left in blue. The shedding of DPP4 from the membrane by indicated matrix metalloproteinases is shown by a scissor symbol in red. The vertical black FIGURE 1 | Domain structure of DPP4 [adapted from Ref. (2)]. Schematic representation of the membrane-bound DPP4 monomer. The extent of the circulating and soluble form of DPP4 is illustrated on the left in blue. The shedding of DPP4 from the membrane by indicated matrix metalloproteinases is shown by a scissor symbol in red. The vertical black FIGURE 1 | Domain structure of DPP4 [adapted from Ref. (2)]. July 2015 | Volume 6 | Article 386 Frontiers in Immunology | www.frontiersin.org 2 Röhrborn et al. DPP4 in diabetes Not only glycosylation and residues within the TMD are impor- tant for the cellular function of DPP4 but also dimerization. DPP4 can be found as monomer, homodimer, or even as homote- tramer on the cell surface of cells. DPP4 needs dimerization for enzymatic activity, and this is the predominant form of DPP4 (15). Dimerization occurs upon interaction with DPP4 itself or with other binding partners, e.g., FAP (16, 17), and occurs via interaction with the cysteine-rich region. Through its interaction with several proteins, DPP4 can act also in an enzymatic activity- independent way. Through this interaction, DPP4 is linked to various mechanisms like immune response and tumor invasion. The heterodimerization and interaction with different binding partners will be discussed in a later section. Potential Receptors for sDPP4 status of the investigated CpGs (41). Two years later, the same group analyzed DPP4 gene methylation levels between obese sub- jects with and without the metabolic syndrome in visceral adipose tissue. They observed no significant difference in the percentage of methylation levels of the CpGs within or near the second exon of the DPP4 gene between non-diabetic severely obese subjects with or without metabolic syndrome. However, they were able to show a correlation between plasma cholesterol levels and the percentage of methylation when the subjects were classified into quartiles (42). This further underpins a link between epigenetic modification of the DPP4 gene and plasma lipid metabolism. Since DPP4 is shedded from the membrane of cells with intact enzymatic and cysteine-rich region, it can also exert biological functions in a paracrine or endocrine manner. These functions might also involve intracellular signaling events in the targeted cells. Therefore, it would be of great importance to know receptors of sDPP4 to better understand the multiple role of sDPP4 on different cells and in different disease conditions where serum levels are elevated. However, there is not much known about DPP4 receptors so far. Ikushima et al. were able to show that DPP4 needs to associate with mannose-6 phosphate/IGF-IIR to exhibit its function as T- cell activator. This is due to the fact that for this activation, internalization of DPP4 is necessary, but DPP4 lacks a signal for exocytosis. The binding with M6P/IGF-IIR occurs via M6P residues in the carbohydrate moiety of DPP4 and the complex is then internalized and able to exert its biological function (38). Aghili et al. analyzed 875 patients with angiographically docu- mented coronary artery disease (CAD), and divided them in two subgroups dependent of their myocardial infarction (MI) status. By a genome-wide association study, loci, which predispose to MI, were assessed and associated with SNPs in the DPP4 gene. They found that polymorphisms in the DPP4 gene increase the risk of MI and progression of atherosclerosis in terms of plaque stability in patients with already existing CAD. Especially, one SNP was identified in both dominant and additive inheritance modes, which associates with low plasma DPP4 levels and which may increase the risk of MI in CAD patients (43). Our group showed that at least in human vascular smooth mus- cle cells, protease-activated receptor 2 (PAR2) might be activated by sDPP4. Potential Receptors for sDPP4 We were able to show that sDPP4-mediated ERK acti- vation and proliferation, as well as upregulation of inflammatory cytokines could be prevented by silencing of PAR2. The same was shown by use of a specific PAR2 antagonist. We propose that sDPP4 acts as an activator of PAR2, since a sequence within the cysteine-rich region of DPP4 is highly homologous to the auto-activating tethered ligand of PAR2 (39). Dyslipidemia, which is characterized by excessive lipids in the blood, is a common feature of T2DM. The status of this risk factor is quantifiable by the measurement of apolipoprotein B (ApoB) in the blood. In a very recent study by Baileys and colleagues, they aimed to identify novel SNPs associated with ApoB level. Especially in South Asians, who tend to develop risk factors for T2DM and MI at younger ages and lower BMI, they found an association of a DPP4 SNP with ApoB level (44). The DPP4 Deficiency in Animal Models To date, there are several studies dealing with the question, which role DPP4 plays in vivo. Animal models are useful tools to study the involvement of DPP4 in different organs. Upon triggering different diseases like insulin resistance (IR) or MI, it is possible to understand the role of DPP4 in these comorbidities of T2DM. In 2009, Bouchard et al. analyzed single nucleotide polymor- phisms (SNPs) in the DPP4 gene and searched for association with blood pressure, lipids, and diabetes-related phenotypes in obese individuals, to verify whether DPP4 gene polymorphisms could explain the individual risks of obese patients to develop metabolic complications. Three of the analyzed SNPs showed sig- nificant association with plasma total-cholesterol levels or plasma triglyceride level or total cholesterol level. But none of the poly- morphisms or cardiovascular disease risk factors showed a sig- nificant correlation with DPP4 mRNA levels in omental adipose tissue. Therefore, the authors concluded that, at least in their studied group, DPP4 gene polymorphisms seem to be unrelated to the inter-individual risk of developing obesity-related metabolic complications (40). Non-Enzymatic Interactions of DPP4 Through its cysteine-rich region, which is separated from the catalytic region, DPP4 is able to interact with different proteins, and further broadens its spectrum of activity and highlights its multifunctional role in different processes. July 2015 | Volume 6 | Article 386 Frontiers in Immunology | www.frontiersin.org 3 DPP4 in diabetes Röhrborn et al. Genetic Alterations of DPP4 and Predisposition to T2DM-Associated Diseases There are only few studies aiming to identify modifications in the DPP4 gene and their association with T2DM. Some of these are reviewed in the following section. Frontiers in Immunology | www.frontiersin.org DPP4 Deficiency in Rats Another effect of the whole-body KO of DPP4 in rats is induction of behavioral changes like a blunted stress phenotype (46, 51) and also effects on the immune-regulatory system like blunted NK cell and T-cell function and differential leukocyte subset composition or altered cytokine levels (46, 47). To really decipher the role of DPP4 in different tissues and their crosstalk with other target tissues, it is of great importance to study tissue-specific KO models. Because of this and because we were the first to describe DPP4 as a novel adipokine linked to parameters of the metabolic syn- drome (4), we decided to develop an adipose tissue-specific KO mouse model. The AT-specific DPP4-KO mouse was generated using a Cre-lox strategy under control of the aP2 promoter on the C57BL/6J background. Interestingly, we found out that KO mice gained significantly more weight, fat, and lean mass under HFD with no effect on energy expenditure or food intake. How- ever, KO mice showed improved HOMA-IR and lower fasting insulin. The observations that within AT, KO mice display a shift toward significantly more smaller adipocytes, and an increased expression of M2 macrophage marker genes points toward a ben- eficial role of DPP4 deletion in adipose tissue remodeling during HFD (57, 58). Enzymatic Function of DPP4 Dipeptidyl-peptidase 4 exerts its enzymatic action by clipping dipeptides from the penultimate position of its substrates. The active center, which is housed in an internal cavity, is surrounded by the β-propeller domain and the catalytic domain. Inhibitors and substrates enter/leave the active center by a so-called “side opening” (59, 60). The following section deals with known sub- strates of DPP4 in respect of T2DM, and with DPP4 as a drug target for T2DM treatment, which will include current knowledge on DPP4 inhibitors and the impact of DPP4 on organs involved in complications of T2DM. DPP4 Substrates In theory, numerous peptides are potential DPP4 substrates since they contain the cleavable amino acid sequence at their penul- timate position, but not for all of them it could be shown that DPP4 is able to cleave them in vivo. There seems to be a size limitation at least for cytokines, where DPP4 is more prone to cleave substrates of around 24 amino acids (aa) length. Fur- thermore, the substrate recognition is also dependent on the aa sequence around the penultimate position (61, 62). It turned out to be difficult to find physiological targets of DPP4 in the literature, reasons of that are excellently summarized in a recent review by Mulvihill and Drucker (6). We decided to focus here on the (potential) substrates of DPP4, which might play a role in T2DM or its complications. The list of DPP4 substrates men- tioned here is not fully complete and aims to highlight the impor- tance of DPP4 in T2DM also beyond its well-known incretin effect. Frontiers in Immunology | www.frontiersin.org DPP4 Deficiency in Mice Most of the observations already described in deficient rats are also true in whole-body DPP4-KO mice. Marguet et al. showed enhanced glucose tolerance, lower plasma glucose, and higher plasma insulin and GLP-1 after a 15 min oral glucose load without further characterizing the diet, age, or sex of the used C57BL/6 DPP4-KO mice (54). Conarello and colleagues found less weight- gain independent of the diet, and marked hypertrophy in the HFD-fed KO mice in epididymal white (eWAT) and brown adi- pose tissue (BAT). Importantly, they admitted that the reduction in caloric intake accounted for ~70% of the observed changes in bodyweight. Although they still observed differences in the bodyweight between KO and WT when they used pair-feeding, they carried out their further analysis in ad libitum fed mice and it is therefore difficult to judge the influence of DPP4 irrespective of bodyweight. However, they found improved insulin sensitivity and islet morphology, and improved liver biology in respect to lipid content and marker gene expression (55). The observation that DPP4 might be involved in the immune-regulatory system was also investigated in DPP4-KO mice, which were treated with pokeweed mitogen that stimulates growth and proliferation of B- cells. DPP4 seems to be involved in maturation and migration of immune cells, cytokine secretion, and percentages of spleen lymphocytes (56). DPP4 Deficiency in Rats A major part of the literature is dealing rather with DPP4-KO in rats than in mice. Most research groups work with the F344/DuCrj (DPP4-deficient) strain. Rats developing IR due to high-fat diet (HFD) feeding showed improved HOMA-IR values and blood glucose levels in oral glucose tolerance test (oGTT) and more active glucagon-like peptide-1 (GLP-1) and insulin in plasma (45). The same improved glucose tolerance with increased GLP- 1 and leptin levels was found in DPP4-depleted Dark Agouti rats with diet-induced obesity (46). Another research group also found improvement in serum lipid profile despite increased vis- ceral fat. They also performed insulin tolerance tests (ITT) in addition to GTT and saw an increased phosphorylation of Akt and reduced expression of gluconeogenic genes, concluding that DPP4-KO improved insulin sensitivity. Furthermore, the KO rats showed increased adipocyte maturation by increased expression of genes involved in triglyceride uptake and in PPARγ expression and increased adiponectin and leptin levels. In addition, adipose tissue is less inflamed illustrated by lower TNFα, IL-6, PAI1, and In another study, visceral adipose tissue DNA of 92 severely obese, non-diabetic female patients was analyzed for methylation rate in the DPP4-promoter CpG island and compared between different DPP4 polymorphisms. These cytosine- and guanine-rich regions are prone to epigenetic modification like methylation, and thus inactivate or activate transcription of certain genes. Different methylation levels of the DPP4 gene were identified in three DPP4 SNPs. Interestingly, the methylation level was nega- tively associated with DPP4 mRNA abundance and positively with plasma total/HDL-cholesterol ratio. These observations suggest that plasma lipid profile is improved by a higher methylation July 2015 | Volume 6 | Article 386 4 Röhrborn et al. DPP4 in diabetes CCL7 levels (47). The observed effects were attributed to elevated glucose-dependent insulinotropic polypeptide (GIP) levels in the KO rats. Furthermore, the same group could also show attenuated liver damage under HFD challenge in the KO rats due to improved bile secretory function. They postulate that the enhanced export of bile acids out of hepatocytes and a reduction of bile acid synthesis via inhibition of CYP7A1, which converts cholesterol to bile acids, were mediated by increased GLP-1 in DPP4-KO rats (48). DPP4 Deficiency in Rats Interestingly, at least Yasuda and colleagues also saw a significant reduced food intake in the KO rats irrespective of the diet (45), which might be due to changed receptor specificity of neuropeptide Y (NPY), which was shown to be more potent in KO rats to influence food intake and feeding motivation (49). Although several independent working groups saw increased NPY levels in KO rats (49–51), the effect on food intake is controver- sial (45, 50). When diabetes is induced via streptozotocin (STZ) treatment in F344/DuCrj-DPP4-deficient rats, onset of hyper- glycemia was delayed, but KO rats showed impaired creatinine clearance and more severe dyslipidemia, which might be caused by a dysregulated expression of factors involved in steroid and lipid metabolism (52, 53). The authors concluded that DPP4 might be responsible for preservation of renal function. Another effect of the whole-body KO of DPP4 in rats is induction of behavioral changes like a blunted stress phenotype (46, 51) and also effects on the immune-regulatory system like blunted NK cell and T-cell function and differential leukocyte subset composition or altered cytokine levels (46, 47). CCL7 levels (47). The observed effects were attributed to elevated glucose-dependent insulinotropic polypeptide (GIP) levels in the KO rats. Furthermore, the same group could also show attenuated liver damage under HFD challenge in the KO rats due to improved bile secretory function. They postulate that the enhanced export of bile acids out of hepatocytes and a reduction of bile acid synthesis via inhibition of CYP7A1, which converts cholesterol to bile acids, were mediated by increased GLP-1 in DPP4-KO rats (48). Interestingly, at least Yasuda and colleagues also saw a significant reduced food intake in the KO rats irrespective of the diet (45), which might be due to changed receptor specificity of neuropeptide Y (NPY), which was shown to be more potent in KO rats to influence food intake and feeding motivation (49). Although several independent working groups saw increased NPY levels in KO rats (49–51), the effect on food intake is controver- sial (45, 50). When diabetes is induced via streptozotocin (STZ) treatment in F344/DuCrj-DPP4-deficient rats, onset of hyper- glycemia was delayed, but KO rats showed impaired creatinine clearance and more severe dyslipidemia, which might be caused by a dysregulated expression of factors involved in steroid and lipid metabolism (52, 53). The authors concluded that DPP4 might be responsible for preservation of renal function. Stromal Cell-Derived Factor-1ααα/CXCL12 Stromal cell-derived factor-1 (SDF-1) is a chemokine that pro- motes angiogenesis and attracts endothelial progenitor cells (EPC) by binding to its receptor C–X–C motif chemokine receptor type 4 (CXCR4). SDF-1 is thus discussed in the literature as important mediator of cardioprotective effects addressed to the use of DPP4 inhibitors (further discussed in “DPP4 Substrates: SDF-1- and BNP-Dependent Effects of DPP4 Inhibitors”). It is a well-known physiological target of DPP4 (68, 69). SDF-1α also plays a role in diabetes itself by protecting stem-cell-derived insulin-producing cells from glucotoxicity under high glucose conditions (70) or promoting pancreatic beta-cell survival in mice via Akt activation (71). Furthermore, it was shown that some genetic variants of SDF-1α are associated with late stage complications in T2DM patients (72, 73). Pituitary Adenylate Cyclase-Activating Polypeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) is very rapidly degraded by DPP4 to the fragments (3–27), (5–27), and (6–27). These fragments lack PACAPs insulinotropic ability, but are no feasible treatment options for T2DM because of their actions on glucose homeostasis and glucagon secretion (85). Sev- eral studies have shown that PACAP is a powerful stimulator of insulin secretion, which enhances glucose uptake in adipocytes and augments antilipolytic action of insulin (86, 87). After DPP4- inhibitor treatment in mice, PACAP-induced insulin secretion was enhanced (88). However, a proof that PACAP also plays a role in humans is lacking so far. Substance P Substance P is a physiological target of DPP4, which is sequentially converted to SP (3–11) and SP (5–11) in vivo in F344–DPP4- positive rats (78). SP is a neurotransmitter and modulator, which is involved in neurogenic inflammation. Serum levels in diabetes are controversially discussed with one study showing a decrease in diabetic patients (79), and another one showing an increase in fasting blood samples with correlation to diabetic risk factors like BMI and blood pressure (80). This discrepancy in serum levels could be addressed to the fact that it is not always stated which form of SP (full length versus truncated) is measured. However, SP was shown to promote IR in vitro in human preadipocytes by interacting with proteins that are involved in the inhibitory phosphorylation of IRS-1. Furthermore, SP can directly inhibit insulin-dependent glucose metabolism in rat adipocytes (81). SP also promotes diabetic corneal wound healing, as shown by Yang and colleagues (82). Glucagon-like peptide-1 is secreted from L-cells of the gut into the bloodstream. Upon binding to G-protein-coupled recep- tors on the beta cells, intracellular cAMP level is elevated and the protein kinases, Epac1 and 2, are activated, which leads to an increase of insulin secretion. Furthermore, GLP-1 enhances beta-cell mass by mediating proliferation and differentiation and inhibiting apoptosis (8). By inhibiting gastric emptying, GLP-1 also improves blood sugar excursion, delays food absorption, and is therefore a regulator of satiety and appetite also through the hypothalamus (63). Glucose-dependent insulinotropic polypeptide is a 42 aa pep- tide, which mainly originates from enteroendocrine K cells (64). Subjects with diabetes or impaired glucose tolerance show sig- nificantly reduced levels of meal-stimulated circulating GIP and the levels are negatively correlated with the severity of IR in the patients (65, 66). GIP has, in contrast to GLP-1, no effect on glucagon secretion, but also regulates fat metabolism in adipocytes (67). Incretin Hormones The incretin hormones account for approximately 50% of the insulin secretion after meal intake and are secreted from the gut within minutes after the meal intake. Through binding to distinct receptors on beta cells in the pancreas, they stimulate insulin secretion and suppress glucagon release depending on the blood glucose level. Most potent in their glucose-lowering action are glucagon-like peptide 1 (GLP-1) and GIP. Both peptides belong All these studies have in common that they use whole-body KO animals. The disadvantage here is that one cannot distinguish between direct effects of the KO and side effects caused, for exam- ple, by different immune cell status or decreased caloric intake. July 2015 | Volume 6 | Article 386 Frontiers in Immunology | www.frontiersin.org 5 Röhrborn et al. DPP4 in diabetes to the same glucagon peptide superfamily and share significant aa character. Brain Natriuretic Peptide Brain natriuretic peptide is responsible for vasodilation, natriure- sis, and suppresses renin secretion. It is so far only a predicted DPP4 substrate, which was cleaved in vitro by DPP4 to BNP (3–32). This truncation was inhibited by a DPP4 inhibitor in a dose-dependent manner (83). Truncated forms of BNP with lower enzymatic activity are discussed as an indicator of heart failure severity. In 2013, dos Santos et al. could show an improved cardiac performance in sitagliptin-treated rats, which they attributed to increased levels of active BNP (84). Since inhibition of DPP4 due to genetic deletion or use of DPP4 inhibitors was shown to elevate GLP-1/GIP levels in numerous studies, this effect is the main focus of developing therapeutic targets for treatment of T2DM. There are numerous reviews, which focus on DPP4- and GLP-1-mediated effects, and this topic will not be further discussed here. Eotaxin/CCL11 Eotaxin mediates mobilization of eosinophils into the blood- stream, which was shown to be increased in DPP4-deficient F344 rats (94). DPP4 cleaves eotaxin to eotaxin (3–74). However, there was no significant correlation of eotaxin seen in patients with T2DM or impaired glucose tolerance in the KORA cohort (95). The six inhibitors have been classified into three classes depending on their different binding modes in the DPP4 active center (10). Class 1 contains vilda- and saxagliptin, which only bind to the S1 and S2 subsites and form a covalent bond with the nitrile group of their cyanopyrrolidine moiety and Ser630 of DPP4. Saxagliptin has a fivefold higher activity in blocking DPP4 than vildagliptin. Group 2 contains alo- and linagliptin, which also interact with the S1′ subsite or even in case of linagliptin with the S2′ subsite. The uracil rings of both gliptins induce a conformational change in the Tyr547 of the S1′ subsite. Because of the additional interaction of linagliptin with S2′ subsite, it has an eightfold higher activity than alogliptin. The third class has the highest inhibitory function toward DPP4, because both sita- and teneligliptin interact with the S2-extensive subsite of the DPP4 active center, and an increasing number of interactions seems to increase the potency of the gliptin (10). Teneligliptin, which is only approved for T2DM treatment in the Japanese and Korean market so far, also has a unique structure characterized by a J-shape and an anchor-lock domain, which explains the strong inhibitory function and the low IC50 value of this drug [for review, see Ref. (97)]. The binding of the DPP4 S2-extensive subsite of some inhibitors also guarantees a high specificity toward DPP4 since other close-related peptidases like DPP8, DPP9, and FAP lack this subsite. All DPP4 inhibitors have in common that they build salt bridges with Glu-residues in the S2 subsite (10). At least for sitagliptin, it is also known that it lowers the level of free fatty acids (FFA) and thereby also comprises insulin- sensitizing properties (98). Furthermore, sitagliptin was shown to have potent anti-inflammatory properties by suppressing expres- sion of pro-inflammatory genes in mouse and humans (98, 99). In patients with renal impairment, which is a very common complication of T2DM, sitagliptin is more suitable than sulfony- lureas (100). DPP4 as a Drug Target for the Treatment of T2DM Deactivation of DPP4 Enzymatic Activity DPP4 inhibitors Major DPP4 substrates are the so-called incretin hormones, which are key regulators of post-prandial insulin release. DPP4 inhibi- tion leads to greater bioavailability of these proteins and therefore prolongs the half-life of insulin action. The majority of effects seen upon DPP4-inhibitor treatment are ascribed to an increase in GLP-1 levels. Because of this, DPP4 became a major target for the treatment of T2DM. This section deals with the most recent knowledge around DPP4 inhibitors, their mode of action – if known – and the newest developments in the inhibition of DPP4 enzymatic activity. There are numerous modifications and poten- tial optimizations of the five so far approved gliptins reported. However, most of them are not in clinical trials yet and not much is known about their advantage in a head to head comparison to established gliptins. Therefore, we decided to focus on the most recent data on approved gliptins in this review. The data are also summarized in Table 1. Dipeptidyl-peptidase 4 inhibitors lower DPP4 activity by 70–90%. They do not pass the blood–brain barrier and have no direct effect on satiety or on altering gastric emptying (8). The benefit for diabetes therapy clearly is their indiffer- ence on bodyweight gain and the low risk of hypoglycemia. There are five gliptins approved so far for clinical use, namely sitagliptin, vildagliptin, saxagliptin, linagliptin, and alogliptin. Three more gliptins, teneligliptin, anagliptin, and trelagliptin are only approved in the Japanese and Korean market. Despite the same mode of action, the different gliptins diverge in their phar- macodynamic and pharmacokinetic properties, which might be clinically relevant for some patients (9, 96). The peptide mimetic compounds, vilda-, saxa, and teneligliptin, were identified by TABLE 1 | Summarized properties of gliptins. Eotaxin/CCL11 Anagliptin, which is only approved since 2012 in the Japanese market, seems to have serum lipid-lowering and anti- atherogenic actions as well, which makes it unique among the gliptins approved so far (101, 102). Anagliptin has an IC50 of 3.3 nM and its main excretion route is renal elimination (101, 103). However, since this gliptin is only on the market since 2012, NPY and PYY Regulated on activation, normal T-cell expressed and secreted (RANTES) recruits leukocytes into inflammatory sites and is cleaved by DPP4 to RANTES (3–68). Due to this truncation, RANTES (3–68) is not able anymore to increase cytosolic calcium concentrations and to induce chemotaxis of human monocytes in vitro. This is explained by a shift in receptor subtype-specificity toward enhanced activation of CC-motif-chemokin-receptor 5 (CCR5) (89). Elevated serum levels of RANTES in T2DM are associated with post-prandial hyperglycemia (90). Interestingly, RANTES and its receptor CCR5 are important mediators of obesity-induced inflammation, which was shown in CCR5-KO mice (91). Levels of RANTES and CCR5 were reduced in adipose tissue of obese patients upon exercise (92). RANTES reduces Neuropeptide Y and peptide YY are members of the polypeptide family. They are highly expressed in the hypothalamus but are also present in peripheral tissues like islets. NPY regulates energy bal- ance, memory, and learning, while PYY reduces appetite, inhibits gastric motility, and increases water and electrolyte absorption in the colon (74). Both NPY and PYY play a role in beta- cell survival and in glucose homeostasis (74). NPY is able to suppress insulin secretion acutely (75). Both polypeptides have in common that DPP4 truncation shifts their receptor speci- ficity and thus alters their biological role in different cellular processes. In vitro experiments in adipocytes could show that DPP4 inhibition has an impact on lipid metabolism mediated by NPY (76, 77). July 2015 | Volume 6 | Article 386 Frontiers in Immunology | www.frontiersin.org Frontiers in Immunology | www.frontiersin.org 6 DPP4 in diabetes Röhrborn et al. glucose-stimulated GLP-1 secretion in vitro and in vivo in mice, by acting most probably through the intestinal glucose transporter SGLT1 (93). replacement experiments of peptide-based substrates, whereas the non-peptide mimetic compounds, sita, alo-, and linagliptin, were derived from initially found inhibitors of random screenings. The diverse chemical structures also explain the unique binding modes of the inhibitors to DPP4 (10). Frontiers in Immunology | www.frontiersin.org DPP4 as a Drug Target for the Treatment of T2DM Deactivation of DPP4 Enzymatic Activity DPP4 inhibitors These controversial discussions are well summarized by the reviews of Nauck and Butler (115, 116). To really assess which medication is of more importance always depends on the special patient characteristic. DPP4 as a Drug Target for the Treatment of T2DM Deactivation of DPP4 Enzymatic Activity DPP4 inhibitors Inhibitor Approved since Binding mode Kind of inhibition Route of excretion IC50 value Reference Sitagliptin 2006 FDA S1, S2, and S2 extensive subsites Competitive inhibition Mostly renal route 19 nM (10, 98–100) Vildagliptin 2007 European medicines agency Only S1 and S2 subsite Substrate–enzyme blocker Mostly renal route 62 nM (10, 209) Saxagliptin 2009 FDA Only S1 and S2 subsite Substrate–enzyme blocker Mostly renal route 50 nM (10, 209) Linagliptin 2011 FDA S1, S2, and S1′ subsites – Through biliary route 1 nM (10) Alogliptin 2013 FDA S1, S2, and S1′ subsites Competitive inhibition Mostly renal route 24 nM (8, 10) Teneligliptin 2012 Japan S1, S2, and S2 extensive subsites Very potent because of unique anchor- lock domain and J-shape of molecule Mostly renal route 0.37 nM (97) 2014 Korea TABLE 1 | Summarized properties of gliptins. July 2015 | Volume 6 | Article 386 Frontiers in Immunology | www.frontiersin.org 7 DPP4 in diabetes Röhrborn et al. system (1) via inhibiting the enzymatic action of DPP4 and thereby upregulating GLP-1 levels physiologically and (2) via increasing GLP-1 levels pharmacologically. While GLP-1 receptor agonists (GLP-1-RA) directly target GLP-1, GLP-1-independent effects are also possible with the use of DPP4 inhibitors (DPP4i). These drugs might also affect the level of other DPP4 substrates and might therefore have a more complex mode of action. comparative head-to-head trials and data on the long-term use are missing at the moment. Shinjo and colleagues demonstrated that anagliptin exerts anti-inflammatory effects on macrophages and adipocytes in vitro and on inflamed mouse livers in vivo. In this very recent study, anagliptin was more potent in its anti- inflammatory actions than sitagliptin (104). To improve the qual- ity of life of T2DM patients, development of novel agents now is more and more focused on long-acting agents. There are two more gliptins, which only have to be applied once weekly, namely SYR-472 (trelagliptin) and MK-3102 (omarigliptin) (105, 106). Trelagliptin is approved in Japan since 2015. g p However, there have been a lot of attempts to compare the effects of GLP-1-RA versus DPP4i in clinical studies. The results of these head-to-head comparisons are summarized in many current reviews (110–114). Most of these comparative studies agree that GLP-1 analogs are more effective in respect of glycemic con- trol. Both incretin-based therapies are equally potent in lowering blood pressure and total cholesterol (110). Alternative modes of DPP4 inhibition Dipeptidyl-peptidase 4 inhibitors exert glucose regulatory actions by prolonging the effects of GLP-1 and GIP, ultimately increas- ing glucose-mediated insulin secretion and suppressing glucagon secretion (117). Beside the glucose-lowering properties of DPP4 inhibitors, emerging evidence suggests that incretin-based ther- apies may also have a positive impact on inflammation, cardio- vascular and hepatic health, sleep, and the central nervous system (118). However, the underlying mechanisms of these effects can- not be fully explained by lower blood glucose levels or increased GLP-1 bioavailability or signaling, and has to be further eluci- dated. Thus, the next section is focused on the role of DPP4 action in T2DM-relevant organs and associated comorbidities. Very recently, Pang and colleagues published a different strategy to inhibit DPP4 activity. They used DPP4-targeted immune therapy by vaccines in a C57BL/6J mouse model and were able to show comparable effects like in treatments with gliptins regarding GLP- 1 plasma levels and post-prandial glucose excursion and insulin sensitivity in HFD-fed mice. Furthermore, they observed no side effects on immune cell activation by the DPP4 vaccine. An advan- tage of this method is the long-lasting effect of the vaccine in the mouse model, which could, if transferable to human patients, be a convenient alternative to the daily intake of gliptins (109). y g Further research in developing alternatives toward Gliptins especially for long-acting medications would be an interesting new approach to improve lifestyle of patients. DPP4 as a Drug Target for the Treatment of T2DM Deactivation of DPP4 Enzymatic Activity DPP4 inhibitors Furthermore, both have the advantage of low incidence of hypoglycemia (110, 111). The results for body-weight lowering effects of DPP4i are heteroge- neous throughout the studies (113), whereas beneficial effects on body-weight are well accepted for GLP-1-RA (110–112, 114). Therefore, some authors tend to prefer GLP-1-RA over the use of DPP4i (112). However, one should be aware of the fact, that GLP- 1-RA have a higher incidence of gastrointestinal adverse events like nausea (110, 112, 114), which might be disadvantageous for elderly people who may be more prone to these side effects (114). Although some authors claim that DPP4 inhibitors are only beneficial in early stages of diabetes, this could be rebutted by the work of Kumar and Gupta (107). They could show beneficial effects of three gliptins (sita-, saxa-, and vildagliptin) in lower- ing HbA1c also in patients with longstanding T2DM for more than 10 years. Thus, DPP4 inhibition also plays an important role irrespective of the duration of diabetes. What has to be mentioned in respect of the beneficial roles of DPP4 inhibitors is that more and more studies about their beneficial pleiotropic effects are upcoming, which are also dis- cussed in the following section of this review dealing with different organs. There are reports that gliptins themselves have effects on lipid profile and blood pressure as well as on inflammatory processes (108). In addition to the incretins, there are some DPP4 substrates, like SDF-1α, which might explain potential cardio- protective effects, which are discussed for gliptins. However, car- diovascular outcomes are still widely debated and controversially evidenced. Ongoing long-term studies will further shed light on the respective role of DPP4i beyond glucose homeostasis. Fur- thermore, one has to keep in mind that also DPP4 has direct effects independent of its enzymatic activity, like activation of downstream signaling events upon receptor binding, which are not well understood so far. Which role DPP4 inhibition plays on T2D relevant organs/comorbidities will be the topic of the following sections. Furthermore, there are reports that DPP4i might also have car- dioprotective effects (113), which will also shortly be discussed in section “Effect of DPP4 Inhibition on the Cardiovascular System” of this review. Despite the clear beneficial effects of incretin-based therapies, there are also concerns reported in respect of the risk for long-term complications like pancreatitis (115). These potential risks might, however, outweigh the benefits. Frontiers in Immunology | www.frontiersin.org Incretin-Based Therapies: Comparing DPP4i and GLP-1 Analogs Adipose tissue is the primary storage organ for excess energy. While the role of adipose tissue as a central source of energy has been recognized for centuries, in the past decade, it has become increasingly clear that adipose tissue also displays char- acteristics of an endocrine organ releasing a number of adipose It is well accepted that incretin-based therapies are able to lower blood glucose levels and are therefore a treatment option for T2DM. There are mainly two approaches to target the incretin July 2015 | Volume 6 | Article 386 Frontiers in Immunology | www.frontiersin.org 8 Röhrborn et al. DPP4 in diabetes Adipose Tissue as Relevant Source of Circulating DPP4 Adipose Tissue as Relevant Source of Circulating DPP4 tissue-specific factors, known as adipokines. During the progres- sion of obesity, the ability of adipocytes to function as endocrine cells and to secrete multiple biologically active proteins is affected (119). Thus, adipose tissue has been shown to be a central driver of T2DM progression, establishing and maintaining a chronic state of low-level inflammation (120). Serum levels of sDPP4 are altered in many pathophysiologic conditions, including different types of cancer, allergic asthma, or hepatitis C (7). Our group was the first analyzing circulat- ing sDPP4 in the context of obesity and the metabolic syn- drome. DPP4 serum levels of morbidly obese men are elevated compared with lean controls and significantly correlated with BMI, the size of adipocytes in subcutaneous and visceral fat, and the adipocyte hormones adiponectin (negatively) and leptin. These data suggest that sDPP4 is related not only to increased body weight but also to other important parameters of adipose tissue physiology. In addition, sDPP4 release and serum con- centration can be reversed to normal levels by surgery-induced weight loss (4). Thus, in obesity, both circulating levels of sDPP4 and sDPP4 release by adipose tissue are increased and correlate strongly with the metabolic syndrome but can be reduced to control levels by substantial weight loss. Thus, indicating that enlargement of visceral adipocytes in obesity may substantially contribute to the augmented level of circulating sDPP4 in obese patients. DPP4 Expression and Release Within Adipose Tissue Recently, we showed that DPP4 is highly expressed in human primary adipocytes (4). Furthermore, DPP4 expression in adipose tissue is increased in obese compared to lean individuals in both subcutaneous and visceral adipose tissue (4, 5). Interestingly, visceral fat of obese patients exhibits the highest DPP4 level. According to the increased expression, we could identify sDPP4 as a novel adipokine released from primary human adipocytes. In vitro, the DPP4 release increased substantially during fat cell differentiation, and comparison with preadipocytes and adipose tissue, macrophages showed that adipocytes most likely represent the major source of DPP4 released from the intact organ to the circulation. Furthermore, the release of sDPP4 was elevated in adipose tissue explants of obese patients compared to lean controls and correlates with various classical markers of the metabolic syn- drome, namely BMI, waist circumference, plasma triglycerides, and HOMA as an index of IR, as well as with fat cell volume and the adipokine leptin (4, 5). Endocrine Effects of Soluble DPP4 Although there is clear evidence that increased circulating levels of sDPP4 are associated with hallmarks of obesity and type diabetes, such as whole-body IR, elevated BMI, and adipocyte hypertrophy, there are only few studies investigating the endocrine effects of sDPP4. We were the first showing that DPP4 consistently impairs insulin signaling at the level of Akt in primary human adipocytes (4). Enzymatic activity of sDPP4 appears to be involved in this process; however, since this work was done in vitro, it is most unlikely that the sDPP4-induced impairment of insulin action is due to an increased bioavailability of any DPP4 substrate. It might rather be that DPP4 inhibitors may also affect the binding properties of sDPP4 to its receptors, namely M6P/IGFII receptor (38) or PAR2 (39). For the latter, it is not only known that PAR2 signaling induces IR in adipocytes (125), but PAR2 might also be a substantial contributor to inflammatory and metabolic dysfunc- tion (126). Although there is a hint that circulating sDPP4 itself might affect adipose tissue function, the exact mechanism has to be further investigated. How DPP4 expression affects adipocyte homeostasis can only be speculated. DPP4 might be involved in adipose tissue lipolysis. DPP4 recruits ADA, a monomeric enzyme catalyzing deamina- tion of adenosine to inosine and ammonia (121, 122). It has been shown that DPP4-bound ADA has a 1000-fold greater activity than free ADA (123), which in turn may modulate the well- established antilipolytic effects of adenosine. Moreover, DPP4 is a strong inhibitor of the antilipolytic activity of NPY (76), which is one of the best peptide substrates of the enzyme (89). In this regard, Rosmaninho-Salgadoa and colleagues demonstrated that DPP4 stimulates lipid accumulation and PPAR-γ expression through cleavage of NPY suggesting that sDPP4 might stimulate adipocyte differentiation (77). However, it is noteworthy that the authors of this study were using tremendously high and non- physiological concentrations of sDPP4. On the contrary, a recent published study showed that DPP4 expression was strongly upreg- ulated during adipocyte dedifferentiation in vitro. Hence, the authors concluded that DPP4 might be a major component in adipose tissue remodeling and cell plasticity (124). Nevertheless, enhanced abundance of DPP4 within adipose tissue of obese subjects may be involved in adipose tissue remodeling and sub- stantially augments the lipolytic activity of enlarged adipocytes (57, 58). Impact of DPP4 Inhibition on Pancreatic Islets Impact of DPP4 Inhibition on Pancreatic Islets Accumulating in vitro and pre-clinical data show that DPP4 inhi- bition has beneficial effects on T2DM induced β-cell dysfunction and apoptosis. Omar and colleagues demonstrated that DPP4 is not only present and active in mouse and human islets, but inhibition of islet DPP4 activity also has a direct stimulatory effect on insulin secretion, which is GLP-1 dependent (131). The same effect could be observed with a 2-week des-fluoro-sitagliptin treatment leading to increased insulin exocytosis by β cells from db/db diabetic mice (133). Furthermore, it could be shown that DPP4 inhibition is clearly associated with significantly increased β-cell mass and function in several models of T2DM (134–136). These beneficial effects were associated with the transcriptional activation of anti-apoptotic and pro-survival genes, as well as the suppression of pro-apoptotic genes in β cells (137). Additionally, Shah and collaborators showed that the DPP4-inhibitor linagliptin protects isolated human islets from gluco-, lipo-, and cytokine- toxicity (132). Accordingly, Akarte et al. reported anti-oxidative properties of vildagliptin shown by a dose-dependent decrease in nitric oxide concentrations in both serum and pancreatic homogenates of vildagliptin-treated diabetic rats (138). With the recognition that adult humans also have BAT, an organ with substantial capacity to dissipate energy, BAT gained considerable interest as a novel target to treat or prevent obesity and its associated diseases. In 2013, the group around Shimasaki was the first reporting that des-fluoro-sitagliptin attenuated body adiposity, without affecting food intake, in C57BL/6 mice with diet-induced obesity (128). The increase in energy expenditure could be explained by enhanced levels of PPAR-α, PGC-1, and uncoupling protein-1 (UCP-1) in BAT as well as elevated lev- els of proopiomelanocortin in the hypothalamus. The beneficial effects of des-fluoro-sitagliptin on energy expenditure could only partly be ascribed to increased GLP-1 levels and have to be further validated. Shortly afterward, Fukuda-Tsuru et al. could confirm these data in the same animal model by administra- tion of teneligliptin (129). Moreover, in this study, teneligliptin also reduces fat mass and suppresses HFD-induced adipocyte hypertrophy. Beside these pre-clinical and in vitro studies, only few are known about the beneficial effect of DPP4 inhibitors on β cells in human. In the short-term, 12-weeks vildagliptin treatment leads to a small increase in the capacity for insulin secretion (139). Pancreatic Islets β-cells play a central role in the etiology of T2DM. Due to failure of β-cell sensitivity to glucose and loss of β-cell mass, insulin secre- tion of these cells is not sufficient to counter balance IR, finally leading to T2DM. Although DPP4 inhibitors are now widely used for glycemic control, many debates are ongoing about their exact mode of action and their beneficial effects on pancreatic β cells. The exact mechanism how DPP4 inhibitors augment insulin secretion and increase β-cell mass in vitro and in vivo is still not fully understood, since not all these effects could be explained by elevated GLP-1 level or improved glycemic control associated with less glucotoxicity. Impact of DPP4 Inhibition on Pancreatic Islets Treatment with vildagliptin over a longer period of time could also confirm an increased β-cell function in humans as a result of improved sensitivity of β cells to glucose (140, 141). However, this effect was not maintained after washout period, indicating that this increased capacity was not a disease modifying effect on beta cell mass and/or function. In the SAVOR-TIMI 53 trial, which was originally performed to assess the cardiovascular safety of saxagliptin, Leibowitz and colleagues recently reported that DPP4 inhibition may attenuate the progression of diabetes (142). This was evidenced by a decreased requirement for intensification of treatment associated with better preservation of glycemic control, as well as better sustained β-cell function as reflected in the fasting HOMA-2β during the 2-year follow-up period. Collectively, there is clear evidence that DPP4 expression and release by adipose tissue play a key role in obesity and T2DM-associated processes, such as inflammation, adipocyte hypertrophy, and IR. However, the underlying mechanism of these beneficial effects is not fully understood and remains unclear in most of the publications. Regulation of DPP4 Expression Within Pancreatic Islets Interestingly, within the pancreatic islets, DPP4 localization dif- fers between species. Islets of rodents showed a near-exclusive expression of DPP4 in β cells, with little expression in α-cells. In contrast, human and pig islets express DPP4 almost exclusively in α cells (130, 131). The species difference in the localization of DPP4 expression, and the possible physiological consequence of that difference, is unclear. Moreover, in a recent published study, it has been demonstrated that DPP4 activity was detectable in the conditioned medium of human islets suggesting that DPP4 is released from human islets as well (132). Under pathological conditions, islets of obese mice chronically fed a HFD that exhibit an increased DPP4 activity. The contrary was found in human islets from type 2 diabetic donors, showing a decreased DPP4 activity (131). Impact of DPP4 Inhibition on Adipose Tissue Impact of DPP4 Inhibition on Adipose Tissue To further investigate the role of DPP4 in adipose tissue, several studies with DPP4 inhibitors were conducted. Interestingly, the administration of the DPP4-inhibitior des-fluoro-sitagliptin ame- liorates linoleic acid-induced adipose tissue hypertrophy in β-cell- specific glucokinase haploinsufficient mice, a model of non-obese T2DM (127). Moreover, des-fluoro-sitagliptin protects against linoleic acid-induced adipose tissue inflammation illustrated by CD8+ T-cell infiltration. Due to the loss of GLP-1 receptors in adipose tissue, the authors exclude the involvement of GLP-1 and claim that the observed effects are due to the huge variety of DPP4 substrates. Thus, DPP4 inhibition might have pleiotropic effects in adipose tissue. A similar outcome has been observed in C57BL/6 mice fed a HFD. After linagliptin treatment, a sig- nificantly lower expression of the macrophage marker F4/80 was Moreover, dendritic cells and macrophages resident in visceral adipose depots exhibit an increased DPP4 expression in response to inflammation or in the obese state (28). Since it is known that DPP4 exerts immunomodulating properties, Zhong et al. showed that membrane-bound DPP4 is co-localized with membrane- bound ADA on human dendritic cells resulting in an increased T- cell proliferation (28). Thus, it can be speculated that DPP4 might also play an important role in the chronic low-grade inflammation taking place in obesity and T2DM. July 2015 | Volume 6 | Article 386 9 DPP4 in diabetes Röhrborn et al. found compared with vehicle treatment. In line with these data, the authors demonstrated an increased insulin sensitivity after linagliptin treatment suggesting that DPP4 and adipose tissue inflammation play a pivotal role in the induction of IR. In 3T3- L1 cells, a murine predipocyte cell line, Rosmaninho-Salgado et al. demonstrated that the DPP4-inhibitor vildagliptin reduces lipid accumulation by inhibiting adipogenesis, without affecting lipolysis through NPY cleavage and subsequent NPY Y2 receptor activation (77). found compared with vehicle treatment. In line with these data, the authors demonstrated an increased insulin sensitivity after linagliptin treatment suggesting that DPP4 and adipose tissue inflammation play a pivotal role in the induction of IR. In 3T3- L1 cells, a murine predipocyte cell line, Rosmaninho-Salgado et al. demonstrated that the DPP4-inhibitor vildagliptin reduces lipid accumulation by inhibiting adipogenesis, without affecting lipolysis through NPY cleavage and subsequent NPY Y2 receptor activation (77). Impact of DPP4 Inhibition on Liver Function Since DPP4 inhibitors are widely used in clinical practice, this drug was also investigated as a potential new therapeutic strategy against the development of liver fibrosis and steatosis. Kaji and collaborators demonstrated that sitagliptin markedly inhibits liver fibrosis development in rats via suppression of hepatic stellate cell proliferation and collagen synthesis (158). These suppressive effects were associated with dephosphorylation of ERK1/2, p38, and Smad2/3 in the hepatic stellate cells. Additionally, hepatic steatosis could be prevented in several different animal models Regulation of DPP4 Expression in the Liver Although DPP4 exhibits a widespread organ distribution, the liver is one of the organs that highly expresses DPP4 (149). In the healthy human liver, intense staining for DPP4 was found in hepatic acinar zones 2 and 3, but not in zone 1. This heterogeneous lobular distribution suggests that DPP4 might be involved in the regulation of hepatic metabolism (150). Furthermore, mRNA expression levels of DPP4 were significantly increased in NAFLD livers compared to that in control livers (151). In accordance to that, DPP4 expression levels of NAFLD patients were negatively correlated with HOMA-IR and BMI, and positively correlated with total cholesterol levels, but not with ALT, lactate dehydro- genase (LDH), or triglyceride levels. Moreover, under conditions of high glucose, DPP4 expression was increased in HepG2 cells. However, other nutritional conditions, such as high insulin or the presence of fatty acids and cholesterol, did not affect DPP4 expres- sion in these cells. Thus, the authors claim that enhanced DPP4 expression in NAFLD liver may rather be associated with IR than triglyceride accumulation and may promote the progression of liver disease via subsequent deteriorations in glucose metabolism. How increased DPP4 expression might affect liver function is still unknown. There are only a few hints that DPP4 might play a role in fibronectin-mediated interaction of hepatocytes with extracellular matrix (2, 36, 152). Beside DPP4 expression, there is only indirect evidence that hepatocytes also release DPP4 to the circulation, which will be further discussed in the next section. Clinical data are very limited; however, several non- randomized trials conducted in small groups of diabetic patients demonstrated that DPP4 inhibitors improved the levels of liver transaminases and liver fat (164–166). Accordingly, Iwasaki et al. found a decrease in ballooning and non-alcoholic steatohepatitis scores in post-treatment liver biopsies (165, 166). Recently, in a comprehensive retrospective review of 459 type 2 diabetic patients, treated with DPP4-inhibitors, it was shown that DPP4 inhibitors improved the abnormality of the liver transaminases AST and ALT independent of HbA1c and body weight (167). Again in the majority of publications, the authors postulate that these beneficial actions were mediated through potentiation of direct GLP-1 actions on hepatocytes; however, it seems unlikely that hepatocytes express the canonical GLP-1 receptor (168). Liver Non-alcoholic fatty liver disease (NAFLD) describes a disorder with excessive deposition of fat within the liver with increasing prevalence in parallel to obesity and diabetes, which are major risk factors for NAFLD (143). Indeed, NAFLD is now the most common cause of chronic liver disease (144) and is present in one-quarter to one-half of diabetes patients (145). In the obese state, elevated triglyceride degradation in adipose tissue causes an increased hepatic uptake of fatty acids leading to fat accu- mulation within the tissue. Furthermore, reactive oxygen species (ROS), produced during lipid oxidation, are assumed to induce hepatocyte death and inflammatory reactions. Liver cirrhosis can be defined as the end stage of chronic liver diseases and is caused July 2015 | Volume 6 | Article 386 Frontiers in Immunology | www.frontiersin.org 10 DPP4 in diabetes Röhrborn et al. by DPP4 inhibition (127, 159, 160). Shirakawa and colleagues studied the effects of sitagliptin in glucokinase ± diabetic mice with diet-induced hepatic steatosis (127). Here, sitagliptin pre- vented fatty liver in both wild-type and glucokinase ± mice paral- leled by decreased expression of sterol regulatory element-binding protein-1c, stearoyl-CoA desaturase-1, and fatty acid synthase, and increased expression of peroxisome proliferator-activated receptor-α in the liver. Furthermore, in a mouse model of non- alcoholic steatohepatitis, further studies indicated that linagliptin improves insulin sensitivity and hepatic steatosis in mice with diet-induced obesity (161) and ameliorates liver inflammation (162). The underlying mechanism of these beneficial effects has been further investigated by Ohyama et al. in ob/ob mice (163). The novel DPP4-inhibitor MK-0626 attenuates hepatic steatosis by enhancing AMPK activity, inhibiting hepatic lipogenic gene expression, increasing triglyceride secretion from liver, and ele- vating serum adiponectin levels. by progressive fibrosis. This process is characterized by excessive accumulation of ECM and activated hepatic stellate cells (146, 147) that ultimately results in nodular regeneration with loss of function (148). Cardiovascular System Cardiovascular complications (CVD) are common in patients with T2DM and a major cause of mortality (169). Atheroscle- rosis is the dominant cause of CVD and usually develops many years before any clinical symptoms are manifested. The underly- ing pathogenesis of atherosclerosis involves an imbalanced lipid metabolism and a maladaptive immune response entailing a chronic low-grade inflammation of the arterial wall. Endothe- lial cells and intimal smooth muscle cells represent the major cell types of the artery wall preserving vessel wall homeosta- sis. Together with leukocytes, they are the major players in the development of this disease. Beside atherosclerosis, T2DM also exacerbates heart failure associated with diastolic heart failure and coronary microangiopathy (170–172). Serum Level of DPP4 in Liver Disease Serum Level of DPP4 in Liver Disease As previously discussed, hepatic DPP4 mRNA expression level in the livers is significantly higher in patients with NAFLD compared to healthy subjects (151). This upregulation of hepatic DPP4 expression is thought to be responsible for elevated DPP4 serum level in patients with liver disease (153–155). In line with this observation, serum DPP4 activity can be correlated with hep- atic steatosis and NAFLD grading (156). Similarly, in patients with NAFLD, DPP4 activity in serum correlates with markers of liver damage, such as serum gamma-glutamyltranspeptidase and ALT levels, but do not correlate with fasting blood glucose levels and HbA1c values (156, 157). Thus, hepatic DPP4 expression in NAFLD may be directly associated with increased DPP4 serum level and may be involved in hepatic lipogenesis and liver injury. Regulation of DPP4 Expression in the Liver In conclusion, accumulating studies indicate that DPP4 inhibitors are clinically useful for patients with T2DM accom- panied by liver dysfunction based on fatty liver, and that DPP4 inhibition affects liver function regardless of diabetic status and obesity. Effect of DPP4 Inhibition on the Cardiovascular System Regarding the vascular system, continuous infusion of the GLP-1 analog exendin-4 reduced monocyte adhesion to aortic endothelial cells, associated with a reduction in atherosclerotic lesion size in non-diabetic C57BL/6 and ApoE−/−mice. Further- more, treatment for 1 h with exendin-4 reduced the expression of the pro-inflammatory cytokines, TNFα and MCP-1, in response to lipopolysaccharide (LPS) (188). In addition, exendin-4 stim- ulates proliferation of human coronary artery endothelial cells through endothelial nitric oxide synthase (eNOS)-, protein kinase A (PKA)-, and PI3K/Akt-dependent pathways (192, 193). Accord- ingly, in humans, preliminary data confirm the ability of GLP-1 to protect from high glucose-induced endothelial dysfunction in the post-meal phase (194). In a model of vascular injury, it has been shown that continuous infusion of exendin-4 reduces neointimal formation at 4 weeks after injury without altering body weight or various metabolic parameters (195). From in vitro studies, Goto et al. suggest that this effect was mediated by the ability of GLP-1 to suppress platelet-derived growth factor (PDGF)-induced prolifer- ation of vascular smooth muscle cells. In contrast, in a pre-clinical study, combining HFD and STZ treatment in ApoE−/−failed to detect evidence for GLP-1R-dependent reduction of lesion size in the thoracic or abdominal aorta (168). The authors discuss that the duration of treatment, the dose of the GLP-1 agonist, or the age of mice might be responsible for the lack of anti-atherogenic activity in this study. y In several in vitro and pre-clinical studies, DPP4 inhibitors have been shown to exert important protective effects on the cardio- vascular system. In this regard, it has been shown that DPP4 inhibitors decrease myocardial infarct size, stabilize the cardiac electrophysiological state during myocardial ischemia, reduce ischemia/reperfusion injury, and prevent left ventricular remod- eling following MI (177, 178). Additionally, DPP4 inhibitors also exert vascular protective properties, including anti-inflammatory and anti-atherosclerotic effects and the ability to induce vascular relaxation (179, 180). To confirm cardiovascular safety or even protection of DPP4 inhibitors in humans, several cardiovascular outcome studies were conducted. However, several clinical trials, namely SAVOR-TIMI 53, EXAMINE, or VIVIDD in patients with established cardiovascular disease failed to confirm a cardio- protective effect (181–183). Even an increased cardiovascular risk for DPP4 inhibitors was discussed, since in the SAVOR-TIMI 53 trial, a significant increased hospitalization due to heart failure in the saxagliptin-treated group was observed. DPP4 Substrates: GLP-1 Dependent Effects of DPP4 Inhibitors Since several studies have identified a role for GLP-1 recep- tor (GLP-1R) signaling in DPP4-dependent cardioprotection, it is suggested that GLP-1 itself has favorable cardiovascular effects. Indeed, mRNA transcripts of the GLP-1R have been detected in the heart of rodents (185, 186) and humans (187). Furthermore, GLP-1R has also been localized to mouse aortic smooth muscle and endothelial cells, as well as monocytes and macrophages (188). Regarding MI and heart failure, pre-clinical studies have demonstrated that DPP4-deficient rats subjected to 45 min of ischemia with 2 h or reperfusion exhibited cardioprotection illus- trated by reduced infarct size, improved cardiac performance, and reduced levels of BNP compared to control rats (189). These ben- eficial effects could be partially reversed by co-administration of the GLP-1R antagonist exendin (9–39). Accordingly, administra- tion of exendin (9–39) reversed the sitagliptin-induced improve- ment in ventricular function in Sprague Dawley rats with tran- sient cardiac ischemia (190). Additionally, in a rat model of chronic heart failure, GLP-1 analogs were able to improve cardiac function and morphology, with a concomitant amelioration of hyperglycemia and hyperinsulinemia (191). Regulation of DPP4 Expression and Release in Vascular Cells Dipeptidyl-peptidase 4 is expressed in both microvascular endothelial cells of different human tissues, such as liver, spleen, July 2015 | Volume 6 | Article 386 Frontiers in Immunology | www.frontiersin.org 11 Röhrborn et al. DPP4 in diabetes substrates or inhibition of direct effects of DPP4 remains unclear and will be assessed in more detail in the following section. lung, brain, heart (170, 172), and in human vascular smooth muscle cells (3). Under conditions of high glucose, DPP4 expres- sion and activity were increased in human glomerular endothelial cells (173). Additionally, in STZ-induced diabetic rats, activity of membrane-bound DPP4 was increased, thereby reducing cardiac SDF-1 concentrations and causing impaired angiogenesis (174). Also hypoxia has been shown to regulate DPP4 expression in vascular cells. Regarding endothelial cells, there are conflicting data on the influence of hypoxia on DPP4 expression. In human microvascular endothelial cells as well as human umbilical vein endothelial cells, Eltzschig and colleagues showed that hypoxia increased DPP4 mRNA and protein level (175), whereas another study by Shigeta et al. observed a decreased protein level of DPP4 under hypoxic conditions in the same cells (174). However, in human vascular smooth muscle cells, we observed an increased DPP4 expression in response to hypoxia (3). In this particular study, we could also show that DPP4 is released from human vascular smooth muscle cells. However, only very little is known about the physiological role of the membrane-bound DPP4 within the vasculature. There is only one study showing that DPP4 forms a complex with ADA capable of degrading extracellular adenosine to inosine in endothelial cells. Increased inosine levels in turn are known to induce vasoconstriction due to mast cell degranulation (176). Frontiers in Immunology | www.frontiersin.org Effect of DPP4 Inhibition on the Cardiovascular System However, in the most recent published outcome study TECOS, the authors could show that among patients with T2DM and established cardiovascular disease, sitagliptin did not appear to increase the risk of major adverse cardiovascular events, hospitalization for heart failure, or other adverse events (184). As sDPP4 is an adipokine upregulated in obesity and T2DM that triggers IR and metabolic complica- tions (4, 5), it might be speculated that the beneficial effects of DPP4 inhibitors would be higher in those early phases of the metabolic disorders previous to the development of established cardiovascular disease. However, in patients with heart failure, pilot studies also suggest cardioprotection by GLP-1 infusion (196, 197). Accordingly, a large retrospective analysis indicates that patients treated with the GLP-1 analog exenatide had a significant 20% reduction of CVD events compared with patients on other glucose-lowering agents (198). Nevertheless, studies showing cardiovascular protective However, whether these beneficial effects observed in pre- clinical settings are due to increased levels of different DPP4 July 2015 | Volume 6 | Article 386 12 Röhrborn et al. DPP4 in diabetes FIGURE 2 | Schematic overview of the impact of soluble DPP4 and DPP4 inhibitors on T2DM-relevant organs/tissues. In the upper panel, direct effects of soluble DPP4 (in red) on different organs/tissues are presented (gray boxes). The lower panel shows known effects of DPP4 inhibitors (in green) in these particular organs/tissues (gray boxes). SMC, smooth muscle cells. presented (gray boxes). The lower panel shows known effects of DPP4 inhibitors (in green) in these particular organs/tissues (gray boxes). SMC, smooth muscle cells. FIGURE 2 | Schematic overview of the impact of soluble DPP4 and DPP4 inhibitors on T2DM-relevant organs/tissues. In the upper panel, direct effects of soluble DPP4 (in red) on different organs/tissues are effects of GLP-1 were carried out using either native GLP-1 or recombinant GLP-1 analogs at high concentrations or in a way that induced supraphysiological GLP-1 signaling. Considering that DPP4 inhibition restores GLP-1 signaling within the phys- iological range, beneficial effects of DPP4 inhibitors might be different to those of GLP-1 analogs. type 2 diabetic patients receiving a 4-week course of therapy with the DPP4-inhibitor sitagliptin show increased SDF-1α plasma concentrations and circulating EPC levels (199). Additionally, SDF-1 engineered to be resistant to DPP4 cleavage, and delivered by nanofibers, improves blood flow in a model of peripheral artery disease (201). Effect of DPP4 Inhibition on the Cardiovascular System Collectively, these studies implicate a rationale to use DPP4 inhibitors for vascular repair through stimulation of EPC and neovascularization. DPP4 Substrates: SDF-1- and BNP-Dependent Effects of DPP4 Inhibitors Brain natriuretic peptide, another substrate of DPP4, plays an important role in regulating body fluid homeostasis and vascular tone through binding and subsequent activation of the cGMP- coupled natriuretic peptide receptor type A (NPR-A) (202). BNP is secreted predominantly by ventricular cardiomyocytes in response to increased wall stress. Thus, elevated BNP is a sensitive marker of heart failure and appears to play a role in cardiac remodeling and healing after acute MI (203–205). DPP4 cleavage of the physiologically active BNP (1–32) to BNP (3–32) effectively lowers plasma cGMP levels, reduces diuresis and natriuresis, and inhibits vasodilatation (83, 202). But beside GLP-1, there are further substrates of DPP4, which might play a role in the favorable cardiovascular effects of DPP4 inhibitors. Two of the most promising candidates are SDF-1α and brain natriuretic peptide (BNP). As already mentioned in section “Stromal Cell-Derived Factor-1α/CXCL12,” SDF-1 is a chemokine promoting stem-cell homing of EPCs by binding to its receptor C–X–C motif chemokine receptor type 4 (CXCR4). EPCs are derived from the bone marrow and are known to pro- mote vascular repair and neoangiogenesis. When vascular dam- age occurs, local growth factors and cytokines signal the bone marrow to release EPC targeted to the injured sites. EPC then differentiate into mature endothelial cells and assist in the recon- struction of the vasculature (199). In mice, genetic deletion or pharmacologic inhibition of DPP4 is able to increase the homing of CXCR4+ EPC at sites of myocardial damage, resulting in a reduced cardiac remodeling and improved heart function and survival (200). In a human study, Fadini et al. demonstrated that Frontiers in Immunology | www.frontiersin.org Conclusion Dipeptidyl-peptidase 4, originally identified as an enzyme nearly 50 years ago, has now been recognized to exert pleiotropic func- tions with substantial impact for a variety of diseases. The com- plexity of DPP4 action stems from (i) a long list of substrates cleaved by the enzyme including hormones, growth factors, and cytokines, (ii) an additional function of this protein being a bind- ing partner at the surface of different cells, specifically immune cells, and (iii) the recent discovery that DPP4 is an adipokine with different endocrine functions. Thus, an integrated view on this molecule is required to more precisely understand its impact for metabolic diseases like type 2 diabetes. For this disease, DPP4 inhibition has gained substantial interest, mostly related to the DPP4 substrate, GLP-1. As shown here, other substrates like SDF- 1 and BNP should also be taken into account and may help to better understand the therapeutic potential of DPP4 inhibitors. In this context, the direct effects of DPP4 inhibitors require to be assessed in more detail, and several aspects like the cardio- protective function of DPP4 inhibition remains controversial. Finally, soluble DPP4 is emerging as a new research line, putting this molecule to the list of adipo-cytokines with pro-inflammatory and proliferative function. Combining the accumulated knowl- edge on DPP4 will lead to an improved understanding of its impact for health and disease. ( ) In human vascular smooth muscle cells, we could show that sDPP4 activates the MAPK and NF-κB signaling cascade resulting in pro-atherogenic changes in human vascular smooth muscle cells illustrated by an increased proliferation, the induction of iNOS and elevated expression, and secretion of pro-inflammatory cytokines (39). Additionally, we observed that all these detrimen- tal effects of sDPP4 were PAR2 mediated, since both a PAR2 antagonist and PAR2 silencing completely prevented the sDPP4- induced effects. In collaboration with the group of Sánchez-Ferrer, we further showed that sDPP4 exhibits direct effects on vascu- lar function illustrated by vascular reactivity of murine mesen- teric arteries (208). sDPP4 impaired the endothelium-dependent relaxation to acetylcholine in a concentration-dependent man- ner by up to 75%, without modifying endothelium-independent relaxation to sodium nitroprusside. Again, enzymatic activity of DPP4 appears to be involved in this process. Similarly, the cyclooxygenase inhibitor indomethacin and the thrombox- ane A2 receptor antagonist SQ29548 abrogated the impairing action of DPP4. 4. Lamers D, Famulla S, Wronkowitz N, Hartwig S, Lehr S, Ouwens DM, et al. Dipeptidyl peptidase 4 is a novel adipokine potentially linking obesity to the metabolic syndrome. Diabetes (2011) 60(7):1917–25. doi:10.2337/db10-1707 Acknowledgments This work was supported by the Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen (Ministry of Science and Research of the State of North Rhine-Westphalia), and the Bundesministerium für Gesundheit (Federal Ministry of Health). Conclusion These data suggest that DPP4 directly impairs endothelium-dependent relaxation through a mechanism that involves cyclooxygenase activation, and likely the release of a vaso- constrictor prostanoid. Since sDPP4 has been reported not only to contribute to monocyte migration and macrophage-mediated inflammatory reactions but also stimulates the proliferation of Endocrine Effects of Soluble DPP4 on Cardiovascular Homeostasis Although it is well established that serum levels of sDPP4 are altered in several pathological conditions and that sDPP4 is released from vascular cells, only a minor part of research has focused on potential endocrine effects of this proteolytic enzyme. July 2015 | Volume 6 | Article 386 Frontiers in Immunology | www.frontiersin.org 13 Röhrborn et al. DPP4 in diabetes Considering that DPP4 is discussed in immunomodulation, it might be speculated that the inhibition of DPP4 modulates responses occurring within early or late atherosclerotic lesions. In low-density lipoprotein receptor-deficient (LDLR−/−) mice, Shah et al. could demonstrate that exogenously injected DPP4 increases monocyte migration in vivo (180). Although these pro-migratory properties of DPP4 could be completely inhib- ited by sitagliptin, the underlying mechanism of these effects remains unclear. Moreover, the combined treatment of sDPP4 and LPS leads to increased expression and secretion of the pro- inflammatory cytokines, TNFα and IL-6. This upregulation was achieved by elevated levels of ERK, c-Fos, NF-κB p65, NF-κB p50, and CUX1, all factors known to bind to the promotor of TNFα and IL-6 (180). In accordance to that, Ikushima and collaborators observed that sDPP4 binds to the M6P/IGF-IIR resulting in enhanced transendothelial T cell migration (206). In a further study, sDPP4 binding to M6P/IGF-IIR leads to elevated ROS levels in HUVECs. In both studies, binding of DPP4 to this particular receptor was completely prevented by a DPP4 inhibitor (207). human coronary artery smooth muscle cells as well as impairs endothelium-dependent vasorelaxation, it might be speculated that sDPP4 itself acts as a risk factor for atherosclerosis. Collectively, this section emphasizes that both membrane- bound and sDPP4 and its inhibition are not only playing an impor- tant role in glucose homeostasis but also in several other processes and organs involved in the pathogenesis of T2DM (Figure 2). This supports the notion that DPP4 exhibits pleiotropic properties that are not fully understood so far and have to be further elucidated in the future. 5. Sell H, Bluher M, Kloting N, Schlich R, Willems M, Ruppe F, et al. Adipose dipeptidyl peptidase-4 and obesity: correlation with insulin resistance and depot-specific release from adipose tissue in vivo and in vitro. Diabetes Care (2013) 36(12):4083–90. doi:10.2337/dc13-0496 7. 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Protease- resistant stromal cell-derived factor-1 for the treatment of experimental peripheral artery disease. Circulation (2011) 123(12):1306–15. doi:10.1161/ CIRCULATIONAHA.110.991786 Copyright © 2015 Röhrborn, Wronkowitz and Eckel. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 202. Boerrigter G, Costello-Boerrigter LC, Harty GJ, Lapp H, Burnett JC Jr. Des- serine-proline brain natriuretic peptide 3-32 in cardiorenal regulation. Am J Physiol Regul Integr Comp Physiol (2007) 292(2):R897–901. doi:10.1152/ ajpregu.00569.2006 203. Kawakami R, Saito Y, Kishimoto I, Harada M, Kuwahara K, Takahashi N, et al. Overexpression of brain natriuretic peptide facilitates neutrophil July 2015 | Volume 6 | Article 386 Frontiers in Immunology | www.frontiersin.org 20
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Pengaruh Teknik Sosiodrama untuk Meningkatkan Keterampilan Komunikasi Interpersonal Siswa Laki-Laki Kelas SMK Ar-Rahman Misriadi Langkat
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Jurnal Counseling Care Volume 3, Nomor 1, Bulan April, 2019 JUDUL ARTIKEL LENGKAP Penulis : Khairina Ulfa Syaimi Sumber : Jurnal Counseling Care, Volume 3, Nomor 1, April-Oktober 2019 Diterbitkan Oleh : Laboratorium Bimbingan dan Konseling, STKIP PGRI Sumatera Barat Copyright © 2019, Jurnal Counseling Care Untuk Mengutip Artikel ini : Syaimi K, U, 2019. Pengaruh Teknik Sosiodrama untuk Meningkatkan Keterampilan Komunikasi Interpersonal Siswa Laki-laki Kelas SMK Ar-Rahman Misriadi Langkat. Jurnal Counseling Care. Volume 3, Nomor 1, bulan April, 2019: 39-45. Untuk Mengutip Artikel ini : Syaimi K, U, 2019. Pengaruh Teknik Sosiodrama untuk Meningkatkan Keterampilan Komunikasi Interpersonal Siswa Laki-laki Kelas SMK Ar-Rahman Misriadi Langkat. Jurnal Counseling Care. Volume 3, Nomor 1, bulan April, 2019: 39-45. Copyright © 2019, Jurnal Counseling Care ISSN : 2581-0650 (Online) Laboratorium Bimbingan dan Konseling STKIP PGRI Sumatera Barat Laboratorium Bimbingan dan Konseling STKIP PGRI Sumatera Barat ABSTRACT This research is intended to observe the effect of through sociodrama technique in improving students’ interpersonal communication skill at the tenth-grade students of SMK Ar-Rahman Misriadi Langkat. In the research methodology, the writer used quasi-experimental design, focusing on non- equivalent control group design. In addition, to decide the sample of the research the writer used non-probability sampling technique in the form of purposive sampling technique. There were twenty four male students who had been chosen as the research sample.The sample chosen was classified based on the interpersonal communication skill in the lower rate. Moreover, the instrument used to decide the sample was the instrument of interpersonal communication skill which provided four options on a Likert scale. The technique of analysis was carried out by using descriptive statistics which provided empirical mean to know the average scores between the pre-test and the posttest in the experimental group and controlled group respectively. The result showed that the score of Asymp. Sig were 0.000 which meant that the interpersonal communication skill of the male students Sig < 0.05. It can be concluded that the high number of increasing of interpersonal communication skill was experienced by the male students in the experimental group than in the controlled group. Therefore, the result of this research was used to improve the interpersonal communication skill of the tenth- grade male students at SMK Ar-Rahman Misriadi Langkat. Kwyword : sociodrama, interpersonal communication, male students Pengaruh Teknik Sosiodrama untuk Meningkatkan Keterampilan Komunikasi Interpersonal Siswa Laki-Laki Kelas SMK Ar-Rahman Misriadi Langkat Khairina Ulfa Syaimi Universitas Muslim Nusantara Al-Washliyah Medan Email : khairinaulfasyaimi12@gmail.com ABSTRAK Penelitian ini bertujuan untuk mengetahui pengaruh teknik sosiodrama untuk meningkatkan keterampilan komunikasi interpersonal siswa. Metode penelitian yang digunakan adalah Quasi ekperimen design nonequivalent control group design. Teknik sampling yang digunakan adalah non-probability sampling dengan bentuk purposive sampling. Siswa laki-laki yang menjadi sampel dalam penelitian ini adalah 24 orang dengan skor keterampilan komunikasi interpersonal berada dalam kategori rendah. Instrumen yang digunakan dalam menentukan sampel penelitian ini adalah instrumen keterampilan komunikasi interpersonal model skala likert. Teknik analisis data statistik dilakukan dengan menggunakan statistik deskriptif dengan menggunakan mean empiris untuk melihat rata-rata skor pretest dan posttest pada kelompok eksperimen dan kelompok kontrol. Hasil pengujian hipotesis menunjukkan bahwa nilai Asymp. Sig sebesar 0.000, yang berarti melalui hasil perhitungan tersebut maka diperoleh kesimpulan bahwa pada keterampilan komunikasi interpersonal siswa laki-laki sig<0.05, Artinya peningkatan keterampilan komunikasi interpersonal siswa laki-laki kelas X SMK Ar-Rahman Misriadi Langkat kelompok treatment lebih tinggi dibandingkan kelompok kontrol. Kata Kunci : Sosiodrama, Keterampilan komunikasi interpersonal, siswa laki-laki Copyright © 2019, Jurnal Counseling Care| 39 Syaimi – Pengaruh Teknik Sosiodrama untuk Meningkatkan... INTRODUCTION / PENDAHULUAN ekspresi yang kuat. Remaja laki-laki (kaum maskulin) tidak memproses informasi panjang dengan gambaran yang terlalu luas. Karena itu, remaja laki-laki seringkali terlihat bosan dengan cerita yang berbelit. Remaja laki-laki cenderung mengatakan apa yang harus mereka katakan, dengan asumsi pesan yang disampaikan jelas (Berko, Wolvin, & Wolvin, 2001). Masa remaja merupakan masa yang penuh konflik karena masa ini periode perubahan dimana terjadi perubahan tubuh, pola perilaku, dan peran yang diharapkan oleh kelompok sosial, serta merupakan masa pencarian identitas untuk mengangkat diri sendiri sebagai individu. Perubahan-perubahan bagi remaja terkadang merupakan situasi yang tidak menyenangkan dan sering menimbulkan masalah. Permasalahan-permasalahan tersebut menuntut suatu penyelesaian agar tidak menjadi beban yang dapat mengganggu perkembangan selanjutnya (Hurlock, 2004). Para remaja laki-laki juga lebih kompetitif dalam kemampuan bicara, dimana para remaja laki-laki telah tersosialiasi untuk memiliki rasa “tanggung- jawab”. Pada kontak verbal remaja laki-laki lebih banyak terlibat pembicaraan publik, remaja laki-laki menggunakan pembicaraan sebagai pernyataan fungsi untuk perintah, menyampaikan informasi, dan meminta persetujuan. Remaja laki-laki lebih kepada menyembunyikan dan menyampingkan perasaan mereka Salah satu ciri perkembangan remaja adalah adanya tekanan teman sebaya yang kuat (peer pressure) (Saputro & Soeharto, 2012). Tekanan teman sebaya ada yang berbentuk positif dan ada pula yang berbentuk negatif. Bentuk positif misalnya ketika remaja memilih hobi yang disukai seperti basket atau futsal. Pemilihan hobi tersebut pada umumnya masih dipengaruhi oleh teman sekitarnya. Sedangkan bentuk negatif dari tekanan teman sebaya seperti perilaku merokok, seks berisiko,narkoba, minum minuman beralkohol, dan tawuran. Bersumber pada wawancara dengan Guru BK SMK Ar-Rahman Misriadi Langkat mengenai keterampilan komunikasi Interpersonal pada siswa laki-laki ditemukan bahwa sebagian besar siswa laki- laki mengalami kesulitan bergaul dengan temannya, yang ditandai dengan sering terjadinya konflik (saling menghina), sulit terbuka dengan orang lain, merasa terasing, pendiam juga gugup, dan takut untuk bertanya pada saat proses belajar. Pada umumnya, permasalahan remaja laki-laki telah mendominasi masyarakat melalui pembicaraan dengan menggunakan Copyright © 2019, Jurnal Counseling Care| 40 Syaimi – Pengaruh Teknik Sosiodrama untuk Meningkatkan... Oleh Sebab itu, teknik sosiodrama siswa dapat mengekpresikan pikiran, perasaan, memecahkan masalah, dan memperjelas nilai-nilai yang ada dalam diri mereka. Pada sosiodrama bukan hanya membahas isu-isu sosial, sosiodrama membuat orang-orang menjelajahi dunia luar dalam aksi mereka dengan topik yang menarik untuk mereka. Saat mereka menjelajahi berbagai masalah, mereka menempatkan diri dalam peran sebagai orang lain untuk memahami diri sendiri dan orang lain dengan lebih baik. INTRODUCTION / PENDAHULUAN Salah satu alasan sosiodrama bekerja dengan baik adalah bahwa sosiodrama mengarahkan seseorang pada kebenaran tentang kemanusiaan bahwa manusia adalah sama (Jacobs, Masson, Harvill, & Schimmel, 2011). bertujuan untuk mengetahui pengaruh variabel dependen (X) yang tercermin dalam perbedaan variabel dependen khususnya O2 dan O4. Penelitian dilakukan sebanyak tujuh kali pertemuan dengan dua kali pertemuan untuk tes dan lima kali pertemuan untuk pelaksanaan eksperimen. Selama pelaksanaan eksperimen peneliti menggunakan teknik sosiorama. Pupulasi dalam penelitian ini adalah seluruh siswa laki-laki kelas X SMK Ar-Rahman Misriadi Langkat yang berjumlah 80 orang. Sementara itu, teknik sampling yang digunakan dalam penelitian ini adalah purposive sampling dengan sampel berjumlah 24 orang. Pada penelitian ini, pengukuran keterampilan komunikasi interpersonal dilakukan dengan menggunakan instrumen yang dikembangkan oleh Josep Devito pada tahun 2013, yang memiliki 5 aspek yaitu keterbukaan, empati, sikap positif, dukungan dan kesetaraan. Copyright © 2019, Jurnal Counseling Care| 41 METODE PENELITIAN Penelitian ini bertujuan untuk mengetahui pengaruh teknik sosiodrama terhadap keterampilan komunikasi interpersonal siswa laki-laki kelas X di SMK Ar-Rahman Misriadi. Metode penelitian yang digunakan ini adalah metode penelitian kuasi (Quasy Experimental Research), desain Nonequivalent Control Group Desain. Oleh sebab itu, penelitian ini akan melibatkan kelompok eksperimen sebagai kelompok yang akan mendapatkan perlakuan. Kedua kelompok akan mendapatkan pretest dan posttest yang Kelo mpok Katego risasi Skor Pre- Test Frek uensi Pre- Test Skor Post- Test Frekue nsi Pre- Test Kelo mpok Eksp erim en Renda h 78- 94 12 78- 94 Sedang 95- 116 95- 116 3 Tinggi 116- 131 116- 131 9 Kelo mpok Kont rol Renda h 78- 94 12 78- 94 12 Sedang 95- 116 95- 116 Tinggi 116- 131 116- 131 Jumlah 12 12 Syaimi – Pengaruh Teknik Sosiodrama untuk Meningkatkan... Perubahan dapat dilihat berdasarkan hasil daripre-test dan post-test yang telah diberikan sebelum dan sesudah diberikan teknik sosiodrama dilakukan pada duabelas siswa SMK kelas X Ar-Rahman Misriadi, didapatkan hasil sebagai berikut: Kualitas skor peningkatan keterampilan komunikasi pada sampel penelitian diketahui melalui pengujian gain skor menggunakan rumus Lei Bao sebagai berikut : Gain ternormalisasi Gain ternormalisasi (g) = Skor posstest−Skor pretest Skor ideal−Skor pretest (g) = Skor posstest−Skor pretest (g) Skor ideal−Skor pretest Pengaruh eksperimen terhadap sampel penelitian diketahui melalui pengolahan data dan analisis data menggunakan Mann Whitney U Testdengan menggunakan bantuan aplikasi SPSS versi 20.0 Berdasarkan tabel diatas, sebelum diberikannya teknik sosiodrama tingkat keterampilan komunikasi interpersonal siswa laki-laki kelas X SMK Ar-Rahman Misriadi Langkat, seluruh siswa berada pada kategoti rendah. Setelah diberikannya teknik sosiodrama, terjadi peningkatan pada rata skor keterampilan komunikasi interpersonal siswa. Sembilan di antarannya berada di kategori tinggi, Tiga orang di kategori sedang dan tidak ada responden yang berada pada tingkat kategori rendah. Sedangkan pada kelompok kontrol, saat pretest dan post test tetap berada pada kategori rendah, dengan kata lain tidak terjadi peningkatan. Skor rata-rata capaian siswa mengalami perubahan setelah diberikannya perlakuan, skor capaian sebelum dan sesudah perlakuan dapat dilihat dalam tabel berikut: DAN PEMBAHASAN Hasil pengujian hipotesis dengan menggunakan Mann Whitney U Testdengan menggunakan aplikasi SPSS versi 20.0 diperoleh Asymp, Sig = 0.000 dan diuji pada signifikan alpha 0,05.Nilai Asymp Sig = 0.000 < Nilai Signifikansi α 0,05 Berdasarkan data diatas dapat disimpulkan bahwa H0 ditolak dan H1 Diterima, ini mengartikan bahwa terjadi peningkatan pada keterampilan komunikasi interpersonal siswa setelah diberikan bimbingan kelompok dengan teknik sosiodrama. Hasil pengujian hipotesis dengan menggunakan Mann Whitney U Testdengan menggunakan aplikasi SPSS versi 20.0 diperoleh Asymp, Sig = 0.000 dan diuji pada signifikan alpha 0,05.Nilai Asymp Sig = 0.000 < Nilai Signifikansi α 0,05 Berdasarkan data diatas dapat disimpulkan bahwa H0 ditolak dan H1 Diterima, ini mengartikan bahwa terjadi peningkatan pada keterampilan komunikasi interpersonal siswa setelah diberikan bimbingan kelompok dengan teknik sosiodrama. Sampel yang digunakan 24 orang yang terpilih sebagai sampel penelitian, yang kemudian dibagi kedalam 2 kelompok, yaitu 12 orang pada kelompok eksperimen, dan 12 orang pada kelompok kontrol. Copyright © 2019, Jurnal Counseling Care| 42 Syaimi – Pengaruh Teknik Sosiodrama untuk Meningkatkan... keterampilan dalam komunikasi pada individu dalam proses interaksi (Aini, Sugiharto, & Sutoyo, 2014). Hal ini akan membantu memperbaiki komunikasi dan pemahaman dengan mendorong diskusi antar peserta. Sosiodrama membantu membangun keterampilan dalam komunikasi, pemecahan masalah, dan kesadaran diri dengan cara memainkan peran dalam menangani situasi (Siregar, 2015). Faktor kedua yaitu dengan memerankan peran seseorang akan belajar melihat peran seseorang dari sudut pandang yang berbeda. dan Faktor yang ketiga sosiodrama menjadi begitu menyenangkan, karena melibatkan anggota kelompok itu sendiri dalam melak sanakan pertunjukandan memutuskan skenario (Ayu, Rosra, & Andriyanto, 2019) Tabel 2. Data Skor Pre-test dan Post-test Keterampilan Komunikasi Interpersonal Per Individu N o Kelompok Eksperimen Kelompok Kontrol Pre Pos t Gai n Pre Pos t Gai n 1 ∑ 10 42 13 52 40 5 ∑ 11 06 11 29 24 x 86, 8 12 1 33, 7 x 92 94 2 Tabel 2. Data Skor Pre-test dan Post-test Keterampilan Komunikasi Interpersonal Per Individu Skor capaian rata-rata sebelum diberikannya perlakuan pada kelompok eksperimen 86,8, setelah diberikan, skor capaian rata-rata siswa meningkat menjadi 121. Hal ini mengindikasikan adanya pengaruh teknik sosiodrama dalam meningkatkan keterampilan komunikasi interpersonal siswa. Sedangkan pada kelompok kontrol, terjadi peningkatan dari 92 menjadi 94. Peningkatan yang terjadi pada kelompok kontrol lebih kecil dibandingkan pada kelompok kelompok eksperimen. CONCLUSION / KESIMPULAN Berdasarkan hasil penelitian diketahui bahwa teknik sosiodrama dapat meningkatkan keterampilan komunikasi interpersonal pada siswa laki-laki kelas X SMK Ar-Rahman Misriadi Langkat. Terdapat perbedaan yang signifikasi 0.000 < 0.05 yaitu antara gain score kelompok eksperimen dan kelompok kontrol dengan kualitas peningkatan skor pada kelompok eksperimen lebih baik dibandingkan peningkatan skor pada kelompok kontrol Jika dilihat dari capaian yang didapatkan oleh siswa setelah melaksanakan teknik sosiodrama dan berdasarkan deskripsi di atas, siswa mulai mengembangkan penilaian positif terhadap orang lain. Ada beberapa faktor yang menyebabkan teknik sosiodrama berpengaruh terhadap interpersonal. Faktor pertama sosiodrama berfokus peningkatan Copyright © 2019, Jurnal Counseling Care| 43 Syaimi – Pengaruh Teknik Sosiodrama untuk Meningkatkan... keterampilan komunikasi interpersonal siswa. Komunikasi interpersonal siswa laki- laki kelas X SMK Ar-Rahman Misriadi Langkat tahun ajaran 2018/2019 secara umum berada pada kategori sedang jumlah siswa 80, artinya siswa sudah dapat menunjukkan keterbukaan tetapi hanya sebatas kepada orang terdekat, menunjukkan sikap empati kepada teman tetapi masih sebatas berempati kepada teman yang dikenalnya. Siswa sudah menunjukkan dukungan kepada orang lain tetapi masih belum mendalam hanya sebatas memberikan dukungan yang sama dilakukan orang lain pada umumnya, siswa sudah menunjukkan sikap yang positif tetapi masih sebatas orang-orang terdekat, dan siswa sudah menunjukkan sikap kesetaraan tapi masih perlu cara mengkomunikasikan kesetaraan agar diterima oleh orang lain. Bagi Peneliti selanjutnya rumusan intervensi yang dirumuskan dan diuji cobakan dipergunakan bagi semua kategori, baik tinggi, sedang maupun rendah bertujuan untuk melihat perubahan yang komprehensif pada keterampilan komunikasi interpersonal. Copyright © 2019, Jurnal Counseling Care| 45 Syaimi – Pengaruh Teknik Sosiodrama untuk Meningkatkan... REFERENCES / DAFTAR PUSTAKA Aini, N., Sugiharto, D. Y. P., & Sutoyo, A. (2014). Pengembangan Model Bimbingan Kelompok dengan Teknik Sosiodrama untuk Meningkatkan Penyesuaian Diri Siswa. Jurnal Bimbingan Konseling, 3(2). Aini, N., Sugiharto, D. Y. P., & Sutoyo, A. (2014). Pengembangan Model Bimbingan Kelompok dengan Teknik Sosiodrama untuk Meningkatkan Penyesuaian Diri Siswa. Jurnal Bimbingan Konseling, 3(2). Ayu, D. N., Rosra, M., & Andriyanto, R. E. (2019). Peningkatan Hubungan Interpersonal Menggunakan Teknik Sosiodrama. ALIBKIN (Jurnal Bimbingan Konseling), 7(3). Teknik sosiodrama memiliki signifikansi terhadap keterampilan kemampuan komunikasi interpersonal siswa, artinya teknik sosiodrama dapat meningkatkan keterampilan komunikasi interpersonal siswa laki-laki. Peningkatan rata-rata skor hubungan interpersonal kepada sasaran intervensi secara keseluruhan pada setiap aspek yaitu keterbukaan, empati, sikap positif, sikap mendukung, kesetaraaan. Berko, R. M., Wolvin, A. D., & Wolvin, D. R. (2001). Communicating: A Social and Career Focus. Houghton Mifflin College Division. Hurlock, E. B. (2004). Psikologi Perkembangan: Suatu Perkembangan Sepanjang Rentang Kehidupan. Edisi Keenam, Jakarta: Penerbit Erlangga. Guru Bimbingan dan Konseling dapat mempergunakan program intervensi melalui teknik sosiodrama untuk meningkatkan Jacobs, E. E., Masson, R. L. L., Harvill, R. Jacobs, E. E., Masson, R. L. L., Harvill, R. Copyright © 2019, Jurnal Counseling Care| 44 Syaimi – Pengaruh Teknik Sosiodrama untuk Meningkatkan... L., & Schimmel, C. J. (2011). Group Counseling: Strategies and Skills. Cengage learning. L., & Schimmel, C. J. (2011). Group Counseling: Strategies and Skills. Cengage learning. Saputro, B. M., & Soeharto, T. (2012). Hubungan antara Konformitas terhadap Teman Sebaya dengan Kecenderungan Kenakalan pada Remaja. Insight, 10(1), 1–15. Siregar, M. M. (2015). Penerapan Metode Sosiodrama Untuk Meningkatkan Kemampuan Interpersonal Siswa Kelas Vii SMP Negeri 5 Depok Sleman Yogyakarta. Jurnal Riset Mahasiswa Bimbingan Dan Konseling, 4(3).
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Lake Surface Area Forecasting Using Integrated Satellite-SARIMA-Long-Short-Term Memory Model
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Lake Surface Area Forecasting Using Integrated Satellite-SARIMA-Long-Short-Term Memory Model Keyvan Soltani  Razi University Arash Azari  Razi University Mohammad Zeynoddin  Laval University: Universite Laval Afshin Amiri  University of Tehran Isa Ebtehaj  Laval University: Universite Laval Taha B.M.J. Ouarda  Institut national de la recherche scienti¦que Bahram Gharabaghi  University of Guelph Hossein Bonakdari  (  hossein.bonakdari@fsaa.ulaval.c Universite Laval https://orcid.org/0000-0001-6169-365 Research Article Keywords: Water resources, stochastic model, SARIMA, Tashk-Bakhtegan Lakes, hybrid model, forecasting. DOI: https://doi.org/10.21203/rs.3.rs-631247/v1 DOI: https://doi.org/10.21203/rs.3.rs-631247/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Lake Surface Area Forecasting Using Integrated Satellite-SARIMA-Long-Short- 1 Term Memory Model 2 3 Keyvan Soltani1, Arash Azari2, Mohammad Zeynoddin3, Afshin Amiri4, Isa Ebtehaj3, Taha B.M.J. Ouarda5, Bahram 4 Gharabaghi6, Hossein Bonakdari3, * 5 1Department of Civil Engineering, Razi University, Kermanshah, Iran 6 2Department of Water Engineering, Razi University, Kermanshah, Iran 7 3Department of Soils and Agri‐Food Engineering, Université Laval, Québec, Canada, G1V 0A6 8 4Department of Remote Sensing and GIS, University of Tehran, Tehran, Iran 9 5Institut National de la Recherche Scientifique, Centre Eau Terre Environnement, INRS-ETE, Québec, G1K 9A9 10 Canada 11 6School of Engineering, University of Guelph, Guelph, Ontario NIG 2W1, Canada 12 *Corresponding author, Phone: +1 418 656-2131, Fax: +1 418 656-3723, E-mail: hossein.bonakdari@fsaa.ulaval.ca 13 14 Lake Surface Area Forecasting Using Integrated Satellite-SARIMA-Long-Short- 1 Term Memory Model 2 3 Keyvan Soltani1, Arash Azari2, Mohammad Zeynoddin3, Afshin Amiri4, Isa Ebtehaj3, Taha B.M.J. Ouarda5, Bahram 4 Gharabaghi6, Hossein Bonakdari3, * 5 1Department of Civil Engineering, Razi University, Kermanshah, Iran 6 2Department of Water Engineering, Razi University, Kermanshah, Iran 7 6School of Engineering, University of Guelph, Guelph, Ontario NIG 2W1, Canada 12 *Corresponding author, Phone: +1 418 656-2131, Fax: +1 418 656-3723, E-mail: hossein.bonakdari@fsaa.ulaval.ca 13 14 14 1 Keywords: Water resources, stochastic model, SARIMA, Tashk-Bakhtegan Lakes, hybrid 33 model, forecasting. 34 Abstract 15 Lake Water Surface Area (WSA) plays a vital role in environmental preservation and future 16 water resource planning and management. Accurately mapping, monitoring and forecasting Lake 17 WSA changes are of great importance to regulatory agencies. This study used the MODIS 18 satellite images to extract a monthly time series of WSA of two lakes located in Iran from 2001 19 to 2019. Following a consequence of image and time series preprocessing to obtain the 20 preprocessed lake surface area time series, the outcomes were modeled by the Long-Short-Term 21 Memory (LSTM) deep learning (DL) method, the stochastic Seasonal Auto-Regressive 22 Integrated Moving Average (SARIMA) method and hybridization of these two techniques with 23 the objective of developing WSA forecasts. After separate standardization and normalization of 24 AL TS and reevaluation of the preprocessed data, the SARIMA (1, 0, 0) (0, 1, 1)12 model 25 outperformed sole LSTM models with correlation index of (R) 0.819, mean absolute error 26 (MAE) of 49.425 and mean absolute percentage error (MAPE) of 0.106. On the other hand, the 27 hybridization (stochastic-DL) enhanced the reproduction of the primal statistical properties of 28 WSA data and caused better mediation. However, the other accuracy indices did not change 29 markedly (R 0.819, MAE 49.310, MAPE 0.105). The multi-step preprocessing and reevaluation 30 also caused all LSTM models to produce their best results by less than 12 inputs. 31 32 Lake Water Surface Area (WSA) plays a vital role in environmental preservation and future 16 water resource planning and management. Accurately mapping, monitoring and forecasting Lake 17 WSA changes are of great importance to regulatory agencies. This study used the MODIS 18 satellite images to extract a monthly time series of WSA of two lakes located in Iran from 2001 19 to 2019. Following a consequence of image and time series preprocessing to obtain the 20 preprocessed lake surface area time series, the outcomes were modeled by the Long-Short-Term 21 Memory (LSTM) deep learning (DL) method, the stochastic Seasonal Auto-Regressive 22 36 2 1. Introduction 37 Accurate mapping of lake Water Surface Areas (WSA) is essential to assess the amount of 38 surface water available [1–5]. WSA is also helpful in determining the relationship between 39 climate and water resources [6–9] and for assessing the impacts of changing water surfaces, 40 which is crucial in water resources management [10-11]. The various methods for the extraction 41 of water surface from remote sensing data fall into two general categories: single-band and 42 multi-band techniques. The single-band technique uses a multispectral image band and identifies 43 other ground-surface phenomena based on a threshold limit for water sources. The multi-band 44 method helps distinguish the water masses from the differences in the reflectance properties of 45 different bands [12]. Monitoring the water dynamics with images taken at different times can 46 show changes in lakes, reservoirs and flood surfaces [13, 14]. 47 Accurate mapping of lake Water Surface Areas (WSA) is essential to assess the amount of 38 surface water available [1–5]. WSA is also helpful in determining the relationship between 39 climate and water resources [6–9] and for assessing the impacts of changing water surfaces, 40 which is crucial in water resources management [10-11]. The various methods for the extraction 41 of water surface from remote sensing data fall into two general categories: single-band and 42 multi-band techniques. The single-band technique uses a multispectral image band and identifies 43 other ground-surface phenomena based on a threshold limit for water sources. The multi-band 44 method helps distinguish the water masses from the differences in the reflectance properties of 45 different bands [12]. Monitoring the water dynamics with images taken at different times can 46 show changes in lakes, reservoirs and flood surfaces [13, 14]. 47 Google Earth Engine (GEE) comprises a considerable amount of satellite and global data types 48 worldwide, making it possible to analyze this data for various purposes such as change detection 49 [15] , mapping [16, 17] and ground level studies [18]. GEE has been widely used in a number of 50 disciplines including reviewing global forest changes [19], estimating crop production [20], 51 ground subsidence monitoring [21], coral reef mapping [22], modeling global surface water 52 change [23, 24], flood risk assessment [25], global urban mapping [26, 27], renewable energy 53 mapping [28], drought monitoring [29], and the reconstruction of the MODIS global vegetation 54 index [30]. 1. Introduction 37 55 Satellite data have been commonly used in hydrological studies [31–35, 36]. Nath and Deb [37] 56 used satellite images to detect and extract the water body of Puyang China. Abou El-Magd and 57 Ali [38] studied surface evaporation from Lake Nasser using high-resolution radiometer satellite 58 images. They demonstrated that robust assessments of lake evaporation can be obtained. Song et 59 Satellite data have been commonly used in hydrological studies [31–35, 36]. Nath and Deb [37] 56 used satellite images to detect and extract the water body of Puyang China. Abou El-Magd and 57 Ali [38] studied surface evaporation from Lake Nasser using high-resolution radiometer satellite 58 images. They demonstrated that robust assessments of lake evaporation can be obtained. Song et 59 Satellite data have been commonly used in hydrological studies [31–35, 36]. Nath and Deb [37] 56 used satellite images to detect and extract the water body of Puyang China. Abou El-Magd and 57 Ali [38] studied surface evaporation from Lake Nasser using high-resolution radiometer satellite 58 images. They demonstrated that robust assessments of lake evaporation can be obtained. Song et 59 3 al. [39] studied water level and lake area in the Tibetan Plateau by extracting time series from 60 Landsat images. Moreira et al. [34] investigated and modelled water balance using satellite 61 images and the evapotranspiration dataset in South America. Veh [40] developed an algorithm to 62 detect the glacial lake outburst floods (GLOFs) in the Himalayas. The algorithm uses satellite 63 images to analyze GLOFs and provide interpretable statistics for risk assessment and hazard 64 prevention planning. 65 The pace of artificial intelligence (AI) models' development and their accuracy is rapidly 66 increasing nowadays. These models are increasingly utilized in various fields of science, 67 including water engineering and hydrology [41–43], since these models produced acceptable 68 results in modelling sophisticated time series. Also, developments in AI and the computer 69 industry played an important role [44] in accelerating this pace. In this field, deep learning 70 methods produced noticeable results in modelling and forecasting hierarchical data [45-47]. The 71 most recent deep learning model, LSTM, can utilize the unlimited historical raw data as inputs to 72 detect the structure of the data and forecast future steps. 1. Introduction 37 The LSTM method is widely used in 73 many fields like natural language understanding and speech recognition [48], image and text 74 survey [49], hydrological data modelling such as precipitation and runoff forecasting [42,50], 75 and modeling climatic and meteorological data [51]. Mohan and Gaitonde [52] used LSTM to 76 model turbulent flow control and its temporal dynamics. Murad and Pyun [53] employed LSTM 77 alongside support vector machine (SVM) and k-nearest neighbours (KNN) for human activity 78 recognition, and they reported a higher performance of the LSTM model compared to other types 79 of AI models. Sahoo et al. [54] used LSTM recurrent neural networks (LSTM-RNN) to model 80 low flow hydrological time series. With a 94 percent correlation and low errors, they reported an 81 acceptable potential of LSTM for modelling hydrological time series. 82 The pace of artificial intelligence (AI) models' development and their accuracy is rapidly 66 increasing nowadays. These models are increasingly utilized in various fields of science, 67 including water engineering and hydrology [41–43], since these models produced acceptable 68 results in modelling sophisticated time series. Also, developments in AI and the computer 69 industry played an important role [44] in accelerating this pace. In this field, deep learning 70 methods produced noticeable results in modelling and forecasting hierarchical data [45-47]. The 71 most recent deep learning model, LSTM, can utilize the unlimited historical raw data as inputs to 72 detect the structure of the data and forecast future steps. The LSTM method is widely used in 73 many fields like natural language understanding and speech recognition [48], image and text 74 survey [49], hydrological data modelling such as precipitation and runoff forecasting [42,50], 75 and modeling climatic and meteorological data [51]. Mohan and Gaitonde [52] used LSTM to 76 model turbulent flow control and its temporal dynamics. Murad and Pyun [53] employed LSTM 77 alongside support vector machine (SVM) and k-nearest neighbours (KNN) for human activity 78 recognition, and they reported a higher performance of the LSTM model compared to other types 79 of AI models. Sahoo et al. [54] used LSTM recurrent neural networks (LSTM-RNN) to model 80 low flow hydrological time series. With a 94 percent correlation and low errors, they reported an 81 acceptable potential of LSTM for modelling hydrological time series. 82 4 4 Stochastic methods are among the most renowned statistical models. 1. Introduction 37 These methods are popular 83 amongst researchers because of their comprehensible principles and easy application. Seasonal 84 Auto-Regressive Integrated Moving Average (SARIMA) uses non-seasonal and seasonal 85 parameters to forecast time series based on historical data linearly [55–58]. Papalaskaris et al. 86 [59] employed the SARIMA model for short-term basin rainfall forecasting in Kavala City, 87 Greece. Mombeni et al. [60] used SARIMA for estimating one-year-ahead water demand in Iran. 88 However, most hydrological time series have complex structures that cannot be efficiently 89 modeled by linear methods like stochastic models or by AI models. Hence, some researchers 90 resorted to the integration of AI and linear models to utilize both their capabilities. Hybridization 91 of AI and linear models is one method that helps catch the complexity in time series and which 92 has produced more accurate results [35,61–64]. Mishra et al. [65] employed a combination of 93 stochastic SARIMA model and ANN to predict droughts in the Kansabati River basin in India. 94 The results indicated that a hybrid model leads to higher accuracy. Shafaei et al. [66] applied 95 wavelet pre-processing to SARIMA, ANN and hybridization of both and modelled monthly 96 precipitation in Iran. They indicated that wavelet-SARIMA-ANN produces better results than 97 wavelet-SARIMA and wavelet-ANN. 98 Greece. Mombeni et al. [60] used SARIMA for estimating one-year-ahead water demand in Iran. 88 A novel methodology based on the integration of remote sensing and deep learning- stochastic 99 modelling for lake surface area forecasting is proposed in the present work. To the best 100 knowledge of the authors, no previous studies have attempted to use such hybrid model for 101 WSA. The satellite images are downloaded, pre-processed and digitized for each time point to 102 obtain changes in the water area. Then the achieved time series is modelled and forecasted by 103 three methods. The modelling methods are deep learning LSTM model, stochastic SARIMA and 104 hybridization SARIMA-LSTM. Prior to modelling, the time series structure is analysed by 105 5 stationarity and normality tests and other statistical and visual tests. If any pre-processing is 106 needed, a standardization and/or normalization of the series is carried out to obtain the optimized 107 modelling results. In the end, statistical and visual tools survey the methods presented in the 108 methodology. 109 stationarity and normality tests and other statistical and visual tests. 1. Introduction 37 If any pre-processing is 106 needed, a standardization and/or normalization of the series is carried out to obtain the optimized 107 modelling results. In the end, statistical and visual tools survey the methods presented in the 108 methodology. 109 The Tashk-Bakhtegan lakes (TB lakes) with a surface area of 540 km2 are Iran's second-largest 114 inland lakes. These lakes are the most important ecological habitats of Iran at an altitude of 1525 115 m above sea level and have a catchment area of 25,000 km2. The maximum depth of Tashk- 116 Bakhtaran lake is 2 m, and the maximum depth of Tashk lake is 3.1 m [66, 67]. These lakes are 117 located between 29° 13'N–29° 48'N and 54° 10'E–53° 23'E. Water inflows to these lakes through 118 the Kor and Syvand rivers. With the construction of three dams in these rivers' upper basin, the 119 inflow of water into these lakes has decreased dramatically, causing a large area to dry out [68]. 120 Fig.1 shows the location of the twin TB lakes in Iran. 121 The Tashk-Bakhtegan lakes (TB lakes) with a surface area of 540 km2 are Iran's second-largest 114 inland lakes. These lakes are the most important ecological habitats of Iran at an altitude of 1525 115 m above sea level and have a catchment area of 25,000 km2. The maximum depth of Tashk- 116 Bakhtaran lake is 2 m, and the maximum depth of Tashk lake is 3.1 m [66, 67]. These lakes are 117 located between 29° 13'N–29° 48'N and 54° 10'E–53° 23'E. Water inflows to these lakes through 118 the Kor and Syvand rivers. With the construction of three dams in these rivers' upper basin, the 119 inflow of water into these lakes has decreased dramatically, causing a large area to dry out [68]. 120 Fig.1 shows the location of the twin TB lakes in Iran. 121 6 6 122 Fig. 1. A) Geographic location of the study area, B) Landsat 5 TM satellite image of TB lakes in 123 false colour composite (7,4,1). 124 122 122 Fig. 1. A) Geographic location of the study area, B) Landsat 5 TM satellite 123 Fig. 1. A) Geographic location of the study area, B) Landsat 5 TM satellite image of TB lakes in 123 Fig. 1. 1. Introduction 37 A) Geographic location of the study area, B) Landsat 5 TM satellite image of TB lakes in 123 false colour composite (7,4,1). 124 false colour composite (7,4,1). 124 false colour composite (7,4,1). 124 Arid and semi-arid regions cover about one-third of the world's land area.. Population growth in 126 such areas caused an increase in the harvesting of groundwater [69]. In arid regions, lakes and 127 wetlands play an indispensable role in the region's ecosystem, including climate change 128 modification and food resources provision in the area. Due to growing water consumption in arid 129 regions, water resources such as lakes ground water and other aquatic ecosystems are 130 increasingly under stress [68]. 131 Arid and semi-arid regions cover about one-third of the world's land area.. Population growth in 126 such areas caused an increase in the harvesting of groundwater [69]. In arid regions, lakes and 127 wetlands play an indispensable role in the region's ecosystem, including climate change 128 modification and food resources provision in the area. Due to growing water consumption in arid 129 regions, water resources such as lakes ground water and other aquatic ecosystems are 130 increasingly under stress [68]. 131 TB lakes are under threat of complete drought due to over-harvesting of groundwater and 132 mismanagement. In the basin of these lakes, two large rivers, Kor and Sivand, flow. Due to the 133 vast area of TB lakes and moisture and water availability, unique plant and animal habitats exist 134 in the surroundings [70]. In the past, TB lakes had a more fertile environment than today due to 135 proper nutrition. At least 220 species of plaants have been identified in the region's environment 136 TB lakes are under threat of complete drought due to over-harvesting of groundwater and 132 mismanagement. In the basin of these lakes, two large rivers, Kor and Sivand, flow. Due to the 133 vast area of TB lakes and moisture and water availability, unique plant and animal habitats exist 134 in the surroundings [70]. In the past, TB lakes had a more fertile environment than today due to 135 proper nutrition. At least 220 species of plaants have been identified in the region's environment 136 7 7 (the third largest from the species number point of view in Iran). More than 100,000 waterfowl 137 migrate to the region in the winter [71]. 1. Introduction 37 There were about 5,000 Marbled Duck in 1990 [71,72]. 138 Due to the diversity of flora and fauna in the wildlife, a refuge and a national park have been 139 identified as protected areas. Their location is shown in Fig. 2. Three important dams that have 140 been built in the upstream area of TB lakes: Sivand dam, Mollasadra dam and Doroodzan 141 (Dariush) dam. The location of these dams is specified in Fig. 2, and their specifications are 142 shown in Table 1. 143 (the third largest from the species number point of view in Iran). More than 100,000 waterfowl 137 migrate to the region in the winter [71]. There were about 5,000 Marbled Duck in 1990 [71,72]. 138 Due to the diversity of flora and fauna in the wildlife, a refuge and a national park have been 139 identified as protected areas. Their location is shown in Fig. 2. Three important dams that have 140 been built in the upstream area of TB lakes: Sivand dam, Mollasadra dam and Doroodzan 141 (Dariush) dam. The location of these dams is specified in Fig. 2, and their specifications are 142 shown in Table 1. 143 144 145 Fig. 2. TB lakes watershed and location of ecological areas and distribution of dams in the area. 146 145 Fig. 2. TB lakes watershed and location of ecological areas and distribution of dams in the area. 146 Table 1. Characteristics of dams located upstream of TB lakes. 148 Dam Type Year3 Vol.2 (M.m3) H.1(m) River Dam A pebble with an impermeable core 1972 960 85 Kor Doroodzan Reservoir (soil with clay core) 2007 440 75 Kor Mollasadra Soil with clay core 2007 255 57 Sivand Sivand 1. Height; 2. Total tank volume (million cubic meters); 3. Year of operation 8 8 149 In Fig. 3 using MODIS satellite, the land cover changes in 2001 and 2018 are compared. This 150 figure was provided using the MODIS Land Cover Type Product (MCD12Q1) satellite. The 151 MCD12Q1 includes a global dataset of land cover types from 2001 to 2018. Its spatial resolution 152 is 500 meters, and six different classification schemes have been used to produce it. The Global 153 Earth Coverage Map provides ecological and physical characteristics of the Earth's surface. 154 In Fig. 3 using MODIS satellite, the land cover changes in 2001 and 2018 are compared. 1. Introduction 37 171 172 Charts seem to indicate the existence of sudden changes around 2006 and 2007, particularly in 173 the Waterbody area, which has declined since 2007 and reached its lowest surface in 2009. This 174 reduction has had significant effects on other uses in the region. It should be noted that this 175 decrease in water bodies in the catchment area of TB lakes has started since the construction of 176 two dams, Mollasadra dam and Sivand dam, i.e., in 2007, and in 2009. These two dams were 177 constructed on the two main rivers of the region, which feed the TB lakes, and resulted in the 178 reduction of these lakes surfaces. Due to the diversity of flora and fauna in the region and 179 protected areas around the TB lakes, these dams have caused severe damage to these genetic 180 resources and the uses of the region. TB lakes increase the humidity of the air, and due to the 181 Fig. 4. a) Land cover changes in 2001-2018, b) changes in TB lakes area. 171 Charts seem to indicate the existence of sudden changes around 2006 and 2007, particularly in 173 the Waterbody area, which has declined since 2007 and reached its lowest surface in 2009. This 174 reduction has had significant effects on other uses in the region. It should be noted that this 175 decrease in water bodies in the catchment area of TB lakes has started since the construction of 176 two dams, Mollasadra dam and Sivand dam, i.e., in 2007, and in 2009. These two dams were 177 constructed on the two main rivers of the region, which feed the TB lakes, and resulted in the 178 reduction of these lakes surfaces. Due to the diversity of flora and fauna in the region and 179 protected areas around the TB lakes, these dams have caused severe damage to these genetic 180 resources and the uses of the region. TB lakes increase the humidity of the air, and due to the 181 11 Charts seem to indicate the existence of sudden changes around 2006 and 2007, particularly in 173 the Waterbody area, which has declined since 2007 and reached its lowest surface in 2009. This 174 reduction has had significant effects on other uses in the region. 1. Introduction 37 This 150 figure was provided using the MODIS Land Cover Type Product (MCD12Q1) satellite. The 151 MCD12Q1 includes a global dataset of land cover types from 2001 to 2018. Its spatial resolution 152 is 500 meters, and six different classification schemes have been used to produce it. The Global 153 Earth Coverage Map provides ecological and physical characteristics of the Earth's surface. 154 In this study, LC_Type 1 band was employed to prepare a land cover map of the areas around 155 TB lakes. This ground cover is based on the International Geosphere-Biosphere Program (IGBP), 156 which is dedicated to styding global changes. The annual land cover maps around TB lakes were 157 extracted from MCD12Q1 data in 2001 and in 2018 and are presented in Fig. 3. The reduction of 158 agricultural coverage, pastures, and water level of the lake in the catchment area of TB lakes and 159 the increase of barrier surface are clearly visible. 160 In this study, LC_Type 1 band was employed to prepare a land cover map of the areas around 155 TB lakes. This ground cover is based on the International Geosphere-Biosphere Program (IGBP), 156 which is dedicated to styding global changes. The annual land cover maps around TB lakes were 157 extracted from MCD12Q1 data in 2001 and in 2018 and are presented in Fig. 3. The reduction of 158 agricultural coverage, pastures, and water level of the lake in the catchment area of TB lakes and 159 the increase of barrier surface are clearly visible. 160 161 9 162 Fig. 3. Map of land cover changes between 2001 and 2018 in TB lakes watershed. 163 Fig. 3. Map of land cover changes between 2001 and 2018 in TB lakes watershed. 163 Fig. 4 shows the changes in five variables: Open shrublands, Grasslands, Barren, Croplands, and 65 Water Bodies between 2001 and 2018. It can be observed that the area covered by Open 66 Shrublands has been relatively stable until 2007, but since 2007, it has been increasing, while 67 grasslands and croplands have declined with a similar trend. 68 169 10 10 170 Fig. 4. a) Land cover changes in 2001-2018, b) changes in TB lakes area. 171 11 170 Fig. 4. a) Land cover changes in 2001-2018, b) changes in TB lakes area. 1. Introduction 37 197 Considering all this background information, the question is raised on how long will the drought 191 process of TB lakes continue, and what will be the changes in their surface in the coming years? 192 To answer this question, we adopt the SARIMA-Long-Short-Term Memory Model to model the 193 lake's surface changes and provide a practical model for future changes in the lake's surface. 194 Hence, using this model, an applied plan for water resources management in a variety of uses in 195 the region can be developed, reducing the water crisis in the region and the abandonment of 196 agricultural land, which has severe environmental and economic consequences in the region. 197 1. Introduction 37 It should be noted that this 175 decrease in water bodies in the catchment area of TB lakes has started since the construction of 176 two dams, Mollasadra dam and Sivand dam, i.e., in 2007, and in 2009. These two dams were 177 constructed on the two main rivers of the region, which feed the TB lakes, and resulted in the 178 reduction of these lakes surfaces. Due to the diversity of flora and fauna in the region and 179 protected areas around the TB lakes, these dams have caused severe damage to these genetic 180 resources and the uses of the region. TB lakes increase the humidity of the air, and due to the 181 11 high altitude of the surrounding mountains, the resulting moisture remains in the atmosphere of 182 the same area. This is referred to as artificial irrigation and causes better fruiting of the plants in 183 this area. 184 high altitude of the surrounding mountains, the resulting moisture remains in the atmosphere of 182 the same area. This is referred to as artificial irrigation and causes better fruiting of the plants in 183 this area. 184 The drought that has been observed in recent years and the significant reduction of TB lakes’ 185 water have affected the region's uses and caused a water crisis in the region. Croplands and 186 grasslands have shown a significant decline, with their area shrinking to less than half its original 187 value. Simultaneously, Shrublands and Barren soils increased, resulting in falling water levels in 188 the region and the release of agricultural land and land-use change due to the lack of water in the 189 area. 190 Considering all this background information, the question is raised on how long will the drought 191 process of TB lakes continue, and what will be the changes in their surface in the coming years? 192 To answer this question, we adopt the SARIMA-Long-Short-Term Memory Model to model the 193 lake's surface changes and provide a practical model for future changes in the lake's surface. 194 Hence, using this model, an applied plan for water resources management in a variety of uses in 195 the region can be developed, reducing the water crisis in the region and the abandonment of 196 agricultural land, which has severe environmental and economic consequences in the region. 2.2. Remote sensing (RS) datasets and pre-processing 198 The MODIS (Moderate Resolution Imaging Spectroradiometer) tools were launched by Terra 199 and Aqua satellites in 1999 and 2002. The MODIS sensor captures images 2230 kilometres wide 200 and generates complete coverage of the earth in 1-2 days. By using Surface Reflectance products 201 and their various bands (MOD09A1), the spectral reflectance of Earth's surface is estimated. 202 12 12 Pre-processing is a vital part of the remote sensing process. One of the problems with remote 203 sensing images is the presence of clouds. Therefore, tools and indices like Google Earth Engine 204 Environment (GEE) for image classification and the NDWI index are required to obtain 205 desirable results. The NDWI index is one of the most commonly used indicators in remote 206 sensing and is calculated from the relationships between bands (equations 1 and 2). Bands are 207 used to obtain the water in which wavelengths have the highest and lowest spectral reflections. 208 The NDWI relationship is computed as follows [73]: 209 G NIR NDWI G NIR    (1) 210 (1) where the G is the green band, and the NIR is the near-infrared band. The modified NDWI 211 where the G is the green band, and the NIR is the near-infrared band. The modified NDWI 211 relationship is as follows [12]: 212 relationship is as follows [12]: 212 G MIR MNDWI G MIR    (2) 213 (2) (2) where MIR is the mid-infrared band (wavelengths 1.2 to 2.2 µm). 214 where MIR is the mid-infrared band (wavelengths 1.2 to 2.2 µm). 214 where MIR is the mid-infrared band (wavelengths 1.2 to 2.2 µm). 214 The resulting image of the MNDWI index has values between -1 and +1. The pixels that indicate 215 the presence of water have positive values. However, due to the presence of mixed pixels that 216 cause errors in the detection of water sources, a threshold limit (MNDWI ≥ 0.3) is used to detect 217 pure pixels with more precision [74,75]. Then, to calculate the area of water bodies in the 218 The resulting image of the MNDWI index has values between -1 and +1. The pixels that indicate 215 the presence of water have positive values. 2.2. Remote sensing (RS) datasets and pre-processing 198 However, due to the presence of mixed pixels that 216 cause errors in the detection of water sources, a threshold limit (MNDWI ≥ 0.3) is used to detect 217 pure pixels with more precision [74,75]. Then, to calculate the area of water bodies in the 218 images, the number of pure pixels identified in each image is multiplied by the area of land cover 219 and the exact area of the water surface can be calculated. 220 images, the number of pure pixels identified in each image is multiplied by the area of land cover 219 and the exact area of the water surface can be calculated. 220 13 13 A series of measurements in equal time intervals is termed time series. Each time series has a 222 stochastic and a deterministic part. Periodical patterns, trends and jumps are the deterministic 223 part and can exist in time series simultaneously or solely. The absence of this part in time series 224 is called stationarity state. For any modeling, the deterministic terms can be removed, and only 225 the stochastic part is required. Therefore, analysis methods are needed to assess the predictable 226 pattern in time series and stationarity [76]. Applying tests to time series to extract interpretable 227 statistics is the analysis of time series. Tests like KPSS, Mann-Whitney, Mann-Kendal, and 228 Jarque-Berra can be employed to investigate stationarity, jump, trends and normality of time 229 series, respectively. 230 A series of measurements in equal time intervals is termed time series. Each time series has a 222 stochastic and a deterministic part. Periodical patterns, trends and jumps are the deterministic 223 part and can exist in time series simultaneously or solely. The absence of this part in time series 224 is called stationarity state. For any modeling, the deterministic terms can be removed, and only 225 the stochastic part is required. Therefore, analysis methods are needed to assess the predictable 226 pattern in time series and stationarity [76]. Applying tests to time series to extract interpretable 227 statistics is the analysis of time series. Tests like KPSS, Mann-Whitney, Mann-Kendal, and 228 Jarque-Berra can be employed to investigate stationarity, jump, trends and normality of time 229 series, respectively. 230 In the KPSS [77] test, a regression equation is fitted to the data. 2.2. Remote sensing (RS) datasets and pre-processing 198 If the variance of the 231 independent variables of the relationship is null the AL, then the series is stationary. The KPSS 232 relationship for trend or level stationarity is as follows: 233 In the KPSS [77] test, a regression equation is fitted to the data. If the variance of the 231 independent variables of the relationship is null the AL, then the series is stationary. The KPSS 232 relationship for trend or level stationarity is as follows: 233 L t t t A = r + +   (3) 234 L t t t A = r + +   (3) 234 (3)   n l n 2 2 l t l t t s t 1 j 1 t j 1 1 2 1 S (t ) e w j,t e e n n n          (4) 235     l l w s,t 1 j/ t 1   (5) 236   n l n 2 2 l t l t t s t 1 j 1 t j 1 1 2 1 S (t ) e w j,t e e n n n          (4) 235     l l w s,t 1 j/ t 1   (5) 236  2 N t 2 2 t 1 l S 1 KPSS n S t            (6) 237   n l n 2 2 l t l t t s t 1 j 1 t j 1 1 2 1 S (t ) e w j,t e e n n n          235 (4) (5)  2 N t 2 2 t 1 l S 1 KPSS n S t            (6) 237 (6) where St = Σ te , lt is the truncation lag, te are the residuals. t t-1 i r r u   and tr is a random walk, 238 where St = Σ te , lt is the truncation lag, te are the residuals. 2.2. Remote sensing (RS) datasets and pre-processing 198 The MT and var (MT) are defined as: 245   N 1 N T L,j L,i i 1 j i 1 M sgn A A       (8) 246   L,j L,i 1 sgn A A    (8)   N 1 N T L,j L,i i 1 j i 1 M sgn A A       (8) 246 (8) i 1 j i 1          g 3 2 T L,j L, j L, j j var M 2N 7N 5N A A 1 2L 5 /18              (9) 247        g 3 2 T L,j L, j L, j j var M 2N 7N 5N A A 1 2L 5 /18              (9) 247        g 3 2 T L,j L, j L, j j var M 2N 7N 5N A A 1 2L 5 /18              (9) 247 (9) where L, j A and L,i A are the lake area time series at the jth and ith group, g is the number of 248 where L, j A and L,i A are the lake area time series at the jth and ith group, g is the number of 248 where L, j A and L,i A are the lake area time series at the jth and ith group, g is the number of 248 identical groups, sgn is the sign function, N is the number of samples and L, j L is the number of 249 the observations at the jth group. 2.2. Remote sensing (RS) datasets and pre-processing 198 t t-1 i r r u   and tr is a random walk, 238 where St = Σ te , lt is the truncation lag, te are the residuals. t t-1 i r r u   and tr is a random walk, 238 iu are independent variables with equal distribution with mean zero and variance σ2, t is the 239 iu are independent variables with equal distribution with mean zero and variance σ2, t is the 239 deterministic term of the trend, and t the stationarity error. 240 deterministic term of the trend, and t the stationarity error. 240 14 In the case of non-stationarity, causing factors are investigated. Trend as a non-stationarity factor 241 In the case of non-stationarity, causing factors are investigated. Trend as a non-stationarity factor 241 is analyzed by the Mann-Kendal test as follows [78]: 242 is analyzed by the Mann-Kendal test as follows [78]: 242 is analyzed by the Mann-Kendal test as follows [78]: 242         0.5 0.5 1 var MK 0 stnd( ) 0 MK 0 1 var MK 0                    (7) 243 (7) (7) where stnd (MT) is the standard of Mann-Kendall statistic, MK is the Man-Kendall statistic, and 244 var (MT) is the variance of MT. 2.2. Remote sensing (RS) datasets and pre-processing 198 The non- 259 parametric Mann-Whitney (MW) test is used to evaluate this factor [79, 80]: 260           1 N 0.5 m1 m1 m2 L, Ordered m1 m2 m1 m2 t 1 N N N 1 MW Dg A / N N N N 1 /12 2               (14) 261           1 N 0.5 m1 m1 m2 L, Ordered m1 m2 m1 m2 t 1 N N N 1 MW Dg A / N N N N 1 /12 2               (14) 261 (14) where AL, Ordered: series sorted by main series AL, Dg (AL, Ordered) the degree of AL, Ordered function, 262 where AL, Ordered: series sorted by main series AL, Dg (AL, Ordered) the degree of AL, Ordered function, 262 Nm1 and Nm2 is the number of members of the main sub-series that Nm1 + Nm2 = Ntotal. A 263 probability related to a test statistic greater than 1% means that AL is jump-less. 264 Periodicity as the third deterministic factor can be surveyed by a time series graph or the auto 265 correlation function (ACF) and the partial auto correlation function (PACF) plots. This term 266 appears as iterative sinusoidal variations in both above graphs. 267 Seasonal standardization is one of the conventional stationarizing methods in hydrology. This 268 method also reduces jumps in time series [81]. By removing the seasonal mean and standard 269 deviation, the AL is transferred to a time series with a zero mean and a standard deviation equal 270 to one as follows: 271 Seasonal standardization is one of the conventional stationarizing methods in hydrology. This 268 method also reduces jumps in time series [81]. By removing the seasonal mean and standard 269 deviation, the AL is transferred to a time series with a zero mean and a standard deviation equal 270 to one as follows: 271 Seasonal standardization is one of the conventional stationarizing methods in hydrology. This 268 method also reduces jumps in time series [81]. 2.2. Remote sensing (RS) datasets and pre-processing 198 The following equation is used for seasonal changes over time, 250 or seasonal trend: 251 identical groups, sgn is the sign function, N is the number of samples and L 249   k k N 1 N k L, kj L, ki i 1 j i 1 S sgn A A       252 (10)     k ω S k k k 1 M S sgn S     (11) 253     k ω S k k k 1 M S sgn S     (11) 253 (11)     k 1 3 2 S ij k k k i 1 j i 1 k var M 2 2N 7N 5N /18            (12) 254 (12) 15 15   k k k 0.5 S S S stnd(M ) M var M   255 (13) 255 where ω represents the seasons, k is the number of months, and σij is the covariance of stationary 256 test in seasons i and j. A probability corresponding to a test statistic higher than 5% means that 257 ALis trendless. 258 where ω represents the seasons, k is the number of months, and σij is the covariance of stationary 256 test in seasons i and j. A probability corresponding to a test statistic higher than 5% means that 257 ALis trendless. 258 where ω represents the seasons, k is the number of months, and σij is the covariance of stationary 256 test in seasons i and j. A probability corresponding to a test statistic higher than 5% means that 257 ALis trendless. 258 Jumps, the second non-stationarity factor, represent sudden steps in the time series. The non- 259 parametric Mann-Whitney (MW) test is used to evaluate this factor [79, 80]: 260 Jumps, the second non-stationarity factor, represent sudden steps in the time series. 2.2. Remote sensing (RS) datasets and pre-processing 198 By removing the seasonal mean and standard 269 deviation, the AL is transferred to a time series with a zero mean and a standard deviation equal 270 to one as follows: 271       L L d std A t, A / S     272 (15) 16 where, stdω represents the outcome of seasonal standardization,   L A t, is the sample at tth 273 year and the ωth season,  L A  is the mean of the ωth season and  d S  is the standard 274 deviation of ωth season. 275 where, stdω represents the outcome of seasonal standardization,   L A t, is the sample at tth 273 year and the ωth season,  L A  is the mean of the ωth season and  d S  is the standard 274 deviation of ωth season. 275 2.4. Long-Short-Term Memory (LSTM) deep learning model 276 Deep learning models are subclasses of artificial intelligence (AI) models enhanced for non- 277 linear sequence solving problems. A renowned deep learning model is the Long Short-Term 278 Memory (LSTM) network. The LSTM architecture is well suited for modelling sequence data 279 like time series and can learn long-term dependencies in series to forecast future steps. A simple 280 LSTM memory block is presented in Fig. 5. The LSTM model is constituted of several gates that 281 control the flow of information and affect the produced results. These gates are the input, the 282 forget, and the output gates which control the data entering to memory blocks tc , which should 283 be forgotten, and which are permitted to continue to further processes. 284 Deep learning models are subclasses of artificial intelligence (AI) models enhanced for non- 277 linear sequence solving problems. A renowned deep learning model is the Long Short-Term 278 Memory (LSTM) network. The LSTM architecture is well suited for modelling sequence data 279 like time series and can learn long-term dependencies in series to forecast future steps. A simple 280 LSTM memory block is presented in Fig. 5. The LSTM model is constituted of several gates that 281 control the flow of information and affect the produced results. These gates are the input, the 282 forget, and the output gates which control the data entering to memory blocks tc , which should 283 be forgotten, and which are permitted to continue to further processes. 2.4. Long-Short-Term Memory (LSTM) deep learning model 276 284 LSTM conducts a mapping [43] from an input sequence x to an output sequence y using the next 285 equations iteratively from t = 1 to t = τ with initial values C0 = 0 and h0 = 0: 286 LSTM conducts a mapping [43] from an input sequence x to an output sequence y using the next 285 equations iteratively from t = 1 to t = τ with initial values C0 = 0 and h0 = 0: 286 L t f f ,t t 1 f f ) A (W U b h     (16) 287 t t t L, t C C C t t 1 C (W U b ) tanh A h     % % % % (17) 288 L t f f ,t t 1 f f ) A (W U b h     (16) 287 (16) (17) where L,t A is the input of the vector at time t, and t 1 h is the hidden cell state at time t−1. The 289 weight matrices are U, W for input-to-hidden, and hidden-to-hidden connections, respectively. ft is 290 a resulting vector with values in the range (0, 1), σ(·) represents the logistic sigmoid function and 291 f W , f U and bf define the set of learnable parameters for the forget gate. Ct % is an update vector 292 where L,t A is the input of the vector at time t, and t 1 h is the hidden cell state at time t−1. The 289 weight matrices are U, W for input-to-hidden, and hidden-to-hidden connections, respectively. ft is 290 a resulting vector with values in the range (0, 1), σ(·) represents the logistic sigmoid function and 291 f W , f U and bf define the set of learnable parameters for the forget gate. Ct % is an update vector 292 where L,t A is the input of the vector at time t, and t 1 h is the hidden cell state at time t−1. The 289 17 with (-1, 1) range for the cell state which calculated form AL, t, tanh (*) is the hyperbolic tangent 293 and t C W% , t C U% and t C b % are other sets of learnable parameters. 2.4. Long-Short-Term Memory (LSTM) deep learning model 276 294 t t i t i i i (W x U b ) h    (18) 295 t t i t i i i (W x U b ) h    (18) 295 i t i U b ) h   (18) (18) i t i U b ) h   (18) ti is the forget gate with range (0,1). i W , i U and bi are a set of learnable parameters, defined for 296 ti is the forget gate with range (0,1). i W , i U and bi are a set of learnable parameters, defined for 296 the input gate. The results of Eqs. 16 to 18 lead to update the cell state: 297 t t t t 1 t c f Oc t OC    % (19) 298 t t t t 1 t c f Oc t OC    % (19) where O denotes element-wise multiplication. The output gate, as the last gate, controls the cell 299 state ct. 300 t o o t 1 o t o W x U h b ) (      (20) 301 t o o t 1 o t o W x U h b ) (      (20) 301 t o o t 1 o t o W x U h b ) (      ( t o o t 1 o t o W x U h b ) (      (20) 301 (20) where to is in the range (0, 1) and o W , o U and o b are a set of learnable parameters, defined for 302 where to is in the range (0, 1) and o W , o U and o b are a set of learnable parameters, defined for 302 the output gate h is calculated as follows: 303 where to is in the range (0, 1) and o W , o U and o b are a set of learnable parameters, defined for 302 the output gate. th is calculated as follows: 303 the output gate. th is calculated as follows: 303 the output gate. th is calculated as follows: 303 (21) 305 18 18 306 Fig. 5. A simple LSTM block. 307 Fig. 5. A simple LSTM block. 307 2.5. Stochastic modelling concepts 309 (1- L) d 320 equals the d-th non-seasonal, and (1- L ω) D equals the D-th seasonal with the lag ω. The L 321 operator helps in modelling the non-stationary series as it removes correlations in time series and 322 changes in mean and variance of the series. To improve the model's accuracy, each forecast is 323 updated with real data, and a 1-step-ahead forecast is carried out. As this model is linear, 324 deterministic terms must be extracted from the series, and data distribution normalized to 325 improve accuracy. To evaluate the distribution's normality, the Jarque-Bera test can be applied to 326 AL time series [84]: 327 where ω is seasonality,  and  are auto-regressive (AR) and seasonal AR (SAR) parameters, θ 319 and Θ are the moving average (MA), L is the differencing operator L (AL(t)) = AL (t-1). (1- L) d 320 equals the d-th non-seasonal, and (1- L ω) D equals the D-th seasonal with the lag ω. The L 321 operator helps in modelling the non-stationary series as it removes correlations in time series and 322 changes in mean and variance of the series. To improve the model's accuracy, each forecast is 323 updated with real data, and a 1-step-ahead forecast is carried out. As this model is linear, 324 deterministic terms must be extracted from the series, and data distribution normalized to 325 improve accuracy. To evaluate the distribution's normality, the Jarque-Bera test can be applied to 326 AL time series [84]: 327 deterministic terms must be extracted from the series, and data distribution normalized to 325 improve accuracy. To evaluate the distribution's normality, the Jarque-Bera test can be applied to 326 AL time series [84]: 327 improve accuracy. 2.5. Stochastic modelling concepts 309 Stochastic models are a subgroup of statistical models. These models are widely used in various 310 fields of science beause of their simplicity of utilization and theory. Seasonal Auto-Regressive 311 Integrated Moving Average (SARIMA) is a stochastic model with seasonal and non-seasonal 312 parameters that allows the model to forecast the future by using historical data [82]. 313 In a SARIMA (p, d, q) (P, D, Q) model, p and q are non-seasonal model parameters; P and Q are 314 seasonal ones. d and D are the order of non-seasonal and seasonal differencing, respectively [83]. 315 The simplified extension of the SARIMA equation for one step ahead forecast is as follows: 316     2 d 1 2 p 1 2 P D km 1 2 p 1 2 P L 1 2 q 1 2 2Q 1 2 q 1 2 Q 1 L L ... L 1 L L L 1 L 1 L A t 1 L L L 1 L L L e t ( ) ( ) ( ) ... ( ) ( )                      (22) 317     2 d 1 2 p 1 2 P D km 1 2 p 1 2 P L 1 2 q 1 2 2Q 1 2 q 1 2 Q 1 L L ... L 1 L L L 1 L 1 L A t 1 L L L 1 L L L e t ( ) ( ) ( ) ... ( ) ( )                      (22) 317 (22) (22)     d D L (B) (B 1 L 1 L A t (B (B e t ) ( ) ) )        318     d D L (B) (B 1 L 1 L A t (B (B e t ) ( ) ) )        318 19 where ω is seasonality,  and  are auto-regressive (AR) and seasonal AR (SAR) parameters, θ 319 and Θ are the moving average (MA), L is the differencing operator L (AL(t)) = AL (t-1). 2.5. Stochastic modelling concepts 309 To evaluate the distribution's normality, the Jarque-Bera test can be applied to 326 AL time series [84]: 327     2 2 k u JB n S / 6 K -3 / 24   (23) 328     2 2 k u JB n S / 6 K -3 / 24   328 (23) where u K is kurtosis k S is skewness; JB is a chi-square distribution with two degrees of 329 ere u K is kurtosis k S is skewness; JB is a chi-square distribution with two degrees where u K is kurtosis k S is skewness; JB is a chi-square distribution with two degrees of 329 freedom that can be used to assume that data is normal. As most of the hydrological time series 330 are non-normal, normalizing transformation should be employed. John-Draper transform is a 331 normalization approach that can transform AL data. The equation is as follows: 332         L L Ln L L A 1 1 sgn A 0 A sgn A log A 1 0             (24) 333 (24)   L L L 1 A 0 sgn A 1 A 0      (25) 334 (25) λ is JD transforming parameters and ALn is the normalized AL series. 335 λ is JD transforming parameters and ALn is the normalized AL series. 335 336 20 2.6. Comparison measures 337 Correlation coefficient (R), Root mean squared error (RMSE), root mean squared relative error 338 (RMSRE), Mean absolute percentage error (MAPE) and Mean absolute error (MAE) are used to 339 evaluate the accuracy of models in time series obtained from pre-processing of ALdata. To 340 compare the stochastic models, corrected Akaike's Information Criterion (AICc) is used. Theil's 341 U coefficients are also used [85–87]. The Theil's U indices compare models based on the 342 simplicity of the model against goodness-of-fit. The lower the index, the better the model results 343 are. 344 simplicity of the model against goodness-of-fit. The lower the index, the better the model results 343 are. 2.6. Comparison measures 337 344 (26)   N 2 2 L, O,i L, P, i i 1 RMSE ( A A ) / N     346 (27) N L,O,i L,P,i i 1 L,O,i A A 100 MAPE N A             347 (28)   N L,O,i L,P,i i 1 1 MAE A A N     348 (29) 2 N L,O, i L, P,i i 1 L,O, i A A 1 RMSRE N A             349 (30)   2 2kn nln( )(n k 1) AICc n k 1        350 (31) 21       0.5 N 2 L, O,i L, P,i i 1 I 0.5 0.5 N n 2 2 L,O,i L, P,i i 1 i 1 A A U A A                            (32) 351   0.5 N 2 L, O,i L, P,i i 1 II 0.5 N 2 L, O,i i 1 A A U A                   (33) 352       0.5 N 2 L, O,i L, P,i i 1 I 0.5 0.5 N n 2 2 L,O,i L, P,i i 1 i 1 A A U A A                            351   0.5 N 2 L, O,i L, P,i i 1 II 0.5 N 2 L, O,i i 1 A A U A                   352 (32) (33) L, O,i A and L, P,i A are the ith value of observed data and predicted AL respectively. N is the number 353 of months,  is the residual's standard deviation, and k is the number of tuned parameters 354 through the modelling process. I U is the accuracy of forecasting, and II U is the forecasting 355 quality. 2.6. Comparison measures 337 Checking the stochastic models' residuals for correlations and white noise state is one of 356 the stochastic modelling steps. For this purpose, the Ljung-Box test can be applied to model 357 residuals as follows [88]: 358 L, O,i A and L, P,i A are the ith value of observed data and predicted AL respectively. N is the number 353 L, O,i A and L, P,i A are the ith value of observed data and predicted AL respectively. N is the number 353 of months,  is the residual's standard deviation, and k is the number of tuned parameters 354 through the modelling process. I U is the accuracy of forecasting, and II U is the forecasting 355 L, O,i L, P,i L y of months,  is the residual's standard deviation, and k is the number of tuned parameters 354 of months,  is the residual's standard deviation, and k is the number of tun 354 of months,  is the residual's standard deviation, and k is the number of tuned parameters 354 through the modelling process. I U is the accuracy of forecasting, and II U is the forecasting 355 quality. Checking the stochastic models' residuals for correlations and white noise state is one of 356 the stochastic modelling steps. For this purpose, the Ljung-Box test can be applied to model 357 residuals as follows [88]: 358   m 2 h h 1 r lbq N 2N N 1      (34) 359   m 2 h h 1 r lbq N 2N N 1      359   m 2 h h 1 r lbq N 2N N 1      359 (34) N is the number of samples, hr is the residual coefficient of the autoregression (εt) in delay h; the 360 value of m is also equal to ln(N). If the probability related to the Ljung-Box test is greater than 361 the α-level (in this case Plbq > α = 0.05), the residues series is white noise. 362 In this research, first in the Google Earth Engine environment, the data were selected, and the 363 necessary pre-processing was performed. MODIS MOD09A1 was used to measure the changes 364 in the area of TB lakes. 2.6. Comparison measures 337 Images with a cloud coverage of less than 10% were selected to continue 365 the process, and then the pixel value was corrected. Due to the area's characteristics, a threshold 366 In this research, first in the Google Earth Engine environment, the data were selected, and the 363 necessary pre-processing was performed. MODIS MOD09A1 was used to measure the changes 364 in the area of TB lakes. Images with a cloud coverage of less than 10% were selected to continue 365 the process, and then the pixel value was corrected. Due to the area's characteristics, a threshold 366 22 for water identification was considered, and with the MNDWI index, water bodies were 367 separated from other zones. Higher threshold (MNDWI ≥0.3) was identified as water bodies. The 368 time series of changes in the extent of the lakes was calculated from 2001 to 2019. Land cover 369 changes were extracted from MODIS MCD12Q1, and the land cover map was prepared. To 370 determine land use, the land cover map was used to identify the changes in the area and their 371 impact on the changes in the lake surface. Then the time series of the WSA data was extracted 372 from the satellite data. Following, the modelling procedure was undertaken. 373 Initially, the WSA time series' structural characteristics were investigated by pre-processed by 374 stationarity and normality tests. If any pre-processing is needed, a standardization and/or 375 normalization to series is carried out to obtain the optimized modelling results. Then deep 376 learning LSTM model, stochastic SARIMA and hybridization SARIMA-LSTM are performed.. 377 The described procedure is depicted in the flowchart of Fig. 6. 378 for water identification was considered, and with the MNDWI index, water bodies were 367 separated from other zones. Higher threshold (MNDWI ≥0.3) was identified as water bodies. The 368 time series of changes in the extent of the lakes was calculated from 2001 to 2019. Land cover 369 changes were extracted from MODIS MCD12Q1, and the land cover map was prepared. To 370 determine land use, the land cover map was used to identify the changes in the area and their 371 impact on the changes in the lake surface. Then the time series of the WSA data was extracted 372 from the satellite data. Following, the modelling procedure was undertaken. 2.6. Comparison measures 337 373 for water identification was considered, and with the MNDWI index, water bodies were 367 separated from other zones. Higher threshold (MNDWI ≥0.3) was identified as water bodies. The 368 time series of changes in the extent of the lakes was calculated from 2001 to 2019. Land cover 369 changes were extracted from MODIS MCD12Q1, and the land cover map was prepared. To 370 determine land use, the land cover map was used to identify the changes in the area and their 371 impact on the changes in the lake surface. Then the time series of the WSA data was extracted 372 from the satellite data. Following, the modelling procedure was undertaken. 373 Initially, the WSA time series' structural characteristics were investigated by pre-processed by 374 stationarity and normality tests. If any pre-processing is needed, a standardization and/or 375 normalization to series is carried out to obtain the optimized modelling results. Then deep 376 learning LSTM model, stochastic SARIMA and hybridization SARIMA-LSTM are performed.. 377 The described procedure is depicted in the flowchart of Fig. 6. 378 23 Fig. 6. Flowchart of the analytical procedures of the study. 379 Fig. 6. Flowchart of the analytical procedures of the study. 380 24 Fig. 6. Flowchart of the analytical procedures of the study. 380 381 3. Results and discussion 382 3.1. RS results 383 24 24 In this study, MODIS data, MOD09A1 version 6 Surface Reflectance (with a resolution of 500m 384 and 8-day from 2000 to 2019) were employed to obtain time-series variations of TB lakes water 385 surface. The MOD09 series is one of the MODIS surface reflection products. This product has 386 seven bands and estimates the spectral reflectance values for each band in the absence of 387 atmospheric absorption or diffusion. 388 In this study, MODIS data, MOD09A1 version 6 Surface Reflectance (with a resolution of 500m 384 and 8-day from 2000 to 2019) were employed to obtain time-series variations of TB lakes water 385 surface. The MOD09 series is one of the MODIS surface reflection products. This product has 386 seven bands and estimates the spectral reflectance values for each band in the absence of 387 atmospheric absorption or diffusion. 388 Table 2 Specifications of MOD09A1 version 6 Band name Band desc. wavelength(nm) Spatial resolution (m) sur_refl_b01 S.R. Band 1 620-670 500 sur_refl_b02 S.R. Band 2 841-876 500 sur_refl_b03 S.R. Band 3 459-479 500 sur_refl_b04 S.R. 2.6. Comparison measures 337 Band 4 545-565 500 sur_refl_b05 S.R. Band 5 1230-1250 500 sur_refl_b06 S.R. Band 6 1628-1652 500 sur_refl_b07 S.R. Band 7 2105-2155 500 Band desc.: Band description; S.R. : Surface Reflectance 390 The necessary pre-processing, including atmospheric corrections, have been made to this 391 product. The workflow for extracting the lake area from the MODIS images includes image 392 preparation, image classification and statistical computation. During the preparation of the 393 images, the location of the lakes was determined. So, at this point in the GEE Environment, 394 images with more than 10% cloud were excluded from the lake extraction process. Images with 395 cloud cover less than 10% were selected, and pixels suitable for classification were identified. 396 The image classification step was also performed in the GEE environment. Fig. 7 illustrates the 397 changes of AL from 2001 to 2019 for April Month. 398 The necessary pre-processing, including atmospheric corrections, have been made to this 391 product. The workflow for extracting the lake area from the MODIS images includes image 392 preparation, image classification and statistical computation. During the preparation of the 393 images, the location of the lakes was determined. So, at this point in the GEE Environment, 394 images with more than 10% cloud were excluded from the lake extraction process. Images with 395 cloud cover less than 10% were selected, and pixels suitable for classification were identified. 396 The image classification step was also performed in the GEE environment. Fig. 7 illustrates the 397 changes of AL from 2001 to 2019 for April Month. 398 The image classification step was also performed in the GEE environment. Fig. 7 illustrates the 397 changes of AL from 2001 to 2019 for April Month. 398 399 25 25 25 Fig. 7. Changes of AL from 2001 to 2019 for April Month. Fig. 7. Changes of AL from 2001 to 2019 for April Month. 401 By using a function, the MNDWI index was applied to the previous step images. Water has high 403 reflectance at the wavelength of 0.5 μm (green band) and absorbs electromagnetic waves at 404 infrared wavelengths and has low reflectance. Therefore, in this study, band 4 (green band) and 405 band 7 (mid-infrared) of MODIS images were used. After applying the threshold limit, the exact 406 By using a function, the MNDWI index was applied to the previous step images. By using a function, the MNDWI index was applied to the previous step images. Water has high 403 reflectance at the wavelength of 0.5 μm (green band) and absorbs electromagnetic waves at 404 infrared wavelengths and has low reflectance. Therefore, in this study, band 4 (green band) and 405 band 7 (mid-infrared) of MODIS images were used. After applying the threshold limit, the exact 406 2.6. Comparison measures 337 Water has high 403 reflectance at the wavelength of 0.5 μm (green band) and absorbs electromagnetic waves at 404 infrared wavelengths and has low reflectance. Therefore, in this study, band 4 (green band) and 405 band 7 (mid-infrared) of MODIS images were used. After applying the threshold limit, the exact 406 26 area of the water surface was obtained. For better change recognition in the lake surface area, the 407 area has been separated from the surrounding environment, and the changes in the TB lakes 408 based on this model are shown in Fig. 8. Based on the calculated areas, the monthly time series 409 of the TB lakes area was achieved. 410 1 Fig. 8. Lake Surface changes per square kilometres from 2001 to 2019 based on MODIS satellite 2 imagery. 3 411 Fig. 8. Lake Surface changes per square kilometres from 2001 to 2019 based on MODIS satellite 412 imagery. 413 27 The results obtained from the annual changes in surface area of TB Lakes are shown in Fig. 9. 414 Surface area changes have decreased dramatically from 2001 to 2019, reaching 709.487 km2 in 415 2001. In 2002, the AL reached 975.64 km2, which shows a 37% increase compared to 2001. In 416 2003, the lake's surface reached 821.55, and in 2004 and 2005, its value reached the highest level 417 among the study years, occupying 1038.47 km2 and 1088.07 km2, respectively. After that, with a 418 steep slope, the lake's surface shows a decrease until 2010 and this year it has reached 481.1 km2. 419 This indicates that between 2005 and 2010, the average level of lake decline was 11.16% per 420 year. In 2011, there was an increase of 74.74 km2 in the lake's water level and it fluctuated in the 421 same range until 2013, and in 2014, it decreased by 132.192 km2 compared to 2013, reaching 422 425,238 km2. With an increase and cache, it reached 389.245 km2 in 2016, which is the lowest 423 number of observations among the study years. In 2017, the AL shows an increase of 34.26%, 424 and in 2018 and 2019, it has reached 379,158 and 480,937 km2, respectively. 425 426 Fig. 9. Annual changes in the surface area of TB lakes (2000-2019) 427 Fig. 9. Annual changes in the surface area of TB lakes (2000-2019) 427 Fig. 9. 2.6. Comparison measures 337 Annual changes in the surface area of TB lakes (2000-2019) 427 28 28 Differences in the AL between the study years confirm the information provided in the case study 428 and can be considered as the main factor in reducing the water level of TB Lakes and changes in 429 the region's ecosystem. Therefore, it is necessary to provide practical and correct solutions in the 430 region to control the ecosystem and prevent further destruction of water resources in the region. 431 Using applied models, the water level of TB Lakes can be modeled for better management in the 432 future. 433 434 3.2. Obtained AL time series attributes and pre-processing 435 The obtained AL time-series statistical characteristics were investigated and the results are 436 presented in Fig. 10. To survey the characteristics of the series and model it, the AL series is 437 divided into train and test parts with 70-30% ratio. From the 224 obtained data points, 157 (from 438 Dec 2000 to Jul Dec 2013) and 67 (from Jan 2014 to Jul 2019) were considered as train and test 439 parts, respectively (Fig. 10a). Regarding the information provided in Table 3 the statistical 440 features of the intervals differ considerably, which can lead to poor modelling results. 441 Differences in the AL between the study years confirm the information provided in the case study 428 and can be considered as the main factor in reducing the water level of TB Lakes and changes in 429 the region's ecosystem. Therefore, it is necessary to provide practical and correct solutions in the 430 region to control the ecosystem and prevent further destruction of water resources in the region. 431 Using applied models, the water level of TB Lakes can be modeled for better management in the 432 future. 433 Using applied models, the water level of TB Lakes can be modeled for better management in the 432 future. 433 3.2. Obtained AL time series attributes and pre-processing 435 The obtained AL time-series statistical characteristics were investigated and the results are 436 presented in Fig. 10. To survey the characteristics of the series and model it, the AL series is 437 divided into train and test parts with 70-30% ratio. From the 224 obtained data points, 157 (from 438 Dec 2000 to Jul Dec 2013) and 67 (from Jan 2014 to Jul 2019) were considered as train and test 439 parts, respectively (Fig. 10a). Regarding the information provided in Table 3 the statistical 440 features of the intervals differ considerably, which can lead to poor modelling results. 441 The obtained AL time-series statistical characteristics were investigated and the results are 436 presented in Fig. 10. To survey the characteristics of the series and model it, the AL series is 437 divided into train and test parts with 70-30% ratio. From the 224 obtained data points, 157 (from 438 Dec 2000 to Jul Dec 2013) and 67 (from Jan 2014 to Jul 2019) were considered as train and test 439 parts, respectively (Fig. 10a). Regarding the information provided in Table 3 the statistical 440 features of the intervals differ considerably, which can lead to poor modelling results. 441 442 29 443 Fig 10 (a) A time series plot and (b) pre-processed data 444 443 443 Fig. 10. (a) AL time series plot and (b) pre-processed data. 444 Fig. 10. (a) AL time series plot and (b) pre-processed data. 444 According to the information provided in Table 3, the highest AL lakes is 1292.32 km2 which is 445 related to Jan 2005 and the lowest value is related to 246.4 which is related to Jul 2018. The 446 minimum values for train and test data are 342.52 km2 and 246.4 km2, respectively, and the 447 maximum values for these two are 1292.32 km2 and 733.39 km2. The average value obtained for 448 224 data is 662.81 km2 and in the train and test stage it is 757.44 and 441.08 km2, respectively, 449 and all data have positive skewness. 450 According to the information provided in Table 3, the highest AL lakes is 1292.32 km2 which is 445 related to Jan 2005 and the lowest value is related to 246.4 which is related to Jul 2018. 3.2. Obtained AL time series attributes and pre-processing 435 The 446 minimum values for train and test data are 342.52 km2 and 246.4 km2, respectively, and the 447 maximum values for these two are 1292.32 km2 and 733.39 km2. The average value obtained for 448 224 data is 662.81 km2 and in the train and test stage it is 757.44 and 441.08 km2, respectively, 449 and all data have positive skewness. 450 Table 3. Statistical attributes of Lakes Area (AL) data Nbr. Min (km2) Max (km2) 1st Q (km2) Median (km2) 3rd Q (km2) Mean (km2) σ (n) γ1 γ2 Total 224 246.40 1292.32 455.76 605.36 882.24 662.81 256.94 0.42 -0.91 Train 157 342.52 1292.32 552.88 735.43 959.65 757.44 241.71 0.08 -1.08 Test 67 246.40 733.39 340.48 451.91 503.65 441.08 116.90 0.30 -0.58 Nbr., Number of data; Min. and Max., Minimum and Maximum of data; 1st Q. and 3rd Q., first and third Quarters; σ(n), Standard Deviation; γ1, Skewness; γ2, Kurtosis. Table 3. Statistical attributes of Lakes Area (AL) data 30 451 The results of the application of statistical tests to the AL time series are provided in Table 4 and 452 Fig. 10. According to MW, MK, SMK, KPSS tests results, the series has jumps and trends and is 453 highly non-stationary. Furthermore, the JB test confirms the non-normality of the data. 454 Therefore, pre-processing of AL time series, prior to AI and stochastic modeling is mandatory. 455 The ACF and PACF values were calculated and the corresponding results are presented in Fig. 456 11 and Fig. 12. The plots plainly demonstrate the non-seasonal and seasonal trends and 457 periodicity with lag 12. The periodicity is also observable in the time series plot (Fig. 10a) as 458 iterative peaks and lows. This lake area data component was foreseeable as the surface water is 459 highly impacted by solar energy’s seasonal flux and earth’s revolutions. Though this periodicity 460 damped after two significant lags, the AL series would be more independent and better results 461 can be obtained by removing it. 462 The results of the application of statistical tests to the AL time series are provided in Table 4 and 452 Fig. 10. According to MW, MK, SMK, KPSS tests results, the series has jumps and trends and is 453 highly non-stationary. Furthermore, the JB test confirms the non-normality of the data. 3.2. Obtained AL time series attributes and pre-processing 435 454 The results of the application of statistical tests to the AL time series are provided in Table 4 and 452 Fig. 10. According to MW, MK, SMK, KPSS tests results, the series has jumps and trends and is 453 highly non-stationary. Furthermore, the JB test confirms the non-normality of the data. 454 Therefore, pre-processing of AL time series, prior to AI and stochastic modeling is mandatory. 455 , p p g L , p g y The ACF and PACF values were calculated and the corresponding results are presented in Fig. 456 11 and Fig. 12. The plots plainly demonstrate the non-seasonal and seasonal trends and 457 periodicity with lag 12. The periodicity is also observable in the time series plot (Fig. 10a) as 458 iterative peaks and lows. This lake area data component was foreseeable as the surface water is 459 highly impacted by solar energy’s seasonal flux and earth’s revolutions. Though this periodicity 460 damped after two significant lags, the AL series would be more independent and better results 461 can be obtained by removing it. 462 463 Fig. 11. AL time series ACF and PACF plots. 464 463 Fig. 11. AL time series ACF and PACF plots. 464 31 31 31 For removing non-stationarity factors, the stdω method (stdω (AL)) was applied to the series 465 (Fig. 10b). After modeling, it was observed that this method only reduced the seasonality to one 466 lag in the series and did not affect other terms. Since the stdω method contained the seasonal 467 parameters, it was expected that it would affect mostly seasonal components. The JB transform 468 was subsequently applied (stdωJD (AL)). The normalization method was able to decrease the JB 469 statistic markedly and normalize data. Also, normalization resulted in a reduction of the non- 470 seasonal correlations from 22 to 18 lags. The corresponding results are presented in table 4 and 471 Fig. 12 for each step. 472 For removing non-stationarity factors, the stdω method (stdω (AL)) was applied to the series 465 (Fig. 10b). After modeling, it was observed that this method only reduced the seasonality to one 466 lag in the series and did not affect other terms. Since the stdω method contained the seasonal 467 parameters, it was expected that it would affect mostly seasonal components. The JB transform 468 was subsequently applied (stdωJD (AL)). 3.2. Obtained AL time series attributes and pre-processing 435 The normalization method was able to decrease the JB 469 statistic markedly and normalize data. Also, normalization resulted in a reduction of the non- 470 seasonal correlations from 22 to 18 lags. The corresponding results are presented in table 4 and 471 Fig. 12 for each step. 472 Table 4 Lakes Area (AL) time-series stationarity and normality tests outcomes 473 Table 4 Lakes Area (AL) time-series stationarity and normality tests outcom 473 Table 4 Lakes Area (AL) time-series stationarity and normality tests 473 Tests Jump Trend Stationarity Norm. PMW PMK PSMK PKPSS JB* AL 0 0.01 0.01 0.01 7.72 stdω(AL) 0.01 0.01 0.01 0.01 10.36 stdωJD(AL) 0.01 0.01 0.01 0.01 2.15 Cons. Diff.** 81.21 53.36 37.30 98.02 1.33 *JB critical :5.99 ; p-value > 5% = acceptable; ** Consecutive 1st order non-seasonal and seasonal differencing 3.3. LSTM Deep learning modelling 475 Almost all the hydrological time series, regarding their nature, have a complex structure. 476 Therefore, studying and involving historical events in the modelling process is of high 477 importance. The LSTM model is an enhanced model produced to cover recurrent neural 478 networks' deficiencies (RNN). The RNNs were limited in using historical data. However, the 479 LSTM model unlimitedly can use long-term dependencies in modelling process. 480 Almost all the hydrological time series, regarding their nature, have a complex structure. 476 Therefore, studying and involving historical events in the modelling process is of high 477 importance. The LSTM model is an enhanced model produced to cover recurrent neural 478 networks' deficiencies (RNN). The RNNs were limited in using historical data. However, the 479 LSTM model unlimitedly can use long-term dependencies in modelling process. 480 Given the seasonal correlations in time series with lag 12, the LSTM model was used for 481 modeling pre-processed data with the hidden cell states of h = 12, 60, 144 and 156 [45,89]. A 482 Given the seasonal correlations in time series with lag 12, the LSTM model was used for 481 modeling pre-processed data with the hidden cell states of h = 12, 60, 144 and 156 [45,89]. A 482 32 piecewise learning rate schedule with Initial learn rate of 0.005 was defined for the model 483 structure. After determining the maximum epochs of 500 and learn rate drop period and drop 484 factor of 125 and 0.2, respectively, the single LSTM layer model was defined. Computational 485 requirements represent an important consideration. In this work, the MATLAB software and a 486 computer with a configuration of CPU core i7, 2500 MHz and 8G RAM were used. The average 487 time spent for modeling each input was around 100 seconds. The results of the models are 488 provided in Table 5. The LSTM model with the seasonal standardized (stdω) data and 12 inputs 489 produced better results than inputs with higher hidden cell states with the same preprocessing. 490 piecewise learning rate schedule with Initial learn rate of 0.005 was defined for the model 483 structure. After determining the maximum epochs of 500 and learn rate drop period and drop 484 factor of 125 and 0.2, respectively, the single LSTM layer model was defined. Computational 485 requirements represent an important consideration. 3.3. LSTM Deep learning modelling 475 The LSTMstdωJD (12) indices are as R = 0.806, RMSE = 109.140, MAE = 91.571, MAPE 503 = 0.229, RMSRE = 0.281, UI = 0.110, UII = 0.239. The Theil’s coefficient also shows slight 504 improvement in the model's quality and power while using normalization and standardization, 505 compared to the single standardization. 506 For further investigation, the pre-processed series with stationarization and normalization 501 (stdωJD) were also modeled. Likewise, the LSTM model with 12 inputs produced the best 502 results. The LSTMstdωJD (12) indices are as R = 0.806, RMSE = 109.140, MAE = 91.571, MAPE 503 = 0.229, RMSRE = 0.281, UI = 0.110, UII = 0.239. The Theil’s coefficient also shows slight 504 improvement in the model's quality and power while using normalization and standardization, 505 compared to the single standardization. 506 For further investigation, the pre-processed series with stationarization and normalization 501 (stdωJD) were also modeled. Likewise, the LSTM model with 12 inputs produced the best 502 results. The LSTMstdωJD (12) indices are as R = 0.806, RMSE = 109.140, MAE = 91.571, MAPE 503 = 0.229, RMSRE = 0.281, UI = 0.110, UII = 0.239. The Theil’s coefficient also shows slight 504 improvement in the model's quality and power while using normalization and standardization, 505 compared to the single standardization. 506 The results show that in stdωJD, as in stdω, the model's accuracy decreases with increasing 507 inputs. In h156 the value of the correlation coefficient is higher than h12 and h144. However, the 508 statistical parameters show better performance for h12 compared to stdωJD model with other 509 hidden cell inputs. As seen in the preprocessed data's correlogram, the seasonal correlation was 510 damped after one seasonal lag and the dependencies were important up to one seasonal lag and 511 few more non-seasonal lags. Therefore, the LSTM models with historical data up to previous 12 512 lags were invistigated. Moreover, the normalization of data distribution enhanced the modeling 513 results and decreased the errors in comparison to lone standardization. The LSTMstdωJD improved 514 the results by R = 2.458%, RMSE = 3.610%, MAE = 0.468%, MAPE = 0.451%, RMSRE = 515 2.720%, UI = 3.428%, UII = 3.610%. This improvement proves the importance of the pre- 516 processing in AI models, regardless of their capability in modeling non-linearity. 3.3. LSTM Deep learning modelling 475 In this work, the MATLAB software and a 486 computer with a configuration of CPU core i7, 2500 MHz and 8G RAM were used. The average 487 time spent for modeling each input was around 100 seconds. The results of the models are 488 provided in Table 5. The LSTM model with the seasonal standardized (stdω) data and 12 inputs 489 produced better results than inputs with higher hidden cell states with the same preprocessing. 490 Table 5 LSTM results for Lake Area (AL) time series Method Inputs R RMSE MAE MAPE RMSRE UI UII stdω h12 0.786 113.227 92.001 0.230 0.289 0.114 0.248 stdω h60 0.790 144.837 124.816 0.317 0.380 0.140 0.317 stdω h144 0.769 181.314 164.596 0.418 0.483 0.169 0.397 stdω h156 0.746 200.116 183.361 0.465 0.532 0.184 0.439 stdωJD h12 0.806 109.140 91.571 0.229 0.281 0.110 0.239 stdωJD h60 0.893 116.363 104.381 0.263 0.304 0.115 0.255 stdωJD h144 0.770 157.532 138.723 0.352 0.416 0.151 0.345 stdωJD h156 0.852 146.578 132.055 0.331 0.380 0.141 0.321 h = hidden states no. Table 5 LSTM results for Lake Area (AL) time series 491 33 In the stdω method, except for h60, where the value of R is improved by 2% and h12 has a better 492 performance in other statistical parameters, and as the number of inputs increases, the accuracy 493 of the model is affected. h156 has the highest error values so that the correlation coefficient has 494 decreased by 5% compared to h12 and the RMSE has increased by 76.7%. RMSRE and MAPE, 495 have increased by more than 100%. These values for LSTM models demonstrated that the 496 models' power and quality were higher while 12 inputs were chosen for modeling, compared to 497 the other models with more inputs. Also, it indicates that the impact of most recent historical data 498 33 is more than the oldest ones. This refers to the capability of the LSTM in modeling dependent 499 data. 500 is more than the oldest ones. This refers to the capability of the LSTM in modeling dependent 499 data. 500 For further investigation, the pre-processed series with stationarization and normalization 501 (stdωJD) were also modeled. Likewise, the LSTM model with 12 inputs produced the best 502 results. 3.3. LSTM Deep learning modelling 475 517 The structure of data should be investigated prior to the preprocessing to assess the impacts of 518 the preprocessing methods. Also, it can be concluded that using more independent inputs causes 519 more variations that impact the final results of the deep learning method. So, limiting the LSTM 520 model inputs to the correlated data is important. 521 34 522 Fig. 12. AL pre-processed time series ACF plots. 523 Fig. 12. AL pre-processed time series ACF plots. 523 Stochastic models are among the most conventional modelling methods in hydrology. These 526 models are noticed for their simple theory and application. As the basis of these models are 527 statistical concepts, some prerequisites should be considered in modelling process. The 528 stationarity and normalization of time series are the two necessities of stochastic models. 529 Concerning the results provided in section 3.2, as the pre-processed data's ACF values are 530 damped after 18 lags and series is normal, modelling can be carried out, but higher orders of 531 parameters are needed. Hence, a consecutive non-seasonal and seasonal differencing was applie 532 35 to series, and it was observed that all non-stationarity factors were removed from series and 533 became stationary. The corresponding results are presented in Table 4 and Fig. 12 for each step. 534 The correlations in ACF plots after consecutive differencing declines considerably to one lag. 535 But for further survey of the model’s capability, the orders of the parameters in SARIMA model 536 are considered as: p = q = P = Q = {0, 1, 2, 3, 4, 5} and d = D = {0, 1} and seasonality ω = 12. 537 After coding the dynamic model in MATLAB software and considering this parameter selection, 538 a total number of 2590 models were produced with the same computer configuration used for the 539 LSTM models. The time spent on stochastic modeling was about two hours. The minimum 540 values of the indices for forecasted AL data in all were R = 0.01, RMSE = 68.70, MAE = 49.42, 541 MAPE = 0.11, RMSRE = 0.14, AICc = 574.80, UI = 0.08, UII = 0.15 and the maximum values 542 were R = 0.85, RMSE = 780.61, MAE = 756.47, MAPE = 1.85, RMSRE = 1.98, AICc = 862.04, 543 UI = 0.47, UII = 1.71. 3.3. LSTM Deep learning modelling 475 With these specifications and after considering the independence of the 544 results, simplicity and goodness of the fit of models, the superior model was chosen as SARIMA 545 (1,0,0)(0,1,1)12. The evaluation results for this model are: R = 0.819, RMSE = 70.217, MAE = 546 49.425, MAPE = 0.106, RMSRE = 0.143, AICc = 574.82, UI = 0.077, UII = 0.154. The model is 547 the most parsimonious and adequate SARIMA model compared to the other 2589 models. It is 548 observed that the model's correlation index is almost in the same range as the LSTM, but other 549 indices like RMSE, MAPE are almost half. This means the linear model could forecast the 550 variation of the AL data better than sole LSTMs after triple preprocessing and removing all the 551 dependencies in the data. However, other model evaluation criteria should be investigated, and 552 there are still opportunities for enhancements. Another step in the evaluation of stochastic 553 modelling is checking the independence of the residuals. This criterion is assessed 554 36 but also has uncorrelated residuals. Therefore, the Ljung-Box test was applied to the stochastic 556 model's residuals for 60 non-seasonal or five seasonal lags. The test indicated the independence 557 of the residuals and the adequacy of the model. The results of the independence test for the 558 superior model are provided in Fig. 12. 559 but also has uncorrelated residuals. Therefore, the Ljung-Box test was applied to the stochastic 556 model's residuals for 60 non-seasonal or five seasonal lags. The test indicated the independence 557 of the residuals and the adequacy of the model. The results of the independence test for the 558 superior model are provided in Fig. 12. 559 560 Fig.13. Ljung-Box residuals test results. 561 Fig.13. Ljung-Box residuals test results. 561 Fig.13. Ljung-Box residuals test results. 561 3.5. Hybrid Deep-learning-Stochastic modelling and disparities 563 Hybridization of models is one of the methods of utilizing non-linear and linear models’ 564 characteristics simultaneously. These methods allow researchers to model data and make 565 predictions by covering the drawbacks of the single models and produce results with lower 566 errors. For this purpose, the linear model residuals that are independent are used as inputs of the 567 AI model. This input is assumed to be the non-linear part of the time series as the stochastic 568 model is also assumed to be able to forecast the linear part [90]. As it can be seen in Fig. 14. The 569 residuals of the linear model are completely independent, and no correlation remains in the 570 residuals. However, they have the circumstances to be modeled by the AI model. Since, no 571 correlation is found in the residuals’ series, the AI model requires less inputs to forecast future 572 steps. However, the previous steps will be followed to provide comparison circumstances. 573 37 574 Fig. 14. Stochastic model residuals auto correlation function plot 575 574 Fig. 14. Stochastic model residuals auto correlation function plot 575 38 574 Fig. 14. Stochastic model residuals auto correlation function plot 575 B integrating SARIMA and LSTM, the superior linear model's residuals were modelled by the 576 LSTM model with the same inputs considered for modelling in previous sections. The residuals 577 are denoted as SARIMAs. The results of the models are provided in Table 6. The SARIMAs- 578 LSTM with 12 inputs outperformed other SARIMAs-LSTM hybrid models. As shown in Fig. 14, 579 the residuals do not have correlations, therefore, the best results with the 12 inputs were 580 expected. Using hidden cells’ inputs less than 12 could also produce these results. 581 582 Table 6 Hybrid models results for Lakes Area (AL) time series Method Inputs R RMSE MAE MAPE RMSRE UI UII SARIMAs - LSTM h12 0.819 70.428 49.310 0.105 0.143 0.077 0.154 h60 0.777 79.138 60.137 0.131 0.165 0.087 0.173 h144 0.754 100.928 82.246 0.198 0.243 0.104 0.221 h156 0.752 104.037 85.689 0.208 0.252 0.107 0.228 h = hidden states no. 583 By comparing the results of the hybrid model and previously presented models, it was observed 584 that the hybridization improved a few characteristics of the results. Compared to the single 585 LSTM models, the Hybrid model increases the correlation of the forecast. 3.5. Hybrid Deep-learning-Stochastic modelling and disparities 563 It improved the 586 mediation of the data by 0.061 compared to the average of the LSTM models. Also, the error 587 574 Fig. 14. Stochastic model residuals auto correlation function plot 575 B integrating SARIMA and LSTM, the superior linear model's residuals were modelled by the 576 LSTM model with the same inputs considered for modelling in previous sections. The residuals 577 are denoted as SARIMAs. The results of the models are provided in Table 6. The SARIMAs- 578 LSTM with 12 inputs outperformed other SARIMAs-LSTM hybrid models. As shown in Fig. 14, 579 the residuals do not have correlations, therefore, the best results with the 12 inputs were 580 expected. Using hidden cells’ inputs less than 12 could also produce these results. 581 B integrating SARIMA and LSTM, the superior linear model's residuals were modelled by the 576 LSTM model with the same inputs considered for modelling in previous sections. The residuals 577 are denoted as SARIMAs. The results of the models are provided in Table 6. The SARIMAs- 578 LSTM with 12 inputs outperformed other SARIMAs-LSTM hybrid models. As shown in Fig. 14, 579 the residuals do not have correlations, therefore, the best results with the 12 inputs were 580 expected. Using hidden cells’ inputs less than 12 could also produce these results. 581 Table 6 Hybrid models results for Lakes Area (AL) time series Method Inputs R RMSE MAE MAPE RMSRE UI UII SARIMAs - LSTM h12 0.819 70.428 49.310 0.105 0.143 0.077 0.154 h60 0.777 79.138 60.137 0.131 0.165 0.087 0.173 h144 0.754 100.928 82.246 0.198 0.243 0.104 0.221 h156 0.752 104.037 85.689 0.208 0.252 0.107 0.228 h = hidden states no. 38 indices were almost reduced to half. However, this improvement, compared to the linear model 588 was less noticeable than lone LSTM models. The hybridization, on the other hand, lowered the 589 MAPE and MAE indices. 590 591 591 Fig.15. Scatter plots of the modeled AL time series. a: LSTMStdω (h12); b: LSTMStdωJD (h12); c: 592 SARIMA(1,0,0)(0,1,1)12; d: HybridS (h12). 593 591 Fig.15. Scatter plots of the modeled AL time series. a: LSTMStdω (h12); b: LSTMStdωJD (h12); c: 592 SARIMA(1,0,0)(0,1,1)12; d: HybridS (h12). 593 39 Since the indices are very close and for better comparison, the scatter plots of the superior 594 LSTM, SARIMA, and hybrid models are provided in Fig. 15. 3.5. Hybrid Deep-learning-Stochastic modelling and disparities 563 From the scatter plots, the 595 dispersion of the modelled data can be observed. The LSTM models predicted data are more 596 dispersed than SARIMA and hybrid models, respectively (Fig. 15 a and b). The linear model 597 (Fig. 15c) has densified the data and brought it closer to the 10% range. However, the hybrid 598 model was more successful than the others in bringing the forecasts closer to the median line and 599 locating data in the 10% intervals (Fig. 15d). In other words, hybridization caused more 600 correlation in the forecasted data and better mediation has occurred by utilizing both methods’ 601 39 Since the indices are very close and for better comparison, the scatter plots of the superior 594 LSTM, SARIMA, and hybrid models are provided in Fig. 15. From the scatter plots, the 595 dispersion of the modelled data can be observed. The LSTM models predicted data are more 596 dispersed than SARIMA and hybrid models, respectively (Fig. 15 a and b). The linear model 597 (Fig. 15c) has densified the data and brought it closer to the 10% range. However, the hybrid 598 model was more successful than the others in bringing the forecasts closer to the median line and 599 locating data in the 10% intervals (Fig. 15d). In other words, hybridization caused more 600 correlation in the forecasted data and better mediation has occurred by utilizing both methods’ 601 39 characteristics. The Box plot of the observed data and superior models are drawn in Fig. 16, and 602 it can be observed that the SARIMA (1, 0, 0) (0, 1, 1)12 and SARIMAS-LSTM model perfectly 603 forecasted the interquartile area of the AL time series and even were able to forecast one of the 604 extreme values of the original series. These methods also predicted the maxima and minima of 605 the data more accurately than other models. A potent model regenerates the statistical 606 characteristics of the studied data. Though the linear model and the hybrid indices were slightly 607 similar, the hybrid SARIMA-LSTM reproduced the primal statistical properties of WSA data 608 better than sole models [91]. The hybrid model performed better in forecasting the mean and 609 other statistical characteristics of the observed data slightly better than the SARIMA model. 3.5. Hybrid Deep-learning-Stochastic modelling and disparities 563 610 Therefore, hybridization was not able to produce noticeable results (Tables 5 and 6) but 611 reproduced the original series statistical attributes. Thus, it can be considered as a superior WSA 612 modelling methodology. 613 616 40 40 . 16. Box plot of the superior models; AL: observed WSA data, Ss: SARIMA (1, 0, 0) (0, 1, ; H: SARIMAS-LSTM; L1: LSTMstdωJD (12), L2: LSTMstdωJD (12); 617 Fig. 16. Box plot of the superior models; AL: observed WSA data, Ss: SARIMA (1, 0, 0) (0, 1, 618 Fig. 16. Box plot of the superior models; AL: observed WSA data, Ss: SARIMA (1, 0, 0) (0, 1, 618 1)12; H: SARIMAS-LSTM; L1: LSTMstdωJD (12), L2: LSTMstdωJD (12); 619 Fig. 16. Box plot of the superior models; AL: observed WSA data, Ss: SARIMA (1 41 Fig. 16. Box plot of the superior models; AL: observed WSA data, Ss: SARIMA (1, 0, 0) (0, 1, 618 1)12; H: SARIMAS-LSTM; L1: LSTMstdωJD (12), L2: LSTMstdωJD (12); 619 620 4. Conclusion 621 Sustainable management of freshwater inland lakes in an arid region plays a vital role in 622 environmental preservation and quality of life. Moreover, monitoring changes in the lake's 623 surface area due to both natural and anthropogenic stressors helps to better plan and manage 624 water resources. Therefore, the accurate mapping and monitoring of lake surface area, and the 625 1)12; H: SARIMAS-LSTM; L1: LSTMstdωJD (12), L2: LSTMstdωJD (12); 619 1)12; H: SARIMAS-LSTM; L1: LSTMstdωJD (12), L2: LSTMstdωJD (12); 619 1)12; H: SARIMAS-LSTM; L1: LSTMstdωJD (12), L2: LSTMstdωJD (12); 41 forecasting of these vital resources future trends are of great importance for planning and 626 management purposes. In this study, the WSA of the TB lakes is studied. To map the lake's 627 surface area, the MODIS satellite images were used to extract a time series depicting changes of 628 the WSA. The images were obtained from MODIS data, MOD09A1 version 6. The pre- 629 processing of the images included image preparation, classification, and statistical computation. 630 The preparation and classification of the images were undertaken in GEE environment. Using the 631 MNDWI index, the water mass was separated from the background, and the lake area was 632 obtained from the chosen images. Finally, by repeating the process for images from 2001 to 2019 633 a monthly time series of lakes areas (AL) was obained. 3.5. Hybrid Deep-learning-Stochastic modelling and disparities 563 The AL time series was examined by 634 stationarity and normality tests to investigate the structure of the timeseries. Periods with 12 lag 635 repetition, trends and jumps with a non-normal distribution were observed in the timeseries. The 636 timeseries was pre-processed with the conventional seasonal standardization (stdω) method and 637 normalized with the John-Draper (JD) transform, two-time series were obtained. 638 forecasting of these vital resources future trends are of great importance for planning and 626 management purposes. In this study, the WSA of the TB lakes is studied. To map the lake's 627 surface area, the MODIS satellite images were used to extract a time series depicting changes of 628 the WSA. The images were obtained from MODIS data, MOD09A1 version 6. The pre- 629 processing of the images included image preparation, classification, and statistical computation. 630 The preparation and classification of the images were undertaken in GEE environment. Using the 631 MNDWI index, the water mass was separated from the background, and the lake area was 632 obtained from the chosen images. Finally, by repeating the process for images from 2001 to 2019 633 a monthly time series of lakes areas (AL) was obained. The AL time series was examined by 634 stationarity and normality tests to investigate the structure of the timeseries. Periods with 12 lag 635 repetition, trends and jumps with a non-normal distribution were observed in the timeseries. The 636 timeseries was pre-processed with the conventional seasonal standardization (stdω) method and 637 normalized with the John-Draper (JD) transform, two-time series were obtained. 638 These timeseries were modelled with the LSTM model with h = {12, 60, 144, 156} number of 639 hidden cell states. The single LSTM models, with the two different preprocessing tasks, required 640 only 12 hidden cell states to obtain the highest accuracy. LSTMstdω (12) with R = 0.786, RMSE = 641 113.227, MAPE = 0.230 and STMstdωJD (12) with R = 0.806, RMSE = 109.140, MAPE = 0.229 642 outperformed others. These results indicated that using multiple preprocessing methods and 643 reevaluating the results of the time series structure tests is necessary since most of the time, the 644 latter part is neglected in the AI modeling procedure. 645 42 A stochastic SARIMA model and hybridization of both deep learning and stochastic models 646 were carried out for further investigation and surveying the possibilities to enhance the 647 forecasting results. 3.5. Hybrid Deep-learning-Stochastic modelling and disparities 563 The superior linear model was chosen as SARIMA with (1, 0, 0) (0, 1, 1)12 648 42 A stochastic SARIMA model and hybridization of both deep learning and stochastic models 646 were carried out for further investigation and surveying the possibilities to enhance the 647 forecasting results. The superior linear model was chosen as SARIMA with (1, 0, 0) (0, 1, 1)12 648 42 parameters based on goodness of fit and model parsimony. The stochastics models' results were 649 better than single LSTM models and the errors were reduced by almost half, R = 0.819, MAE = 650 49.425, MAPE = 0.106. To utilize both models’ capabilities, residuals of the stochastic model 651 were modelled by LSTM. 652 parameters based on goodness of fit and model parsimony. The stochastics models' results were 649 better than single LSTM models and the errors were reduced by almost half, R = 0.819, MAE = 650 49.425, MAPE = 0.106. To utilize both models’ capabilities, residuals of the stochastic model 651 were modelled by LSTM. 652 Results indicate that the hybrid model indices were marginally better than others,. The scatter 653 and Box plots of the models revealed that the hybridization did not produce noticeable better 654 error indices but improved the statistical characteristics and made them closer to observational 655 data. The hybrid SARIMA-LSTM reproduced the primal statistical properties of WSA data and 656 caused better mediation as observed in scatter plots and the Box plot of the data compared to sole 657 models. 658 In conclusion, the hybridization can reproduce model forecasts that better preserve the observed 659 timeseries's statistical attributes compared to single models. Therefore, it is suggested that the 660 undertaken methodology of AL time series modelling be applied to other AL time series and other 661 AI methods like Extreme Learning Machine (ELM), LSTM developments like Genetic 662 Algorithm (GA)-LSTM and a combination of these models with linear models be investigated. 663 Results indicate that the hybrid model indices were marginally better than others,. The scatter 653 and Box plots of the models revealed that the hybridization did not produce noticeable better 654 error indices but improved the statistical characteristics and made them closer to observational 655 data. 3.5. Hybrid Deep-learning-Stochastic modelling and disparities 563 The hybrid SARIMA-LSTM reproduced the primal statistical properties of WSA data and 656 caused better mediation as observed in scatter plots and the Box plot of the data compared to sole 657 models. 658 Results indicate that the hybrid model indices were marginally better than others,. The scatter 653 and Box plots of the models revealed that the hybridization did not produce noticeable better 654 error indices but improved the statistical characteristics and made them closer to observational 655 data. The hybrid SARIMA-LSTM reproduced the primal statistical properties of WSA data and 656 caused better mediation as observed in scatter plots and the Box plot of the data compared to sole 657 models. 658 In conclusion, the hybridization can reproduce model forecasts that better preserve the observed 659 timeseries's statistical attributes compared to single models. Therefore, it is suggested that the 660 undertaken methodology of AL time series modelling be applied to other AL time series and other 661 AI methods like Extreme Learning Machine (ELM), LSTM developments like Genetic 662 In conclusion, the hybridization can reproduce model forecasts that better preserve the observed 659 timeseries's statistical attributes compared to single models. Therefore, it is suggested that the 660 undertaken methodology of AL time series modelling be applied to other AL time series and other 661 AI methods like Extreme Learning Machine (ELM), LSTM developments like Genetic 662 In conclusion, the hybridization can reproduce model forecasts that better preserve the observed 659 timeseries's statistical attributes compared to single models. 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https://openalex.org/W1900770471
https://wjso.biomedcentral.com/track/pdf/10.1186/1477-7819-4-23
English
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Primary mediastinal hemangiopericytoma
World journal of surgical oncology
2,006
cc-by
2,845
Background Hemangiopericytoma is a rare mesenchymal neoplasm, accounting for about 1% of vascular tumors [1]. Heman- giopericytoma is known to be derived from the vascular pericyte and was first reported by Stout and Murray in 1942 [2]. The tumor occurs most commonly in the skin, subcutaneous soft tissues, muscles of the extremities, ret- roperitoneum but rarely in the lung, trachea or mediasti- num [3]. Herein, a surgical case of primary mediastinal hemangiopericytoma is presented. Case report Primary mediastinal hemangiopericytoma A Chnaris1, N Barbetakis*2, A Efstathiou3 and I Fessatidis3 * Corresponding author Published: 27 April 2006 World Journal of Surgical Oncology2006, 4:23 doi:10.1186/1477-7819-4-23 Received: 26 December 2005 Accepted: 27 April 2006 This article is available from: http://www.wjso.com/content/4/1/23 © 2006Chnaris et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 26 December 2005 Accepted: 27 April 2006 © 2006Chnaris et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creative which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cit ; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Hemangiopericytoma is a rare mesenchymal neoplasm, accounting for about 1% of vascular tumors The tumor occurs most commonly in the skin, subcutaneous soft tissues, muscles of the extremities, retroperitoneum but rarely in the lung, trachea or mediastinum. Case presentation: A rare case of primary mediastinal hemangiopericytoma is presented. A 72- year-old woman was treated by complete surgical resection of the tumor. Details of the clinical and radiographic feature are presented. The patient's postoperative course was uneventful with no evidence of recurrence 9 months after the operation. Conclusion: Hemangiopericytoma is an uncommon, potentially malignant tumor originating from pericytes in the small vessels and surgical radical excision is the treatment of choice, although the criteria for determining the area of resection have not been established. International literature has demonstrated that recurrent disease usually occurs within 2 years and therefore a long-term careful follow-up is required. orthopnea and use of accessory respiratory muscles. Lab- oratory studies were essentially within normal limits. A chest X-ray revealed a homogenous opacity occupying lower two-thirds of the left hemithorax and causing con- tralateral shifting of the mediastinum and heart (Figure 1). Thoracentesis was performed in order to palliate respi- ratory problems. One thousand eight hundred (1800) ml of serosanguinous fluid were aspirated. Cytologic exami- nation revealed an exudate with no signs of malignancy. Culture of the aspirated pleural fluid was also negative. A computed tomographic scan (CT) of the chest revealed a large solid tumorous mass measuring 7.3 × 2.3 cm located in the posterosuperior mediastinum on the left side asso- ciated with pleural effusion (Figure 2). In order to perform preoperative staging of the tumor, the patient underwent World Journal of Surgical Oncology Open Access Case presentation A 72-year-old woman was referred to our institution com- plaining of dyspnea, cough and chest tightness for the last two months. Physical examination indicated tachypnea, Page 1 of 6 (page number not for citation purposes) Page 1 of 6 (page number not for citation purposes) World Journal of Surgical Oncology 2006, 4:23 http://www.wjso.com/content/4/1/23 Chest X-ray showing an abnormal shadow associated with ipsilateral pleural effusion Figure 1 Chest X-ray showing an abnormal shadow associated with ipsilateral pleural effusion. Chest X-ray showing an abnormal shadow associated with ipsilateral pleural effusion Figure 1 Chest X-ray showing an abnormal shadow associated with ipsilateral pleural effusion. Chest X-ray showing an abnormal shadow associated with ipsilateral pleural effusion Figure 1 Chest X-ray showing an abnormal shadow associated with ipsilateral pleural effusion. CT scans of brain, upper abdomen, a bone scan and bron- coscopy. All were normal. The tumor markers alpha-feto- protein (AFP), carcinoembryonic antigen (CEA), CA 19-9, neuron-specific enolase (NSE) and squamous cell carci- noma antigen were within normal limits. The mass seemed to be resectable and surgical approach was sug- gested to the patient. measured 7 × 3 × 2.5 cm and weighed 210 g. Grossly the resected specimen was a smooth, friable encapsulated mass with focal hemorrhages. The cut surface was smooth, elastic and pale brown. Microscopic examination showed round and spindle cells surrounded by thin-walled, endothelium-lined vascular channels, giving a "staghorn" appearance to the vessels as typically seen in hemangi- opericytoma (Figure 3). The low-mitotic activity and the absence of clear nuclear pleomorphism were suggestive of a low-grade malignant tumor. The tumor cells were immunoreactive only for anti-smooth-muscle-actin pro- tein (Figure 4). The patient underwent left posterolateral thoracotomy through 5th intercostal space. During the operation, the mass appeared to have mediastinal origin and was highly vascularized. There was no involvement of vital mediasti- nal structures and the surrounding lung parenchyma was compressed by the tumor. Six hundred ml of sanguinous pleural fluid were aspirated. Despite the persistent bleed- ing during dissection the tumor was resected. The mass The postoperative course was uneventful and the patient was discharged home on postoperative day 9. Case presentation Postopera- tive chemoradiotherapy was recommended but the Page 2 of 6 (page number not for citation purposes) (page number not for citation purposes) World Journal of Surgical Oncology 2006, 4:23 http://www.wjso.com/content/4/1/23 Preoperative computed tomographic scan demonstrated a tumor mass located in the mediastinum next to descending aorta and associated with pleural effusion Figure 2 Preoperative computed tomographic scan demonstrated a tumor mass located in the mediastinum next to descending aorta and associated with pleural effusion. Preoperative computed tomographic scan demonstrated a tumor mass located in the mediastinum next to descending aorta and associated with pleural effusion Figure 2 Preoperative computed tomographic scan demonstrated a tumor mass located in the mediastinum next to descending aorta and associated with pleural effusion. patient denied any further treatment. Nine months after the operation the patient is alive and well without evi- dence of recurrence of the disease. Hemangiopericytoma has no uniform clinical or radio- graphic features, usually affects older individuals, and mostly presents as an asymptomatic, non-calcified soli- tary mass on chest X-ray. These tumors are composed of closely-packed spindle cells and prominent vascular chan- nels. The histological differential diagnosis includes many mesenchymal tumors, such as the solitary fibrous tumor and the synovial sarcoma [3]. No single clinical or histo- logical feature including histological type or DNA ploidy allows prediction of biologic aggressiveness [8]. Malig- nant hemangiopericytoma is recognized by its increased mitotic rate, tumor size and foci of hemorrhage and necrosis [3]. Hemangiopericytoma has no uniform clinical or radio- graphic features, usually affects older individuals, and mostly presents as an asymptomatic, non-calcified soli- tary mass on chest X-ray. These tumors are composed of closely-packed spindle cells and prominent vascular chan- nels. The histological differential diagnosis includes many mesenchymal tumors, such as the solitary fibrous tumor and the synovial sarcoma [3]. No single clinical or histo- logical feature including histological type or DNA ploidy allows prediction of biologic aggressiveness [8]. Malig- nant hemangiopericytoma is recognized by its increased mitotic rate, tumor size and foci of hemorrhage and necrosis [3]. Discussion Hemangiopericytoma is an uncommon, potentially malignant tumor originating from pericytes in the small vessels. Intrathoracic hemangiopericytoma usually arises from pericytes that surround the basement membrane of capillaries and small venules within the lung parenchyma [3]. Our case was an intrathoracic mediastinal hemangi- opericytoma, which is extremely rare. Only a few isolated case reports are available in the literature [4-7], whereas the intrapulmonary variety of the same tumor is relatively more common. Immunohistochemically, hemangiopericytomas are known to show a positive response to antibodies against vimentin and type IV collagen and a negative response to Page 3 of 6 (page number not for citation purposes) Page 3 of 6 (page number not for citation purposes) World Journal of Surgical Oncology 2006, 4:23 http://www.wjso.com/content/4/1/23 Round and spindle cells surrounded by thin-walled, endothelium-lined vascular channels, giving a "staghorn" appearance to the vessels as typically seen in hemangiopericytoma (Hematoxylin and eosin stain ×400) Figure 3 Round and spindle cells surrounded by thin-walled, endothelium-lined vascular channels, giving a "staghorn" appearance to the vessels as typically seen in hemangiopericytoma (Hematoxylin and eosin stain ×400). Round and spindle cells surrounded by thin-walled, endothelium-lined vascular channels, giving a staghorn appearance to the vessels as typically seen in hemangiopericytoma (Hematoxylin and eosin stain ×400) Figure 3 Round and spindle cells surrounded by thin-walled, endothelium-lined vascular channels, giving a "staghorn" appearance to the vessels as typically seen in hemangiopericytoma (Hematoxylin and eosin stain ×400). extended surgery [12]. During the resection, it is impor- tant to look for invasion of the surrounding lung tissue and to avoid intrathoracic spread of tumor cells by man- ual examination. With respect to adjuvant therapy, chem- otherapy or radiotherapy have been recommended but is considered to be almost ineffective [3]. On the other hand Rusch et al., reported that combination therapy or single therapy with adriamycin was effective against metastases [13]. Jalal and Jeyasingham reported that preoperative radiotherapy of large hemangiopericytomas on the chest wall significantly reduced the vascularity of the tumor and made complete resection much easier [14]. Some authors have proposed an innovative approach of treatment, which includes complete surgical resection along with intraoperative and postoperative radiotherapy, whereas others have recommended that radiotherapy may be used palliatively for local tumor recurrence or superior vena VIII-related antigen, S-100 protein, neuron specific eno- lase, carcinoembryonic antigen, desmins, laminin and cytokeratins [9]. A special consideration concerns the preoperative diagno- sis. Page 4 of 6 (page number not for citation purposes) Discussion When a mass appears to be radiologically resectable, many authors perform a thoracotomy without histologi- cal diagnosis. Previous reports propose an attempt to obtain a preoperative diagnosis even in tumors that are clearly resectable if high vascularization is suspected on imaging techniques [10,11]. Surgical radical excision is the treatment of choice for hemangiopericytomas, although the criteria for determin- ing the area of resection have not been established. Hansen and colleagues stated that it was necessary to con- sider all hemangiopericytomas as malignant and perform Page 4 of 6 (page number not for citation purposes) Page 4 of 6 (page number not for citation purposes) World Journal of Surgical Oncology 2006, 4:23 http://www.wjso.com/content/4/1/23 Tumor cells clearly positive for a-SMA (anti-smooth-muscle-actin) protein (×400) Figure 4 Tumor cells clearly positive for a-SMA (anti-smooth-muscle-actin) protein (×400). World Journal of Surgical Oncology 2006, 4:23 http://www.wjso.com/content/4/1/23 http://www.wjso.com/content/4/1/23 Tumor cells clearly positive for a-SMA (anti-smooth-muscle-actin) protein (×400) Figure 4 Tumor cells clearly positive for a-SMA (anti-smooth-muscle-actin) protein (×400). Tumor cells clearly positive for a SMA (anti smooth muscle actin) protein ( 400) Figure 4 Tumor cells clearly positive for a-SMA (anti-smooth-muscle-actin) protein (×400). Conclusion cava obstruction [13,15,16]. Morandi et al., recom- mended preoperative percutaneous embolization of hypervascular mediastinal tumors, in order to allow a safe complete removal of the lesion later [17]. cava obstruction [13,15,16]. Morandi et al., recom- mended preoperative percutaneous embolization of hypervascular mediastinal tumors, in order to allow a safe complete removal of the lesion later [17]. Hemangiopericytoma is rare vascular slow-growing tumor with high local recurrence and the long-term prognosis is poor because of its propensity to recur. Surgical radical excision is the treatment of choice despite the fact that the risk of intraoperative uncontrollable bleeding is high. Local or distant recurrence is commonly seen and a long- term careful follow-up is required. The 5-year survival of patients with hemangiopericytoma originating in any organ has been reported to be 85%, whereas the survival of patients with a tumor of pulmo- nary origin is 30–35%. Approximately 50% of hemangi- opericytomas have been reported to recur within 5 years [3,12]. It has been demonstrated that recurrent disease usually occurs within 2 years after initial treatment and recurrences are commonly found in the thorax, either in the pulmonary parenchyma or in the pleura. Distant metastases to liver, brain and bone have also been reported [12]. Competing interests The author(s) declare that they have no competing inter- ests. References 1. Hart LL, Weinberg JB: Metastatic hemangiopericytoma with prolonged survival. Cancer 1987, 60:916-920. 1. Hart LL, Weinberg JB: Metastatic hemangiopericytoma with prolonged survival. Cancer 1987, 60:916-920. prolonged survival. Cancer 1987, 60:916-920. 2. Stout AP, Murray MR: Hemangiopericytoma: a vascular tumor featuring Zimmerman's pericytes. Ann Surg 1942, 116:26-33. 3. Espat NJ, Lewis JJ, Leung D: Conventional hemangiopericytoma: modern analysis of outcome. Cancer 2002, 95:1746-1751. 4. Simonton SC, Swanson PE, Watterson J, Priest JR: Primary medias- tinal hemangiopericytoma with fatal outcome in a child. Arch P h l L b M d 1995 119 839 841 2. Stout AP, Murray MR: Hemangiopericytoma: a vascular tumor featuring Zimmerman's pericytes. Ann Surg 1942, 116:26-33. 3 Espat NJ Lewis JJ Leung D: Conventional hemangiopericytoma: featuring Zimmerman's pericytes. Ann Surg 1942, 116:26-33. 3. Espat NJ, Lewis JJ, Leung D: Conventional hemangiopericytoma: modern analysis of outcome. Cancer 2002, 95:1746-1751. featuring Zimmerman s pericytes. Ann Surg 1942, 116:26-33. 3. Espat NJ, Lewis JJ, Leung D: Conventional hemangiopericytoma: modern analysis of outcome. Cancer 2002, 95:1746-1751. modern analysis of outcome. Cancer 2002, 95:1746 1751. 4. Simonton SC, Swanson PE, Watterson J, Priest JR: Primary medias- tinal hemangiopericytoma with fatal outcome in a child. Arch Pathol Lab Med 1995, 119:839-841. 5. Hayashi A, Takamori S, Tayama K, Mitsuoka M, Tamura K, Shirouzu K, Fujimoto K, Watanabe J: Primary hemangiopericytoma of the superior mediastinum: a case report. Ann Thorac Cardiovasc Surg 1998, 4:283-284. 6. Mori M, Nakanishi N, Furuya K: Hemangiopericytoma of the mediastinum causing spontaneous hemothorax. Ann Thorac Surg 1994, 58:1525-1527. g 7. Gomez Finana MS, Paya Perez L, Parede Osaelo JR, Aranda Lopez I, Massuti Sureda B, Talavera Sanchez J: Hemangiopericytoma of the soft palate and mediastinum: a case report. Acta Otorri- nolaringol Esp 1994, 45:465-468. g p 8. Fukunaga M, Shimoda T, Nikaido T, Ushigom S, Ishikawa E: Soft tis- sue vascular tumor. A flow cytometric DNA analysis. Cancer 1993, 71:2233-2241. 9. Yoshida M, Morita M, Kakimoto S, Kawakami M, Sasaki S: Primary hemangiopericytoma of the trachea. Ann Thorac Surg 2003, 76:944-946. 10. Baldo X, Sureda C, Gimferrer JM, Belda J: Primary mediastinal lei- omyoma. Eur J Cardiothorac Surg 1997, 11:574-576. y J g 11. Fiumara E, D'Angelo V, Florio FP, Nardella M, Biscelia M: Preopera- tive embolization in surgical treatment of spinal thoracic dumpbell schwannoma. J Neurosurg Sci 1996, 40:153-156. p J g 12. Hansen CP, Francis D, Bertelsen S: Primaryhemangiopericytoma of the lung. Authors' contributions AC, NB, AE took part in the care of the patient and con- tributed equally in carrying out the medical literature search and preparation of the manuscript. IF participated Page 5 of 6 (page number not for citation purposes) (page number not for citation purposes) http://www.wjso.com/content/4/1/23 World Journal of Surgical Oncology 2006, 4:23 in the care of the patient and had the supervision of this report. All authors approved the final manuscript. in the care of the patient and had the supervision of this report. All authors approved the final manuscript. Acknowledgements Written consent of the patient was obtained for publication of this case report. References Scand J Thorac Cardiovasc Surg 1990, 24:89-92. g J g 13. Rusch VW, Shuman WP, Schmidt R, Laramore GE: Massive pulmo- nary hemangiopericytoma. An innovative approach to eval- uation and treatment. Cancer 1989, 64:1928-1936. 14. Jalal A, Jeyasingham K: Massive intrathoracic extrapleural hemangiopericytoma: deployment of radiotherapy to reduce vascularity. Eur J Cardiothorac Surg 1999, 16:378-381. y J g 15. Mira JG, Chu FCH, Fortner JG: The role of radiotherapy in the management of malignant hemangiopericytoma – report of 11 cases and review of the literature. Cancer 1975, 39:1254-1259. 16. Jha N, McNeese M, Barkley HT Jr, Kong J: Doesradiotherapy have a role in hemangiopericytoma management? Int J Radiat Oncol Biol Phys 1987, 13:1399-1402. y 17. Morandi U, Stefani A, De Santis M, Paci M, Lodi R: Preoperative embolization in surgical treatment of mediastinal hemangi- opericytoma. Ann Thorac Surg 2000, 69:937-939. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 6 of 6 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." 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Same Same but Different: Exploring the Effects of the Stroop Color Word Test in Virtual Reality
Lecture notes in computer science
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To cite this version: Romina Poguntke, Markus Wirth, Stefan Gradl. Same Same but Different: Exploring the Effects of the Stroop Color Word Test in Virtual Reality. 17th IFIP Conference on Human-Computer In- teraction (INTERACT), Sep 2019, Paphos, Cyprus. pp.699-708, ￿10.1007/978-3-030-29384-0_42￿. ￿hal-02544574￿ Same Same but Different: Exploring the Effects of the Stroop Color Word Test in Virtual Reality Romina Poguntke, Markus Wirth, Stefan Gradl To cite this version: Romina Poguntke, Markus Wirth, Stefan Gradl. Same Same but Different: Exploring the Effects of the Stroop Color Word Test in Virtual Reality. 17th IFIP Conference on Human-Computer In- teraction (INTERACT), Sep 2019, Paphos, Cyprus. pp.699-708, ￿10.1007/978-3-030-29384-0_42￿. ￿hal-02544574￿ Distributed under a Creative Commons Attribution 4.0 International License 1 University of Stuttgart, Germany {firstname.lastname}@vis.uni-stuttgart.de 2 Friedrich-Alexander-Universit¨at Erlangen-N¨urnberg (FAU), Germany {firstname.lastname}@fau.de 1 University of Stuttgart, Germany {firstname.lastname}@vis.uni-stuttgart.de 2 Friedrich-Alexander-Universit¨at Erlangen-N¨urnberg (FAU), Germany {firstname.lastname}@fau.de Abstract. Virtual reality (VR) is used for different trainings e.g. for pi- lots, athletes, and surgeons. Dangerous and difficult situations are often focused in such simulations in VR, targeting to learn how to perform well under stress. However, there has been little work on understanding stress perception in VR compared to the real-world situation. In this paper we present an investigation of how users experience a stressful task in VR compared to in a classic office environment. Specifically, we investigate the subjective stress experience and physiological arousal with 15 partic- ipants performing the Stroop color word test either on a regular desktop screen, in VR, or in VR requiring head movements. Our findings suggest that stressful tasks are perceived less stressful when being performed in VR compared to the real environment as long as there is no additional stress factor, such as head movement involved. Our work indicates that it may be valuable to transfer stressful tasks, currently done in traditional office environments into VR. Keywords: Virtual Reality · Stress · Human-Computer Interaction. HAL Id: hal-02544574 https://inria.hal.science/hal-02544574v1 Submitted on 16 Apr 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Same Same But Different: Exploring the Effects of the Stroop Color Word Test in Virtual Reality Romina Poguntke1, Markus Wirth2, and Stefan Gradl2 Romina Poguntke1, Markus Wirth2, and Stefan Gradl2 1 University of Stuttgart, Germany {firstname.lastname}@vis.uni-stuttgart.de 2 Friedrich-Alexander-Universit¨at Erlangen-N¨urnberg (FAU), Germany {firstname.lastname}@fau.de 1 Introduction and Background Virtual reality (VR) is becoming increasingly popular for researchers in differ- ent domains and for various purposes, e.g. rehabilitation [6, 18, 25], sports [24], and gaming [5]. Moreover VR has been proven to be an important tool for sim- ulating stressful and cognitively demanding scenarios in e.g. military trainings [3, 5], pilot flight trainings [13, 14], other dangerous working environments, such as petrol refinery [7]. Aiming to train soldiers, flight pilots, and also firefighters stress elicitation in VR has pointed towards an important research gap. To not interrupt immersion in the VR scenario and elicit stress at the same time, there is a need for effectively stress inducing tasks that can be applied in VR. Aiming to elicit stress in VR, Legkov et al. [12] asked participants to react on approach- ing objects in a space shooter scenario. Measuring the Galvanic Skin Response (GSR) and the subjective stress level, they found that their dual task paradigm increased physiological arousal and affected certain stress dimensions. J¨onsson et al. [10] validated if the Trier Social Stress Test (TSST) [11], a standardized R. Poguntke et al. 2 and validated task mainly used for laboratory studies, performed in VR also induces stress. They found that the cortisol level in the participants’ saliva had increased by 88% and also the heart rate and heart rate variability showed a solid indication of experienced social stress. Based on these results, stress induction in VR has been studied [1, 4, 17]. However, there has been little work on the role of stress in VR. Particularly the investigation of the effects of stress elicitation in virtual environments compared to classic desktop based scenarios has been neglected so far. VR represents a promising approach to be applied as a training technique for learning to deal with heavy stressors. To address this research gap, we explore the transferability of stressful tasks from office to virtual environ- ments using the Stroop color word test [21], a well established stress elicitation task [26], to observe stress perception in VR. With this work, we contribute the first investigation of transferring it from its classic desktop screen version into VR. 2 Implementation Subsequently, we will explain what the Stroop color word test is and how we implemented it in our three experimental conditions. 1 Introduction and Background Through the stress assessment based on a three dimensional structure comprising engagement, worry, and distress, and the evaluation of heart rate variability, we provide first insights in the exploration of stressful tasks in VR and prospective effects of transferring these tasks from reality into virtual environments. 2.1 Stroop Color Word Test Among the classical version of the Stroop color word test [21] and several dis- tinct versions that exist, we took the Stroop color word version for this work presenting both, the congruent and incongruent condition (cf. Figure 2). This task is commonly used in HCI [26] and intensifies physiological reactions [20], moreover it can be easily implemented in VR which is an advantage when con- sidering the transferability of a stress inducing task. While ’congruent’ means that words are displayed in the color that they signify, ’incongruent’ refers to (a) Desktop cond. (b) VR cond. (c) VR head movement cond. Fig. 1: Depicts the three conditions compromising our independent variable. (c) VR head movement cond. (a) Desktop cond. (b) VR cond. Fig. 1: Depicts the three conditions compromising our independent variable. The Stroop Test in VR 3 the incongruence between the color of the word and in its actual meaning. For example, in the congruent condition the word ’red’ is being presented in red and in the incongruent conditions it is painted in blue (cf. Figure 1). Hereby the participant’s task is to name the word’s color and neglect the color that the word is designating. 3.1 Measures As independent variables, we designed three different conditions how the Stroop color word test should be performed: (a) sitting in front of a desktop screen and all stimuli presented in the centered field of view, (b) in VR and the stimuli also presented in the centered field of view, and (c) in VR but the stimuli appeared as described above in the visual field of the participant. The latter condition was introduced based on the observations that the vestibular system being involved in head movements, was found to have an influence on the susceptibility to motion sickness [19]. Thus, we were interested if requiring head movements to accomplish the task would increase stress perception. As dependent variables we assessed the subjectively perceived stress level employing the Short Stress State Questionnaire (SSSQ) [8]; also used by Legkov et al. [12] to observe stress reactions induced in VR. Likewise, we recorded physiological data, i.e. heart rate (HR) and heart rate variability (HRV) to monitor if the physiological arousal corresponding to stress also varies among our conditions as could be shown in related work for VR experiences in general [2, 18]. For recording physiological data we used the Nexus Kit 4 by MindMedia4. 2.2 Implementation of the Stroop Test in VR For our user study setup, we utilized the HTC Vive virtual reality system con- nected to a PC consisting of a 3.7 GHz Intel Xeon processor, 16 GB RAM and a GeForce GTX 970 graphics card with a display size of 17,3 inch (Full-HD, 1.920x1080) (cf. Figure 1a). To render the stimuli of the Stroop color word test, we employed the Unity 3D engine3. For each condition, two sequences of 120 randomly selected Stroop items were generated at the beginning. Randomiza- tion was based on the subject ID, thus an individual sequence was generated for each participant. To get an equal distribution of Stroop items among all partic- ipants, two initial static buckets containing all items were created: one for the incongruent and one for the congruent test. The incongruent bucket contained each possible color combination ten times, the congruent bucket each stimulus 30 times. For each participant, the sequence of Stroop items that was presented, was randomly drawn from these buckets until they were empty. For the desktop screen and the VR condition, all stimuli were displayed in the centered field of view. For the VR head movement condition, the Stroop items were displayed at a pseudorandom position in the field of view of the participants in the virtual reality. This means that a random position was selected in either the left/right (50 degrees from the center field of view) or lower/upper (50.5 degrees from the center field of view) hemisphere of the subject with the constraint that no hemisphere could be selected two subsequent times. Fig. 2: Study design showing the sequence of trials including the three conditions (desktop screen, VR, VR requiring head movements). We let the participants perform a practice trial before each of the two trials (incongruent, congruent) started, resulting in four trials in total preceded by a five minutes resting period. Fig. 2: Study design showing the sequence of trials including the three conditions (desktop screen, VR, VR requiring head movements). We let the participants perform a practice trial before each of the two trials (incongruent, congruent) started, resulting in four trials in total preceded by a five minutes resting period. 4 R. Poguntke et al. 4 R. Poguntke et al. R. Poguntke et al. 3 User Study For our user study we invited 15 participants and randomly split them into three equally sized groups, resulting in an in-between-subject design. Whereas one group experienced the Stroop color test in VR and another group performed head movements in VR, the third group conducted the test on a regular desktop screen positioned on an office desk. We randomized the sequence of three conditions for each participant according to Latin square. 4 https://www.mindmedia.com/de/produkte/nexus-4/ 3.2 Procedure and Data Collection Before we started with the evaluation, participants were explained the study’s purpose and procedure. After giving their written consent, we asked each one to place the three gel electrodes connected to a two channel ExG sensor for assessing HR and HRV to themselves; meaning the negative (black) electrode was attached at the right collar bone, the positive (red) electrode on the midaxial line on the lateral aspect of the chest, and the ground electrode near the right leg on the chest. Inspired by previous study designs [22], we specified the sequence of trials as depicted in Figure 2. Before the initial resting phase of five minutes, baseline measurements started, each participant was asked to fill in the first part (’At the moment’) of the Short Stress State Questionnaire [8] for assessing the baseline stress level. Then, there was a one minute practice phase; by this we ensured that everyone understood the task. It was followed by an eight minute incongruent phase. During that time the participants were presented 120 words, either on the desktop screen or in VR, for 3 seconds intermitted by one second. After another 5 The Stroop Test in VR five minutes of resting and a one minute practice trial, the congruent phase lasted eight minutes followed, presented in the same manner as the incongruent condition. When the last Stroop color test trial was completed, all participants were asked to complete the second part of the Short Stress State Questionnaire referring to their stress perception ’During the task’. Fig. 3: Participant wearing the HTC Vive VR glasses performing the Stroop test in VR while measuring heart rate and heart rate variability. Fig. 3: Participant wearing the HTC Vive VR glasses performing the Stroop test in VR while measuring heart rate and heart rate variability. 5 https://www.mindmedia.com/en/products/biotrace-software/ 3.3 Participants Our sample consisted of 15 participants (M = 23.5, SD = 2.5 years), among these were seven females – in each group at least two females. The majority (ten people) were VR novices, while three stated to have some VR experience and two others said they had been using VR ”a lot”. Recruiting our participants via uni- versity mailing lists and personal acquisition, we had eleven students, one PhD student, one teacher and two engineers among our sample. The experimental procedure had been approved by the ethics committee of our institution. 4 Results For the physiological data analysis, we removed the first and last 30 seconds of the baseline and the experimental (incongruent, congruent) sessions to avoid primacy effects. Prior to the statistical calculations, we normalized the data ac- cording to each participant’s baseline values. We focused on the physiological measures HR and HRV using the standard formula provided by the manufac- turers processing software 5 for HRV value calculation. R. Poguntke et al. 6 HR HRV incongr. con. incongr. con. Desktop1.00 (0.02) 1.00 (0.06) 1.12 (0.37) 1.26 (0.66) VR 1.04 (0.23) 1.01 (0.22) 0.99 (0.60) 1.25 (0.45) VR-hm 1.08 (0.07) 1.04 (0.06) 0.93 (0.36) 1.05 (0.56) Table 1: Table shows means and standard deviations of the incongruent and congruent trials for heart rate and heart rate variability according to the three different conditions. While high values indicate increased arousal for HR, lower values are associated with high arousal for HRV [23]. Table 1: Table shows means and standard deviations of the incongruent and congruent trials for heart rate and heart rate variability according to the three different conditions. While high values indicate increased arousal for HR, lower values are associated with high arousal for HRV [23]. Physiological Measures During the incongruent trial, participants showed slightly higher HR values in the VR condition and also for the head movement condi- tion, compared to performing the Stroop test in front of a desktop screen (cf. Table 1). For the congruent trial, the results were similar to those for the in- congruent one, but overall the values were little lower as can be obtained from Table 1. Regarding the HRV, we observed that during the incongruent condition in VR, participants had lower HRV values in both conditions, namely VR and VR requiring head movements, indicating physiological arousal (cf. Table 1). In front of the desktop screen, the HRV was higher with an average of 1.12. Again, these results are similar to the HRV values recorded during the congruent trial showing that this trial resulted in lower physiological arousal (cf., Table 1). Performance As performance measures, we recorded the error rate. Most errors in the incongruent condition were made in VR requiring head movements (M = 1.4, SD = 2.2) followed by the Stroop at a desktop screen (M = 1.0, SD = 1.0) and in VR-only the average errors were 0.6 (SD = 1.3). 4 Results In the congruent condition there occurred only one error in VR requiring head movements. Subjective Measures For the SSSQ results, we calculated the difference between the pre-test and post-test SSSQ scores ((post-score −pre-score) / pre-score standard deviation) [15] resulting in a so-called change score. Hereby positive scores signify a higher stress rating after the task was accomplished and nega- tive change scores mean that stress was higher before the task. Since the SSSQ has an underlying three factorial structure divided into Disstress, Worry, and Engagement [9] we present the results according to these factors in Figure 4. The desktop screen condition induced the most distress with an average change score of 12.45 (SD = 12.90), while for the Stroop test performed in VR, the participants felt almost not stressed at all (M = −0.57, SD = 1.27). When head movements in VR were required, the distress increased to an average score of 3.50 (SD = 9.37). Referring to the sub-dimension worry, there was no change when the Stroop test was performed in front of a desktop screen (M = 0.02, The Stroop Test in VR 7 Fig. 4: Results from the SSSQ [8] according to its three dimensions Engagement, Disstress, and Worry showing that the Stroop test in VR does not have been perceived as stressful in VR as in the desktop screen condition. Fig. 4: Results from the SSSQ [8] according to its three dimensions Engagement Disstress, and Worry showing that the Stroop test in VR does not have been perceived as stressful in VR as in the desktop screen condition. Fig. 4: Results from the SSSQ [8] according to its three dimensions Engagement, Disstress, and Worry showing that the Stroop test in VR does not have been perceived as stressful in VR as in the desktop screen condition. SD = 7.39). And for both VR conditions we observed that worry was even de- creasing compared to the baseline measurement, namely to an average of -1.81 in VR (SD = 1.93) and to -3.48 (SD = 3.78) when head movements were required in VR. For engagement there was no difference in the VR condition (M =-0.42, SD = 3.65) and for the desktop screen condition (M =-1.40, SD = 4.51) as well as the head movement requiring one (M =-1.28, SD = 2.48), there were only minor changes signifying that engagement was lower after the Stroop test. 4 Results Inferential Statistical Analysis Since our data was not normally distributed, which is required for parametric tests, we used non-parametric tests. Thus, we performed a Kruskal-Wallis test aiming to reveal differences among our three conditions. No significant results could be found here. We further investigated correlations between the different variables. For this, we used the Spearman rank coefficient which is also robust against outliers in our data. We found a strongly positive correlation between the stress assessing SSSQ overall score and it’s two underlying dimensions distress (r = .862, p = .000) and worry (r = .601, p = .018). 5 Discussion Our results show that the participants subjectively perceived the task on a desk- top screen as the most stressful (M = 12.45), whereas the same task in VR has been rated almost not stressful at all with an average of -0.57 signifying that there has been almost no difference between the stress level before and after the task. The participants rated the VR condition requiring head movements R. Poguntke et al. 8 more stressful (M = 3.50), which suggests that the involvement of motor skills acts as an additional stress factor. Whereas the standard deviation of distress perception in VR had been low with an average of 1.27, it was exceeded by the two other conditions. Particularly in the desktop screen variant, there were two participants for whom there was almost no change between the distress level before and after the Stroop test, while three other participants felt enormously stressed having a change score of 14.14 and respectively 19.80. These differences underline the subjective perception of stress that is challenging [26]. Moreover, our results are supported by prior work [12] where the SSSQ was applied and dis- tress increased with the presentation of a stressful task, while worry decreased. In contrast, during the two most stressful conditions, desktop screen and VR re- quiring head movements, engagement was low after performing the Stroop test (M = −1.40, M = −1.28) indicating that stress dominated then. This is fur- ther strengthened by the correlational analysis revealing that engagement was the only dimension of the SSSQ that didn’t correlate significantly with its total score and thus insufficiently reflected the participant’s stress perception. Again, in VR there was almost no difference before and after the task (M = −0.42). For worry we found almost no difference when being performed on a desktop screen (M = 0.02). In the VR conditions it even decreased after finishing the task (M = −1.81, M = −3.48) what can be explained with a feeling of relief after having accomplished the test. Regarding the physiological data we recorded, the results show that our participants had lower arousal values in HR (M = 1.00) and HRV (M = 1.12) during the desktop screen task. 5 Discussion While arousal had been mild but slightly higher in the VR condition for HR (M = 1.04), and HRV (M = 0.99), there was a greater rise in HR (M = 1.08) and respectively a decrease in HRV (M = 0.93). These findings show that the participants experi- enced higher physiological arousal in both VR conditions, what is supported by the results for the subjective measures. However, performing motor skills seems to increase only the subjective stress perception but does not affect physiological arousal. Thus, the Stroop color word test seems not suitable for inducing stress in participants when it is transfered into VR. To successfully evoke subjectively perceived stress, there is the requirement of moving the head as an additional factor. This is in line with the findings from research on the reason for why head- mounted displays (HMDs) used for VR are causing visual stress. Mon-Williams et al. [16] stated the that vertical gaze angle is a crucial factor and that there- fore the HMD needs to be placed in the correct vertical position for each user individually. Consequently, the presentation of the stimuli in the virtual space shifted on both, the x- and y-axis, could have provoked a level of stress in the user that is perceived only subjectively. Although we believe, that this piece of work yields important insights in the perception and the transfer of stress in VR, we have to acknowledge that due to our limited number of participants, future work should repeat this experi- ment involving a greater sample so that the observed tendencies can be verified statistically. Nevertheless, our results show that inducing stress in VR cannot 9 The Stroop Test in VR The Stroop Test in VR be adopted on a one to one basis for VR and thus could benefit from further investigations, particularly focusing on the design of stressful tasks for VR. 6 Conclusion and Future Work In this paper, we explored whether a stressful task can be transferred into VR. The results show that participants felt higher distress and lower engagement when the test was performed in the office environment compared to the VR condition. Likewise, the involvement of motor skills in the virtual environment led also to higher distress and lower engagement, what could only be observed in the subjective data. Hence, our findings suggest that the Stroop color word test is not suitable for inducing stress when being performed in VR and when being adopted one to one. To successfully evoke subjectively felt stress, e.g. as part of an VR flight simulation scenario to practice reactions under pressure, an additional requirement is needed, e.g. to perform motor skills. Consequently future work should focus on the exploration and determination of suitable motor skill tasks in VR to elicit stress. Through the initial exploration of the transferability of stressful tasks into VR, we believe to provide a valuable starting point for further investigations in the underlying mechanics, to ultimately design effective training scenarios for VR. Acknowledgements This work was partly conducted within the Amplify project funded from the European Research Council (ERC) (grant agreement no. 683008). 5. Fong, G.: Adapting cots games for military simulation. In: Proc 2004 Int Conf Virtual Reality Continuum Applications Industry. pp. 269–272. VRCAI ’04, ACM, New York, NY, USA (2004) References Psychoneuroendocrino 35(9), 1397 – 1403 (2010) 11. Kirschbaum, C., Pirke, K.M., Hellhammer, D.H.: The ’trier social stress test’ - a tool for investigating psychobiological stress responses in a laboratory setting. Neuropsychobiology 28 (1993) 12. Legkov, P., Izdebski, K., K¨archer, S., K¨onig, P.: Dual task based cognitive stress induction and its influence on path integration. In: Proc 23rd Symp Virtual Reality Software Technology. pp. 41:1–41:5. ACM, New York, NY, USA (2017) 13. Loftin, R.B., Kenney, P.: Training the hubble space telescope flight team (1995) 14. Markov-Vetter, D., Moll, E., Staadt, O.: Evaluation of 3d selection tasks in parabolic flight conditions: Pointing task in augmented reality user interfaces. In: Proc 11th Int Conf Virtual Reality Continuum Applications Industry. pp. 287–294. VRCAI ’12, ACM, New York, NY, USA (2012) ( ) 15. Matthews, G., Joyner, L., Gilliland, K., Campbell, S., Falconer, S., Huggins, J.: Val- idation of a comprehensive stress state questionnaire: Towards a state ’big three.’. In: Personality Psychology Europe. pp. 335–350. Tilburg University Press (1999) ( ) 16. Mon-Williams, M., et al., A.P.: Gaze angle: a possible mechanism of visual stress in virtual reality headsets. Ergonomics 41(3), 280–285 (1998) 17. 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J¨onsson, P., Wallerg˚ard, M., ¨Osterberg, K., Hansen, r.M.e.a.: Cardiovascular and cortisol reactivity and habituation to a virtual reality version of the trier social stress test: A pilot study. References Wirth, M., Gradl, S., Poimann, D., Schaefke, H., Matlok, J., Koerger, H., Es- kofier, B.M.: Assessment of perceptual-cognitive abilities among athletes in virtual environments: Exploring interaction concepts for soccer players. In: Proc 2018 De- signing Interactive Sys Conf. pp. 1013–1023. ACM, New York, NY, USA (2018) ( ) 25. Yeh, S.C., Rizzo, A., Zhu, W., Stewart, J., McLaughlin, M., Cohen, I., Jung, Y., Peng, W.: An integrated system: Virtual reality, haptics and modern sensing tech- nique (vhs) for post-stroke rehabilitation. In: Proc ACM Symposium Virtual Real- ity Software Technology. pp. 59–62. VRST ’05, ACM, New York, NY, USA (2005) 26. Zhai, J., Barreto, A.: Stress recognition using non-invasive technology. Proc Int Florida Artificial Intelligence Research Society Conf. pp. 395–400 (2006)
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Bounded Responsibility and Bounded Ethics of Science and Technology
Axiomathes
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Axiomathes (2020) 30:597–616 https://doi.org/10.1007/s10516-020-09514-7(0123456789().,-volV)(0123456789().,-volV) Axiomathes (2020) 30:597–616 https://doi.org/10.1007/s10516-020-09514-7(0123456789().,-volV)(0123456789().,-volV) https://doi.org/10.1007/s10516-020-09514-7(0123456789().,-volV)(0123456789().,-volV) ORIGINAL PAPER & Gu¨nter Abel abel@tu-berlin.de 1 Institute of Philosophy, Technical University Berlin (TUB), Sekr. H 72; Room H 7150/51, Straße des 17. Juni 135, 10623 Berlin, Germany Bounded Responsibility and Bounded Ethics of Science and Technology Gu¨nter Abel1 Received: 4 August 2020 / Accepted: 31 August 2020 / Published online: 16 September 2020  The Author(s) 2020 1 What is at Stake? In what follows, what will be at stake is responsibility in science and technology. More precisely, what is at stake in these fields are, on the one hand, specific questions regarding e.g. the different types of responsibility in science and technology, the basic rules of proper scientific practice, or the validity of scientific standards. On the other hand, there is the more fundamental question of where the normativity of the ethics of science and technology comes from and how it functions. This Where-from-question can be completed by the two further questions of why we need an ethics of science and technology at all and what exactly it is for. These three W-questions are basal. They reach beyond the specific questions mentioned above. The challenge that the answers to these latter questions face can be emphasized as follows: Where does normativity come from when the classical and metaphysical reservoirs of convenience are no longer at our disposal the way they used to be? Such reservoirs traditionally used to be available, e.g., in religious instances or in a realm of previously secured ethical values and norms, in other words: in a metaphysical universalism. How do we free ourselves from the predicament of requiring normativity but not being able to import it from some external domain or external authorities? But fortunately there are starting points that can help us out here. In this paper, I will particularly elucidate the following three starting points with focus on the ethics of science and technology. First (a) we develop the conception of a human-oriented bounded responsibility and bounded ethics focused on domain-specific as well as concrete problem solving (Sects. 2 and 3). Then (b) we can, from a reflective point of view, go back into what I call practice-internal normativity (Sects. 3 and 4). With this term I want to address that type of normativity that should not be conceived in the sense of external rules and criteria, but in the sense of human and practice-internal rule following. That is the kind of normativity that we are interested in under the sketched conditions of the loss of a metaphysical comfort zone. The thesis is that this normativity is always already presupposed in precisely those human life, knowledge, science, and technology practices in which it is internally embodied and relied upon. The normativity we are interested in here is always internal, not external normativity. Abstract The leading question of this paper is: Where does the normativity of the ethics of science and technology come from? This is a challenging question given that the traditional reservoirs of convenience (like metaphysical universalism) are no longer at our disposal the way they used to be. The paper is divided into eight sections: (1) It is specified what challenges a non-foundationalist justification and normativity has to meet. (2) A three-dimensional conception of responsibility is developed based on the human triangular I–We–World relations. (3) The concepts of bounded responsibility and bounded ethics of science and technology are formulated. (4) The principle of reflective equilibrium is introduced as a principle of rationality, and it is shown how this principle generates rational and reasonable justifications in the ethics of science and technology. (5) Against this background, a reconception of internal and external responsibilities of science is given. (6) The type of responsi- bility demanded is exemplified by today’s climate research. (7) The paper argues for a hand-in-hand model of uncertainties in the sciences and for ethical obligations to preserve the conditions of human life on earth. The ethical argument is spelled out in terms of ethical care, preservation, and precaution. (8) Additionally, some arguments are developed to answer the question of why it is reasonable at all to preserve human life on earth. Keywords Normativity in ethics of science  Bounded responsibility  Bounded ethics  Principle of reflective equilibrium  Internal and external responsibilities  Uncertainties in the sciences  Climate research 123 123 598 Axiomathes (2020) 30:597–616 1 What is at Stake? Finally (c) we are, as finite human beings, systematically (not only contingently) cut off from a realm of universalistic norms and values. But at the same time we can shape and justify the relation between, e.g., our considered moral judgments and our general ethical principles in such a way that the validity as well as the justification of the values and norms is warranted. This is where the Principle of Reflective Equilibrium comes into play. I would like to apply this principle in view of the above-mentioned predicament, i.e. the loss of metaphysical comfort zones, as well as in view of the foundation of bounded responsibility and bounded ethics (Sect. 4). Trivially, in this altered perspective everything depends on the realization that it is ourselves, as humans, who actively, in an evidence-based and problem-solving 123 Axiomathes (2020) 30:597–616 599 way, and in virtue of our practice-internal normativities try to maintain our human life practices and expand our competencies and our scope of action. way, and in virtue of our practice-internal normativities try to maintain our human life practices and expand our competencies and our scope of action. Likewise, we can name further areas of the image and concept of man illustrated in this paper. Furthering these areas is, humanely viewed, a central task of the ethics of science and technology. These areas include the possibilities of: (a) taking an active part in shaping and orienting our life practices; (b) furthering milieus in which our human capacities can develop; (c) opening spaces for the generation of something new, i.e. milieus of creativity; (d) warranting human education in the age of scientific and technological civilization; (e) opening, securing, expanding, and strengthening the realms of human autonomy; and (f) keeping open and strengthening our human disposition to make rational and reasonable decisions. This latter point must be emphasized particularly in view of irrational and unreasonable decisions, which are always possible. Reasonability is a human disposition, not a natural property. We can choose rationality and reasonability or not. The humane ethics of science and technology must not lose sight of these crucial points. But let us first focus on the concepts of three-dimensional responsibility (Sect. 2), bounded responsibility, and bounded ethics (Sect. 3). In all following deliberations (Sects. 2 to 8), I will also make reference to the three major challenges of our time: climate change, pollution, and pandemics. 1 What is at Stake? We will be able to elucidate the conception of the responsibility of science and technology using the example of climate research (Sect. 6). These elucidations will be carried out in such a way that we, on the one hand, not only do not deny uncertainties in the sciences and its modelings, but rather make them more precise and spell them out explicitly (Sect. 7). On the other hand, however, it is emphasized that the ethical obligation to preserve the conditions of human life by means of appropriate measures of preservation, precaution, and care is independent from the remaining uncertainties in scientific theories, models, and chaotic developments of dynamic systems. Some deliberations on the questions of why it is rational and reasonable at all to preserve the conditions of human life on this planet constitute the conclusion of this paper (Sect. 8). Throughout these points and sections the above-mentioned normativity plays the ultimately decisive role in the ethics of science and technology. 2 Three-dimensional Responsibility In what follows, I want to make the case for a three-dimensional scenario that constitutes a significant expansion of the classical field of ethical responsibility, care, preservation, and precaution. In this view, the transition from an ethics particularly focused on subjectivity and intersubjectivity into the triangular responsibility and ethics of the I–We–World/Nature relations, i.e. into a, in this sense, three-dimensional responsibility and three-dimensional ethics, is imperative. Responsibility is a complex and multilayered phenomenon. In sum, the word means to stand up for one’s own actions, ascribe them to oneself or other persons or institutions, and account for them. And thus it means to be held accountable for the consequences of one’s own actions (or one’s omission to act). This aspect of 12 3 600 Axiomathes (2020) 30:597–616 standing up for one’s actions is completed by the aspect of taking responsibility in the sense of actively participating in the development of solutions to concrete problems. The latter aspect stands out especially when we have to face the challenges of life, society, and nature with future-oriented solutions. Facing responsibility and contributing to solutions to those challenges is what is at stake. The second sense of the talk of responsibility is currently important in view of the challenges in terms of climate change, environmental pollution, and pandemics. Responsibility in the first sense means that science and technology must be accountable for their actions and artifacts. In the second sense of the talk of responsibility we expect science and technology to provide future-oriented and sustainable solutions (e.g., to develop and provide a vaccine in the case of the current coronavirus pandemic). As human beings we do not live secondarily, but primordially in our above- mentioned triangular human I–We–World/Nature relations. In this sense, man is the relational being par excellence. Obviously, the single components of these triangulations cannot be strictly isolated against one another. I deliberately chose the triangular model. We know the triangle as a musical instrument. One of this instrument‘s characteristic features is that when one of its three sides is struck, the other two sides chime as well. Transferring this triangular model, we can assume that the inseparability of our human I–We–World/Nature relations also has consequences for our moral and ethical responsibilities in the world, towards other persons, ourselves, and nature. If one of the relations becomes topical or even conflictive, the other two are always already at play. 3 Bounded Responsibility and Bounded Ethics The human triangular I–We–World/Nature relations can also be examined in terms of their interconnections, entanglements, situatedness, embodiments, and entrench- ments, i.e. in terms of their different types of boundedness. This internal boundedness to concrete contexts, challenges, situations, and problems requires, when it comes to responsibility and ethics, what we can call bounded responsibility and bounded ethics.1 These two conceptions are not defined by or dependent on abstract, formal, and universalistic principles. Rather they can be seen as conceptions deeply rooted in our human life practices themselves. This fundamental change of perspective also bears consequences for the answers to our three W-questions regarding the normativity of the ethics of science and technology. The vector of attention is shifting. The consequence is that we should not expect or look for the solutions to problems of our life, knowledge, and science practices in a realm of a universalistic responsibility, ethics, and quasi-eschatolog- ical theories. We should rather, being the finite thinking subjects we are, focus our attention on the concrete problems and challenges right in front of our eyes that require sustainable solutions (e.g. environmental or climate- and pandemic-related problems). Whether or not the concepts of bounded responsibility and bounded ethics can provide ethically responsible, justified, and justifiable solutions to the respective domain-specific and concrete problems is thus of crucial importance. This is the first sense of the talk of bounded responsibility and bounded ethics. A second sense of this talk lies in the fact that the concrete, considered, and ethically responsible solutions to problems must have the status of justified or, if necessary, justifiable solutions. Consequently, justified and considered judgments and actions are of particular importance in our life practices. Given that scenario, we have to ask whether there is a principle which could take up the functions of the former metaphysical comfort zone and science-related ethical universalism. I would like to propose ‘‘The Principle of Reflective Equilibrium’’ as an appropriate candidate for these functions (see upcoming Sect. 4). A third sense of the talk of ‘bounded responsibility’ and ‘bounded ethics’ results from the irreducible but heterogeneous variety of concrete and highly diverse challenges in the context of ethical responsibilities. Thus it is important to know what kind and domain of concrete challenges in terms of responsibility and ethics we are dealing with, for example: (a) with responsibility in the domain of human health (e.g. 2 Three-dimensional Responsibility The triangular model transports another important message. We are always, even in view of ever trans-subjective ethics, dealing with not only a bipolar boundedness of I–We relations, but with a tripolar boundedness in the sense of our human I–We– World/Nature relations. This tripolar understanding of the talk of trans-subjective is important in this context because the bipolar talk of an inter-subjective ethics (e.g., an inter-subjective discourse ethics) primarily focuses on the relations between the subjects involved and thus, so to speak, runs the risk of leaving out nature. In the proposed three-dimensional model of the I–We–World/Nature relations, however, nature is included from the start (which is particularly relevant these days in view of the current environmental, climate-, and pandemic-related challenges). We do not have to bring nature into play in a second step (or by means of an additional material argument). Nature is always already included, whether we like it or not, and it has ethical values of its own. In the light of this expansion, I would like to argue the case for this kind of three- dimensional conception of responsibility and ethics. This point is crucial not only in view of the current debates on climate change, environmental issues, and pandemics. It is also relevant particularly because from now on the question of the normativity of the ethics of science and technology does not only refer to each one of us as an individual first-person subject. And neither does it simply refer to our fellow human beings as co-subjects. Instead, nature, surroundings, and environment as well as human physical health are included as legitimate recipients of responsibility and ethics. They are conceived as new and equal residents in the house of ethics. If these findings seem trivial,—then all the better. 123 Axiomathes (2020) 30:597–616 601 1 I use these two expressions with reference to Herbert A. Simon’s concept of ‘‘bounded rationality’’. See Simon (1956). The concept of bounded rationality was significantly extended and expanded in Gigerenzer and Selten (2001). 3 Bounded Responsibility and Bounded Ethics a doctor’s decision for or against performing a high-risk surgical procedure); (b) with responsibility for a section of nature and environment (e.g. with questions concerning the use of chemicals in farming and agriculture); (c) with a specific responsibility in view of animal welfare (animal ethics); or (d) with the effects a certain type of diet can have on our health and wellbeing. Simply put, the profiles, problems, challenges, and solutions of moral and ethical responsibility can present themselves very differently in the various fields, respects, and perspectives 12 123 602 Axiomathes (2020) 30:597–616 depending on whether we are dealing with bounded responsibility in cases of, e.g., medical, ecological, animal-related, dietary, or other problems and challenges. Likewise, the responsibilities of, e.g., a pilot, a teacher, a climatologist, a football coach, a conductor, a prosecutor, or a member of another profession are obviously very different. Hoping for a universalistic authority, i.e. the one and only ultimate authority, or a ‘tertium comparationis’ in these cases would not only be misleading. It would be ultimately irresponsible and could not be reasonably justified. y p y j We certainly can and must ask and determine what characteristics the different types of profession-bounded responsibility have in common. But our experiential realities and real life practices require (first of all and basically) multi-dimensional, concrete, and complex responsibilities of a cooperative and problem-solving kind, not one-dimensional, abstract, and universalistic ones. Critically viewed, it simply cannot be made plausible to conceive of the irreducibly many different types of responsibility merely as special cases of the one and only universalism, without typical individualities and differences among one another, and to try to subsume them under such a construction like ‘the one universalistic concept of responsibil- ity’. The alternatively proposed idea of bounded responsibility and bounded ethics thus also aims at bringing the individuality of challenges concerning ethics and responsibility as well as the individuality of ethos- and ethics-bounded problem solving into the focus of attention. Bounded responsibility and bounded ethics are, so to speak, about nothing less than staying true and committed to humankind and the preservation of the conditions of human life. It must be noted here that the sketched sense of domain-specific, problem-related, and bounded ethics is not simply to be understood in the sense of the talk of ‘applied ethics’. Even the term ‘applied ethics’ is slightly misleading. 3 Bounded Responsibility and Bounded Ethics For this wording presupposes the distinction between ‘pure and universalistic ethics’ on the one hand and an ‘application of this ethics to concrete situations and challenges’ on the other hand. Precisely this dualism, however, is what the concept of bounded responsibility and ethics is not about. Rather, the talk of the boundedness and justification of, e.g., considered moral judgments, ethical principles, actions, and concrete solutions has to do with those considered (and thus not random and arbitrary) judgments, decisions, and actions required in the concrete human practices, situations, problems, and challenges themselves (as, e.g., in the intensive care unit of a hospital). Hence, we could also speak of ‘situated responsibility and ethics’. 4 The Principle of Reflective Equilibrium As soon as we need justification in a situation where questions or even conflicts in the sketched I–We–World/Nature triangulation occur, we search for a method of justification and rational conflict resolution. Against the background of the described scenario (Sect. 3), this needs to be a method and principle that can perform the task without recourse to an abstract universalism and without universalistic and ultimate justifications. I believe that the most promising principle 123 603 Axiomathes (2020) 30:597–616 for this task is the Principle of Reflective Equilibrium.2 This principle seeks (by means of reciprocally adjusting and adapting the respective components in question) to either prove a given state of balance, of equilibrium (and thus of coherence) of the entire system to be already justified, or to create new justifications and secure them for the time being. for this task is the Principle of Reflective Equilibrium.2 This principle seeks (by means of reciprocally adjusting and adapting the respective components in question) to either prove a given state of balance, of equilibrium (and thus of coherence) of the entire system to be already justified, or to create new justifications and secure them for the time being. The method of reflective equilibrium can successfully be applied in the following domains: (1) in the domain of inductive logic (as illustrated by Nelson Goodman in view of the relation between considered judgments and deductions on the one hand, and general rules on the other); (2) in the domain of the sciences (regarding the relation of single scientific judgments to the general rules of scientific theories, which Goodman has also illustrated); (3) in the domain of moral philosophy and ethics (regarding the relation of everyday moral judgments on the one hand and general moral and ethical rules and principles on the other hand). I explicitly want to include in this list (4) the points of overlap between ethics and science and thus the domain of the ethics of science and technology. Accordingly, I would like to use the principle of reflective equilibrium in view of the question of which normative requirements the ethics of science must meet in order to contribute to the orientation of humans and their actions. Furthermore (5), I believe that we can, with recourse to the principle of reflective equilibrium, provide a satisfying characterization of the triangular dynamics and states of our human I–We–World/ Nature relations. 2 This principle was formulated by Nelson Goodman and John Rawls and then further developed by Catherine Z. Elgin. See Goodman (1983) and Rawls (1971). Rawls (1971, p. 20) explicitly makes reference to Goodman’s method in order to emphasize that making reciprocal adjustments between general principles and considered judgments is not only characteristic of moral and legal philosophy, but fundamental to the entire field of deductive and inductive rules and conclusions. On the epistemological aspect of this principle, see Elgin (1996, chap. IV) and (2003). 3 On this last type of equilibrium, particularly the equilibrium of comprehension and the equilibrium of action, see Abel (2018a). 4 The Principle of Reflective Equilibrium The principle can in my opinion also (6) successfully be applied in view of the processes and states of successful and/or unsuccessful and conflicting human communication and cooperation. The two latter domains (5) and (6) primarily have to do with the important role of the principle of reflective equilibrium in the field of language-, sign-, and action-based equilibria of comprehension and action.3 The relation between a generally accepted everyday moral judgment on the one hand and an (also accepted) general ethical principle on the other hand can help illustrate what the principle of reflective equilibrium is about. Think of, e.g., the relation between the moral judgment ‘The fact that Peter pays for the oranges he put in his basket at the grocery store makes it a morally good action’ and the general ethical principle ‘Things that are for sale have to be paid for’. As long as the relation between both sides functions directly, fluently, connectively, and (for the time being) unquestionably, there is no problem. But questions, irritations, disturbances, or conflicts regarding the individual judgment or the general principle and/or their relation can always arise. Think of, e.g., the relation between the moral judgment ‘Isolating the elderly in order to protect them from contracting the coronavirus is a morally good action’ (which judgment has actually been made in the context of the 12 3 604 Axiomathes (2020) 30:597–616 corona pandemic) and the general ethical principle ‘Humans must not be confined’. In this case, the relation between the moral judgment and the general ethical principle does not function fluidly anymore. As a consequence, we no longer view the judgment as justified, but rather exclude it from the category of considered and justified judgments. Such conflicts not only occur in the relations between moral judgments and ethical principles. As mentioned earlier, they can also be found within the sciences. Think of, for instance, the relation between physical judgments of the type ‘‘The astronomical observations XYZ and the calculations ABC show that the earth revolves around the sun’’ and the (pre-Copernican) principle ‘‘The earth is the static center of the universe that is orbited by the sun’’. 4 Goodman (1983, p. 64). John Rawls, who coined the term ‘‘reflective equilibrium’’, explains his choice as follows: ‘‘It is an equilibrium because at last our principles and judgments coincide’’. And it deserves to be called ‘‘reflective since we know to what principles our judgments conform and the premises of their derivation.’’ See Rawls (1971, p. 20). 4 The Principle of Reflective Equilibrium In this perspective, the principle of equilibrium embodies two different performance profiles at the same time: on the one hand (a) the principle includes the request to make explicit a so far assumed 12 3 Axiomathes (2020) 30:597–616 605 state of being justified; on the other hand (b) the principle helps, if necessary, to generate (or reject) new justifications in the face of new challenges. The first case has to do with being able to make explicit, when requested, the state we are usually in (in the above-mentioned example: trusting the considered judgment just as much as the general principle). That also means having to show that both (in our example: judgment and principle/rule) are valid and well-justified in their relation to each other and thus in an equilibrium we accept. Such a state deserves to be called a (for the time being) satisfying and justified state. ‘‘For the time being’’ in this context means as long as no critical requests arise and the directness, fluency, connectiveness, and self-evidence of the assumption of being justified are warranted. But as soon as questions, disturbances, irritations, and conflicts occur, we apply the principle of reflective equilibrium. If in the course of the then made adjustments, balances, and improvements no equilibrium is achieved, we are usually willing to modify and revise both judgment and rule and sometimes even abandon them and replace them with different and new judgments and rules. We find ourselves in a quasi-revolutionary situation when not just one of the two sides (sentence or theory; judgment or principle), but the entire construction itself cannot be maintained anymore. In this case, new paradigms and new background assumptions are required. In both respects (making explicit a given being justified; generating required justifications) the principle of reflective equilibrium can be conceived as a human principle of rationality (see Abel 2016). The principle is also important in view of our appropriate reactions to entirely new and challenging situations as well as in view of the opening of a new space of possible and creative solutions. I would like to bring creativity into play here in the sense that the processes of reciprocal adjustments (conceived as processes of improvements of balance regarding the change and interplay of the components involved) can lead to creative, new, and innovative improvements. 5 On the role of creativity in this context, see Abel (2009). 4 The Principle of Reflective Equilibrium In this case of conflict between considered judgment and general principle (and in contrast to the above-mentioned conflict between moral judgment and general principle), it is a well-known historical and systematic fact that not the object-related propositional scientific sentence, but the (up to that point justified) general theory of the universe lost its validity. In cases where questions, disturbances, and conflicts in the relation between moral judgment and ethical principle arise (or between a physical sentence and a scientific theory), we search for a method to either restore or rearrange the fluid functioning of the relations (for the time being). This is the crucial achievement of the principle of reflective equilibrium. In the reflective process characteristic of this principle, the considered judgments/sentences are pondered against each other and, if possible, brought into a coherent balance, a reflective equilibrium, by means of adjustments. Both the general principle’s and the considered judgment’s being justified, in ethics as well as in science, derive from the prevailing circumstances, i.e. from those life or knowledge practices under which they are established, justified, and accepted. I would like to call this the pragmatic dimension of the principle of reflective equilibrium. Nelson Goodman illustrated the crucial mechanism of the dynamic and adjusting processes of this relation and thus was the first one to formulate the principle of reflective equilibrium: ‘‘A rule is amended if it yields an inference we are unwilling to accept; an inference is rejected if it violates a rule we are unwilling to amend. The process of justification is the delicate one of making mutual adjustments between rules and accepted inferences; and in the agreement achieved lies the only justification needed for either.’’4 At this point I would like to emphasize the internal relation between the principle of reflective equilibrium and the talk of bounded responsibility and bounded ethics (introduced in Sect. 3). As finite thinking subjects—God does not need a principle of reflective equilibrium!—, we are committed to the humane principle of equilibrium as well as to the humane boundedness, situatedness, and entanglement in the circumstances of our life practices. 4 The Principle of Reflective Equilibrium Such improvements can, e.g., also be required in the sciences (and thus in, e.g., the climate, environmental, and pandemic sciences). In the sciences, an improve- ment usually consists in an increase in the precision of the used models, theories, and simulations. Such improvements can be imperative in the face of new data or new empirical observations. The new data and observations are from then on included as components in the (nowadays primarily mathematical and statistical) models. The activities involved in these processes can be conceived as modeling- relevant creativities.5 But the principle of reflective equilibrium can not just be conceived of as a principle of rationality. It can, at the same time, be understood as a principle of humanity. This is possible simply because the principle is a crucial human (and not a divine) method. And the processes of reciprocal adjusting as well as the states of successful balance are characteristic of and desirable to us humans across the entire spectrum of our triangular I–We–World/Nature relations and thus across the entire spectrum of our human experience, perception, speech, thought, action, and creativity. Accordingly, the idea of an ethics of science and technology is essentially 123 123 606 Axiomathes (2020) 30:597–616 a human-related, human-bounded, human-based, human-oriented, and human- orienting matter. In other words: it is a humane matter. Humanity, rationality, and normativity go hand in hand. An ethics of science and technology must be ultimately grounded and anchored in human life, i.e. in our human life practices and circumstances, if such an ethics is supposed to have normative power for us. And it must, at the same time, refer to those life, knowledge, science, and technology practices if it indeed wants to be relevant and orienting for the human triangulation of the I–We–World/Nature relations (and not just get lost in intellectual finger exercises). My answer to the three W-questions posed in Sect. 1 regarding the normativity of the ethics of science and technology (Where does it come from?, Why normativity at all?, What is normativity for?) is thus the following: The required normativity and ethical responsibility can, critically viewed, only be searched for and only be found where we are dealing with what I earlier called the practice-internal normativity and responsibility of our human life, communication, cooperation, action, and orientation practices. 6 On the concept of practice-internal normativity, see Abel (2018b). This paper examines the question of the practice-internal normativity particularly in view of (a) the fluid functioning of speaking a language and (b) successful knowing-how (e.g. riding a bicycle or tying a necktie). In the present paper, I expand this concept of practice-internal normativity to our human life practices in total. 4 The Principle of Reflective Equilibrium The required normativities and responsibilities are not to be found in a separate and external realm of metaphysical and universalistic ideas, but internally in our triangularly bounded life practices themselves. We can reflect ourselves into the individual rules, logics, and mechanisms of these practices. And we do so with the intention of finding and elucidating the human origins and goals of the normativities and responsibilities and making them a guideline for the ethics of science and technology. The application of the principle of reflective equilibrium makes it possible and also necessary to examine whether or not the background assumptions brought to light by such a reflection are justified and whether or not they can be justified. The principle of reflective equilibrium thus explicitly includes the principle of critique. Critically viewed, however, this critique and examination itself can always and only be internal, not external critique.6 7 On this distinction see Lenk (1991, p. 56 ff). 5 The Internal and External Responsibilities of Science Within the described scenario (Sects. 1 to 4), we can now address other and more specific aspects in terms of responsibility in science and technology. These include the distinction between two types of responsibility, (a) the science- and technology- internal responsibility and (b) the science- and technology-external responsibility.7 This distinction also constitutes the crucial difference between the ethos of individual scientists on the one hand, and the ethics of the sciences as specific scientific disciplines on the other hand. Science-internal/ethos responsibility means the obligation towards the rules of proper scientific work with the goal of generating, expanding, and securing knowledge as opposed to merely subjective opinions and ideologies. Internal/ethos 123 Axiomathes (2020) 30:597–616 607 responsibility in the sense of, e.g., scientific best practice, a code of norms, and an ethics of professions inside the scientific community includes, among others, the following values and norms: the obligation to justify beliefs, hypotheses, theories, and models; the obligation not to manipulate data; including new data and observations in the examination; ensuring the transparency of data collection and data evaluation; revising and modifying previous models and theories in the face of new data and observations; the obligation to subject one’s own theories and models to discussion and criticism; the willingness to consider alternative models and theories; the obligation to reexamine previously used models in the light of new insights, data, and observations; making improvements in terms of an increase in the precision of models and theories; the willingness to extend, modify, revise, and, if necessary, abandon a hypothesis, theory, model, or simulation. In terms of science-external/ethical responsibility of scientists and the sciences, different types of responsibility can be distinguished as well. The following four types help us meet some of the challenges of modern societies. (1) A first type of responsibility can be found in the area of actively intervening research (e.g. in scientific and technological experiments, in which human beings are the immediate test subjects and thus the direct objects of research, like in the coronavirus vaccination tests). (2) A second type of responsibility can be found in those areas of scientific research that deal with consequences in the areas of, e.g., biotechnology, gene technology, nuclear technology, or nanomedicine. (3) A third type refers to responsibility in terms of consequences of certain man-made scientific and technological developments themselves. 8 On the latter see, e.g., the recommendations of the Leopoldina (German National Academy of Sciences; April 13th, 2020) under the title Coronavirus-Pandemie—Die Krise nachhaltig u¨berwinden (Coronavirus Pandemic—Sustainably Overcoming the Crisis). See on this also the statement of researchers of the Helmholtz Association under the title Systemische Epidemiologische Analyse der COVID-19-Epidemie (Systemic Epidemiological Analysis of the COVID-19 epidemic, also April 13th, 2020). 5 The Internal and External Responsibilities of Science Think of, e.g., the consequences of scientific or technological developments for climate change and environmental pollution. (4) Finally, as already mentioned, a fourth type of responsibility science and technology have to face must be emphasized. Science and technology have an obligation to make substantial and sustainable contributions to (a) the preservation, precaution, and improvement of the conditions of human life on earth and to (b) the shaping of the future of modern societies. (4) Finally, as already mentioned, a fourth type of responsibility science and technology have to face must be emphasized. Science and technology have an obligation to make substantial and sustainable contributions to (a) the preservation, precaution, and improvement of the conditions of human life on earth and to (b) the shaping of the future of modern societies. It must be emphasized, however, that the remarks regarding the fourth type of responsibility by no means imply a desire to maneuver science and technology into the role of political decision makers. That is definitely not the case. The social and political decisions regarding concrete measures and programs lie in politics, not in science. Science and technology must, however, face the responsibility of providing contributions and recommendations in view of rational, intelligent, and reasonable decisions in the political realm. And they do so in various ways and by providing various scientific and technological competencies, disciplines, practices, and professions. The handling of questions concerning climate change and the coronavirus pandemic are current examples of this.8 Making a recommendation in no way means entering the field of political consulting. Rather, a 12 3 3 608 Axiomathes (2020) 30:597–616 recommendation from the sciences or technologies is, so to speak, the spelling out of an answer to the (inner-scientific and inner-technological) question of what the respective scientific or technological analyses, results, and predictions mean and what consequences follow from them. It would be unscientific and ethically irresponsible if the sciences tried to keep their findings contained to themselves (e.g. in the case of climate change or pandemic research). Earlier we made a distinction between internal and external responsibilities of the sciences. But the four illustrated types of responsibility show that, across the board, internal and external responsibilities are at the same time tightly connected and interwoven. 5 The Internal and External Responsibilities of Science It should also have become obvious that the distinction of these two ways of responsibility does not suggest that science and technology can be viewed as ethically neutral affairs. Science and technology must take a perspectival responsibility for their own practices, processes, and products and justify them if requested. Neutrality can, strictly speaking, neither be directly achieved here nor is it the crucial strategic goal and norm. Rather, the epistemic, the epistemological, and the ethical goal and norm is constituted of the preservation, protection, and improvement of the human life practices and the conditions of human life on earth. On more detailed examination of this task, we must distinguish a variety of different types, tasks, and conflict situations of science-internal as well as of science-external responsibilities and their overlaps. This variety cannot be presented and discussed in detail here. Hans Lenk has addressed this subject and made some fine-grained conceptual distinctions in this context (see Lenk, e.g., 1991, p. 61 ff. and p. 64 and recently 2020). Using the question of responsibility in climatology as an example, I would like to point out just one aspect. As Lenk correctly emphasizes, we are not only dealing with direct responsibilities, but also with ‘‘indirect’’ responsibilities. Indirect responsibilities are not immediately given by the action situation itself. Examples of ‘‘science-induced long distance effects’’ can be used to illustrate what is meant by indirect responsibilities. Think of the delayed and remote effects of interventions in nature, e.g. of the use of ecologically harmful pesticides or plastic materials. But the sciences view themselves as obligated to the spirit of enlightenment and thus essentially embody three key obligations that must be explicitly emphasized. First (1), there is the obligation to provide the best scientific results possible and to maintain their priority over mere opinions and politically motivated ideologies. Second (2), there is an obligation to provide the best possible rationality in terms of intellectually convincing and empirically rich arguments. And the third (3) obligation, as I would like to emphasize once more, is to contribute to the concrete improvement of life and to the solving of problems, crises, and unforeseen situations in the age of science and technology by using the scientific and technological tools and resources available at the time. The legitimacy, normativity, and benefit of science and technology are based on these three pillars. 123 6 Responsibility Exemplified by Climate Research In the current (and often heated) discussions on climate research and its prognoses, the reference to uncertainties concerning scientific explanations, theories, model- ings, and forecasts often plays an important role. The situation is analogous in the case of pandemic-related debates. Uncertainties in the sciences are particularly brought up when, as in the case of so-called climate change sceptics, attempts to stabilize and positively influence climate development are rejected because of uncertainties regarding the theory. This is the case despite the fact that overwhelming majority of scientists not only recommend the respective analyses, diagnoses, therapies, and measures, but regard them as postulates that need to be realized as soon as possible. Otherwise, there could be devastating consequences for human life on earth and the planet itself. Regardless of these concerning findings, many climate change sceptics misinterpret the reference to uncertainties in the sciences as an excuse for inactivity or even as an ideological weapon. It must be noted here that the Intergovernmental Panel on Climate Change (IPCC) explicitly discusses the question of how reliable, precise, and resilient the current models of climate science are. But even if we cannot definitively eliminate uncertainties from science and technology, this does not mean, in terms of ethical responsibility, that we are not committed to the ethical principle of prevention, precaution, and care. This ethical obligation towards our own human conditions of life receives its legitimacy from a different source than the degree of reliability of statistical methods and scientific (mathematical) modelings. Legitimacy rather arises from the ethical commitment to prevention, precaution, and care particularly from two directions: firstly (1), from the answer to the question of what actions (and omissions of actions) are ethically imperative in order to preserve and secure the conditions of our functioning human life practices; and secondly (2), from the answer to the question of what is imperative under the rule of human rationality, of everyday practical prudence (phro´nesis), and of reasonableness and what is not. The rule of prudence must by no means be misunderstood as a capitulating retreat to ethically mediocre slogans such as ‘‘The ends justify the means’’ or ‘‘The justification of moral judgments and ethical principles is ultimately a matter of cost– benefit calculation’’. We have heard the latter slogan particularly in connection with ethical utilitarianism and the min–max rule in the context of the rational choice theory. 5 The Internal and External Responsibilities of Science And the obligation of scientists to actively take part in public debates (e.g. currently in debates on climate change, environmental issues, and the coronavirus pandemic) is based on these pillars, too. The sciences must, according to their own self-conception, always be involved in order to not just be a vacuous 123 123 Axiomathes (2020) 30:597–616 609 ceremony of hypotheses, models, theories, and simulations, but beneficial to our human life, knowledge, science, and technology practices.9 9 On the role and task of philosophy in the public, social, and political domain, see Abel (2020/forthcoming). 6 Responsibility Exemplified by Climate Research In contrast however, the rule of prudence does not formulate a mediocrity, but a human peak performance. In book VI of his Nicomachean Ethics, Aristotle emphasizes the practical prudence (phro´nesis) in its own relevance towards and in addition to theoretical knowledge (episteme). Practical prudence means the ability to appropriately act in concrete individual situations with regard to the factors, 12 123 610 Axiomathes (2020) 30:597–616 goals, and insights internally related to our human conduct of life and thus to our life practices in the sense of a pragmatic knowledge. Practical prudence is, in this sense, always bounded prudence. And the question of the legitimacy of this type of obligation to provide care, precaution, and prevention (given, e.g., the results of today’s climate research) is an everyday relevant practical and thus ethical question. Moreover, this ethical question is primarily not a question of the sciences and hence cannot be answered with scientific means, methods, and models. I would like to illustrate this point using the example of the obligations of a physician. Kant used this example in his Critique of Pure Reason (Kant 1956, B 852f). A slightly modified version of this example can help illustrate the point. A patient suffering from a severe headache consults a doctor. The doctor’s initial diagnosis is migraines, but he is not entirely sure. He tries to comfort (or even reject) the patient by telling him that there still are too many uncertainties regarding his diagnosis. And it could very well be the case that another doctor might make a more positive diagnosis. Thus he, the doctor, is still too far away from an absolutely certain and perfect knowledge required in advance in order to confirm the diagnosis and treat the headache appropriately. And thus the doctor, in his own opinion, does justifiably (sic!) not believe that it is his duty to actively care and take precautions in the given case. He feels sorry for the patient, but his obligation to provide absolute certainty regarding his knowledge forces him to inaction, in ethical terms, to the omission of a treatment. In view of this example we have to explicitly emphasize that the doctor obviously is, due to his medical ethos and due to the ethics of care, precaution, and prevention, obligated to act. What do we learn from this example? 6 Responsibility Exemplified by Climate Research First of all, it is needless to say that the physician from our example would likely be prosecuted for failure to provide assistance. This way, the entire process turns from a process of ethics into a legal matter. For as a doctor, he must act. He may not act from a position of absolute certainty (since such certainty is never actually available anyway). Rather, his actions are based on what we can, with reference to Kant, call the ‘‘pragmatic belief’’ (Kant 1956, B 852). This term describes the pragmatic interaction of sufficiently subjective and sufficiently justified belief in order to enter into the action (even if the doctor does not have absolute knowledge in this case). Neither the doctor nor the patient need any further justification. 7 Uncertainties in the Sciences Uncertainties and susceptibility to error in the sciences (and thus in climate, environmental, and pandemic science, too) cannot be entirely eliminated. All science is subject to uncertainties. And there is also the fact that the individual sciences themselves are primarily defined by their limits and boundaries. But at the same time, all sciences constantly try to improve their hypotheses, models, and theories and try to avoid problems that can arise from limited perspectives. In what follows, we will therefore focus on illustrating the inner connection of (a) the quasi- natural uncertainties in the sciences and (b) the measures of prevention, precaution, and care that are ethically imperative in order to prevent damage to or even the Axiomathes (2020) 30:597–616 611 destruction of the conditions and foundations of human life. With this question, we are obviously dealing with an important point of overlap of scientific and theoretical analysis and ethical obligation. destruction of the conditions and foundations of human life. With this question, we are obviously dealing with an important point of overlap of scientific and theoretical analysis and ethical obligation. In terms of the sciences, we can, according to Dagfinn Føllesdal, distinguish at least three types of uncertainties: (a) uncertainties in theories, (b) uncertainties in models, and (c) uncertainties in chaotic systems.10 On (a): Uncertainty of theories:—In scientific theories (e.g. theories on climate change), hypotheses are made and then tested in terms of whether or not they match our empirical observations. Uncertainty exists here in the sense that it is always possible that the prognoses made by means of mathematical models and statistical methods do not match our empirical observations. Also, competing (and sometimes even mutually exclusive) theories, in the sense of Willard Van Orman Quine’s thesis of underdeterminacy, always remain possible. According to this thesis (which I strongly agree with), every theory is systematically underdetermined in terms of the data base it receives its input from (e.g. an astrophysical theory on the beginning and development of the entire universe). There is always a relation between a ‘‘meager output’’ and a ‘‘torrential input’’. The question following from this finding is the question of ‘‘in what ways one’s theory of nature transcends any available evidence’’ (Quine 1969, p. 83). This transcending is always given and necessary, no matter how large an amount of data we are talking about. 10 See Føllesdal (2011). This paper has only been published in Norwegian language so far. I would like to thank Rune Nyrup for translating it to German for my personal use. I would also like to thank Dagfinn Føllesdal for proofreading and authorizing Nyrup’s translation. In what follows, I will quote this German version of Føllesdal’s paper. The quotation page numbers refer to this so far unpublished German version by Nyrup. I would very much welcome a published English and German version of this important paper. 7 Uncertainties in the Sciences from R = 1 to R = 3, and this new figure is incorporated in the equations of the model, this can result in significant alterations in terms of the previously predicted number of new infections. The related curve of the predicted course of the pandemic would rise exponentially instead of linearly. Against this background it becomes clear that models and modelings are always accompanied by uncertainties. ‘‘If there are enough equations and appropriate parameters, a model can be matched with almost any data’’ (Føllesdal, p. 3). The calculated predictions made by means of the model are supposed to match our most recent data and observations. In this sense, scientific modelings are always dynamic. As Føllesdal (p. 4) says, ‘‘we are more likely to trust a model that matches observations that were not known yet at the time the model (or theory) was formulated’’. This certainly also holds true for today’s models of climate change and pandemic development. We could even go one step further. Because no matter how tight a modeling’s restrictions in terms of the relation between model and reality may be, there always remains the possibility of various equally legitimate relations between model and reality. And thus there are always (as Hilary Putnam has shown) various equally valid satisfaction relations and satisfaction objects.11 This degree of uncertainty internally connected to models themselves cannot be eliminated, neither empirically (no matter how much data we possess) nor theoretically (no matter how strict the formal restrictions of our models may be). On (c): Uncertainty due to the chaotic development of systems:—Føllesdal (p. 6 f) reminds us of the famous butterfly scenario formulated by the meteorologist Edward Lorenz (1917–2008). As is well-known, Lorenz simply wanted to re-run a weather simulation on his computer. But Lorenz did not start this second run at the beginning of the simulation, but at a later stage at which he entered the numbers his printer had printed out for this stage. To Lorenz’s surprise, the second run lead to results that were significantly different from the ones of the initial simulation. And the reason for this was as surprising as it was simple: Lorenz’s computer had calculated to six decimal places while his printer had only printed out the first three decimal places. Thus, a number like 0,506127 was abbreviated and used in the calculations as 0,506. 7 Uncertainties in the Sciences For, according to Quine, it is not an underdetermination that can ultimately be empirically corrected somehow. It is rather a logical underdeterminacy. And within this open realm of underdeterminacy, two different (and maybe even mutually exclusive) but equally valid theories can be formulated. It must be noted, however, that this finding does by no means lead to a complete and terminal uncertainty in the sciences. Rather, it is crucial that we still can and must be able to compare and distinguish between good and less good hypotheses, models, and methods. The crucial criterion for preference cannot be the recourse to absolute and perfect knowledge. The criterion is rather provided by the answer to the question of whether or not a hypothesis, model, or theory is able to explain our observations more precisely and coherently. If a theory succeeds to do so, we are justified in our belief that it is a good theory (for the time being). The ethos- as well as ethics-based obligation in terms of responsibility resulting from this scenario of uncertainties is the obligation to constantly test the hypotheses, models, and theories with new empirical observations and new data and thus modify and improve them. On (b): Uncertainty of models:—A model is, as Føllesdal correctly emphasizes, a representation of something that highlights certain characteristics and disregards others. Today, particularly mathematical and statistical models are predominant in the sciences. They operate with various parameters and items that are considered relevant and incorporated in the equations of the model as well as in the modeling. In many cases, however, these are not items we can empirically observe, but 12 12 3 3 3 612 Axiomathes (2020) 30:597–616 estimated items that we adopt from other relevant fields and then incorporate and apply in our models. Obviously even the smallest alterations of these items can have great effects on the course and results of mathematical and statistical modeling. In the current epidemiological modeling of the course of the coronavirus pandemic, e.g., this is the case depending on whether the reproduction factor R (i.e. the number indicating how many new infections are statistically caused by a single infected person) is increased or decreased. If the parameter R increases, e.g. 11 See Putnam (1983). See also Abel (2008), in which I illustrate the relation of model and reality as a sign-based and interpretation-determined relation. On this, see also the index entry ‘‘Modell’’ (‘‘model’’) in Abel (2018c). 7 Uncertainties in the Sciences And this relatively small difference between the two numbers lead to an entirely different course and meteorological result of the simulation. This way Lorenz had demonstrated that even the smallest alteration of the initial 123 613 Axiomathes (2020) 30:597–616 conditions can have great consequences and that even an optimal model cannot accurately predict the weather for more than a short period of time. conditions can have great consequences and that even an optimal model cannot accurately predict the weather for more than a short period of time. In his famous paper, Lorenz writes: If the flap of a butterfly’s wings can be instrumental in generating a tornado, it can equally be instrumental in preventing a tornado. (…) Since we do not know exactly how many butterflies there are, nor where they are all located, let alone which ones are flapping their wings at any instant, we cannot, if the answer to our question is affirmative, accurately predict the occurrence of tornados at a sufficiently distant future time. (Lorenz 1972, p. 1f) This butterfly effect is an example of a phenomenon that occurs in non-linear and dynamic systems. We know uncertainties of the described type as well as the associated measures of care and precaution from our everyday life. Føllesdal (p. 12 f) uses the example of getting fire insurance to illustrate this point. The risk of my house burning down is relatively small, but if it actually happened, it could possibly be devastating for me. That is why it is obvious to me that I get appropriate insurance and believe that my decision is rational and reasonable. In situations like this, in which what is at stake is the prevention of real possible future catastrophes or even the destruction of the conditions of human life, the illustrated principle of ethical care, prevention, and precaution, and thus the responsibility of care, prevention, and precaution, come into play (Lenk 1991, p. 64). Føllesdal emphasizes an aspect that is crucial in this context. When it comes to the possibility of harm, there must be a realistic possibility, not just a merely theoretical possibility. He uses a nice example to illustrate this. It may be compatible with scientific theories and theoretically possible that I will be hit by a large meteorite in the near future. 7 Uncertainties in the Sciences But it would not be rational, prudent, or reasonable to get an insurance for this case, since the probability of such a collision actually happening is extremely small. That would be just as unreasonable as the even further-reaching decision to live my entire life under the assumption that I have to avoid such a collision at any cost. 8 Epilogue: Why is it Reasonable at all to Preserve the Conditions of Human Life on Earth? Imagine a doubting Thomas comes along and poses the question of why it is reasonable and rational to preserve the conditions of human life on earth at all. The answer to this question seems instantaneous and obvious. Who honestly does not know why we should protect and preserve ourselves, other persons, our environ- ment, as well as planet earth?! As soon as we have to explicitly spell out this self- evidence, however, it seems that we (in Wittgenstein’s words) do not know the answer anymore. In what follows, I would like to illustrate some aspects that concern our self-interest as human beings and can be understood, directly or indirectly, as an affirmative answer to this provocative question. It is presupposed in this answer that this affirmation of life and not self-destruction is a primordial 12 3 123 614 Axiomathes (2020) 30:597–616 characteristic and presupposition of human life itself. This life-internal presuppo- sition needs no proof. Rather, it is always already internally included in what we call living our lives. Demanding any additional proof would be what Kant rightfully called a ‘‘scandal of philosophy and human reason in general’’ (Kant 1956, B XXXIX). (1) If we do not take measures of care and precaution in terms of, e.g., climate change (and accordingly in terms of viral pandemics), the threat of harm becomes a real possibility that not only affects individual and isolated areas of the conditions of human life on earth. It rather affects the conditions of life of the human species itself as a whole and thus ultimately the survival of humanity as a whole. Putting that on the line would not exactly be a sign of reasonableness now, would it? (2) (2) Humankind survives in the course of nature’s survival. This finding is trivial. At the same time, we must recall another triviality, i.e. the fact that humankind needs nature, but nature does not need humankind. Do we really want to abandon our human self-manifestation on earth in light of these findings? By the way, in this context antique Stoicism introduced the concept of ‘‘oikeiosis’’, i.e. the survival instinct. In the Stoa, this concept describes the reasonable attitude and wish to bring one’s own life and one’s own self- preservation as well as one’s self-care into a well-proportioned balance with nature. 8 Epilogue: Why is it Reasonable at all to Preserve the Conditions of Human Life on Earth? What is at stake in these activities is certainly not just the mere existence and continuation of biological, organic human life. It is rather the internally connected pursuit of a good life (which idea was established by Aristotle). The punchline of this idea of a successful and good life can pointedly be formulated as follows: from the moment on I (as a biological, organic individual) wish to preserve my own biological, organic existence, I am always already tied to the idea of living a good life. One could say that we orient ourselves towards well-proportioned relations as well as successful states of equilibrium within the illustrated triangulation of our human I–We–World/Nature relations. The idea of a good life is bounded to its embodiment in this triangulation. Good life in this sense is always bounded good life. It is a triangulation-based, triangulation-bounded, and triangulation-oriented life. And a good life always already includes other people, the world, nature, the environment, and the entire planet earth. Why then should we unnecessarily put this reality of life at stake or even abandon it entirely?! (3) There is a lot of evidence that suggests that the active shaping of our relations to ourselves, others, and the world is characteristic of human life. What is at stake in these activities is certainly not just the mere existence and continuation of biological, organic human life. It is rather the internally connected pursuit of a good life (which idea was established by Aristotle). The punchline of this idea of a successful and good life can pointedly be formulated as follows: from the moment on I (as a biological, organic individual) wish to preserve my own biological, organic existence, I am always already tied to the idea of living a good life. One could say that we orient ourselves towards well-proportioned relations as well as successful states of equilibrium within the illustrated triangulation of our human I–We–World/Nature relations. The idea of a good life is bounded to its embodiment in this triangulation. Good life in this sense is always bounded good life. It is a triangulation-based, triangulation-bounded, and triangulation-oriented life. And a good life always already includes other people, the world, nature, the environment, and the entire planet earth. Why then should we unnecessarily put this reality of life at stake or even abandon it entirely?! 8 Epilogue: Why is it Reasonable at all to Preserve the Conditions of Human Life on Earth? As is well known, this important principle of the Stoa can be understood as an invitation to live one’s life ‘‘in appropriate harmony with nature (homologoumenos te physei zen)’’. It is safe to say that this principle is as relevant today as ever. as relevant today as ever. (3) There is a lot of evidence that suggests that the active shaping of our relations to ourselves, others, and the world is characteristic of human life. What is at stake in these activities is certainly not just the mere existence and continuation of biological, organic human life. It is rather the internally connected pursuit of a good life (which idea was established by Aristotle). The punchline of this idea of a successful and good life can pointedly be formulated as follows: from the moment on I (as a biological, organic individual) wish to preserve my own biological, organic existence, I am always already tied to the idea of living a good life. One could say that we orient ourselves towards well-proportioned relations as well as successful states of equilibrium within the illustrated triangulation of our human I–We–World/Nature relations. The idea of a good life is bounded to its embodiment in this triangulation. Good life in this sense is always bounded good life. It is a triangulation-based, triangulation-bounded, and triangulation-oriented life. And a good life always already includes other people, the world, nature, the environment, and the entire planet earth. Why then should we unnecessarily put this reality of life at stake or even abandon it entirely?! (3) (3) There is a lot of evidence that suggests that the active shaping of our relations to ourselves, others, and the world is characteristic of human life. What is at stake in these activities is certainly not just the mere existence and continuation of biological, organic human life. It is rather the internally connected pursuit of a good life (which idea was established by Aristotle). The punchline of this idea of a successful and good life can pointedly be formulated as follows: from the moment on I (as a biological, organic individual) wish to preserve my own biological, organic existence, I am always already tied to the idea of living a good life. (3) There is a lot of evidence that suggests that the active shaping of our relations to ourselves, others, and the world is characteristic of human life. 8 Epilogue: Why is it Reasonable at all to Preserve the Conditions of Human Life on Earth? (4) (4) Kant formulated his famous categorical imperative for human beings as follows: Act in such a way that the maxims of your actions were to become a general law. On the grounds of the sketched triangulation of the human I– (4) Kant formulated his famous categorical imperative for human beings as follows: Act in such a way that the maxims of your actions were to become a general law. On the grounds of the sketched triangulation of the human I– 123 Axiomathes (2020) 30:597–616 615 We–World/Nature relations, this imperative always also includes, next to the responsibility for oneself, the responsibility towards other persons and nature. This imperative is directly relevant with regard to the internal as well as the external responsibility of science and technology. With special focus on today’s scientifically and technologically determined realities of life, the following version of the categorical imperative can be formulated: ‘Act in such a way that the maxims of your actions were to become a general law that takes into account the preservation and expansion of human life practices and conditions of life and provides possibilities for living a good life on earth.’12 Funding Open Access funding provided by Projekt DEAL. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creativecommons.org/licenses/by/4.0/. 12 Hans Jonas (1984, p. 11) has proposed an imperative of responsibility for the age of technology. It reads as follows: ‘‘Act so that the effects of your actions are compatible with the permanence of genuine human life.’’ I agree with this formulation as it is. Jonas, however, bases this imperative on a number of presuppositions that require further discussion. I explicitly disagree with Jonas’ ontological justification of our norms and values as well as with his general strategy of basing ethics in metaphysics. But this subject cannot be discussed here. Funding Open Access funding provided by Projekt DEAL. References Abel G (2008) Modell und Wirklichkeit. In: Dirks U, Knobloch E (eds) Modelle. Peter Lang Verla Frankfurt am Main, pp 31–46 Abel G (2009) The riddle of creativity. In: Meusburger P, Funke J, Wunder E (eds) Milieus of creativity (Proceedings of the 2nd international symposium on knowledge and space, Heidelberg 2006). Springer, Dordrecht, pp 53–72 Abel G (2016) Rethinking rationality: the use of signs and the rationality of interpretations. In: Wagner A, Ariso JM (eds) Rationality reconsidered (Berlin Studies in Knowledge Research, vol 9). De Gruyter, Berlin, pp 15–29 Abel G (2018a) Verstehensgleichgewichte und Horizont-Entschmelzung. Replik zum Beitrag von Marco Brusotti. In: Dirks U, Wagner A (eds) Abel im Dialog. Perspektiven der Zeichen- und Interpretationsphilosophie, vol 1. De Gruyter, Berlin, pp 204–223 Abel G (2018b) Die praxis-interne Normativita¨t des Sprechens, Denkens und Handelns. Replik zum Beitrag von Catherine Z. Elgin. In: Dirks U, Wagner A (eds) Abel im Dialog. Perspektiven der Zeichen- und Interpretationsphilosophie, vol 1. De Gruyter, Berlin, pp 499–511 Abel G (2018c) In: Dirks U, Wagner A (eds) Abel im Dialog. Perspektiven der Zeichen- un Interpretationsphilosophie, vol 2. De Gruyter, Berlin Abel G (2020) Philosophie und O¨ ffentlichkeit. In: Olay C (ed) O¨ ffentlichkeit und Partizipation. Zwei Schlu¨sselbegriffe demokratischer Praxis. Verlag Alber, Freiburg Elgin CZ (1996) Considered judgment. Princeton University Press, Princeton 96) Considered judgment. Princeton University Pre Elgin CZ (2003) Erkenntnistheoretisches Gleichgewicht. In: Vogel M, Wingert L (eds) Wissen zwischen Entdeckung und Konstruktion. Erkenntnistheoretische Kontroversen. Suhrkamp, Frankfurt am Main, pp 193–217 12 Hans Jonas (1984, p. 11) has proposed an imperative of responsibility for the age of technology. It reads as follows: ‘‘Act so that the effects of your actions are compatible with the permanence of genuine human life.’’ I agree with this formulation as it is. Jonas, however, bases this imperative on a number of presuppositions that require further discussion. I explicitly disagree with Jonas’ ontological justification of our norms and values as well as with his general strategy of basing ethics in metaphysics. But this subject cannot be discussed here. 12 3 616 Axiomathes (2020) 30:597–616 Føllesdal D (2011) Klimaforskning, usikkerhet og forskningsformidling. In: Roll-Hansen N (ed) Status i klimaforskningen (The state of Climate Research), (Vortrag an der Norwegischen Akademie der Wissenschaften, 20, Januar 2010). Novus, Oslo, pp 47–65 Gigerenzer G, Selten R (eds) (2001) Bounded rationality: the adaptive toolbox. The MIT Press, Cambridge g Goodman N (1983) Fact, fiction, and forecast, 4th edn. Translated from German by Hadi Nasir Faizi. References Harvard University Press, Cambridge Goodman N (1983) Fact, fiction, and forecast, 4th edn. Harvard University Press, Cambridge Jonas H (1984) The imperative of responsibility: in search of an ethics for the technological age. University of Chicago Press, Chicago Goodman N (1983) Fact, fiction, and forecast, 4th edn. Harvard University Press, Cambridge Jonas H (1984) The imperative of responsibility: in search of an ethics for the technological U i it f Chi P Chi Jonas H (1984) The imperative of responsibility: in search of an ethics for the technological ag University of Chicago Press, Chicago University of Chicago Press, Chicago Kant I (1956) Kritik der reinen Vernunft (Second Edition B), ed by Raymund Schmidt, Meiner, Hambu Kant I (1956) Kritik der reinen Vernunft (Second Edition B), ed by Raymund Schmidt, Meiner, Hamburg Lenk H (1991) Zu einer praxisnahen Ethik der Verantwortung in den Wissenschaften. In: Lenk H (ed) Wissenschaft und Ethik. Reclam, Stuttgart Lenk H (1991) Zu einer praxisnahen Ethik der Verantwortung in den Wissenschaften. In: Lenk H (ed) Wissenschaft und Ethik. Reclam, Stuttgart g Lenk H (2020) Verantwortlichkeit in der Wissenschaft. In: Mieg HA, Lenk H (eds) Wissenschaftsver- antwortung (Wissenschaftsforschung Jahrbuch 2019; Gesellschaft fu¨r Wissenschaftsforschung). Wissenschaftlicher Verlag Berlin (wvb), Berlin, pp 45–95 Leopoldina. Nationale Akademie der Wissenschaften (April 13th, 2020) Coronavirus-Pandemie—D Krise nachhaltig u¨berwinden Lorenz E (1972) Predictability: Does the flap of a butterfly’s wings in Brazil set off a Tornado in Texas? In: Talk presented Dec 29, 1972, at the American Association for the Advancement of Science (AAAS), Section on Environmental Sciences, New Approaches to Global Weather: GARP (The Global Atmospheric Research Progam) Sheraton Park Plaza Hotel, Boston, MA. Lorenz‘s talk can be downloaded here: http://eaps4.mit.edu/research/Lorenz/Butterfly_1972.pdf Putnam H (ed) (1983) Models and reality. In: Realism and reason. Cambridge University Pres Cambridge, pp 1–25 Quine WVO (1969) Epistemology naturalized. In: Quine WVO (ed) Ontological relativity and other essays. Columbia University Press, New York, pp 69–90 Rawls J (1971) A theory of justice. Harvard University Press, Cambridge Simon HA (1956) Rational choice and the structure of environments. Psychol Rev 63:129–138 Simon HA (1956) Rational choice and the structure of environments. Psyc Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published map and institutional affiliations. Translated from German by Hadi Nasir Faizi. Translated from German by Hadi Nasir Faizi. 123 123
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Reflexiones sobre los aspectos que influyen en la popularidad de las ciencias
Conciencia Digital
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ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org Reflexiones sobre los aspectos que influyen en la popularidad de las ciencias Reflections on the factors influencing the popularity of sciences 1 2 3 4 Ramón Antonio Abancin Ospina https://orcid.org/0000-0002-2417-6671 Escuela Superior Politécnica de Chimborazo (ESPOCH), Facultad de Ciencias, Carrera de Matemática, Grupo CIDED, Riobamba, Ecuador, Panamericana Sur, Km 11/2. Universidad Simón Bolívar (USB), Valle de Sartenejas, Venezuela. ramon.abancin@espoch.edu.ec. Zenaida Natividad Castillo Marrero https://orcid.org/0000-0002-4424-8652 Escuela Superior Politécnica de Chimborazo (ESPOCH), Facultad de Ciencias, Carrera de Matemática, Grupo CIDED, Riobamba, Ecuador, Panamericana Sur, Km 11/2, Universidad Central de Venezuela, Facultad de Ciencias. zenaida.castillo@espoch.edu.ec. Andrés Eloy Salazar Domínguez https://orcid.org/0000-0001-7310-2241 Universidad Central de Venezuela, Los Chaguaramos, Caracas, Venezuela. andres.salazar.d@ucv.ve Paulina Elizabeth Valverde Aguirre https://orcid.org/0000-0003-0458-7083 Escuela Superior Politécnica de Chimborazo (ESPOCH), Facultad de Ciencias, Carrera de Matemática, Grupo CIDED, Riobamba, Ecuador, Panamericana Sur, Km 11/2. Artículo de Investigación Científica y Tecnológica Enviado: 07/01/2022 Revisado: 22/01/2022 Aceptado: 19/02/2022 Publicado:20/03/2022 DOI: https://doi.org/10.33262/concienciadigital.v5i1.3.2104 Cítese: Abancin Ospina, R. A., Castillo Marrero, Z. N., Andrés Eloy Salazar Domínguez, & Valverde Aguirre, P. E. (2022). Reflexiones sobre los aspectos que influyen en la popularidad de las ciencias. ConcienciaDigital, 5(1.3), 148-167. https://doi.org/10.33262/concienciadigital.v5i1.3. CONCIENCIA DIGITAL, es una revista multidisciplinar, trimestral, que se publicará en soporte electrónico tiene como misión contribuir a la formación de profesionales competentes con visión humanística y crítica que sean capaces de exponer sus resultados investigativos y científicos en la misma medida que se promueva mediante su intervención cambios positivos en la sociedad. https://concienciadigital.org La revista es editada por la Editorial Ciencia Digital (Editorial de prestigio registrada en la Cámara Ecuatoriana de Libro con No de Afiliación 663) www.celibro.org.ec Esta revista está protegida bajo una licencia Creative Commons Attribution Non Commercial No Derivatives 4.0 International. Copia de la licencia: http://creativecommons.org/licenses/by-nc-nd/4.0/ Innovando P á g i n a 148 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org Palabras Resumen claves: En torno a las ciencias, concurren aspectos que influyen en su popularidad dentro del ámbito educativo y, también en el social, repercutiendo en forma directa en las plazas vacantes dentro del campo laboral. Bajo esta perspectiva, en el presente artículo se analizan los eventos originados en el contexto académico, vinculados al proceso de enseñanza y aprendizaje, que influyen negativamente en la percepción de las ciencias, y que han ido desfavoreciendo y condicionando su popularidad. El estudio fue abordado bajo una orientación cualitativa, de tipo descriptivo/exploratorio, con diseño de investigación documental, apoyado en la realidad estudiantil, y basado en las experiencias relacionadas con la construcción del conocimiento de las ciencias. Se reconocen y estudian algunos aspectos que influyen en la percepción y popularidad de las ciencias. Adicionalmente, se presentan eventos reconocidos de la temática planteada, tales como: primer contacto, desconocimiento, desinterés, doble efecto vocacional, minoría y déficits de profesionales, todos concernientes y relacionados con las ciencias. ciencias, enseñanza y aprendizaje en ciencias, popularidad de las ciencias, vacantes. Abstract Keywords: job openings, science, science popularity, teaching and learning in sciences. Aspects that influence the popularity of science in the educational and social spheres have a direct impact on job vacancies in the labor market. From this perspective, the purpose of this article is to analyze the events originating in the academic context, related to the teaching, and learning process, which have a negative influence on the perception of science, and have been disfavoring and conditioning its popularity. The study was conducted under a qualitative orientation, of a descriptive/exploratory type, with a documentary research design, supported by the students' reality, and based on experiences related to the construction of knowledge of science. Some aspects that influence the perception and popularity of the sciences are recognized and studied. In addition, we present recognized events of the proposed topic, such as: first contact, ignorance, disinterest, double vocational effect, minority, and deficits of professionals, all concerning and related to the sciences. Innovando P á g i n a 149 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org Introducción El contacto de los individuos con actividades inherentes a un área del conocimiento les permite obtener información, que, de alguna manera, induce percepciones respecto al área, y en particular, les proporciona ideas que pueden ser correctas, aproximadas, o erradas, sobre su respectivo ámbito de acción. En este sentido, las ciencias representan un área privilegiada, una vez que, los miembros de la sociedad las comienzan a conocer desde muy jóvenes a través de sus asignaturas escolares más elementales. Específicamente, en la educación primaria se dan cita actividades estrechamente relacionadas con biología y matemática, ya que son estas las que despiertan mayor interés en los primeros momentos de la escolaridad. A lo largo de esta primera etapa los conocimientos en el área de ciencias se van haciendo más complejos, de acuerdo con el programa curricular de cada nivel educativo. Luego, en la educación media, comienzan a emerger otras áreas como la física y la química. En todos los casos, la matemática se erige como el eje articulador de un importante grupo de contenidos de otras disciplinas del conocimiento; por tal razón, su presencia es de incuestionable valía dentro de la trayectoria académica de los aprendices. Por otra parte, si se analiza el contexto social, a pesar de que no siempre es explicita la aplicabilidad de las ciencias en el entorno común de los individuos, es innegable que, en el transcurrir de la vida misma, nos encontramos expuestos a los resultados de la investigación científica y a los avances indetenibles de las ciencias. Los avances en medicina, infraestructura, y tecnología, así como el entendimiento de fenómenos naturales, son muestras del contacto humano con la ciencia, aun cuando esto pase desapercibido. En los dos aspectos mencionados anteriormente (educativo y social), existe un distanciamiento latente entre los conocimientos académicos adquiridos y la utilidad que esto representa para el entorno. Esta situación apunta a preconcebir a las ciencias como un área del saber poco útil y, por ende, carente de cualquier atractivo que dinamice la profesionalización de los individuos en el área. El panorama anterior plantea un dilema para los estudiantes en torno al área de ciencias, el cual se ve reflejado en el desinterés por las asignaturas que componen estos saberes y; a largo plazo, la inminente carencia de motivación por seguir estas profesiones. Resumidamente, la desidia académica de la cual se suele hacer referencia desencadena Innovando P á g i n a 150 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org eventos desafortunados que afectan directa o indirectamente la popularidad de las carreras en ciencias, repercutiendo finalmente en la falta de profesionales cualificados para desarrollar estas profesiones. Precisamente, ese tipo de desinterés se gesta en los estudiantes por la falta de atractivo en las profesiones que componen el área; en parte, por la ingente cantidad de inversión en tiempo y esfuerzo intelectual que se requiere para aprender y dominar los contenidos científicos, y, que muchas veces, estos no se llegan a conocer a profundidad con la aplicabilidad practica que exigen. En cierto modo, esto coloca a los estudiantes en un escenario de incertidumbre con respecto a la cobertura y alcance de las profesiones de ciencias. Aunado a esto, la histórica fama desfavorable (área difícil) de las ciencias, la cual conduce al rechazo o desaprobación de estas, ha sido la consecuencia directa de la de la enseñanza del área en las instituciones educativas. Todo esto desemboca en un distanciamiento del estudiante con las ciencias, en el que se descarta a temprana edad la posibilidad del estudio de alguna de estas carreras. A este respecto, la literatura se organiza tentativamente en tres grupos, los cuales no son excluyentes ni exhaustivos. Primero un conglomerado de libros e investigaciones se concentran en mejorar y motivar el proceso de enseñanza y aprendizaje de las ciencias en los diferentes niveles y sistemas educativos. En esta dirección, se pueden encontrar diseño de unidades didácticas para optimizar la enseñanza de las ciencias (Flórez & González, 2021); así como también didácticas de las ciencias para la praxis docente (Álvarez & Valls, 2019), entre otros. Segundo existen estudios de carácter reflexivo sobre la importancia de las ciencias, en los cuales se busca explicar la contextualización y funcionalidad de los aprendizajes, para acercar la realidad académica de los alumnos, a la experiencia cotidiana de los mismos (Martín, 2002). Tercero existen escasas investigaciones orientadas en indagar lo que sucede alrededor de la búsqueda, selección y elección de una profesión de las carreras de ciencias. A modo de ilustración se tienen los trabajos de Martínez et al. (2021) y Chvanova & Garbín (2017), focalizados en la disciplina de matemática. No obstante, no existe una postura critico-reflexiva desde el ámbito académico que esté orientada a puntualizar, desde el inicio, el conjunto de eventos en torno al proceso de enseñanza y aprendizaje de las ciencias, a los cuales se enfrentan los estudiantes en las Innovando P á g i n a 151 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org aulas de clases y; que pueden estar inoculando el rechazo y desinterés por el área, devastando la popularidad de las ciencias. En este trabajo se discuten algunos aspectos involucrados en el contexto, como lo son: el continuismo tradicionalista de la academia, en cuanto a la enseñanza y aprendizaje de las ciencias, y el surgimiento de situaciones aisladas que influyen perniciosamente en la percepción preconcebida y simplista del individuo por las ciencias. Estos aspectos influyen directamente en la selección, elección y decisión de los estudiantes por el estudio de alguna carrera de las ciencias. El punto clave del estudio es identificar y discutir eventos que se suceden en la trayectoria de los estudiantes desde el Bachillerato hasta la Educación Superior, en torno a la adquisición de conocimientos científicos. Se investigan propuestas de algunos autores que señalan cómo los alumnos deben aprender ciencia, aprender a hacer ciencia y aprender sobre la ciencia (Hodson, 1994); cómo diseñar actividades de enseñanza y aprendizaje que permitan educar científicamente a la población, para que sea consciente de los problemas del mundo, y, de la posibilidad de su actuación sobre los mismos, además, de la capacidad de modificar situaciones, incluso las ampliamente aceptadas (Martín, 2002). Usando como antesala estas ideas, el propósito del presente ejercicio reflexivo, fue analizar eventos originados en el proceso de enseñanza y aprendizaje de las ciencias, con el fin de detectar los aspectos que desfavorecen el área, tanto académica como profesionalmente. Abordar estos eventos claves permitirá tomar acciones preventivas o remediales, tendientes a minimizar el rechazo de los aprendices por las ciencias, no solo de la arista de lo académico, sino también desde lo social, en aspectos intrínsecos del ser como: el recreacional (e.g., clubes), el entretenimiento (e.g., gamificación) y la emocionalidad (e.g., satisfacción de resolver problemas) de vivir, hacer y sentir las ciencias como propias cuando se estudian con técnicas alternativas. Metodología El estudio fue abordado bajo una orientación cualitativa, de tipo descriptivo/exploratorio, con diseño de investigación documental, debido a que la naturaleza inductiva de esta Innovando P á g i n a 152 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org aproximación metodológica ayuda a entender la realidad social como fruto de un proceso histórico de construcción (Sandoval, 1996). Además, se desea observar y registrar un fenómeno sin introducir modificaciones, tal como se presenta en una situación de espacio y de tiempo dado (Rojas, 2015). Se revisó y recopiló información para sustentar el estudio y familiarizarse con los conocimientos existentes dentro del campo al que pertenece el objeto de estudio (Cabezas et al., 2018). Concretamente, se persiguió analizar y generar una discusión y reflexión en torno a los eventos que suceden en el proceso de enseñanza y aprendizaje y que pueden estar impulsando una popularidad parcializada y no favorable para toda el área. En este sentido, la ruta metodológica seguida por el estudio comprendió: 1) Un proceso de revisión documental que contemplo la indagación, identificación, recolección y selección de trabajos disponibles en la Internet en repositorios académicos como Google académico, relacionados con contenidos oportunos y relevantes sobre el ámbito académico y profesional de las ciencias. 2) Análisis de documentos procedieron y creación de eventos (Primer contacto, Desconocimiento, Desinterés, Doble efecto vocacional, Minoría y Déficits de profesionales, todas con respecto a las ciencias) que facilitaron la organización, comparación, contrastación e interpretación de la información recabada. 3) Reflexión y análisis de la información, definiendo la postura crítica propia de los autores, contrastadas con referentes teóricos. Resultados y Discusión Eventos entorno al área de las ciencias En este apartado se abordarán algunos de los más sobresalientes con la finalidad de ilustrar y obtener un panorama de la temática planteada: ciencia y popularidad. Sin ser exhaustivos, ni establecer un orden estricto, se intenta en primera instancia establecer un escenario susceptible para las ciencias, basado en las vivencias que tienen las personas con el área, como producto de la interacción con su entorno educativo. Luego ofrecer un espacio de discusión y reflexión, sobre cómo el ámbito educativo puede influir en la reputación del área. Innovando P á g i n a 153 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org Primer contacto con el área de ciencias El primer encuentro a nivel formal con las ciencias se da en la etapa escolar, a través de las clases tradicionales en asignaturas elementales, entre la que destacan temas vinculado con la biología, la física, la matemática y la química; las cuales van tomando forma progresivamente, incrementando su grado de dificultad hasta llegar a la Educación Superior. En este sentido, es importante destacar la acción del educador que imparte conocimientos en Ciencias Básicas a través de los cursos escolares, puesto que ésta será la primera impresión que tendrán los educandos sobre la “ciencia”. Esta gran responsabilidad que asumen docentes y/o profesores del área, que también recae sobre las instituciones de educación (pública y privada), los planes, proyectos y directrices de desarrollo del sector educativo y los entes a los que se encuentran adscritos. Esto presupone que, los estudiantes al verse expuestos durante todos sus años de escolaridad a una serie de conocimientos y disciplinas que resultan poco menos que ineludibles, estarán influenciados de manera negativa, neutra o positiva, no por la ciencia como tal, sino por las concepciones subjetivas de los alumnos desde sus primeros contactos con el área. De acuerdo con lo anterior, los docentes y profesores siguen estrechamente ligados a rutinas escolares monolíticas, propias de la profesión, las cuales están previstas por obligatoriedad dentro de los currículos escolares oficiales de cada sistema educativo. Estas clases, por lo general magistrales, están basadas de forma casi absoluta en aprendizajes por transmisión, o el método partiendo de la teoría, que consiste en que el profesor exponga de manera rigurosa, clara y precisa, los resultados de la actividad científica; cuya intención es la aplicación del conocimiento en la resolución de problemas cerrados y cuantitativos (Pozo, 1999). Una forma alterna a este modelo consiste en presentar a los alumnos una teoría a estudiar, para después asignarles una lista de problemas y/o ejercicios a solucionar, presuponiendo que cada problema tiene una única solución correcta y el profesor la conoce con anterioridad (Sgibnev, 2013). Bajo esta perspectiva, cualquier estudiante inscrito en el sistema educativo, pasa por la experiencia de este tipo de clases propias de las actividades escolares relacionadas con ciencias que, sin duda, lo ayudará en las formulaciones de sus propias concepciones Innovando P á g i n a 154 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org epistemológicas acerca de esta área. Estas últimas, se refieren propiamente a las ideas acerca del conocimiento en general, en tanto que pretende responder cómo se estructura, cómo evoluciona y cómo se produce (Hammer, 1994), lo que guarda estrecha relación con la forma como se concibe la ciencia, y cómo se aprende el conocimiento científico (Campanario & Moya, 1999). Concretamente, el proceso de interacción y experimentación de los estudiantes con las ciencias permite cristalizar una concepción epistemológica individual sobre el área, de acuerdo con las experiencias que previamente se obtengan en pro de la búsqueda del conocimiento. Por tanto, si las clases de ciencias están planificadas bajo un esquema simple de aprendizaje por transmisión o partiendo de la teoría, entonces es inevitable encontrarse con: clases estructuradas sobre contenidos sin progresión lineal, descontextualizados y muchas veces desactualizados por parte de los profesores y autores de libros de textos elementales. Esta forma de distribución del conocimiento hace hincapié únicamente en definiciones y resultados relevantes tales como: teoremas, propiedades, entre otros; que, al ser reducidos a su mínima expresión por medio reglas mnemotécnicas, facilitan la memorización y aplicación, pero dejan de lado la interpretación, la comprensión, el análisis profundo y sistemático que requiere el estudio de las ciencias. El inconveniente o la dificultad de esta forma de aprender ciencias para los estudiantes, surge cuando es necesario articular varios de estos aprendizajes en la aplicación de algún ejercicio y/o problema, con el propósito construir la solución. Cuestión que no resulta fácil, cuando no hay una comprensión profunda de los conocimientos que se requieren para tal fin; además, de las bien requeridas competencias, habilidades y destrezas para resolver problemas, los cuales son adquiridas expandiendo las fronteras de las simples aplicaciones de un recetario de fórmulas descontextualizadas. Entonces, la ausencia o descoordinación de algunas de estas cualidades, desemboca irremediablemente en intentos fallidos de alcanzar la solución a un problema; sembrando en el educando frustración, desaliento, resignación y abandono; a la postre de generar una matriz de opinión negativa en la que se estigmatiza la ciencia como área difícil e imposible de aprender. Esta reputación se extiende a medida que los estudiantes avanzan en los estudios académicos, proyectándose fuera de las fronteras del ámbito educativo. Es en este punto, Innovando P á g i n a 155 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org donde los medios de comunicación (e.g. prensa, radio, televisión, redes sociales), lejos de promover el estudio de la ciencia para el progresivo desarrollo de la sociedad, refuerzan la visión simplificada de un área complicada y solo para genios, a través de chistes, parodias y sátiras. Finalmente, estas circunstancias, conducen a una desvalorización de la ciencia y de la adquisición del conocimiento científico (Justi, 2006). Un punto que destacar es que, aquellos responsables de la enseñanza de las ciencias involuntariamente muestran solo la arista de la labor docente, dejando de lado otros campos de acción de los profesionales en el área, tales como: la investigación y aplicabilidad. Esto responde a la considerable carga académica de la que son objeto los profesores, quienes deben presentar excesivo contenido “obligatorio” en el menor tiempo posible. Lo que no da cabida para planificar actividades investigativas tendientes a mostrar a los aprendices, la aplicabilidad de las ciencias, tanto dentro de las propias disciplinas como en otras áreas del conocimiento. Se resalta pues, que las actividades de carácter investigativo son tareas de los alumnos, vinculadas a la búsqueda de respuestas para problemas de índole creativo o de investigación, sin una solución conocida de antemano y que supone la presencia de las etapas básicas de una investigación validada por la comunidad científica (Alekseev et al., 2002). Por otro lado, aun cuando las actividades extracurriculares tienen un carácter opcional, resultan una alternativa factible de enseñanza y aprendizaje de las ciencias, en virtud que no se encuentra como mediador, la responsabilidad de una calificación. Entre uno de sus propósitos, se encuentra incentivar el acercamiento a las ciencias, y, de alguna manera, minimizar los obstáculos latentes dentro de las clásicas clases académicas, buscando engendrar motivación, interés, gusto y disfrute en la realización de actividades relacionadas con el área. Estas dinámicas comúnmente son viables dentro de clubes, campamentos, festivales, ferias, revistas de ciencias, competencias escolares, concursos, entre otros. Los resultados de estas actividades son beneficiosos, cuando se trata de despertar interés y motivación por el aprendizaje en ciencia, y cuando los alumnos tengan la oportunidad de participar. Aquellos estudiantes que logran participar en este tipo de experiencias tienen la oportunidad de conocer alternativas de enseñanza y aprendizaje de las ciencias. En el caso de la enseñanza, resolución de problemas y desarrollo las capacidades metacognitivas; y Innovando P á g i n a 156 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org en cuanto al aprendizaje, se encuentran estrategias que determinan condiciones favorables para el descubrimiento, el cambio conceptual como punto de partida de las ideas constructivistas y la investigación dirigida (Campanario & Moya, 1999). Además, se puede incluir el desarrollo de competencias a través del método partiendo del problema. Este último, consiste en presentar a los estudiantes situaciones problemáticas, inquietudes o curiosidades de interés que ellos mismos planteen, con la finalidad de que sean ellos, quienes comiencen a formularse preguntas con respecto al fenómeno a estudiar, e intenten buscar respuestas a sus interrogantes, planteando hipótesis que luego serán aceptadas o rechazadas (Sgibnev, 2013). Aun cuando este tipo de actividades extraescolares también refleja la faceta docente de un profesional de ciencias, suele ser más comprometida y cercana con la búsqueda de resultados satisfactorios en el proceso de enseñanza y aprendizaje de los alumnos por el área. Además, en condiciones ideales, el uso de algunas de las propuestas alternativas mencionadas genera cierta aproximación del estudiante a la faceta investigativa, lo que, sin lugar a duda, eleva el nivel de abstracción y comprensión de los fenómenos e incluye al alumno como una pieza fundamental vinculada con su aprendizaje. El desarrollo de actividades escolares y extraescolares ofrece un primer contacto directo de los estudiantes con las ciencias que, hasta cierto punto no llega a mostrarles concreta y explícitamente la labor de un profesional en esta área, pero si puede ofrecerles ciertos vestigios de otras facetas como, por ejemplo: la investigación. Todo esto evidencia, la desconexión por desconocimiento que tiene el estudiante, acerca de la verdadera labor y panorama de desarrollo intelectual, con el que cuenta un individuo que ha decidido profesionalizarse en el área de ciencias. Desconocimiento por la labor de los profesionales de ciencias Existe un marcado desconocimiento acerca de la totalidad de quehaceres ejecutados por estos profesionales de las ciencias. Esto se refuerza al usar como base de referencia las tradicionales clases en las instituciones educativas. Es decir, en ese primer contacto con el área, los alumnos frecuentemente ven a sus diferentes profesores de ciencias, solamente impartiendo clases. En tal sentido, no es descabellado suponer que la única labor que desempeña un profesional en ciencias sea la docencia. Esto conduce a etiquetar a priori Innovando P á g i n a 157 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org a todos los profesionales de ciencias bajo este abreviado rol; aun cuando, esta sea un área que influye y se yuxtapone con la mayoría de las otras áreas del conocimiento. Si bien los estudiantes tienen la oportunidad de ahondar en otras facetas de la ciencia por medio de actividades extraescolares donde se fortalezcan otras áreas, no es menos cierto que solo un grupo selecto es el que puede acceder a ellas. En estas actividades, probablemente conocen a pequeña escala esta otra faceta de la profesión. Sin embargo, las probabilidades de participación de los alumnos en este tipo de eventos son relativamente bajas y dependen de diversas razones, entre las cuales se destacan: el interés, los recursos económicos, el tiempo y el esfuerzo intelectual, tanto individual como colectivo, por parte de las instituciones educativas y de las comunidades donde estos se desarrollen. Esto conlleva, a que sea una minoría privilegiada la que tiene acceso a este tipo de actividades, generando que la docencia siga siendo la cara visible y predominante del área de las ciencias. Es así, como la gran mayoría de estudiantes están privados de conocer los continuos avances de la ciencia, y sobre todo las múltiples aplicaciones dentro del ámbito sociolaboral del área. En relación con esto, y a la par de las plazas encontradas en el campo de la docencia e investigación, se pueden hallar una diversidad de roles dentro de la faceta de la aplicabilidad, los cuales pueden ir desde un maestro cervecero en una empresa de elaboración de cervezas (profesional de química), hasta un especialista en análisis numérico en una industria petrolera (profesional de matemática), entre otros. Por lo tanto, es preciso puntualizar que, la docencia de corte tradicional da pie a la creencia de que las personas estudiosas de las ciencias, solo pueden impartir clases; por otro lado, la investigación a nivel escolar es realmente efectiva en el desarrollo cognitivo de los estudiantes, al demostrar aproximaciones o simulaciones de la labor que persigue únicamente la obtención del conocimiento y; finalmente, la aplicabilidad en la industria, debe ser un puntal para rebatir el argumento de la docencia, como punto único de desarrollo profesional, sin embargo, estos debates solo tienen lugar en contexto más interno. Desinterés por las carreras de ciencias Innovando P á g i n a 158 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org La falta de interés por carreras en el marco de las ciencias puede atribuirse a la carencia de información fidedigna del quehacer propio de cada disciplina. En particular, lo importante e influyente que puede llegar a ser la labor de los profesionales para el desarrollo de su área, y para las otras áreas del conocimiento. Esto se debe, en parte, a que solo se cuenta con información parcial y/o vaga para difundir, lo que conlleva a malas interpretaciones, y conduce a un sesgo pernicioso que influye sobre el interés del estudiante por las carreras de ciencias. Este desinterés aumenta cuando se observa a los profesores de ciencias impartir muchas horas de clases, a la par de planificar, organizar, preparar clases y evaluar contenidos programáticos, que, a su vez, requieren del diseño, implementación y corrección. Sumado a esto está el tema de la investigación, que, al estar estrechamente vinculado con las ciencias, capitaliza el desgano, porque requiere de un esfuerzo intelectual importante por parte del profesor, que se dificulta más aun, cuando no se dispone de los conocimientos necesarios para llevar a cabo una pesquisa por muy básica que esta sea. Por otra parte, las instituciones educativas, quizás por cuestiones de tiempo o comodidad, no se preocupan por actualizar y articular contenidos, haciendo parecer a las ciencias un área del conocimiento desolada e infértil, en cuanto a asuntos de aplicabilidad y utilidad para la sociedad. La combinación de estas consideraciones sin duda influye en las concepciones de los estudiantes con respecto a las profesiones de ciencias. Hecho que repercute a la hora del proceso de búsqueda, selección, elección y decisión por un programa de estudio que le permita convertirse en profesionales en algún área del conocimiento. Todo lo anterior, funciona como un tejido conectivo que minimiza la factibilidad de las carreras del área de las ciencias, por considerarlas improductivas, difíciles y con un panorama laboral bastante reducido, lo que hace que no sean tomadas en cuentas seriamente como opción, durante el proceso mencionado al inicio de este parágrafo. Doble efecto de la deseabilidad vocacional Es un problema latente que se presenta constantemente dentro de los procesos de admisión para los programas de estudio de las carreras de ciencias, principalmente en las instituciones públicas de Educación Superior. En efecto, como las carreras en ciencias no Innovando P á g i n a 159 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org gozan de una alta popularidad entre los estudiantes como futura profesión, esto las hace susceptible de ser utilizadas como carreras puente para acceder a la carrera deseada. Esta forma de ingreso se conoce como el efecto trampolín. También es posible observar casos como los que se detallan a continuación, cuando los estudiantes no logran el propósito de ubicarse en la carrera que desean, por ejemplo: resignación con tal de obtener un título universitario (tercer nivel); deserción escolar voluntaria (abandono) u obligatoria. Esta última, es considerada como la expulsión de los programas de estudio, de acuerdo con las normas y reglamentos de permanencia de la institución de Educación Superior. Existe entonces la posibilidad de que los cupos ofrecidos por las instituciones de Educación Superior cada nuevo año para estudiar alguna carrera en el área de las ciencias, sean cubiertos en su totalidad en los procesos de admisión. Pero, esto no garantiza que todos estudiantes seleccionados o admitidos, tengan la verdadera intención de estudiar o, al menos de terminar con éxito, una carrera en esta área del conocimiento. Adicionalmente, los aspirantes que usan esta forma de ingreso a los programas de estudio en las instituciones de Educación Superior lo hacen por el doble efecto de la deseabilidad vocacional, ascendente y descendente. El efecto ascendente, explica la tendencia a enfocarse y alistarse en determinadas profesiones de alto rango (cotizadas), con el propósito de lograr situarse en ellas; y el efecto descendente, cuando aceptan ocupaciones de menor rango o puntaje de ingreso, en ocasiones con matices de resignación y frustración (Rivas, 1995). Ejemplos de esta situación, se muestran en los estudios realizados por Abancin & Strauss (2013a y 2013b). Minoría de interesados por las carreras de ciencias A pesar de la poca motivación por las carreras en el área, siempre surgen individuos interesados en estudiar las ciencias, decantar este grupo resulta una tarea un tanto compleja. En este sentido, con el tiempo y el transcurso de los lapsos académicos se consolidan progresivamente los posibles candidatos a profesionales de ciencias. No obstante, obtener un título académico en el área, requiere de un arduo trabajo y méritos académicos suficientes que certifiquen las capacidades que se requieren para el ejercicio de estas profesiones. Esto explica, en parte, la insuficiencia de estos profesionales en el campo laboral, traduciéndose en un déficit y una sobre oferta de plazas de trabajo para Innovando P á g i n a 160 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org los profesionales de ciencias, lo que irónicamente pudiera incrementar el desencanto para aquellos que siguen la moda o las tendencias. Déficit de profesionales de ciencias El déficit de profesionales es un problema de vieja data dentro del campo laboral, agravándose más ante la falta de personal cualificado para desenvolverse en alguna de las tres facetas principales que revisten el desarrollo de las ciencias. Es frecuente contrarrestar la insuficiencia de docentes de ciencias, contratando a estudiantes en proceso de formación (no graduados), o profesionales en otras áreas de formación que posean algún conocimiento con respecto al área de ciencias (e.g., ingenieros). En el primer caso, es posible que los estudiantes no graduados no alcancen las competencias básicas necesarias para el puesto y, además de ello, se encuentra latente la posibilidad de que no culminen con éxitos sus estudios. Esta solución paliativa bien puede aliviar la insuficiencia de profesores en la Educación Media; sin embargo, lo que puede representar una solución a corto plazo para las instituciones educativas, puede originar otro problema; ya que el empleo desacertado de técnicas de enseñanza y aprendizaje puede traer como consecuencia la animadversión, el desinterés y la falta de motivación por el estudio de estas profesiones. Con respecto a los profesionales de otras áreas, aun cuando poseen ciertos conocimientos del área de ciencias, precisan de una formación extra capaz de otorgarle saberes especializados para el ejercicio de la profesión docente, indispensables en el background de un profesional que se desempeñe bajo la figura de profesor. Al respecto Álvarez & Valls (2019), consideran que la interdisciplinariedad del área hace que para los docentes de la didáctica de las ciencias no sea suficiente con tener una buena formación científica y en contenidos científicos; además, deben poseer formación proveniente de la filosofía y de la historia de las ciencias; así como, estar familiarizados con aquellos recursos, estrategias y habilidades que permitan la eficacia de los procesos de enseñanza y aprendizaje, los cuales se adquieren a través de áreas como la psicología y la pedagogía. Entonces, contrario a lo que se suele suponer, la labor docente requiere de unos conocimientos y habilidades técnico-prácticas para lograr con éxito el proceso de la enseñanza; ya que no resulta lo mismo saber que enseñar. Por ejemplo, un ingeniero Innovando P á g i n a 161 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org puede saber matemática, pero esto por antonomasia no lo convierte un profesional calificado para la enseñanza del área. Por otro lado, para el desarrollo de las actividades académicas a nivel de Educación Superior, es frecuente la constante búsqueda de profesionales que estén dispuestos a impartir clases del área de ciencias. La solución es similar a la empleada en la Educación Media, solo que en este caso se emplean individuos recién egresados de sus estudios de pregrado, o profesionales de otras áreas del conocimiento. Las consecuencias de esta solución alternativa apuntan en la misma dirección que las consideradas en párrafos ut supra, pero en este caso, el daño en los estudiantes universitarios puede ser incalculable; ello sin contar la pertinencia de los métodos de enseñanza empleados y la escasa o nula actividad investigativa que soporta la docencia a nivel universitario. Además, esta solución tiene fuertes limitaciones debido a que es solo aplicable para los cursos de los niveles básicos; a niveles superiores, por el grado de exigencia, se necesitan profesionales altamente calificados con aptitudes demostrables en docencia e investigación. En cuanto a los profesionales de ciencias involucrados con la investigación, nos encontramos con una comunidad aún más pequeña que la de los profesores, cuyo déficit implica un constante problema dentro del área. Los profesionales de ciencias que desean realizar actividades de investigación requieren de una adecuada formación académica, derivada de años de estudios dedicados a la preparación académica a través de los diferentes niveles de Educación Superior (pregrado y postgrado). Sin lugar a duda, esos años de estudio a los que se hace mención, consolidan una o más líneas de investigación que, que contribuyen a reforzar el área y al individuo con logros académicos, tales como: trabajos de titulación de grado y posgrado, disertaciones, ponencias, artículos científicos y/o libros, en los que se exponen y divulgan ideas y planteamientos de orden inédito. Concretamente, los años de formación per se no son suficientes para convertirse en un investigador exitoso; para ello se requiere de mucho trabajo extra, dedicación, compromiso y creatividad para investigar, así como proponer, plantear y abordar soluciones a problemas diversos. Al mismo tiempo, se precisa formar nuevas generaciones de profesionales que continúen apoyando las líneas de investigación existentes, o generen nuevas líneas, con miras a ampliar las fronteras del conocimiento. Innovando P á g i n a 162 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org En este sentido, es común que la investigación científica se vea impulsada por instituciones de Educación Superior. En resumen, el déficit de profesionales en ciencias es alarmante. La situación se agrava aún más, cuando la gran mayoría se dedica casi exclusivamente a la labor docente. Sin embargo, no todo está perdido, aún hay un valioso grupo que se encuentra dedicado a la investigación, así como aquellos que optan por realizar aplicaciones de sus hallazgos o los de otros investigadores. Adicionalmente, existe un tercer grupo de profesionales concentrados en actividades relacionadas directamente con industrias y/o empresas, y distanciados de la docencia y la investigación, los cuales aportan con sus conocimientos al desarrollo del aparato productivo y tecnológico de las naciones. La figura1 ilustra de forma resumida la cadena de eventos e implicaciones existentes en torno a la impopularidad de las ciencias, todas relacionadas con lo que posiblemente sucede en torno al área académica circunscrita a esta. Figura 1 Cadena de eventos en torno al área de ciencias Innovando P á g i n a 163 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org Conclusiones El bagaje de ideas esbozado a lo largo de esta investigación permite abrir un espacio para la discusión y reflexión sobre aspectos y acontecimientos que subyacen dentro del ámbito educativo, tales como: el primer contacto, el desconocimiento, el interés, el doble efecto de la deseabilidad vocacional, la minoría y el déficit de profesionales en las ciencias, los cuales influyen en la escasa o baja popularidad de las ciencias. Estos condicionantes, que visiblemente desestiman la importancia de las ciencias frente a otras ramas del conocimiento, son originados a lo largo del proceso de enseñanza y aprendizaje, en parte, por el monolítico tradicionalismo que reviste el área, desde óptica educativa. Principalmente se busca con esta discusión, que la enseñanza y aprendizaje de las ciencias pueda darse desde la perspectiva del integracionismo colaborista, entendiendo que las ciencias, como cualquier rama del saber, tiene su nivel de complejidad; pero también tiene un sinnúmero de aplicaciones adicionales, a las que la simplista preconcepción, no tiene acceso. Por otra parte, comparados con otras profesiones, los profesionales en ciencias tienen una desventaja adicional, no contemplada en investigaciones en el área. Aun cuando se encuentran vinculados con su profesión desde cualquiera de las tres facetas descritas en parágrafos anteriores, no logran presentar sus ramas del conocimiento desde la óptica de las bondades, facilidades y privilegios. Esta falta de interés de los profesionales del área, por demostrar la magnanimidad de las ciencias, abre la brecha para el desprestigio y el descredito por parte de aquellos que no la conocen a profundidad, y, que tampoco están vinculados con ella desde ningún punto de vista. La vigencia de este ejercicio reflexivo estará demarcada por las adecuaciones que se hagan en pro de mejorar las técnicas de enseñanza y aprendizaje de las ciencias, así como las demostraciones de la ciencia en lo cotidiano, toda vez que nos encontramos en presencia de una generación que, al encontrarse fuertemente influenciada por la tecnología (nativos digitales), se resiste a aprender como históricamente lo hemos venido haciendo. Se espera entonces un cambio de paradigma en el proceso de enseñanza y aprendizaje de las ciencias mediante el cual se presente a las ciencias en forma atractiva haciendo uso del lenguaje tecnológico. Esto ayudaría a entender y aceptar el papel relevante e influyente que juegan las ciencias en los progresos tecnológicos, sociales y Innovando P á g i n a 164 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org culturales Todo esto contribuirá a la captación de individuos para el estudio de las ciencias, hecho que a futuro permitirá un abastecimiento significativo de profesionales cualificados para cubrir la demanda constante dentro del campo laboral, dentro y fuera de la academia. Finalmente, es preciso tomar en cuenta que el proceso de enseñanza y aprendizaje de las ciencias debe ser visto desde una perspectiva articulada e integracionista de contenidos que prevean aspectos tales como la contextualización y la funcionalidad. Recordemos que, muchas veces, durante el proceso de enseñanza y aprendizaje, resulta difícil salir del contexto exclusivamente académico, y, colocar ejemplos y/o actividades útiles que transciendan más allá de la barrera académica intramuros, pero es un esfuerzo que merece la pena realizar Referencias Bibliográficas Abancin, R. & Strauss, V. (2013a). Trayectoria académica de los estudiantes de las licenciaturas en matemáticas de la Universidad Simón Bolívar durante la última década. En A. Ramírez e Y. Morales. (Eds.), Memorias. I CEMACYC (pp. 15021504). Red de Educación Matemática de América Central y El Caribe. http://ciaemredumate.org/memorias-icemacyc/memorias_completo.html Abancin, R. & Strauss, V. (2013b). Perfil e inclinación vocacional en matemáticas de los estudiantes del Programa Ciclo de Iniciación Universitaria de la Universidad Simón Bolívar. En A. Ramírez e Y. Morales. (Eds.), Memorias. I CEMACYC (pp. 1112-1125). Red de Educación Matemática de América Central y El Caribe. http://ciaemredumate.org/memorias-icemacyc/memorias_completo.html Alekseev, N. G., Leontovich, A. V., Obujov, A. S. & Fomina, L. F. (2002). El concepto del desarrollo de las actividades de carácter investigativo con estudiantes. Issledovatelskaya rabota shkolnikov (Actividades de carácter investigativo de estudiantes), 1, 24-33. Álvarez, J. & Valls, C. (2019). Didáctica de las ciencias, ¿de dónde venimos y hacia dónde vamos? Universitas Tarraconensis. Revista de Ciències de l’Educació, 2, 5-19, https://raco.cat/index.php/UTE/article/view/369759. Cabezas, E., Naranjo, D. & Torres, J. (2018). Introducción a la metodología de la investigación científica. Universidad de las Fuerzas Armadas ESPE. Campanario, J. & Moya, A. (1999). ¿Cómo enseñar ciencias? Principales tendencias y propuestas. Enseñanza de las ciencias: revista de investigación y experiencias didácticas, 17(2), 179-192. https://raco.cat/index.php/Ensenanza/article/view/21572. Innovando P á g i n a 165 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org Chvanova, A. & Garbín, S. (2017). La formación matemática y la resolución de “problemas para investigar”: Una aproximación según el enfoque integral de Ken Wilber. Revista Paradigma, 38(1), 353–379. DOI: 10.37618/PARADIGMA.1011-2251. 2017.p353–379.id 614. Flórez, E. & González, M. (2021). Diseño de unidades didácticas mediante el aprendizaje basado en problemas para la enseñanza de las ciencias. Revista Científica, 41(2), 134-149. https://doi.org/10.14483/23448350.17472. Hammer, D. (1994). Epistemological beliefs in introductory physics. Cognition and instruction, 12, 151-183. Hodson, D. (1994). Hacia un enfoque más crítico del trabajo de laboratorio, Enseñanza de las Ciencias, 12, 299-313. Justi, R. (2006). La enseñanza de las ciencias basada en la elaboración de modelos. Enseñanza de las ciencias, 24(2), 173-184. Martín, M. (2002). Enseñanza de las ciencias ¿Para qué? Revista Electrónica de Enseñanza de las Ciencias, 1(2), 57-63. http://reec.uvigo.es/volumenes/volumen1/REEC_1_2_1.pdf. Martínez, G., Cervantes. & Jiménez, L. (2021). Experiences of Mexican teenage students when choosing a math degree: A mathematical narrative identity study. Uniciencia, 35(1), 245-264. DOI: http://dx.doi.org/10.15359/ru.35-1.15. Pozo, J. (1999). Sobre las relaciones entre el conocimiento cotidiano de los alumnos y el conocimiento científico: del cambio conceptual a la integración jerárquica. Enseñanza de las Ciencias, número extra, 15-29. Rivas, F. (1995). Manual de asesoramiento y orientación vocacional. Síntesis. Rojas, M. (2015). Tipos de Investigación científica: una simplificación de la complicada incoherente nomenclatura y clasificación. REDVET. Revista Electrónica de Veterinaria, 16(1), 1-14. https://www.redalyc.org/articulo.oa?id=63638739004. Sandoval, C. (1996). Investigación cualitativa. Instituto Colombiano para el Fomento de la Educación Superior. Bogotá, Colombia. Sgibnev, A.I. (2013). Problemas de carácter investigativo para principiantes. Moskovski centr neprerivnogo obrazovaniya (Centro de educación continua de Moscú), Moscú, Rusia. Innovando P á g i n a 166 | 167 ISSN: 2600-5859 Vol. 5 No. 1.3, pp. 148– 167, marzo 2022 www.concienciadigital.org El artículo que se publica es de exclusiva responsabilidad de los autores y no necesariamente reflejan el pensamiento de la Revista Conciencia Digital. El artículo queda en propiedad de la revista y, por tanto, su publicación parcial y/o total en otro medio tiene que ser autorizado por el director de la Revista Conciencia Digital. Innovando P á g i n a 167 | 167
https://openalex.org/W2732932618
https://bmcanesthesiol.biomedcentral.com/track/pdf/10.1186/s12871-017-0379-2
English
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Case report: anaesthetic management of radical gastrectomy for gastric cancer associated with anti-N-methyl-D-aspartate receptor encephalitis
BMC anesthesiology
2,017
cc-by
4,313
Open Access © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a rare neurological disorder that is caused by the production of antibodies against NMDARs. As many anaesthetic drugs interact with NMDARs and may worsen the disease and because the disease poses risks, such as cardiovascular events, hyperthermia and respiratory insufficiency, while under anaesthesia, administering anaesthesia to patients with this disorder is clinically challenging. Case presentation: A 55-year-old man with gastric cancer associated with anti-NMDAR encephalitis who was diagnosed 8 months prior was admitted to Peking University Cancer Hospital for tumour resection. Before surgery, the patient’s symptoms had been successfully controlled via aggressive immunotherapy. Radical gastrectomy was performed under general anaesthesia induced with remifentanil, propofol, and cisatracurium and maintained with sevoflurane and remifentanil. The patient had a favourable recovery without any adverse symptoms or post-operative complications. Conclusions: Adequate preparation for surgery is essential for the anaesthetic management of patients with anti-NMDAR encephalitis. These rare patients may benefit from general anaesthesia induced using remifentanil, propofol and cisatracurium and maintained using sevoflurane and remifentanil. Additionally, the use of NMDA antagonists, such as ketamine, nitrous oxide and tramadol, should be avoided. Keywords: Case report, Anaesthetic management, Anti-N-methyl-D-aspartate receptor encephalitis, Gastric cancer that tumour resection in combination with aggressive immunotherapy (corticosteroids and/or immunoglobulin i.v.) can facilitate earlier functional recovery [6–8], but associated with a high rate of recurrence and mortality [9, 10]. Moreover, the interactions of various anaesthetic drugs with NMDARs may aggravate this disease and lead to increased risk with respect to general anaesthesia for tumour resection, such as cardiovascular events, hyperthermia and respiratory insufficiency; this risk poses a novel challenge for anaesthetic management during surgical procedures. This report provides the first description of anaesthetic management in a male patient with anti-NMDAR encephalitis who presented with gastric cancer. We discuss the specific considerations and certain principles related to the planning of general Case report: anaesthetic management of radical gastrectomy for gastric cancer associated with anti-N-methyl-D-aspartate receptor encephalitis Case report: anaesthetic management of radical gastrectomy for gastric cancer associated with anti-N-methyl-D-aspartate receptor encephalitis Lei Ding1, Hongyu Tan1* , Ziyu Li2, Jiafu Ji2 and Xuejun Song1 Lei Ding1, Hongyu Tan1* , Ziyu Li2, Jiafu Ji2 and Xuejun Song1 * Correspondence: hongyutan0062@sina.com 1Key laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Anaesthesiology, Peking University Cancer Hospital & Institute, Beijing 100142, China Full list of author information is available at the end of the article Ding et al. BMC Anesthesiology (2017) 17:90 DOI 10.1186/s12871-017-0379-2 Ding et al. BMC Anesthesiology (2017) 17:90 DOI 10.1186/s12871-017-0379-2 Background Anti-N-methyl-D-aspartate receptor (NMDAR) enceph- alitis is recognized as a rare autoimmune-induced disorder in which autoantibodies generated in the body bind NMDA receptors in the brain, causing a series of neurological dysfunctions, such as psychosis, involuntary movements, autonomic nervous instability, central hypoventilation, seizure and hyperthermia [1–3]. Studies have demonstrated that these disorders commonly affect females in association with the presence of a mature NMDAR ovarian teratoma [2, 4, 5]. It has been reported * Correspondence: hongyutan0062@sina.com 1Key laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Anaesthesiology, Peking University Cancer Hospital & Institute, Beijing 100142, China Full list of author information is available at the end of the article Page 2 of 5 Ding et al. BMC Anesthesiology (2017) 17:90 (1 L·min−1), air (1 L·min−1), sevoflurane (1.5–2.0%) and remifentanil (0.3 μg·kg−1·min−1). Neuromuscular blockade was maintained with intermittent cisatracurium. The monitored parameters included ECG, ABP, capnography, pulse oximetry and the bispectral index (BIS). The patient’s intraoperative systolic blood pressure was 100–135 mmHg, his heart rate was 60–80 beats·min−1 and his BIS was 40–60. Surgery was completed without any complications. The durations of surgery and anaesthe- sia were 2 h 22 min and 3 h 18 min, respectively. Intraop- eratively, total blood loss was 80 ml, urine output was 200 ml, and total infusion volume was 1600 ml. The patient was then transferred to the ICU with a tracheal tube and mechanical ventilation, administered a low dose of propofol for sedation and monitored closely. Consider- ing that the patient was in convalescence from anti- NMDAR encephalitis, the neurologist suggested that there was no need to use corticosteroids or immunoglobulin. The patient was stable and successfully extubated two days after surgery. No evidence of post-operative complications or worsened neurological symptoms were observed. With an uneventful recovery and disappearance of the quiver, the patient was discharged on the 26th post- operative day after the risk of recurrence of anti-NMDAR encephalitis was evaluated. At the 2-month follow-up, he was symptom-free and exhibited no signs of any post- operative complications. anaesthesia for the population of patients with anti- NMDAR encephalitis. Case presentation The subject was a 55-year-old man (162 cm, 64 kg) with a 5-year history of stomach-ache that had worsened during the prior year. He also complained of hypologia, depression, insomnia and hypomnesis during the previ- ous 2 years. Eight months prior to surgery at our hos- pital, he was admitted to Peking Union Medical College Hospital for medical evaluation immediately after he developed left upper limb twitching. Cerebrospinal fluid analysis revealed the following: cells 280 × 106 L−1 (normal values 0–8 × 106 L−1); lymphocyte 75% (normal values 49–73%); neutrophil 24% (normal values 0–2%); glucose 2.9 mmol·L−1 (normal values 2.8–4.5 mmol·L−1); protein 0.8 g·L−1 (normal values 0.1–0.4 g·L−1); IgG 61.30 mg·L−1 (normal values 10.00–40.00 mg·L−1); and positive antigen-specific oligoclonal bands and NMDAR antibody. Tests for paraneoplastic antibodies, including Hu, Yo, Ri, amphiphysin, CV2 and Ma2, were all negative. A previous PET scan and electronic gastroscopy did not show obvious lesions. The patient was diagnosed with anti-NMDAR encephalitis and treated with intravenous methylprednisolone and immunoglobulin for 8 days, followed by oral prednisone for 2 months. Eventually, his symptoms gradually improved. Two months later, gastros- copy and pathology revealed a malignant lesion in the gastric fundus and cardia (0.6 × 0.7 cm2). Based on previ- ous reports, a neurologist suggested that the perioperative risk for this patient with anti-NMDAR encephalitis was extremely high if the patient underwent gastric cancer sur- gery, even if this surgery occurred during the recovery period, and recommended that the surgery be performed in our hospital—one of the most advanced and authorita- tive hospitals on gastric cancer surgery in China—by an experienced perioperative medical management team. Discussion inhibition of the NMDA pathway and worsen the clinical presentation of anti-NMDAR encephalitis. However, in most cases, a detailed anaesthetic procedure was not pro- vided, and the post-operative course was not described. Therefore, the choice of anaesthetics for patients with anti-NMDAR encephalitis requires additional study. Furthermore, due to the complex condition of patients with anti-NMDAR encephalitis, including high fever, auto- nomic dysfunction, and central ventilation dysfunction, attention should be paid to adverse reactions, such as car- diovascular events, hyperthermia, respiratory insufficiency and delayed (or difficult) extubation, during the induction and maintenance of general anaesthesia and after surgery. Anaesthesiologists should also be prepared for PSH, which is frequently observed in combination with this disorder. The readily available vasopressors, beta-blockers, anti- hypertensives, and anti-cholinergics during a case are pru- dent to ensure that any autonomic instability can be dealt with in a timely manner. Because PSH has been described in patients suffering from severe traumatic brain injury, the management principles for PSH should be followed for anti-NMDAR encephalitis patients [31, 32]. Patients undergoing surgical resection of tumours commonly require general anaesthesia. However, anti- NMDAR encephalitis presents anaesthesiologists with many risks and challenges. First, many anaesthetic drugs inhibit NMDAR and can therefore induce the same symptoms as those observed in anti-NMDAR encephalitis [13, 18]. N2O reduces NMDAR-mediated excitatory currents in the basolateral amygdala, an area associated with anaesthesia-induced amnesia and the formation of aversive memories, fear, and addictive behaviour [19]. Ketamine is a well-known NMDAR antagonist that inhibits glutamate-triggered calcium influx [20]. The effects of certain anaesthetic drugs on NMDARs remain unclear. Research has demon- strated that halogenated anaesthetics, such as sevoflurane, inhibit NMDA-gated currents and NMDA-induced mitochondrial membrane depolarization; however, their enhancing effects on GABA receptors may be dominant and may blunt the autonomic hyperactivity observed in patients with anti-NMDAR encephalitis [1, 21–25]. Propofol acts by enhancing GABAergic transmission and may also inhibit NMDARs in vitro [4, 26, 27]; how- ever, the clinical relevance of this inhibition has not been established. Moreover, drugs that primarily act on GABA receptors, including pentobarbital, diazepam, and midazolam, may also have indirect interactions with NMDARs. Sufentanil and cisatracurium appear to have no significant effects on NMDARs [28]. The conditions of cases reported by Chen W et al. [29] and Lapedbie et al. [30] all deteriorated to some degree after anaesthesia, although all cases ultimately survived. Discussion Anti-NMDAR encephalitis was first identified as a type of autoimmune limbic encephalitis in 2007. This disease has been commonly associated with malignancies such as germ cell tumours of ovarian teratomas and breast cancer but rarely gastric cancer, with unclear aetiology and incidence [2, 6–8, 11, 12]. Although there are several case reports on the anaesthetic management of anti- NMDAR encephalitis associated with ovarian teratomas, this report is the first description of the anaesthetic management of a patient with gastric cancer that led to this condition. Radical gastrectomy was scheduled after the adminis- tration of chemotherapy for 4 months. Prior to surgery, the patient still exhibited a slight but very rare quiver in the left upper limb, but blood tests were all negative for NMDAR antibodies. Therefore, the patient was consid- ered to be in convalescence. Dexamethasone (10 mg) was administered as a pre-anaesthetic medication. Upon the patient’s arrival to the operating room, invasive blood pressure monitoring was established. The patient’s blood pressure was 150/75 mmHg, his heart rate was 90 beats·min−1, and his arterial oxygen saturation was 97% when breathing room air. To ensure stable vital signs, vasopressors, beta blockers, anti-hypertensives and anti-cholinergics were readily available. General anaesthe- sia was intravenously induced with remifentanil (100 μg), cisatracurium (20 mg) and propofol (150 mg) to facilitate tracheal intubation and was maintained with oxygen Anti-NMDAR encephalitis is classified as a paraneo- plastic syndrome and involves the production of auto- antibodies against NMDARs induced by nerve tissue containing the NMDAR subunits in the tumour [2, 13]. Dysregulation of NMDARs has been linked to schizophre- nia, Alzheimer’s disease and Parkinson’s disease [14, 15]. Inactivation of inhibitory gamma-amino butyric acid (GABA)-ergic interneurons, which express high concen- trations of NMDARs, also plays a key role in the patho- physiology of this disease [13]. Clinical manifestations during the early stage are characterized by psychiatric symptoms. Late stages are often accompanied by par- oxysmal sympathetic hyperactivity (PSH) that includes Page 3 of 5 Page 3 of 5 Ding et al. BMC Anesthesiology (2017) 17:90 hyperthermia, tachycardia or hypertension, hypoventilation, and motor or complex seizures [13, 16, 17]. Various treat- ment modalities, including first-line immunotherapy (corti- costeroids and/or IV Ig and/or plasma exchange) and the early removal of an underlying tumour, may be associated with a good prognosis. However, recovery may require 3–4 months of hospitalization followed by several months of rehabilitation. Moreover, 12% of patients relapse [9, 10]. Discussion In another hospital, 3 patients with similar conditions who recovered after immunotherapy unfortunately relapsed several days after anaesthesia for ovarian teratoma resec- tion and did not survive to discharge (not reported). This outcome suggests that certain forms of anaesthesia may play significant roles in inducing recurrence and death among patients with anti-NMDAR encephalitis. In our patient, we used remifentanil, cisatracurium and propofol to induce anaesthesia and sevoflurane, remifentanil and cisatracurium for maintenance. Vaso- pressors, beta-blockers, anti-hypertensives and anti- cholinergics were readily available. As Lapebie et al. [30] reported a case who was administered sevoflurane and propofol during the operation and a high dose of propofol for post-surgery sedation. We hypothesized that his deteri- oration after surgery may have been attributed not only to the combination of sevoflurane and propofol but also to the high dose of propofol. Thus, we used a low dose of propofol only during induction and chose sevoflurane as the single anaesthetic during maintenance. All medica- tions were given at the minimum effective dose and stopped as soon as the operation was completed to reduce potential reactions with NMDARs. As mentioned above, patients with anti-NMDAR encephalitis may also exhibit autonomic dysfunction and central ventilation dys- function, and some anaesthetic drugs may induce or aggravate cardiovascular events, respiratory insufficiency and delayed (or difficult) extubation. Several cases deterio- rated after extended periods of time following surgery. Therefore, when administering anaesthesia to patients with anti-NMDAR encephalitis, it is wise to avoid the use of drugs that act via NMDARs, including ketamine, N2O and tramadol. Medications that indirectly interact with NMDARs can still be considered for use. Several important medications, including fentanyl, sufentanil, remifentanil, propofol, sevoflurane, isoflurane, desflur- ane, vecuronium, rocuronium and cisatracurium, have been well tolerated during surgery in certain reported cases. Chen W et al. [29] reported three cases that underwent anaesthesia for ovarian teratoma resection with midazolam, fentanyl, propofol and rocuronium for induction and sevoflurane, fentanyl and rocuronium for maintenance. All patients survived the surgery and were discharged with mild psychiatric symptoms. Lapebie et al. [30] hypothesized that anaesthesia using both pro- pofol and sevoflurane simultaneously may facilitate Ding et al. BMC Anesthesiology (2017) 17:90 Page 4 of 5 Page 4 of 5 Ding et al. Ethics approval and consent to participate Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Funding Not applicable. Received: 23 December 2016 Accepted: 14 June 2017 Received: 23 December 2016 Accepted: 14 June 2017 Competing interests Th h d l h Competing interests The authors declare that they have no conflicts of interest to disclose. Discussion BMC Anesthesiology (2017) 17:90 Page 4 of 5 Early extubation without adequate time of observation, assessment or medication in some cases may induce or worsen cardiovascular events or result in reintubation if the patient relapsed. To ensure adequate time to nurse and monitor the patient and to provide safe and stable post-operative recovery, we kept the patient intubated and sedated with a low dose of propofol, as propofol is a short-acting drug that may not affect NMDARs suffi- ciently to worsen neurologic symptoms. The patient was extubated after observation for one day, and we ensured there was no sign of deterioration or dysfunction of spon- taneous breathing. In the report by Chen W et al. [29], the three patients were extubated on the 1st, 5th and 90th day and discharged on the 3rd, 5th and 8th week, respectively, after surgery, depending on the status of anti-NMDAR encephalitis. However, all patients developed obvious neurological and psychiatric symptoms and received immunoglobulin after surgery. By contrast, our patient was extubated and discharged early and recovered well without any neurologic symptoms, recurrence or worsen- ing post-surgery, even in the absence of immunotherapy after surgery. We suspect that this result may be attributed to the reasonable management of anaesthesia (without midazolam) and the effective control of his disease prior to surgery. Availability of data and materials The datasets supporting the conclusions of this article are available in the case repository of Peking University Cancer Hospital & Institute. Because the medical records include identifying/confidential patient data, we cannot deposit datasets in publicly available repositories. Author details 1 In this case report, we present successful anaesthetic management of radical gastrectomy for gastric cancer associated with anti-NMDAR encephalitis and highlight specific aspects of anaesthetic management for this rare type of encephalitis. For patients with this encephalitis, NMDA antagonists, such as ketamine, nitrous oxide and tramadol, should be avoided, whereas benzodiazepines, opioids, muscle relaxants and curares, which have been demonstrated to have no significant effects on the NMDA pathway, are preferred. Medications that have indirect interactions with NMDARs can be considered but should be used with caution. Finally, adequate prep- aration, including ensuring the availability of vasoactive agents and monitoring throughout the perioperative period, particularly after surgery, is of vital importance. 1Key laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Anaesthesiology, Peking University Cancer Hospital & Institute, Beijing 100142, China. 2Key laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Gastrointestinal Surgery, Peking University Cancer Hospital & Institute, Beijing 100142, China. 1Key laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Anaesthesiology, Peking University Cancer Hospital & Institute, Beijing 100142, China. 2Key laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Gastrointestinal Surgery, Peking University Cancer Hospital & Institute, Beijing 100142, China. Abbreviations BIS: Bispectral index; GABA: Gamma amino acid butyric acid; NMDAR: N-methyl- D-aspartate receptor; PSH: Paroxysmal sympathetic hyperactivity Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements We thank all the doctors and nurses of Peking University Cancer Hospital & Institute who assisted with the anaesthesia and post-operative care of the case patient. References 1. Pryzbylkowski PG, Dunkman WJ, Liu R, Chen L. Case report: anti-N-methyl-D- aspartate receptor encephalitis and its anesthetic implications. Anesth Analg. 2011;113:1188–91. 2. Dalmau J, Gleichman AJ, Hughes EG, Rossi JE, Peng X, Lai M, et al. Anti- NMDA-receptor encephalitis: case series and analysis of the effects of antibodies. Lancet Neurol. 2008;7:1091–8. 2. Dalmau J, Gleichman AJ, Hughes EG, Rossi JE, Peng X, Lai M, et al. Anti- NMDA-receptor encephalitis: case series and analysis of the effects of antibodies. Lancet Neurol. 2008;7:1091–8. 3. Sansing LH, Tuzun E, Ko MW, Baccon J, Lynch DR, Dalmau J. A patient with encephalitis associated with NMDA receptor antibodies. Nat Clin Pract Neurol. 2007;3:291–6. There is scarce literature to date describing anaes- thetic management for patients with anti-NMDAR encephalitis, and further investigations are necessary to develop detailed guidelines for anaesthesiologists to assess surgery-related risk factors of anti-NMDAR encephalitis and to develop a stable perioperative anaesthetic plan for patients with this condition. 4. Kawano H, Hamaguchi E, Kawahito S, Tsutsumi YM, Tanaka K, Kitahata H, et al. Anaesthesia for a patient with paraneoplastic limbic encephalitis with ovarian teratoma: relationship to anti-N-methyl-D-aspartate receptor antibodies. Anaesthesia. 2011;66:515–8. 5. Kamei S, Kuzuhara S, Ishihara M, Morita A, Taira N, Togo M, et al. Nationwide survey of acute juvenile female non-herpetic encephalitis in Japan: relationship to anti-N-methyl-D-aspartate receptor encephalitis. Intern Med. 2009;48:673–9. 6. Iizuka T, Sakai F, Ide T, Monzen T, Yoshii S, Iigaya M, et al. Anti-NMDA receptor encephalitis in Japan: long-term outcome without tumour removal. Neurology. 2008;70:504–11. Authors’ contributions LD contributed to the collection of data and writing of the manuscript. HT contributed to the collection of data and anaesthesia administration. ZL and JJ performed the surgery. XS contributed to revising the manuscript. All authors read and approved the final manuscript. Consent for publication Written informed consent was obtained from the patient for publication of this case report. A copy of the written consent is available for review by the Editor of this journal. Abbreviations BIS Bi t l i d 7. Dalmau J, Tuzun E, Wu HY, Masjuan J, Rossi JE, Voloschin A, et al. 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Brain. 2000;123:1481–94. 13. Dalmau J, Lancaster E, Martinez-Hernandez E, Rosenfeld MR, Balice-Gordon R. Clinical experience and laboratory investigations in patients with anti- NMDAR encephalitis. Lancet Neurol. 2011;10:63–74. 14. Mony L, Kew JN, Gunthorpe MJ, Paoletti P. Allosteric modulators of NR2B- containing NMDA receptors: molecular mechanisms and therapeutic potential. Br J Pharmacol. 2009;157:1301–17. 15. Hughes EG, Peng X, Gleichman AJ, Lai M, Zhou L, Tsou R, et al. Cellular and synaptic mechanisms of anti-NMDA receptor encephalitis. J Neurosci. 2010; 30:5866–75. 16. Pruss H, Dalmau J, Harms L, Holtje M, Ahnert-Hilger G, Borowski K, et al. Retrospective analysis of NMDA receptor antibodies in encephalitis of unknown origin. Neurology. 2010;75:1735–9. 17. Granerod J, Ambrose HE, Davies NW, Clewley JP, Walsh AL, Morgan D, et al. 31. Perkes I, Baguley IJ, Nott MT, Menon DK. A review of paroxysmal sympathetic hyperactivity after acquired brain injury. Ann Neurol. 2010; 68:126–35. 32. Bower RS, Sunnarborg R, Rabinstein AA, Wijdicks EF. Paroxysmal sympathetic hyperactivity after traumatic brain injury. Neurocrit Care. 2010;13:233–4. Abbreviations BIS Bi t l i d Causes of encephalitis and differences in their clinical presentations in England: a multicentre, population-based prospective study. Lancet Infect Dis. 2010;10:835–44. 18. Dilger JP. The effects of general anaesthetics on ligand-gated ion channels. Br J Anaesth. 2002;89:41–51. 19. Ranft A, Kurz J, Becker K, Dodt HU, Zieglgansberger W, Rammes G, et al. Nitrous oxide (N2O) pre- and postsynaptically attenuates NMDA receptor-mediated neurotransmission in the amygdala. Neuropharmacology. 2007;52:716–23. 20. Weiner AL, Vieira L, McKay CA, Bayer MJ. Ketamine abusers presenting to the emergency department: a case series. J Emerg Med. 2000;18:447–51. 21. Martin DC, Plagenhoef M, Abraham J, Dennison RL, Aronstam RS. 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Macrokinetic analysis of blockade of NMDA-gated currents by substituted alcohols, alkanes and ethers. Brain Res. 2004;1015:107–13. 25. Fodale V, Santamaria LB. In clinical practice, coadministration of sevoflurane or propofol could antagonize remifentanil stimulation of N-methyl-D- aspartate receptors. Anesthesiology. 2005;102:695–6. 25. Fodale V, Santamaria LB. In clinical practice, coadministration of sevoflurane or propofol could antagonize remifentanil stimulation of N-methyl-D- aspartate receptors. Anesthesiology. 2005;102:695–6. 26. Kingston S, Mao L, Yang L, Arora A, Fibuch EE, Wang JQ. Propofol inhibits phosphorylation of N-methyl-D-aspartate receptor NR1 subunits in neurons. Anesthesiology. 2006;104:763–9. Ding et al. BMC Anesthesiology (2017) 17:90 31. Perkes I, Baguley IJ, Nott MT, Menon DK. A review of paroxysmal sympathetic hyperactivity after acquired brain injury. Ann Neurol. 2010; 68:126–35. 32. Bower RS, Sunnarborg R, Rabinstein AA, Wijdicks EF. Paroxysmal sympathetic hyperactivity after traumatic brain injury. Neurocrit Care. 2010;13:233–4. Abbreviations BIS Bi t l i d • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: 27. Orser BA, Bertlik M, Wang LY, MacDonald JF. Inhibition by propofol (2,6 di-isopropylphenol) of the N-methyl-D-aspartate subtype of glutamate receptor in cultured hippocampal neurones. Br J Pharmacol. 1995;116: 1761–8. 28. Sato Y, Kobayashi E, Murayama T, Mishina M, Seo N. Effect of N-methyl-D- aspartate receptor epsilon1 subunit gene disruption of the action of general anesthetic drugs in mice. Anesthesiology. 2005;102:557–61. 29. Chen W, Sang N, Luo A, et al. Anesthetic management for ovarian cystectomy in patients with anti-N-methyl-D-aspartate receptor encephalitis undergoing general anesthesia. Chin J Anesthesiol. 2014;34(9):1069–72. 30. Lapebie FX, Kennel C, Magy L, Projetti F, Honnorat J, Pichon N, et al. Potential side effect of propofol and sevoflurane for anesthesia of anti-NMDA-R encephalitis. BMC Anesthesiol. 2014;14:5. 30. Lapebie FX, Kennel C, Magy L, Projetti F, Honnorat J, Pichon N, et al. 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Microstructure and defects in a Ni-Cr-Al-Ti γ/γ’ model superalloy processed by laser powder bed fusion
Materials & design
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⁎ Corresponding authors. E-mail addresses: anthony.deluca@empa.ch (A. De Luca), christoph.kenel@northwestern.edu (C. Kenel). 1 both authors contributed equally to this work. Materials and Design journal homepage: www.elsevier.com/locate/matdes H I G H L I G H T S a b s t r a c t • Solidification and liquation cracking are eliminated in a model Ni-Cr-Al-Ti alloy. • Ductility-dip cracking of high-angle- grain-boundaries is the active mechanism. • The inherent weakness of grain bound- aries in Nickel superalloys is evidenced. • Trace O in the powder lead to the in-situ formation of Al2O3 oxide dispersoids. • Trace O in the powder lead to the in-situ formation of Al2O3 oxide dispersoids. a b s t r a c t a b s t r a c t Article history: Received 14 September 2020 Received in revised form 14 January 2021 Accepted 24 January 2021 Available online 28 January 2021 Keywords: Additive manufacturing Laser powder bed fusion Nickel superalloy Cracking mechanism Article history: Received 14 September 2020 Received in revised form 14 January 2021 Accepted 24 January 2021 Available online 28 January 2021 Additive manufacturing (AM) of non-weldable high-γ’ Ni base superalloys is challenging due to various issues, but notably because of their inherent cracking propensity. Typically, the segregation of melting point- depressant elements to grain boundaries (GB) drastically increases the solidification interval, allowing the high processing-induced stresses in the parts to pull apart the liquid film at GBs. To achieve a better understanding of the consolidation process of nickel superalloys as well as the origin of defects and cracks, a simplified model γ/γ’-strengthened Ni-Cr-Al-Ti alloy with reduced solidification interval, related to the commercial CM247LC alloy, is investigated under a large parameter survey. The consolidation behavior is typical of nickel superalloys produced by AM, with the optimal condition being a compromise between cracking and porosity. The cracking mechanism is, however, changed to solid-state cracking, localized at high-angle GBs, and likely due to the lack of GB strengthening phases and the inherently low strength of this simplified alloy. Transmission electron mi- croscopy and atom probe tomography reveal elemental segregation of Ti, and to a lower extent Cr and Al, to the solidification cell boundaries, in agreement with Calphad calculations. No γ’ precipitates are observed in the as-processed condition, indicating that all elements remain in solid solution. No chemical differences are ob- served between cracked and non-cracked boundaries. Trace amounts of oxygen contained in the powder lead to Al2O3 slag formation, as well as nano oxide dispersoid incorporation. Sulfur, a critical contaminant in superalloys, is detected but rendered harmless by the formation of TiS nanoprecipitates. Keywords: Additive manufacturing Laser powder bed fusion Nickel superalloy Cracking mechanism © 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). Contents lists available at ScienceDirect Contents lists available at ScienceDirect Microstructure and defects in a Ni-Cr-Al-Ti γ/γ’ model superalloy processed by laser powder bed fusion Microstructure and defects in a Ni-Cr-Al-Ti γ/γ’ model superalloy processed by laser powder bed fusion Anthony De Luca a,⁎,1, Christoph Kenel b,⁎,1, Seth Griffiths a, Shreyas S. Joglekar a, Christian Leinenbach a, David C. Dunand b a Empa – Swiss Federal Laboratories for Materials Science and Technology, Überlandstrasse 129, 8600 Dübendorf, Switzerland b Department of Materials Science and Engineering, McCormick School of Engineering, Northwestern University, 2220 Campus Drive, Evanston, IL 60208, USA Materials and Design 201 (2021) 109531 Materials and Design 201 (2021) 109531 1. Introduction Additive manufacturing (AM) of γ’-containing Ni-base superalloys has attracted much interest due to potential replacement of convention- ally cast parts to achieve optimized designs, including internal cooling https://doi.org/10.1016/j.matdes.2021.109531 0264-1275/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). A. De Luca, C. Kenel, S. Griffiths et al. Materials and Design 201 (2021) 109531 present study, thus has the potential to shed light on the validity of alloying concepts that have been established decades ago on complex commercial alloys for processes quite different from the capabilities of modern AM machines. Such concepts include: i) the critical influence of the Al + Ti content on processability, ii) the importance of grain- boundary strengthening elements, iii) the solidification challenges asso- ciated with heavily segregating elements, often also acting as grain boundary strengtheners, iv) the detrimental nature of large solidifica- tion intervals, v) the importance of controlling powder contamination, vi) the formation of dispersoids during L-PBF from strong oxide forming elements present in the alloy. Individually, all these effects have been observed on various commercial alloys processed by L-PBF, depending on alloy composition and processing conditions. In order to deepen the understanding of the key factors and also interactions between these challenges, studying selected model alloys can provide insight into the alloy behavior without interference due to the compositional complexity of commercial alloys. and repair [1]. Based on their alloy design optimized for slow casting into polycrystalline, directionally solidified or even monocrystalline parts, such alloys are typically highly alloyed to maximize strengthening contributions from solid solution, carbide formation and a high volume fraction of γ’-Ni3(Al,Ti) precipitates [2,3]. However, these alloys are typ- ically difficult to weld and, consequently, have proven to be challenging for AM processing. Typically, cracks are observed and classified as (i) solidification cracks due to segregation in the liquid and rupture of remaining liquid films, (ii) liquation cracks by melting of low-melting point phases previously formed by segregation, (iii) ductility-dip cracks formed in the solid-state when the alloy has minimal ductility and can- not withstand residual stresses and (iv) strain-age cracking occurring during post-process heat treatment due to the formation of second phases and an increase in yield stress [1]. 1. Introduction p y [ ] Research on AM of γ/γ’ Ni-base superalloys has predominantly fo- cused on the commercial alloys Inconel 738LC [4–17], due to strong commercial interest for industrial gas turbines, as well as CM247LC, originally developed for directional solidification casting, and its parent MarM247 [1,18–25]. Further alloys studied for AM include CMSX-4, Rene 104, and others [26–35]. Being the final product of decades of alloy development and process optimization, all the above alloys are highly alloyed and have complex microstructures. Solidification cracks in laser powder bed fusion (L-PBF) processed IN738LC were attributed to strong Zr segregation upon solidification [4], in agreement with cast- ing literature [36]. Additionally, higher Si content of the powder was ob- served to lead to increased solidification cracking [5]. Recent atom probe studies reveal that Si and Zr segregation is only observed at high-angle grain boundaries, while C and B segregate to every grain boundary [9]. High-angle grain boundaries have been identified as sus- ceptible to solidification cracking in several alloys [4,10,33]. As a result of several studies into the segregation behavior and resulting cracking, reduction of Zr and Si has been adopted as a successful alloy modifica- tion strategy to reduce solidification cracking in IN738LC processed by L-PBF [5,9]. However, atom probe data on modified IN738LC with lowered Zr and Si suggest that the actual grain boundary segregation level of Zr and Si is unchanged by the alloy modification, indicating that the actual cracking mechanism is more complex [9]. Liquation cracking is observed as a second cracking mechanism, typically after di- rected energy deposition, and is related to re-melting of carbides or for- mation of γ’/γ eutectics [10,14–16]. Additionally, Al-Si-W-based oxide formation during L-PBF has been reported as another cause for cracking in IN738LC with 70 ppm oxygen [13]. In CM247LC, solidification crack- ing has been studied in connection to its Hf content [24]. Interestingly, both an increase [24] and a decrease [37] in Hf lead to lower cracking after laser powder bed fusion (L-PBF), suggesting a complex interplay between Hf and other alloying additions. High dislocation densities in solidification cell walls are attributed to localized crack formation due to resulting thermal stresses [18,19]. Similar to IN738LC, high angle grain boundaries are generally found to be susceptible to cracking across various alloys [1,33,38]. 1. Introduction Apart from alloy-specific adaptions, higher levels of B, Zr, Si, and S are generally observed to increase crack- ing in γ/γ’, as well as solid solution strengthened, Ni-base alloys during AM, in agreement with existing welding literature [4,9,12,24,33,39–43]. While B and Zr are deliberately added to increase the creep perfor- mance, Si and S are impurity elements. Here, the laser powder bed fusion of a Ni-8Cr-5.5Al-1Ti (wt%) model alloy is studied. This model alloy inherits its Cr, Al and Ti content from CM247LC but eliminates all other alloying elements. This provides a model Ni-base superalloy with a high Al + Ti content, high γ’ fraction and solvus temperature and a narrow freezing range free of any addi- tional phases. It provides insight into the processing characteristics and defect formation with reduced interference from the complex com- position of established Ni-base superalloys. L-PBF processing maps are created, taking into account resulting material density, crack formation and melt pool metrics. The formed defects are characterized in detail to study their origin, nature and location. In-depth electron microscopy provides insight into the potential cracking mechanism and formation of native oxide and sulfide dispersoids in the alloy. Atom probe tomog- raphy is employed to study the formation of enriched zones within the supersaturated γ matrix present after processing. The observed behav- ior is compared to commercial Ni-base alloys and conclusions for future alloy and process development are drawn. 2.1. Pre-alloyed powder and laser powder bed fusion The Ni-Cr-Al-Ti experimental pre-alloyed powder was inert-gas at- omized by Nanoval (Berlin, Germany) with its composition indicated in Table 1. The powder also contained trace amounts of O and S whose concentration, as determined by melt combustion methods, is also re- ported in Table 1. The pre-alloyed powder size distribution has an aver- age size d50 = 25 μm (d10-d90 = 12–48 μm). Micrographs of the powder are presented in Supplementary Fig. 1. While the powders are typically spherical, with smooth surfaces, a cross-sectional investigation reveals the presence of internal porosity in some powders, which will ulti- mately lead to porosity in the printed parts. A 63 μm sieve was employed to remove powder agglomerates prior to printing. The acqui- sition of a X-ray powder diffraction pattern, allowed the identification of the diffraction peak position and thus the lattice parameter a = 3.506 Å. Considering the FCC crystal structure of nickel, and the alloy's composi- tion, a density of 8.399 g/cm3 for the model alloy is estimated. Cubic 5 × 5 × 5 mm samples were fabricated using a L-PBF machine Sisma MySint 100 (Sisma S.p.A., Italy) equipped with a 200 W, 1070 nm fiber-laser operating in continuous wave mode, with a 55 μm spot size. The cubes were built on 34.5 mm diameter stainless steel build plates, using a bidirectional scan strategy (90 degree rotation between layers), Despite the success of alloy modification to suppress cracking in se- lected alloys, this approach requires extensive studies into each alloy. Identification of the problematic alloy component, adaption of alloy specifications and processing of custom-produced alloy batches, followed by in-depth analysis, is a lengthy process. Additionally, the complex nature of highly alloyed Ni-base superalloys gives rise to a multitude of interactions in the optimized alloys, making the generali- zation of results difficult. Interestingly, processing studies of model al- loys are scarce in the literature, which is largely focused on existing commercial alloys originally developed for casting, with varying de- grees of weldability. Studying simplified model alloys, as we do in the 3.1. Model alloy characteristics Fig. 1 shows the calculated solidification characteristics of the Ni-Cr- Al-Ti model alloy using the TCNI5 database. The alloy is a prototype Ni-base superalloy featuring a high γ’ fraction and a high γ’ solvus tem- perature (Fig. 1a). No phases beyond γ, γ’ and liquid are predicted to be stable. The predicted equilibrium freezing range is 13 °C (Fig. 1b). The theoretical freezing range remains <100 °C under Scheil conditions as- suming no diffusion within the solid but perfect mixing in the liquid. This continuously removes elements preferentially partitioning to the solid from the remaining melt, which then becomes enriched in segre- gating elements. In the model alloy, Ti is predicted to partition most strongly to the liquid upon rapid solidification (Fig. 1c). Ni is depleted in the liquid and preferentially partitions to the solid. Cr weakly parti- tions to the liquid. Al increases in the liquid by 50% in the course of solidification while Ti increases 4-fold. While the Scheil conditions pro- vide insight into potential segregation, it remains a simplification of a complex process. The assumption of an infinite diffusion coefficient in the liquid and perfect mixing does not necessarily hold in rapid solidifi- cation, as experienced in L-PBF, where there is little time for concentra- tion equilibration throughout the melt pool. The Calphad calculations are compared to DSC measurements on L-PBF specimens (Table 2). The experimentally obtained γ’ solvus is higher than predicted (+36 °C), whereas the extrapolated solidus is slightly lower (−13 °C). The experimentally measured liquidus is in good agreement (−2 °C) with the predicted value. The determined freezing range for the used alloy is 24 °C. Scanning electron microscopy (SEM) was conducted on a FEI NanoSEM 230 in backscatter mode (BSE), or with the Through the Lens Detector (TLD) to image crack surfaces. The composition of ob- served phases was measured by EDX using an Oxford Instruments de- tector. Electron backscatter diffraction (EBSD) was performed on a FEI Quanta 650 ESEM equipped with an Oxford Aztec EBSD detector, with step sizes of 1 μm and 0.4 μm. Data post-processing was performed in AztecCrystal. y A lamella for transmission electron microscopy (TEM) analysis was extracted from the region at the tip of a crack (Supplementary Fig. 2) located in the specimen center, with a FEI Helios NanoLab 600i focused ion beam (FIB). The lamella was extracted perpendicular to the build di- rection. 2.3. Phase transformation temperatures The layer thickness t (30 μm) and hatch spacing h (75 μm) were kept constant throughout the study, and the laser power P and scanning speed v were varied from 100 to 175 W, and from 100 to 1250 mm/s, re- spectively. Argon shielding gas kept oxygen content in the build cham- ber below 0.01% during processing. The parts were removed from the build plates by electro discharge machining (EDM). The phase transformation temperatures of the model alloy produced by L-PBF, in as-printed condition, were determined by differential scan- ning calorimetry (DSC) measurements, conducted using a NETZSCH DSC 404C Pegasus thermal analyzer. Two samples of 13.7 and 20.1 mg were used, with their surfaces ground with SiC grinding paper to P4000 to ensure a better thermal conduction with the crucible. The ex- periments were carried out under high-purity Ar atmosphere (99.9999% Ar) with a flow rate of 40 ml/min. The Al2O3 crucibles were heated at a rate of 10 K/min from room temperature to 1430 °C and cooled down at the same rates. Only the heating thermograms are con- sidered in this study and compared with Thermocalc simulations, con- ducted using the TCNI5 database. Time-transformation-temperature diagram calculations are performed using TC Prisma with the TCNI5 and MOBNI5 databases. 2.4. Microhardness The microhardness of samples with various printing parameters was measured with a Fischerscope HM2000 hardness tester, with a load of 1.8 N, for 60 s. Between 10 and 20 indentations were made per sample, typically in one line parallel to the build direction to asses any effect of build height on mechanical properties. No significant trend in the inden- tation position (i.e. top or bottom of the sample) was observed, nor be- tween samples printed with various conditions. Across all samples tested, we measured an average microhardness of 3130 ± 160 MPa. A Leica VZ700C optical microscope was employed to measure the crack density of the samples, and approximately 250–300 images at 200× magnification were stitched together to generate one uncom- pressed image of the cube's vertical cross-section. The image analysis was performed using ImageJ. The uncompressed stitched images were filtered with a 3 pixels median filter, followed by manual thresholding to generate a binary image. The analyze particle function of ImageJ was applied to the binarized images. Particles smaller than 20 pixels, touching edges, or with a circularity above 0.35 (pores) were filtered. The cracks were fitted by ellipses, with the long dimension taken as the crack length. Additionally, horizontally inclined ellipses (± 20 de- grees) were filtered, as lack of fusion defects and slags were not re- moved by the circularity filter, and cracks typically propagate vertically through the part. Table 1 Model alloy nominal composition and impurity elements in the pre-alloyed powder. O and S determined by melt combustion (*). Ni Cr (wt%/at.%) Al (wt% /at.%) Ti (wt% /at.%) O (wt.ppm)* S (wt.ppm)* Bal 8 / 8.4 5.5 / 11.1 1 / 1.1 180 40 Ni Cr (wt%/at.%) Al (wt% /at.%) Ti (wt% /at.%) O (wt.ppm)* S (wt.ppm)* Bal 8 / 8.4 5.5 / 11.1 1 / 1.1 180 40 2 Materials and Design 201 (2021) 109531 A. De Luca, C. Kenel, S. Griffiths et al. followed by a contour strategy (with the same parameter than in the part). A large parameter field was surveyed to investigate the effects of printing parameters on consolidation quality. The volumetric energy density E (J/mm3) referenced throughout this manuscript is calculated using the following formula: laser atom probe tomography was performed with a CAMECA LEAP 5000XS Atom-Probe Tomograph (Cameca Instruments, USA) at a pulse frequency of 500 kHz, specimen temperature of 40 K and 25 pJ pulse energy using an ultraviolet laser (355 nm) focused to the diffrac- tion limit. Data processing was performed in IVAS 3.8.5. Data plotting is performed in Python (Anaconda, Continuum Analyt- ics). Perceptually uniform colormaps are retrieved from the cmocean package [44]. E ¼ P v  h  t ð1Þ E ¼ P v  h  t ð1Þ 2.2. Microstructure and defect characterization The consolidated parts densities were measured by the Archimedes method in ethanol. The specimens were then cold mounted in epoxy, ground, and polished down to 1 μm diamond suspension. The final polishing was done with 50 nm colloidal silica. All specimens were ground to reveal the X-Z plane, allowing to image the top melt pools. Specimens for Electron Backscatter Diffraction (EBSD) measurements were extensively lapped with silica and Ar ion beam polished (Leica EM TIC 3X). Selected specimens were etched with Glyceregia (15 ml HCl, 10 ml glycerol, 5 ml NHO3) for melt pool and microstructure anal- ysis by optical microscopy. 3.2. Laser powder bed fusion correlated to an increased crack frequency and crack length. Despite the very high energy density used to consolidate certain specimens, no keyhole porosity was observed in the investigated parameter field. Although the inhomogeneous distribution of the lack of fusion defects prevents us to precisely estimate the true porosity from micrographs, the crack density could be measured in a smaller parameter field (125–175 W, 500–1000 mm/s) (Fig. 2b). A strong relationship is ob- served between crack density and energy input, also displayed in Fig. 2f. At 56 J/mm3, no crack was observed in an entire cross-section, and the crack density reaches a saturation at ~2 mm/mm2, in the inves- tigated field. Additionally, slag is incorporated into the consolidated ma- terial (Fig. 2c). With increasing energy input, less slag is observed within the parts (cf. Fig. 2c and f). Fig. 2 shows the consolidated results from material density, crack density, slag fraction and melt pool overlap measurements. Multiple se- ries of cubes were consolidated by L-PBF, and cover a wide range of pa- rameters from 100 to 175 W and 100 to 1250 mm/s, with an energy density input as low as 44 J/mm3 and as high as 556 J/mm3. Hatch spac- ing (75 μm) and layer thickness (30 μm) are kept constant. Fig. 2a shows a part density map, as measured by the Archimedes method, as a func- tion of scanning speed and laser power. To highlight regions of low and high energy input in this map, calculated isocontours of energy density are overlaid. The highest density achieved is 7.965 ± 0.006 g/cm3 (150 W and 1000 mm/s, equivalent to 66.7 J/mm3), the lowest is 7.800 ± 0.001 g/cm3 (125 W and 1250 mm/s, equivalent to 44 J/mm3). Although not as prominent, a decrease of density is also observed in the top left corner of the map. The melt-pool widths and depths were measured on the top layers of etched cross-sections and converted to vertical and horizontal over- laps, between layers and lines respectively, reported in Fig. 2d and Supplementary Fig. 5. The lateral and vertical overlaps are calculated as OL = 100∙(1-hatch/width) and OV = 100∙(1-layer thickness/depth), re- spectively. The melt pool width-to-depth ratio is around 3 for high- density specimens, comparable to observations on commercial CM247LC investigated in previous studies on the same L-PBF machine [37]. 3.2. Laser powder bed fusion The highest densities are achieved for melt pool overlaps between 30 and 50% vertically and 55 to 65% horizontally (Supplementary Fig. 5). At low vertical and/or horizontal overlap, the lack of fusion de- fects reduce the material density. Crack density increases with increased melt pool overlap (Fig. 2e). Deeper and wider melt pools are observed for higher energy inputs, enhancing the columnar growth of grains across many layers, and thus correlate with the occurrence of cracks. Material density and crack density are compared based on the volumet- ric energy density (Fig. 2f). Upon achieving maximum density, cracks start to appear while the incorporation of slag is reduced. Higher energy inputs lead to more cracks being formed and thus an overall lower den- sity. The maximum density is achieved at 66.6 J/mm3. Based on Fig. 2a, a laser power of 100 W is generally insufficient to consolidate the powder, leading to outliers in the density curve in Fig. 2f. The upper envelope of density is formed by values obtained at 125 to 175 W. Further studies into the detailed defect characteristics are conducted on specimens pro- duced at 150 W, 1000 mm/s (highest density, low cracking) and 175 W, 750 mm/s (near peak density, increased cracking). For comparison, a porosity value of 0.34% was determined on a cube full cross section using optical microscopy and pore thresholding for the sample with highest density (150 W, 1000 mm/s), Supplementary Fig. 4. The observed porosity is identified as gas porosity, as all pores are spherical. The discrepancy between Archimedes method and optical imaging of cross-sections can be explained by i) the high porosity con- tent of the powder (cf. Supplementary Fig. 1), estimated to be at least 2%, and ii) the presence of contour porosity. As the study herein was fo- cused on the bulk consolidation, the contouring strategy is not opti- mized, which leads to the formation of vertically aligned pores, typically at one hatch distance of the part's edges, and expected to rep- resent a significant amount of the total porosity. All samples are ex- pected to be equally affected, regardless of the printing parameters. Additionally, surface roughness is known to affect density estimation by Archimedes method. The Archimedes method thus underestimates the true material density in the core of all specimens. 3.1. Model alloy characteristics Scanning transmission electron microscopy (STEM) was per- formed on a FEI Titan Themis microscope operated at 300 kV and equipped with a probe spherical aberration corrector and a SuperEDX system (ChemiSTEM technology) with four silicon drift detectors for energy-dispersive X-ray (EDX) spectroscopy. A convergence semi angle of 25 mrad was used in combination with an annular dark field (ADF) detector with inner and outer collection semiangles of 53 and 200 mrad, respectively. The semi-quantitative EDX data processing was performed in Velox 2.12, with a pre-filtering applied to improve the counting statistic per pixel. Atom probe sample tips were prepared with a FEI Helios Nanolab 600 FIB, the wedge being sampled from the specimen center. Pulsed- 3 Fig. 1. Ni-Cr-Al-Ti model alloy design and characteristics. a) Phase fraction, b) Scheil solidification and c) elemental enrichment in the liquid calculated for the Ni-8.4Cr-11.1Al-1.1Ti (at. %) model alloy using the Calphad approach. Only γ-(Ni, Cr), γ’-Ni3(Al,Ti) and liquid are stable, with >50% γ’ below 800 °C. The alloy has a low predicted equilibrium solidification range of 13 °C. Under Scheil conditions, Ti shows the strongest segregation tendency to the liquid, reducing the solidus temperature together with Al. A. De Luca, C. Kenel, S. Griffiths et al. Materials and Design 201 (2021) 109531 A. De Luca, C. Kenel, S. Griffiths et al. Materials and Design 201 (2021) 109531 Fig. 1. Ni-Cr-Al-Ti model alloy design and characteristics. a) Phase fraction, b) Scheil solidification and c) elemental enrichment in the liquid calculated for the Ni-8.4Cr-11.1Al-1.1Ti (at. %) model alloy using the Calphad approach. Only γ-(Ni, Cr), γ’-Ni3(Al,Ti) and liquid are stable, with >50% γ’ below 800 °C. The alloy has a low predicted equilibrium solidification range of 13 °C. Under Scheil conditions, Ti shows the strongest segregation tendency to the liquid, reducing the solidus temperature together with Al. 3.2. Laser powder bed fusion To assess the origin of the density variation in the investigated parameter field, optical microscopy was conducted on all samples (cf. Supplementary Fig. 4). Overall, the strong decrease in part density in the bottom right corner of the map is due to a higher frequency of lack of fusion defects, while in the top left corner of the map, it is Table 2 Thermal properties determined by DSC (Supplementary Fig. 3) and by Calphad calculated using Thermocalc software with the TCNI5 database. γ’ solvus (°C) Lowest melting (°C) Solidus (°C) Liquidus (°C) ΔTL-S (°C) DSC 1111 1368 1381 (onset) 1405 24 Calphad 1074 1394 1407 13 3.3. Microstructure and defects Grain columns extend over multiple build layer thicknesses (layer thickness: 30 μm)). No secondary phases in the grain, at the grain boundaries, nor any dendritic structure is observed by SEM, consistent with minimal segregation upon solidification, in agreement with Calphad predictions. Four types of defects are observed: i) cracks, ii) lack of fusion defects, iii) gas pores and iv) alumina slag. Cracks follow grain boundaries and ap- pear along the build direction (Fig. 3b). Locally, the cracks appear jagged when propagating through an area with small grain columns. When fol- lowing a vertically aligned grain boundary, the cracks appear straight. High-magnification imaging shows smooth crack faces, indicating solid-state grain boundary decohesion as the main cracking mechanism. Liquid phase cracking is excluded based on the absence of segregating liquid, the lack of observation of any secondary phases and the absence of a dendritic structure on the crack surfaces. where they form upon a subsequent melting, and are incorporated in the host matrix due to strong melt pool convection. Such embedded slag lenses with sharp corners are likely to reduce the fatigue and high-temperature strength of the material due to crack initiation. The presence of alumina slag at the interfaces of lack of fusion defects addi- tionally indicates that once the slag is present it effectively separates the molten alloy. This is consistent with the observation of lack of fusion de- fects at high energy densities (cf. Supplementary Fig. 4). While typi- cally formed due to insufficient heat input and incomplete melting, here the alumina slags hinder coalescence of adjacent melt tracks, creat- ing a similar defect as classical lack of fusion. Fig. 4 shows EBSD data obtained on a specimen processed at 150 W and 1000 mm/s, achieving maximum density. The microstructure is highly columnar with individual grains spanning over 500 μm vertically (in the build direction), indicating epitaxial growth of grains through >15 layer thicknesses (layer thickness: 30 μm). The inverse pole figure coloring shows no strong preferred orientation alignment with the build direction. Grains close to 100 (red), 110 (green) and 111 (blue) orientations grow into large columns. Inverse pole figure colored maps for the x and y directions are shown in Supplementary Fig. 6, along with the unit cell orientation. After processing, the alloy contains Lack of fusion defects are observed even at the process conditions where maximum density is achieved (Fig. 3c). 3.3. Microstructure and defects Fig. 3 shows the grain microstructure and characteristic defects ob- served in our L-PBF -processed Ni-Cr-Al-Ti model alloy. As cracks are wider at higher energy density, the crack surface analysis was con- ducted on the sample printed at 175 W - 750 mm/s. In general, a fine- 4 Materials and Design 201 (2021) 109531 A. De Luca, C. Kenel, S. Griffiths et al. . 2. Laser powder bed fusion processing. Maps of a) material density, b) crack density, c) incorporated slag area fraction, and d) horizontal melt pool overlap as a function of laser wer and scan speed at a constant layer thickness of 30 μm and hatch of 75 μm. Maps are linearly interpolated between measured conditions. For a), isoenergy density lines are own overlaid. e) Crack density as a function of vertical and horizontal melt pool overlap. f) Material density, crack density and slag fraction as a function of volumetric energy nsity. Cracking occurs as soon as maximal densification is achieved at 66.6 J/mm3. Slagging decreases with increased energy input. De Luca, C. Kenel, S. Griffiths et al. Materials and Design 201 (2021) 109531 Fig. 2. Laser powder bed fusion processing. Maps of a) material density, b) crack density, c) incorporated slag area fraction, and d) horizontal melt pool overlap as a function of lase power and scan speed at a constant layer thickness of 30 μm and hatch of 75 μm. Maps are linearly interpolated between measured conditions. For a), isoenergy density lines are shown overlaid. e) Crack density as a function of vertical and horizontal melt pool overlap. f) Material density, crack density and slag fraction as a function of volumetric energy 3 Fig. 2. Laser powder bed fusion processing. Maps of a) material density, b) crack density, c) incorporated slag area fraction, and d) horizontal melt pool overlap as a function of laser power and scan speed at a constant layer thickness of 30 μm and hatch of 75 μm. Maps are linearly interpolated between measured conditions. For a), isoenergy density lines are shown overlaid. e) Crack density as a function of vertical and horizontal melt pool overlap. f) Material density, crack density and slag fraction as a function of volumetric energy density. Cracking occurs as soon as maximal densification is achieved at 66.6 J/mm3. Slagging decreases with increased energy input. grained, columnar microstructure is observed (Fig. 3a). 3.3. Microstructure and defects Gas pores from hollow powder particles also persist in the microstructure, appearing spherical. The free surfaces of lack of fusion defects, as well as the part's surface (Fig. 3c inset), are partially coated with alumina slag. Slag lenses embed- ded in the metallic matrix are likely drawn away from the free surfaces 5 Materials and Design 201 (2021) 109531 A. De Luca, C. Kenel, S. Griffiths et al. Fig. 3. Microstructure and processing-related defects. a) Columnar grain structure spanning over multiple layers (layer thickness: 30 μm) revealed by BSE SEM. The build direction z is indicated and valid for all figures. b) Cracks formed aligned with the columnar grain structure. Magnified view (right) shows smooth crack faces indicating solid-state cracking. c) Lack of fusion defects and gas pores from hollow powder particles. Magnified view (right) shows the formation and incorporation of alumina slag lenses at free interfaces and within the metallic matrix. Fig. 3. Microstructure and processing-related defects. a) Columnar grain structure spanning over multiple layers (layer thickness: 30 μm) revealed by BSE SEM. The build direction z is indicated and valid for all figures. b) Cracks formed aligned with the columnar grain structure. Magnified view (right) shows smooth crack faces indicating solid-state cracking. c) Lack of fusion defects and gas pores from hollow powder particles. Magnified view (right) shows the formation and incorporation of alumina slag lenses at free interfaces and within the metallic matrix. (Supplementary Fig. 2) to provide insight into the local microstructure of susceptible grain boundaries (Fig. 5a). The crack plane is perpendicu- lar to the lamella and opens horizontally in mode I along a grain bound- ary. A high dislocation density is observed within the grains consistent with the material exceeding its yield strength and being subjected to high residual stresses (Fig. 5b). Closer inspection reveals the presence of small dispersoids in the alloy, which further pin the dislocations. Local chemical analysis confirms two types of dispersoids present after processing: Al2O3 and Ti-,Cr- and S-rich particles (Fig. 5c), the Cr con- taining particles being relatively rare. The Ti:S ratio of 1:1, suggests (Ti,Cr)S. These precipitates are found to form as individual Al2O3 and (Ti,Cr)S dispersoids, and regularly also as co-precipitates where both types occur together. Co-precipitates have a globular shape with distinct parts being either Al2O3 or (Ti,Cr)S type. 3.3. Microstructure and defects Large area mapping shows the presence of O and S at distinct precipitate locations, where the occur- rence of both elements indicates co-precipitation (Fig. 5d). The two types of dispersoids being comparable in size, shape and contrast, it is difficult to differentiate them without measuring their composition. Counting 200 particles, the radius of both Al2O3 and (Ti,Cr)S precipitates is estimated at 11.6 ± 5.6 nm. The Al2O3 nanoparticles are, however, more prevalent than (Ti,Cr)S. Neither γ’ nor any other phase are ob- served, indicating that the cooling rate upon L-PBF is sufficiently high (and that subsequent temperature excursions are short enough) to sup- press γ’ precipitation and preserve a supersaturated fcc Ni-Cr-Al-Ti ma- trix. The initial cooling rate on solidification is very high, and is estimated to be 1.1 ± 0.4 106 K/s using [45]: many small angle grain boundaries with 2–15° misorientation (Fig. 4b). They appear inhomogeneously distributed, forming vertical bands with a distance comparable to the hatch spacing used for processing (75 μm). Pockets of small, misoriented grains are formed locally be- tween large grain columns (Fig. 4c). The length of these pockets is in the order of the layer thickness of 30 μm. The abrupt change in grain orientation and size indicates a pronounced layer-by-layer effect upon building, leading to nucleation and termination of grains. The nucleation of grains and their growth over at least one layer, again, in- dicates no dominant selected crystal direction upon solidification. Cracks are correlated with high angle grain boundaries present in the alloy (Fig. 4d) and intragranular fracture is not observed. Cracks are clearly identified from the band contrast map (Supplementary Fig. 6d). The disorientation angle between the grains adjacent to the cracks, in Fig. 4d, is >40° across all cracks. The overall texture of the alloy is rather weak without any distinct fiber textures observed (Fig. 4e). A tendency for alignment of 110 and 100 directions close to the build direction is observed, whereas 111 appears to be compa- rably unfavorable. This is also visible by a comparably low number of blue grains in Fig. 4a. Inverse pole figures along the principal direc- tions also show this slight alignment of 110 with the build direction, alignment of 100 with the transverse x direction, and alignment of 100 and orientations close to 〈112〉and 〈113〉with the transverse y di- rection (Supplementary Fig. 6e). many small angle grain boundaries with 2–15° misorientation (Fig. 3.3. Microstructure and defects The build direction (z) is indicated and valid for (a-c); the layer height (~30 μm) is also ustrated. (d) Magnified view (shown in a)) of selected cracked grain boundaries visualized by a spatial map of the Euler angle Φ. Disorientation angles across the cracks are indicated. (e) le figures derived from the full area shown in (a) for 100, 110, and 111 directions along the build orientation z. De Luca, C. Kenel, S. Griffiths et al. Materials and Design 201 (2021) 109531 Fig. 4. Grain structure and orientation (a) Inverse pole figure map along the build direction (z). Cracked high angle grain boundaries (GB) are marked with arrows. (b) Grain boundary map with high angle grain boundaries (>15°) in black and small-angle grain boundaries (2–15°) in blue. Small-angle grain boundaries are concentrated in vertical bands along the build direction. The crack locations are indicated with arrows. (c) Higher resolution of the center area in (a) showing pockets of misoriented grains between large columnar grains growing in different orientations overlaid with the grain boundary map (small angle: gray, high angle: black). The build direction (z) is indicated and valid for (a-c); the layer height (~30 μm) is also illustrated. (d) Magnified view (shown in a)) of selected cracked grain boundaries visualized by a spatial map of the Euler angle Φ. Disorientation angles across the cracks are indicated. (e) Pole figures derived from the full area shown in (a) for 100, 110, and 111 directions along the build orientation z. are observed in the top-most layer that did not experience any further thermal cycles preventing further grain boundary movement. Based on the performed TEM measurements, no differences (neither struc- tural nor chemical) between the cracked and intact grain boundaries are observed. λ1 ¼ 97  5 ∂T ∂t  −0:360:01 λ1 ¼ 97  5 ∂T ∂t  −0:360:01 ð2Þ ð2Þ with a measured cell spacing λ1 = 0.65 ± 0.070 μm. p g 1 μ Chemical mapping (Fig. 5 d,e) also reveals enrichment of Ti, from ~1 at.% in the cells, to ~2 at.% in the solidification cell boundaries. Enrich- ment in Cr and Al in the cell boundary areas is also observed, while Ni is depleted. The measured Ti content is close to the segregation values es- timated from DSC and Scheil calculations, however, intrinsic heat treat- ment during L-PBF likely reduced the maximum amount of segregation prior to observation. 3.3. Microstructure and defects 4b). They appear inhomogeneously distributed, forming vertical bands with a distance comparable to the hatch spacing used for processing (75 μm). Pockets of small, misoriented grains are formed locally be- tween large grain columns (Fig. 4c). The length of these pockets is in the order of the layer thickness of 30 μm. The abrupt change in grain orientation and size indicates a pronounced layer-by-layer effect upon building, leading to nucleation and termination of grains. The nucleation of grains and their growth over at least one layer, again, in- dicates no dominant selected crystal direction upon solidification. Cracks are correlated with high angle grain boundaries present in the alloy (Fig. 4d) and intragranular fracture is not observed. Cracks are clearly identified from the band contrast map (Supplementary Fig. 6d). The disorientation angle between the grains adjacent to the cracks, in Fig. 4d, is >40° across all cracks. The overall texture of the alloy is rather weak without any distinct fiber textures observed (Fig. 4e). A tendency for alignment of 110 and 100 directions close to the build direction is observed, whereas 111 appears to be compa- rably unfavorable. This is also visible by a comparably low number of blue grains in Fig. 4a. Inverse pole figures along the principal direc- tions also show this slight alignment of 110 with the build direction, alignment of 100 with the transverse x direction, and alignment of 100 and orientations close to 〈112〉and 〈113〉with the transverse y di- rection (Supplementary Fig. 6e). Fig. 5 shows detailed TEM micrographs of the alloy close to a crack tip. The TEM lamella is extracted by FIB in front of the open crack 6 A. De Luca, C. Kenel, S. Griffiths et al. A. De Luca, C. Kenel, S. Griffiths et al. Materials and Design 201 (2021) 109531 g. 4. Grain structure and orientation (a) Inverse pole figure map along the build direction (z). Cracked high angle grain boundaries (GB) are marked with arrows. (b) Grain boundary ap with high angle grain boundaries (>15°) in black and small-angle grain boundaries (2–15°) in blue. Small-angle grain boundaries are concentrated in vertical bands along the build ection. The crack locations are indicated with arrows. (c) Higher resolution of the center area in (a) showing pockets of misoriented grains between large columnar grains growing in ferent orientations overlaid with the grain boundary map (small angle: gray, high angle: black). 3.3. Microstructure and defects The (Ti,Cr)S particles are predominantly observed in the solidification cell boundaries, highlighted by their higher Ti con- tent (Fig. 5c). (Ti,Cr)S appears as dark spots in the Ti map. The Al2O3 pre- cipitates are found throughout the metal matrix and are visible as dark spots in the Al map. Detailed tracing of the grain boundaries observed in Fig. 5a and overlay with the chemical map reveals grain boundary movement (Fig. 5e). Several solidification cells can share a common ori- entation and later be part of the same grain, but different cell colonies are expected to have a grain boundary along their common solidifica- tion cell boundary. However, the resulting grain boundary location does not coincide with the original solidification cell boundaries, with an estimated movement 0–0.5 μm. This indicates a slight grain growth during the complex thermal history during L-PBF including several rapid heating and cooling cycles. This is occurring most likely after the cracking occurred along those Ti-enriched grain boundaries, as cracks 3.4. Local chemical structure Atom probe measurements were conducted to study the local chem- ical structure, and are shown in Fig. 6. Tip compositions are indicated in Table 3, and the mass-to-charge spectrum of the largest tip (No. 2) is shown in Supplementary Fig. 7. In agreement with TEM, no second phases or γ’ are observed within the tip (Fig. 6a). Ti enrichment is ob- served within a ~ 110 nm wide zone across the APT tip (Fig. 6b). Based on TEM observations (Fig. 5), this region is identified as a solidifi- cation cell boundary. Zone lines dominate the observed compositional fluctuations for Ni, Cr and Al (Cr and Al evaporate preferentially from zone lines increasing the apparent local concentration). A compositional profile along the tip axis is obtained from a reduced cylinder volume (20 nm diameter) placed in the tip center, outside of the poles and zone lines (Fig. 6c). Ti increases from ~1 to ~1.9 at.% (+90%) while Cr in- creases from ~7.5 to 9 at.% (+20%). Ni compensates for the local enrich- ment and consequently shows a drop. No enrichment is observed for Al. The observed decrease in Al concentration is an artefact of the diverging zone lines, leading to a reduced contribution of the high-evaporation in- tensity areas to the cylinder volume further down the tip. The 7 Materials and Design 201 (2021) 109531 A. De Luca, C. Kenel, S. Griffiths et al. Fig. 5. STEM HAADF microstructural characterization. a) Low magnification image of a cracked boundary ahead of the crack tip (central). The build direction is out of plane. b) High dislocation densities are observed within the grains. Dispersoids embedded in the matrix (arrows) lead to dislocation-dispersoid interaction. c) EDS maps of Al2O3 and (Ti,Cr)S co- precipitates. d) EDS maps for Ti, Cr, Al, Ni, O and S along the cracked grain boundary. e) Overlay of large area EDS Ti map and traced grain boundaries identified from HAADF micrographs. Grain boundaries are shifted by ~0–0.5 μm from the original solidification cell boundaries. EDS maps are adjusted for visualization, data extends beyond the displayed range (e.g. in precipitates). Fig. 5. STEM HAADF microstructural characterization. a) Low magnification image of a cracked boundary ahead of the crack tip (central). The build direction is out of plane. b) High dislocation densities are observed within the grains. Dispersoids embedded in the matrix (arrows) lead to dislocation-dispersoid interaction. 3.4. Local chemical structure Based on the calculated TTT (Time-Temperature-Trans- formation) diagram for Ni-8Cr-5.5Al-1Ti (wt%) and the absence of γ’ in the center of the produced parts, the alloy experienced less than 3 ms at 970 °C, the temperature of fastest γ’ formation (Supplementary Fig. 8). This is consistent with the observed high cooling rate of 1.1 ± 0.4 106 K/s at solidification and also shows that the subsequent thermal history does not exceed this threshold. Residual heat buildup in the part during the 15 min of processing on top of a cen- ter position (total processing time: 30 min) also does not lead to γ’ for- mation and can thus be estimated to not exceed ~520 °C. continuation of zone lines and poles throughout the full tip indicates similar crystal orientations above and below the enrichment zone. This is in agreement with TEM measurements showing several solidifi- cation cells belonging to the same grain. g g g A comparison of compositional profiles from APT and TEM EDS across the solidification cell boundary is shown in Fig. 7. Both tech- niques are subject to different limitations and artefacts, e.g. TEM-EDS is dependent on the lamella thickness while APT is sensitive to peak overlap in the mass spectrum. Thus, they can deliver complementary in- formation. Fitting of Gaussian profiles allows to estimate the 95% per- centile width of the zone as 4σ. Both techniques reveal a Ti enriched zone with a width of 110–120 nm. The TEM-EDS profiles measure a higher matrix level of Ti (~1.4 at.%), due to the limited integration width of each profile. Manual integration of each cell improves the EDX counting statistic, allowing to estimate a cell core composition of ~1 at.% Ti, comparable to APT (~1.1 at.%). Both techniques deliver a sim- ilar maximum enrichment of ~1.9 at.% Ti. Chromium is also observed to segregate to the solidification cell boundaries, as revealed by TEM-EDS and APT. TEM-EDS suggests a similar width of the Cr distribution (~120 nm) compared to the Ti enrichment, while APT suggests a zone width of ~300 nm. Cr is heavily affected by the presence of zone lines and poles in APT, artificially widening the zone with a low-Cr tail; thus the TEM measurement is considered more reliable. 3.4. Local chemical structure Although Al is observed to segregate as well to the cell boundaries, the precise quanti- fication of its segregation appears particularly challenging by TEM, as the thickness of the sample drastically affects the absorption of the Al K-line (1.5 keV). It should be remembered that any ex-situ measure- ments underestimate segregation at the final stage of solidification due to a certain amount of diffusion of solutes in the solid-state dur- ing cooling. Additionally, in beam-based AM processing, the material is cyclically heated and cooled, and may also experience elevated tem- peratures for prolonged times due to build-up of residual heat in the 3.4. Local chemical structure Mean and standard deviations are weighted using the tip ion count. Table 3 Atom probe tip composition. Concentrations of alloying elements (Ni, Cr, Al, Ti), Co (from Ni metal), impurities (V, Fe, Cu) as well as trace elements (C and Si, the latter measured using the Si2+ signal). Contributions from Ga, O and H are ignored. B is below the detection limit of 3 ppm. No S peaks were detected in the mass spectra due to overlap with Ni. Tip #3 is shown in Fig. 6. Tip #2 fractured in the Ti-enriched zone. Mean and standard deviations are weighted using the tip ion count. Table 3 Atom probe tip composition. Concentrations of alloying elements (Ni, Cr, Al, Ti), Co (from Ni metal), impurities (V, Fe, Cu) as well as trace elements (C and Si, the latter measured using the Si2+ signal). Contributions from Ga, O and H are ignored. B is below the detection limit of 3 ppm. No S peaks were detected in the mass spectra due to overlap with Ni. Tip #3 is shown in Fig. 6. Tip #2 fractured in the Ti-enriched zone. Mean and standard deviations are weighted using the tip ion count. Tip No. Ion count (M) Ni (at.%) Cr (at.%) Al (at.%) Ti (at.%) Co (at.%) V + Fe + Cu (at.ppm) C (at.ppm) Si2+ (at.ppm) 1 26 77.3 8.7 12.4 1.1 0.4 427 41 81 2 102 78.3 8.7 11.6 1.2 0.1 200 71 142 3 50 77.6 8.7 12.3 1.3 0.1 192 50 145 4 32 78.1 8.8 11.4 1.3 0.3 354 62 187 Weighted mean Weighted SD 78.0 8.7 11.9 1.2 0.2 250 61 142 0.4 0.1 0.4 0.1 0.2 100 13 32 produced part. Based on the calculated TTT (Time-Temperature-Trans- formation) diagram for Ni-8Cr-5.5Al-1Ti (wt%) and the absence of γ’ in the center of the produced parts, the alloy experienced less than 3 ms at 970 °C, the temperature of fastest γ’ formation (Supplementary Fig. 8). This is consistent with the observed high cooling rate of 1.1 ± 0.4 106 K/s at solidification and also shows that the subsequent thermal history does not exceed this threshold. Residual heat buildup in the part during the 15 min of processing on top of a cen- ter position (total processing time: 30 min) also does not lead to γ’ for- mation and can thus be estimated to not exceed ~520 °C. produced part. 3.4. Local chemical structure c) EDS maps of Al2O3 and (Ti,Cr)S co- precipitates. d) EDS maps for Ti, Cr, Al, Ni, O and S along the cracked grain boundary. e) Overlay of large area EDS Ti map and traced grain boundaries identified from HAADF micrographs. Grain boundaries are shifted by ~0–0.5 μm from the original solidification cell boundaries. EDS maps are adjusted for visualization, data extends beyond the displayed range (e.g. in precipitates). Fig. 6. Local chemical structure measured by atom probe tomography. a) Volumetric reconstruction of a tip showing the distribution of Ni, Cr, Al and Ti atoms. No second phase or small clusters are observed. b) Concentration contour plots averaged over a slice thickness of 10 nm from the tip center. Ti and Cr are enriched in a solidification cell boundary. For Ni, Cr and Al the presence of zone lines dominates over the compositional change. c) Concentration profile along the tip center indicated in a), spanning a solidification cell boundary but outside the poles and zone lines. Ti and Cr are enriched, Ni is depleted. Al is dominated by the diverging zone lines. Fig. 6. Local chemical structure measured by atom probe tomography. a) Volumetric reconstruction of a tip showing the distribution of Ni, Cr, Al and Ti atoms. No second phase or small clusters are observed. b) Concentration contour plots averaged over a slice thickness of 10 nm from the tip center. Ti and Cr are enriched in a solidification cell boundary. For Ni, Cr and Al the presence of zone lines dominates over the compositional change. c) Concentration profile along the tip center indicated in a), spanning a solidification cell boundary but outside the poles and zone lines. Ti and Cr are enriched, Ni is depleted. Al is dominated by the diverging zone lines. 8 Materials and Design 201 (2021) 109531 A. De Luca, C. Kenel, S. Griffiths et al. Table 3 Atom probe tip composition. Concentrations of alloying elements (Ni, Cr, Al, Ti), Co (from Ni metal), impurities (V, Fe, Cu) as well as trace elements (C and Si, the latter measured using the Si2+ signal). Contributions from Ga, O and H are ignored. B is below the detection limit of 3 ppm. No S peaks were detected in the mass spectra due to overlap with Ni. Tip #3 is shown in Fig. 6. Tip #2 fractured in the Ti-enriched zone. 4.1. Model alloy characteristics The present model alloy is inspired by CM247LC, a complex indus- trial Ni-base superalloy with a nominal composition of Ni-9.5 W- 9.2Co-8.1Cr-5.6Al-3.2Ta-1.4Hf-0.7Ti-0.5Mo-0.075C-0.015Zr-0.015B (wt%). This alloy, optimized for casting, is highly alloyed with W, Ta and Hf to promote high-temperature strength and carbide formation, com- bined with Al and Ti to form a high volume fraction of γ’. IN738LC (Ni-16Cr-8.5Co-1.75Mo-2.6 W-1.75Ta-0.9Nb-3.4Al-3.4Ti-0.01B-0.05Zr wt%) is a casting alloy of similar complexity and similar high Al + Ti content (>6 at.%). Additive manufacturing of CM247LC, IN738LC and other high γ’-containing Ni base superalloys remains challenging due to the segregation tendency of alloy constituents, especially Zr, B and Hf, leading to cracking along grain boundaries with impurity elements such as Si [4,5,9,24]. Eliminating heavily segregating elements and studying a simplified model alloy thus provides insight into the process- ing characteristics and defect formation with reduced interference from the complex composition of these established Ni-base superalloys. Fig. 7. Comparison of Ti enrichment in solidification cell boundaries. a) Ti and b) Cr concentration profiles derived from APT and TEM EDS. TEM data is averaged over 91 measured boundaries. The full segregation zone width is estimated as 4σ based on a Gaussian fit of the data. The width from APT data is corrected for the non-orthogonal measurement direction of the line profile. The large Cr distribution width measured by APT is influenced by the presence of poles. Fig. 7. Comparison of Ti enrichment in solidification cell boundaries. a) Ti and b) Cr concentration profiles derived from APT and TEM EDS. TEM data is averaged over 91 measured boundaries. The full segregation zone width is estimated as 4σ based on a Gaussian fit of the data. The width from APT data is corrected for the non-orthogonal measurement direction of the line profile. The large Cr distribution width measured by APT is influenced by the presence of poles. 9 A. De Luca, C. Kenel, S. Griffiths et al. Materials and Design 201 (2021) 109531 boundaries and liquation cracking. The absence of any strong segrega- tion thus rules out solidification and liquation as the cause of cracking for the model alloy. The present Ni-Cr-Al-Ti model alloy has a narrow freezing range of only 13 °C under equilibrium conditions. This is significantly smaller than for commercial Ni-base superalloys (IN738LC: 117 °C, CM247LC: 130 °C, calculated with the TCNI5 database). 4.1. Model alloy characteristics Additionally, it has a low content of C (~60 at.ppm by APT) and Si (~140 at.ppm) and no measur- able Zr or B. The model alloy thus combines the experimentally ob- served beneficial characteristics that have been attributed to enhanced AM processability and reduced cracking while maintaining a high γ’ vol- ume fraction: i) low segregation tendency of major alloying elements eliminating solidification and liquation cracking, ii) low combined (or even absence) of B and Zr, and iii) minimal Si. The smooth crack surfaces are indicative of cracking in the solid state during processing by decohesion along grain boundaries. The grain boundaries are natural weak points in the structure, especially if they are oriented perpendicular to the predominant tensile stresses [46]. Under AM processing conditions high thermal stresses are induced in the material, with the highest tensile stresses occurring parallel to the build plane [47]. This renders the columnar microstructure inherently susceptible to cracking, as the grain boundaries are subjected to normal tensile loads. The high internal stresses are consistent with the observed high-density dislocation networks formed within grains. If the fracture strength of the grain boundary is lower than the internal stress, a crack will form and propagate. Due to the simplified nature of the model alloy, no grain boundary strengthening phase was created upon solidification. The observed cracking is thus considered to be a type of ductility-dip cracking. Despite these characteristics, the processing maps (Fig. 2) reveal the typical behavior of high-γ’ nickel superalloys processed by L-PBF: a trade-off between densification and cracking [22]. As cracks are ob- served in samples fabricated with volumetric energy densities for which full densification is not yet achieved, such an alloy cannot be suc- cessfully processed in a crack-free condition without further measures, such as hot isostatic pressing to close cracks in post-processing steps. In the literature, several mechanisms are discussed that can cause transient low ductility at elevated temperature such as i) embrittlement by impurities, especially S, ii) formation of precipitates that increase high-temperature strength but reduce ductility and iii) grain boundary sliding where the grain boundaries do not withstand a persistent high temperature creep load and fracture [48]. The model alloy studied in thisworkcontains40ppmS,whichhasbeenshowntoleadtoasignificant reduction of hot ductility in pure nickel [40,49]. However, in technical al- loys, S is efficiently gettered by Ti, Zr and lanthanoids forming stable sul- fides [40]. 4.2. Microstructure and crack formation Typical of nickel alloys produced by additive manufacturing, direc- tional growth is prominent along the build direction, leading to a colum- nar microstructure with layer-to-layer epitaxy. The BSE and EBSD micrographs highlight the columnar nature of the grain growth, with grains that can be longer than 0.5 mm. However, no pronounced fiber texture is clearly identified for the model alloy. The predominant grain orientations are 100 and 110, with a tendency to grow into large col- umns. Published research on IN738LC typically reports 100 textures in the studied parameter fields [4,6,7]. In CM247LC, mixed 100/110 tex- tures along the build direction have been observed after L-PBF using a unidirectional scan strategy; typically, higher energy inputs lead to re- duced texture as melt pool sizes increase and thermal gradients are re- duced [21]. In our simplified alloy, cracks appear to propagate only along high-angle grain boundaries, as it has been observed abundantly in related literature. Over the complete cross-section, all grains with cracked boundaries have disorientation angles larger than 30°. Based on the EBSD data on the x-z plane, the material contains 77 mm/mm2 high angle grain boundaries (>15°) of which 15% have disorientation angles over 50°, 23% over 40° and 22% over 30°. This provides plenty of susceptible grain boundaries for crack formation. However, this crite- rion is not sufficient to predict crack formation, as most high angle grain boundaries do not crack. The comparison between Fig. 4a, b, and d indi- cates that cracks are also preferentially formed in areas close to a large number of small-angle grain boundaries with locally high stored energy and dislocation densities indicating high stress in the solid state. The TEM micrographs did not reveal significant differences between the structures of a cracked and nearby non-cracked grain boundaries, both exhibiting similar Ti segregation in adjacent solidification cell bound- aries, and Al2O3/(Ti,Cr)S nanoparticle distribution within the grains. A reduction of ductility by the formation of coherent precipitates such as γ’ during processing and the following cyclic heating can be ruled out as no precipitates were detected by TEM or APT, within the so- lidification cells or at their Ti-enriched boundaries. The cell boundaries would be particularly expected to form γ’ due to a higher γ’ solvus at higher Al and Ti contents. The atom probe tip was extracted from the center of a 5x5x5 mm3 SLM L-PBF specimen. 4.2. Microstructure and crack formation As such, the volume ob- served is buried ~2.5 mm (i.e., ~ 83 layers) within the part. The observed volume has thus experienced a complex thermal history after initial melting and solidification, including several solid-state heating cycles with decaying magnitude, as well as a low-intensity heat treatment through heat accumulation within the part during the L-PBF process. For the tested specimen size, this cyclical heat treatment is insufficient to achieve γ’ formation. The third mechanism, grain boundary sliding leading to high-temperature fracture, is considered the most likely cause for crack formation in this model alloy. In the absence of grain boundary strengthening elements, such as Zr and B, or any grain boundary phases, such as carbides or borides, the model alloy provides little resistance to such a cracking mechanism. Additionally, the alloy has an increased yield strength due to supersaturation of Al and Ti within the γ matrix during L-PBF and suppression of γ’ formation. The solid solution strengthening contribution ΔσSS in nickel can be estimated using The crack surfaces of the model alloy appear smooth in nature, un- like those observed in CM247LC, from which the current alloy is derived [18,37]. Due to the heavy segregation of melting point depressant ele- ments such as Hf, CM247LC suffers from solidification cracking as well as from liquation cracking. In this case, the grain boundary is being pulled apart while a liquid remains, leading to crack surfaces with den- dritic features. While Ti appears to segregate to solidification cell boundaries in the model alloy (up to 2 at.%), it does not lead to solidifi- cation cracking. This local segregation of Ti is expected to reduce the local melting point only slightly. Using the composition of the solidifica- tion cell boundaries determined by APT (Ni-8.7Cr-12Al-2Ti, at.%), the calculated solidus is 1375 °C (TCNI5). This is only 7 °C higher than the lowest melting detected by DSC (Table 2). However, the calculated local solidus reduction compared to the bulk value of only −19 °C is considered unlikely to lead to remelting of the solidification cell ΔσSS ¼ ∑ki 1 n∙ci  n ð3Þ ΔσSS ¼ ∑ki 1 n∙ci  n ð3Þ with kithe element-specific strengthening coefficient, ci the solute con- centration, and n ¼ 1 2 [50,51]. The estimated ΔσSS for the model alloy is 148 MPa for the bulk concentration, and 167 MPa in the Ti-rich solidifi- cation cell boundaries. 4.1. Model alloy characteristics In the studied model alloy, S indeed reacts with Ti, and to a lesser extent Cr, forming stable (Ti,Cr)S particles. While these particles act as obstacles for dislocations and thus increase the yield strength (see 4.4), they prevent free S in the matrix and its detrimental effects. 4.2. Microstructure and crack formation 4.3. Slag formation in oxygen-containing nickel alloy Slag located on the freshly solidified melt tracks has is present on the last layer (top surface) of all printed parts (cf. inset of Fig. 3c), regardless of the printing parameters. Additionally, as the successive melt tracks partially overlap, the subsequent laser track remelts the metal alloy as well as the slag. Depending on processing parameter, the slag remains in place or floats again to the fresh surface. If the energy input is high enough, the higher melt pool temperature, and the associated stronger melt flow and larger melt pool sizes, potentially partially re-dissolves or breaks up the slag to create more dispersoids. At low energy input, the slag is covered by the fresh melt track, forming lenses which are embed- ded in the matrix after solidification. Lack of fusion defects are likely due to the poor wettability of the metallic melt on the oxide lenses, locally hindering consolidation. Additionally, a higher fraction of oxide slag might be ejected from the melt pool, alongside metal spatters, as energy density increases. This hypothesis is consistent with the estimated slag area fraction, which reaches values as high as ~0.06% at 55 J/mm3, close to the estimated total Al2O3 in the alloy, and decreases to under 0.01% above 140 J/mm3. In terms of successful processing strategies, the need for excessively high energy input to prevent embedded slag is however not compatible with the need for low energy input to pre- vent cracking. In a slag-forming alloy, optimal processing conditions thus become a compromise between cracking and slag/porosity forma- tion (see Fig. 2f). The formation of Al2O3-based slag is observed in all processed spec- imens (Figs. 2 and 3). The O content of the Ni-Cr-Al-Ti model alloy pow- der is 180 ppm, comparable to the lower concentration range reported by Engeli et al. for IN738LC powder batches (160–1000 ppm) [5]. How- ever, oxide slag formation has been reported in IN738LC with only 70 ppm oxygen after L-PBF [13]. Hastelloy X and In718 have been found to form Cr and Al-rich surface oxides on powders recovered after EBM [53]. The presence of Al2O3 and TiN in the powder later leads to defects in EBM-manufactured In718 parts with increased defect frequency with increasing powder recycling cycles [54]. 4.3. Slag formation in oxygen-containing nickel alloy Considering the O content of the powder, the theoretical matrix den- sity of 8.4 g/cm3, and the density of Al2O3, the as processed material can contain up to 0.08 vol% of oxide, assuming all measured O in the powder is scavenged into Al2O3. To rule out oxygen pick up from the process chamber atmosphere during L-PBF, control experiments were con- ducted in an atmosphere containing up to 0.1% oxygen. No noticeable differences were observed in terms of slag formation, as compared to the normal processing condition with 0.01% oxygen. The oxide exists in two forms: as separated slag and dispersed as nanoparticles. In cast- ing, any slag formed on the top of the melt is skimmed off before casting, but this is inherently impossible to do in additive manufacturing. In the absence of a dedicated in-situ monitoring experiment, the precise mechanism behind the slag formation remains unclear. However, based on the current experimental evidence, a possible mechanism is il- lustrated in Fig. 8. 4.2. Microstructure and crack formation Additional strengthening of ~150 MPa is pro- vided by natively formed dispersoids through the Orowan mechanism (see 4.4 and Fig. 5). In combination, these effects increase the yield strength of the as-processed material by ~300 MPa, at room tempera- ture. With increased yield stress, the material can build up high residual 10 A. De Luca, C. Kenel, S. Griffiths et al. Materials and Design 201 (2021) 109531 available as free solute. The concept of O gettering and native disper- soid formation has been successfully used to create in-situ oxide and nitride dispersion strengthened steels processed by wire-fed arc melting [57] and L-PBF [58]. For the present Ni base alloy, however, a significant amount of the formed Al2O3 particles accumulate at the melt pool surface and form a buoyant, low-density Al2O3 slag. The bad wettability between Ni and Al2O3 then potentially prevents re- introduction of the oxide into the melt pool, leading to steady slag agglomeration at the melt pool sides, which then is incorporated as slag lenses, as shown in Fig. 3. stresses that may then cause grain boundary cracking along the most vulnerable direction, i.e., the long columnar high angle grain boundaries oriented perpendicular to the horizontal tensile stress. This is in agree- ment with welding literature, where straight, precipitation-free grain boundaries were observed to lead to cracks, while a small amount of grain boundary carbides, as well as tortuous grain boundaries, provide higher resistance [52]. 4.4. Role and potential of natively formed dispersoids While molten Ni can dissolve a high concentration of oxygen (>10 at.% at 1700 °C) [55], the model alloy contains three very potent oxide-forming elements: Cr, Al and Ti. According to the experimental evidence, Al appears to have the strongest affinity with O during the melting of this model alloy. Ti and Cr were found in the slag at ~1 at.%, and Ni at ~4%. Excess O thus predominantly reacts with Al to form Al2O3 dispersoids, that separate from the molten alloy. The newly formed nanoparticles are then dispersed throughout the melt pool, based on the strong Marangoni convection driven by the surface tension gradients. Both sulfur and oxygen have been shown to reduce or even invert the surface tension gradients in liquid Fe and Ni [56], thus possi- bly affected surface segregation. As demonstrated by TEM, both O and S are gettered by Al and Ti, respectively, forming dispersoids and are not The presence of oxygen and sulfur in the powder, and their strong affinity to aluminum and titanium, respectively, leads to the in situ formation of Al2O3 and (Ti,Cr)S dispersoids within the melt pool upon L-PBF. Due to the strong Marangoni flow in the melt pool, the nanopar- ticles appear evenly distributed, as evidenced by TEM (Fig. 5). The pres- ence of a second phase in the melt during solidification can induce grain nucleation, and grain refinement when present in sufficiently high number density, as observed with Al3Zr/Al3Sc in aluminum alloys, their crystal structure promoting grain inoculation [59,60]. However, this does not appear to be the case with Al2O3 in nickel nor with in situ ODS steels [57]. Based on TEM-EDX mapping, the solidification cell boundaries are revealed by their higher Ti content. Comparing the Fig. 8. Slag formation and particle incorporation. a) Upon laser melting, liquid oxide slag is formed on top of the melt pool due to local heating by the laser. The low-density ionic liquid separates from the metallic melt and is carried to the melt pool side by the Marangoni convection. b) After solidification, oxides are present as trapped nanoparticles (not to scale) within the metallic matrix and as μm-size slag lenses. Fig. 8. Slag formation and particle incorporation. a) Upon laser melting, liquid oxide slag is formed on top of the melt pool due to local heating by the laser. 4.4. Role and potential of natively formed dispersoids The low-density ionic liquid separates from the metallic melt and is carried to the melt pool side by the Marangoni convection. b) After solidification, oxides are present as trapped nanoparticles (not to scale) within the metallic matrix and as μm-size slag lenses. 11 A. De Luca, C. Kenel, S. Griffiths et al. Materials and Design 201 (2021) 109531 under service conditions and negative effects on processability needs to be achieved. Ti map to the distribution of O and S shows that larger Al2O3 and (Ti,Cr)S nanoparticles are regularly found at, or close to, grain and cell bound- aries. This indicates that the dispersoids formed in the melt are at least partially rejected by the solidification front and pushed into the re- maining liquid. The location of dispersoids at the grain boundaries, which have moved slightly away from the solidification cell boundaries due to the thermal history imposed during L-PBF, indicates that these particles also can lead to grain boundary pinning. This might improve high temperature creep properties or impede grain growth during solutionizing and heat treatment required to obtain the final γ/γ’ microstructure. The current GB cracking mechanism highlights the inherent weakness of high angle grain boundaries subjected to large tensile residual stresses. One option to mitigate GB cracking would be the control of the grain microstructure to prevent excessive columnar growth of grains, and achieve an equiaxed microstructure, which however decreases creep resistance. This has been achieved in electron-beam melting controlling the thermal gradients [35]. How- ever, this is challenging for nickel alloys due the high thermal gradi- ent involved in L-PBF manufacturing, and the difficulty to induce grain inoculation by a second phase. Controlled additions of GB strengthening elements, i.e. to form carbides and borides, is thus an interesting potential area for alloy modification. Considering that carbide formers are usually heavily segregating elements such as Hf and Zr, this creates potentially conflicting requirements re- quiring a trade-off and optimized additions. To further improve the strength of the matrix, solid solution strengthening elements, such as Co and Fe [2,3], can be added to increase the yield strength of the alloy above the residual tensile stress in the part, which is the expected source of cracking in the material. 4.4. Role and potential of natively formed dispersoids This is similar in magnitude to the effect from solid- solution strengthening of Cr, Al and Ti in the supersaturated γ phase (see 4.2). 5. Conclusions To investigate the defect formation in additively-produced high- γ’ nickel-base superalloys, the commercial CM247LC alloy composi- tion was simplified to contain only four of its core components: Ni, Cr, Al, and Ti. The model alloy successfully prevented the typical so- lidification/liquation cracking which often affect L-PBF processing. The simplified composition comes, as expected, at the cost of re- duced alloy strength which leads to the cracking of high angle grain boundaries in the solid state due to the high internal stress from thermal gradients. A successful high-γ’ superalloy for L-PBF ap- plications can thus safely contain Cr, Al and Ti, but will require a combination of grain boundary strengthening phases and solid solu- tion strengthening elements to resist cracking under stress. Segre- gating elements such as Hf and Zr are ideally avoided, or added in controlled amounts. Additionally, there is a need to identify contam- ination levels in reported studies on AM more accurately in order to assess their criticality. Here, the presence of slag is an indication of excess O, which can lead to lack of fusion defects and potentially cracking. Interestingly, if the slagging issue can be mitigated, the trace oxygen in the powder allows for the in-situ formation of oxide dispersoids, which provide additional strength to the mate- rial. Ultimately, alloy modifications to increase processability and to mitigate the various defect-forming mechanisms of high-γ’ Ni- base superalloys seem required. However, such changes need to have the ultimate applications and the required mechanical proper- ties of these alloys in mind. If such changes are implemented in existing, optimized alloys, inferior mechanical properties would be a likely result, albeit better processability would be achieved. 4.4. Role and potential of natively formed dispersoids While the volume fraction of dispersoids remains low in the studied alloy, it can be expected that they will lead to dispersoid strengthening of the matrix, as evidenced by the observed dislocation-dispersoid in- teraction (Fig. 5b). While Al2O3 is a less stable dispersoid in Ni–Cr alloys compared to other oxides such as Y2O3 [61], its coarsening is still ex- pected to be slow during following heat treatments. The dispersoids may also have the potential to interact with the high volume fraction of γ’ precipitates that can form during subsequent heat treatments. The strengthening contribution of the dispersoids from the Orowan mechanism ΔσOr can be calculated using Lastly, nanometric dispersoids, either formed natively or added de- liberately, allow to strengthen the metal matrix independent of the presence of efficient solid solution strengthening elements. This concept is classically used in oxide dispersion strengthened alloys but has been recently revisited in light of the potential to process ODS alloys by AM [66–72]. If the dispersoids can be distributed homogenously at a small size, without slag formation, this approach might allow designing alloys for AM that do not rely on high additions of elements deleterious to the AM process but rather maintain a simple, low segregation alloy matrix with a narrow freezing range. ΔσOr ¼ M 0:4 π Gb ffiffiffiffiffiffiffiffiffiffiffi 1−ν p ln 2 ffiffiffiffiffiffiffiffiffiffiffi 1:5∙〈R〉 p b   λ ð4Þ ð4Þ where M = 3.07 [62], b = 0.249 nm [63], G = 78.9 GPa [63], ν=0.296 [64], 〈R〉is the mean dispersoid radius and λ is the interparticle distance [65]. Based on the measured 〈R〉= 11.6 ± 5.6 nm and λ = 191 ± 114 nm (uncorrected) or λ = 213 ± 116 nm (corrected for TEM lamella thickness of 100 nm) the estimated ΔσOr is between 133 and 149 MPa. This is similar in magnitude to the effect from solid- solution strengthening of Cr, Al and Ti in the supersaturated γ phase (see 4.2). where M = 3.07 [62], b = 0.249 nm [63], G = 78.9 GPa [63], ν=0.296 [64], 〈R〉is the mean dispersoid radius and λ is the interparticle distance [65]. Based on the measured 〈R〉= 11.6 ± 5.6 nm and λ = 191 ± 114 nm (uncorrected) or λ = 213 ± 116 nm (corrected for TEM lamella thickness of 100 nm) the estimated ΔσOr is between 133 and 149 MPa. 4.5. Future alloy development and modification The present investigation conducted on a model Ni-Cr-Al-Ti su- peralloy, as well as the results reported in the literature on more complex superalloys, highlight some specific alloy design criteria that need to be fulfilled to achieve defect-free parts of γ/γ’ Ni base superalloys by L-PBF. p y y The results reported in this paper indicate that defects like porosity, slag formation and – as a result – lack-of-fusion defects are related to the powder quality. The experimental powder batch was not optimized, like commercial alloy powders, with regard to internal gas porosity. While its quality is sufficient for the purpose of this study, the detected amount of internal porosity is assumed to be detrimental to part integrity. More importantly, the accurate control of trace contaminants in the powder is required. A tight control on the oxygen and oxide content of the powder is important to prevent Al2O3 slag formation. In general, slag formation in high Al/Ti containing Ni alloys during L-PBF needs to be further stud- ied, as it is only scarcely reported in the literature, but some results in- dicate that it can occur at low oxygen contents (< 100 ppm) [13]. The presence of trace amounts of S does not appear to be critical in this sys- tem, as it is gettered by Ti and Cr forming nanometer-sized (Ti,Cr)S. Other elements detected in the alloy by atom probe are Co, Fe, V, Cu, Si, and C but they are deemed innocuous at their low concentrations. 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Tadalafil attenuates ischemic damage as well as reperfusion injury in the rat ovary
Journal of the Turkish-German Gynecological Association
2,020
cc-by
4,391
Çağdaş Şahin1, Nuri Yıldırım1, İsmet Hortu1, Ali Akdemir1, Serdar Özşener1, Gürkan Yiğittürk2 Oytun Erbaş3 1Department of Obstetrics and Gynecology, Ege University Faculty of Medicine, İzmir, Turkey 2Department of Histology and Embryology, Muğla Sıtkı Koçman University Faculty of Medicine, Muğla, Turkey 3Department of Physiology, Demiroğlu Bilim University Faculty of Medicine, İstanbul, Turkey Address for Correspondence: Çağdaş Şahin e.mail: cagdasdr@yahoo.com ORCID: orcid.org/0000-0001-7346-3987 ©Copyright 2020 by the Turkish-German Gynecological Education and Research Foundation - Available online at www.jtgga.org Journal of the Turkish-German Gynecological Association published by Galenos Publishing House. DOI: 10.4274/jtgga.galenos.2019.2018.0121 35 35 Original Investigation Original Investigation Tadalafil attenuates ischemic damage as well as reperfusion injury in the rat ovary Nuri Yıldırım1, İsmet Hortu1, Ali Akdemir1, Serdar Özşener1, Gürkan Yiğittürk2, Oytun Erbaş3 Abstract Objective: Tadalafil is a selective phosphodiesterase type-5 inhibitor with a long half-life. It has a dual function in ischaemic and re-perfused tissues, i.e. vasodilatation and anti-oxidant effects. These features of tadalafil distinguish it from other anti-oxidants. We investigated the dual effect of tadalafil on ischaemia and reperfusion injury in the rat ovary. Material and Methods: We established five study groups. Group 1 (n=6): sham-operated; group 2 (n=6): torsion; group 3 (n=6): torsion and Tadalafil; group 4 (n=6): torsion/de-torsion; and group 5 (n=6): torsion/de-torsion and tadalafil. Ovarian samples were harvested from animals and evaluated in terms of histopathologic changes, tissue malondialdehyde (MDA) concentrations, lactate production, and plasma cyclic guanosine monophosphate (cGMP). Results: Follicular degeneration, oedema, haemorrhage, and inflammatory cells were significantly decreased in group 5 in comparison with group 4. Group 2 and group 3 were compared in terms of vascular congestion and haemorrhage; these parameters were significantly decreased in group 3. In addition, significantly decreased MDA and lactate concentrations were observed in group 5 in comparison with group 4. Increased cGMP concentrations were detected in group 3 and group 5. Conclusion: We conclude that tadalafil might be useful in protecting the ovary against ischaemia and reperfusion injury. In the evet of ovarian torsion, it will provide a greater therapeutic effect than only performing de-torsion of the ovary or using other anti-oxidant agents. (J Turk Ger Gynecol Assoc 2020; 21: 35-40) Keywords: Phosphodiesterase type-5, tadalafil, ischemia-reperfusion injury, vasodilatation; anti-oxidant Received: 24 September, 2018 Accepted: 8 May, 2019 Animals Thirty mature, female Sprague-Dawley albino rats were used in this study. All rats were aged 12 weeks and weighed 200- 220 g. Animals were housed in steel cages maintained in a temperature-controlled room (21±1 °C) under a 12-h light/ dark cycle and fed ad libitum. This study was approved by the intuitional animal care committee, and all procedures were performed according to the experimental guidelines of Ege University (SPK-HADYEK 34562/2015/24). Patient approval has not been obtained as it is performed on animals. The oestrous stage was determined by taking a vaginal smear from all animals, and cell types were identified under a microscope using the Papanicolaou staining procedure. Oestrous was confirmed in the smear specimens from 30 rats, which were included in the experiment. The rate of vascular congestion, stromal haemorrhage and oedema, follicular degeneration, and infiltration of inflammatory cells in sections for each animal were scored from 0 to 3 according to the severity of injury. A score of 0 represents normal histology, and <33%, 33-66%, and >66% pathologic findings in the ovarian sections were scored as 1, 2, and 3, respectively (8). Histopathologic evaluation of tissue samples Ovarian samples were evaluated using a light microscope. Specimens were fixed in 10% buffered formalin, then an increasing alcohol series was used to dehydrate samples. Subsequently, samples were cleared with xylene and embedded in paraffin. Tissue sections were sliced at 4-µm thickness and slides were stained with haematoxylin and eosin (H&E) prior to histologic analysis. An Olympus BX51 microscope connected to an Olympus C-5050 digital camera (Olympus Corp., Tokyo, Japan) was used for the analysis and photography of sections. Histologic sections were evaluated in terms of primordial and developing follicles. In the histology of the ovary, primordial follicles are located beneath of the cortex, surrounded by a single layer of granulosa cells. Primary follicles are encircled by a single layer of cuboidal granulosa cells. The identification of stratified granulosa cells indicates secondary follicles, and stratum granulosum and antral space are major features of tertiary follicles. The pyknotic appearance of the nucleus and collapsed ooplasm, with or without irregular granulosa cells, indicate a degenerated follicle. Tadalafil has a dual function as a vasodilator and antioxidant. These characteristics distinguish it from other antioxidants used to prevent IR injury. In the current study, we investigated the effect of tadalafil on the ischemic stage and on reperfusion injury. Introduction The skin of the abdominal area was trimmed and cleaned with povidone iodine. A 2 cm midline incision was performed on the abdomen, and the uterus and ovaries were detected. brings another problematic condition, reperfusion damage. The reoxygenation of ischemic tissues results in the production of reactive oxygen species (ROS), including superoxide anions (O2-), hydrogen peroxide, and hydroxyl radicals (OH-), among others. These ROS damage phospholipids and proteins of the cell membrane and promote mitochondrial permeability which can cause a reduction in ATP and lead to cell death (2). At this stage, the use of antioxidant agents helps to support the self-antioxidant defence mechanism of cells. In the sham-surgery group (group 1, n=6), laparotomy was performed and the abdomen was closed 1 min later without performing any surgical procedures. In the torsion group (group 2, n=6), ischemia was created for 3 h by applying atraumatic vascular clips to the vascular pedicles of ovaries on both sides. The incision was subsequently closed with 3/0 silk sutures. In the torsion and tadalafil group (group 3, n=6), ischemia was performed, as described for group 2, 30 min after administering 20 mg/kg of tadalafil [(Cialis, Lilly, IN, United States of America (USA)] via oral gavage. In the torsion/detorsion group (group 4, n=6), 3 h of ischemia was created, as in group 2, followed by 3 h of reperfusion. In the torsion/detorsion and tadalafil group (group 5, n=6), torsion was created for 3 h, followed by tadalafil administration 30 min prior to 3 h of detorsion/reperfusion. At the end of the reperfusion stage, both ovaries were harvested for histologic and biochemical evaluation. One of the phosphodiesterase type-5 (PDE-5) inhibitors is tadalafil which has been used in the treatment of erectile dysfunction (3). As PDE-5 catalyses the hydrolysis of 3'5'-cyclic guanosine monophosphate (cGMP), PDE-5 inhibitors cause an increase in cGMP. Nitric oxide (NO) is a potent vasodilator, and works via the secondary messenger cGMP. PDE-5 inhibitors facilitate the accumulation of cGMP within cells, and also facilitate NO-mediated vasodilation of vascular smooth muscle in mammals. Recent studies have demonstrated a potential beneficial effect of PDE-5 inhibitors on IR injury in the brain (4), kidney (5), and heart (6). These authors found that increasing cGMP attenuates lipid peroxidation (7) and nicotinamide- adenine dinucleotide phosphate [NAD(P)H] oxidase activity, which are the main sources of ROS production in oxidative stress (6). Introduction When the ischemic period is extended, the associated cell damage becomes irreversible. During the ischemic stage, the production of adenosine triphosphate (ATP) decreases and anaerobic glycolysis begins. Decreasing ATP concentrations leads to the cessation of sodium-potassium pump channel function and subsequent water influx into cells, resulting in cell swelling. If ischemia persists, cells proceed to an irreversible stage. At this stage, severe swelling is seen in the mitochondria, as well as cell membrane damage. Cell death usually results in necrosis. Ovarian torsion is an emergency condition in gynaecology practice. It is particularly important when diagnosed in the reproductive period. Ischemia-reperfusion (IR) injury can damage ovarian tissue and also reduce the ovarian reserve (1). The time of diagnosis is important due to the diminishing ovarian reserve. However, it usually takes some time to achieve a diagnosis given the non-specific diagnostic symptoms. In addition, although restoration of the blood supply of ovarian tissue can reduce ischemic damage, it can also cause reperfusion injury. Reinstating the blood supply of ischemic tissue leads to recovery of cells in the reversible stage. However, this situation Şahin et al. Tadalafil attenuates ischemic damage as well as reperfusion injury in the rat ovary J Turk Ger Gynecol Assoc 2020; 21: 35-40 36 The skin of the abdominal area was trimmed and cleaned with povidone iodine. A 2 cm midline incision was performed on the abdomen, and the uterus and ovaries were detected. In the sham-surgery group (group 1, n=6), laparotomy was performed and the abdomen was closed 1 min later without performing any surgical procedures. In the torsion group (group 2, n=6), ischemia was created for 3 h by applying atraumatic vascular clips to the vascular pedicles of ovaries on both sides. The incision was subsequently closed with 3/0 silk sutures. In the torsion and tadalafil group (group 3, n=6), ischemia was performed, as described for group 2, 30 min after administering 20 mg/kg of tadalafil [(Cialis, Lilly, IN, United States of America (USA)] via oral gavage. In the torsion/detorsion group (group 4, n=6), 3 h of ischemia was created, as in group 2, followed by 3 h of reperfusion. In the torsion/detorsion and tadalafil group (group 5, n=6), torsion was created for 3 h, followed by tadalafil administration 30 min prior to 3 h of detorsion/reperfusion. At the end of the reperfusion stage, both ovaries were harvested for histologic and biochemical evaluation. Statistical analysis Mann-Whitney U-test was used to compare the biochemical data and histopathologic scores between groups. The results were considered statistically significant if the p-value was <0.05. Statistical analyses were performed using SPSS software version 16.0 (Chicago, IL, USA). Measurement of plasma cGMP and lactic acid concentrations Measurement of plasma cGMP and lactic acid concentrations After administering tadalafil, plasma samples were collected and stored at -80 °C until use for the cyclic nucleotide assay. Enzyme-linked immunosorbent assay (Cusabio, Biotech Co. Ltd. Wuhan, China) was used to determine the plasma cGMP level. The ultraviolet detection method was used to measure the L-lactic acid concentrations in plasma samples. Increased MDA concentrations were found in the torsion (group 2) and torsion/detorsion (group 4) groups compared with the sham-surgery group (group 1, p<0.01). Decreased MDA concentrations were measured in the torsion/tadalafil (group 3) and torsion/detorsion/tadalafil (group 5) groups, but this decrease only reached statistical significance in the torsion/detorsion/tadalafil group (group 5, p<0.001). Similar findings were also observed for the lactate results. Increased tissue lactate concentrations were found in the torsion (group 2) and torsion/detorsion (group 4) groups. Importantly, lactate concentrations were normalised in the groups that received tadalafil (groups 3 and 5). Plasma cGMP concentrations reflected the effect of tadalafil treatment, and a statistically significant increase in cGMP concentrations was found in the groups that were administered tadalafil (groups 3 and 5, p<0.01). The biochemical results are presented in Table 2. Determination of lipid peroxidation and total protein concentrations (p<0.05), oedema (p<0.05), vascular congestion (p<0.001), haemorrhage (p<0.05), and infiltration of inflammatory cells (p<0.05) were observed in the torsion group (group 2). When tadalafil was administered before torsion, a statistically significant decrease in vascular congestion (p<0.05) and haemorrhage (p<0.05) was found for group 3 compared with the torsion-only group (group 2). Tissue samples were homogenised in KCl (150 mM) and centrifuged at 5000 g for 10 min. Lipid peroxidation was determined by analysis of the supernatant, with malondialdehyde (MDA) concentrations measured as thiobarbituric acid-reactive substances in each tissue sample (9). Trichloroacetic acid and thiobarbituric acid reactive substances reagents were mixed with tissue samples and incubated at 100 °C for 60 min. The samples were centrifuged at 3000 rpm for 20 min after cooling on ice, and the absorbance of the supernatant was read at 535 nm. The standard calibration curve obtained using tetraethoxypropane was used to calculate tissue MDA concentrations, which were expressed as nmol/µg protein. Bradford’s method was used to determine the total protein level in the tissue samples, with bovine serum albumin used as the standard (10). When we evaluated the reperfusion effect, severe tissue damage was observed after restoration of the ovarian blood supply in group 4. Follicular degeneration and infiltration of inflammatory cells were more prominent in the torsion/ detorsion-only group (group 4), and all abnormal findings were significantly decreased after adding tadalafil (group 5) when compared with group 4. A comparison of scores between groups in terms of histopathologic abnormalities is shown in Table 1. Experimental design Animals were anaesthetised with 50 mg/kg ketamine and 7 mg/ kg xylazine hydrochloride (Alfasan Int. BV, the Netherlands). Şahin et al. Tadalafil attenuates ischemic damage as well as reperfusion injury in the rat ovary Şahin et al. Tadalafil attenuates ischemic damage as well as reperfusion injury in the rat ovary J Turk Ger Gynecol Assoc 2020; 21: 35-40 37 Determination of lipid peroxidation and total protein concentrations Discussion The histopathologic results of ovarian tissue samples of the five groups are presented in Figure 1, with no histopathologic changes observed in the group that received sham surgery (group 1). In contrast, increased follicular degeneration The aim in cases of ovarian torsion is to reduce the extent of necrotic tissue and preserve ovarian function. For this purpose, the ischemic period should be shortened and reperfusion Table 1. Histopathologic findings in the groups Group 1 Group 2 Group 3 Group 4 Group 5 Follicular degeneration 0.16±0.16 1.0±0.25* 0.5±0.22 2.66±0.21 0.66±0.21†† Vascular congestion 0.33±0.21 2.66±0.21** 1.5±0.22# 2.5±0.34 1.83±0.30 Oedema 0.5±0.22 1.16±0.27* 1.33±0.21 2.16±0.30 1.16±0.16† Haemorrhage 0.16±0.16 1.83±0.30* 0.66±0.33# 2.33±0.33 1.0±0.25† Infiltration by inflammatory cells 0.16±0.16 0.83±0.16* 1.0±0.25 1.66±0.21 0.83±0.16† Group 1: Sham operation, Group 2: Torsion, Group 3: Torsion and tadalafil, Group 4: Torsion/detorsion, Group 5: Torsion/detorsion and tadalafil, *: P<0.05, group 1 vs group 2, **: P<0.001, group 1 vs group 2, #: P<0.05, group 2 vs group 3, ##: P<0.001, group 2 vs group 3, †: p<0.01, group 4 vs group 5, ††: P<0.001, group 4 vs group 5 Table 1. Histopathologic findings in the groups Group 1: Sham operation, Group 2: Torsion, Group 3: Torsion and tadalafil, Group 4: Torsion/detorsion, Group 5: Torsion/detorsion and tadalafil, *: P<0.05, group 1 vs group 2, **: P<0.001, group 1 vs group 2, #: P<0.05, group 2 vs group 3, ##: P<0.001, group 2 vs group 3, †: p<0.01, group 4 vs group 5, ††: P<0.001, group 4 vs group 5 Şahin et al. Tadalafil attenuates ischemic damage as well as reperfusion injury in the rat ovary J Turk Ger Gynecol Assoc 2020; 21: 35-40 38 inhibitor in ischemic ovaries was also revealed in our results. Tadalafil has a similar function to other PDE-5 inhibitors like sildenafil; however, it is more selective than sildenafil for the binding of PDE-5 (16) and has a longer half-life (17.5 vs 4.5 h, respectively) (17). These features mean that tadalafil is longer acting. Tadalafil augments the effect of NO by decreasing cGMP degradation. NO plays a major role in inhibiting NAD(P) H oxidase and limiting neutrophil and platelet adhesion, aggregation, and activation (18). injury should be prevented. Many antioxidant drugs have been investigated for their efficiency in preventing oxidative damage during the reperfusion period (11,12). One of them, tadalafil, has antioxidant activity, and attenuates the generation of ROS under oxidative stress (6). Discussion In addition, tadalafil also possess a vasodilation effect, leading to an increase in cGMP concentrations. These functions provide a valuable effect during the ischemic period of certain organs, such as the heart and kidney (13,14). In the current study, we demonstrated decreased vascular congestion and haemorrhage in ischemic ovaries with the use of tadalafil. Our findings are similar to those obtained by Lledo-Garcia et al. (15), who studied the use of sildenafil for renal ischemia and reported the effectiveness of administering sildenafil in warm ischemic kidneys during the immediate post-transplant period. The effectiveness of a PDE-5 Tadalafil has a similar function to other PDE-5 inhibitors like sildenafil; however, it is more selective than sildenafil for the binding of PDE-5 (16) and has a longer half-life (17.5 vs 4.5 h, respectively) (17). These features mean that tadalafil is longer acting. Tadalafil augments the effect of NO by decreasing cGMP degradation. NO plays a major role in inhibiting NAD(P) H oxidase and limiting neutrophil and platelet adhesion, aggregation, and activation (18). Increased tissue MDA indicates lipid peroxidation because MDA concentrations increase under conditions of oxidative stress (19). MDA impairs the ionic transport and enzymatic activity of cells, causing major changes in cell membrane permeability, resulting in damage and breakages that separate cell and organelle contents (20). In the event of ischemia, energy Figure 1. a. No pathologic changes were detected in the sham-operated animals. (v) vessel, (sf) secondary follicle, (pf) primary follicles, (cl) corpus luteum. b. Haemorrhaging (h) and oedema (*) were detected in the 3-h torsion group. c. Decreased vascular congestion (vc), and Haemorrhaging (h) were observed in the torsion and tadalafil group. d. Vascular congestion (vc), haemorrhaging (h), and oedema (*) were observed in the torsion/de-torsion group. e. Decreased oedema (*) and haemorrhaging were detected in the torsion/de-torsion tadalafil group. Haematoxylin and eosin staining was performed. Scale bars represent 250 µm Figure 1. a. No pathologic changes were detected in the sham-operated animals. (v) vessel, (sf) secondary follicle, (pf) primary follicles, (cl) corpus luteum. b. Haemorrhaging (h) and oedema (*) were detected in the 3-h torsion group. c. Decreased vascular congestion (vc), and Haemorrhaging (h) were observed in the torsion and tadalafil group. d. Vascular congestion (vc), haemorrhaging (h), and oedema (*) were observed in the torsion/de-torsion group. e. Decreased oedema (*) and haemorrhaging were detected in the torsion/de-torsion tadalafil group. Haematoxylin and eosin staining was performed. References 1. Bayer AI, Wiskind AK. Adnexal torsion: can the adnexa be saved? Am J Obstet Gynecol 1994; 171: 1506-10. 1. Bayer AI, Wiskind AK. Adnexal torsion: can the adnexa be saved? Am J Obstet Gynecol 1994; 171: 1506-10. 2. Toyokuni S. Reactive oxygen species-induced molecular damage and its application in pathology. Pathol Int 1999; 49: 91-102. 2. Toyokuni S. Reactive oxygen species-induced molecular damage and its application in pathology. Pathol Int 1999; 49: 91-102. The effectiveness of PDE-5 inhibitors on reperfusion injury in ovaries has been confirmed by two studies (21,22). The investigators demonstrated an antioxidant effect of PDE-5 inhibitors on reperfusion injury. The vasodilator feature of PDE-5 inhibitors also contributes to this effect. Therefore, in the current study, we investigated the vasodilator effect of tadalafil during the ischemic stage and found attenuated vascular damage in the ischemia groups that were administered tadalafil. These findings indicate that tadalafil has a dual function to reduce ischemia and reperfusion damage in the ovary. 3. Özkıdık M, Gökce Mİ, Yaman Ö. Efficacy of tadalafil treatment on erectile dysfunction in patients under dutasteride treatment: A prospective non- randomized comparative study. Turk J Urol 2018; 44: 294-7. 3. Özkıdık M, Gökce Mİ, Yaman Ö. Efficacy of tadalafil treatment on erectile dysfunction in patients under dutasteride treatment: A prospective non- randomized comparative study. Turk J Urol 2018; 44: 294-7. 4. Altaş M, Aras M, Meydan S, Nacar E, Ulutaş KT, Serarslan Y, et al. Effects of tadalafil on ischemia/reperfusion injury in rat brain. Acta Neurol Belg 2014; 114: 33-40. 5. Küçük A, Yucel M, Erkasap N, Tosun M, Koken T, Ozkurt M, et al. The effects of PDE5 inhibitory drugs on renal ischemia/reperfusion injury in rats. Mol Biol Rep 2012; 39: 9775-82. 6. Koka S, Das A, Salloum FN, Kukreja RC. Phosphodiesterase-5 inhibitor tadalafil attenuates oxidative stress and protects against myocardial ischemia/reperfusion injury in type 2 diabetic mice. Free Radic Biol Med 2013; 60: 80-8. A limitation of this study is the absence of long-term observations for both the biochemical and histologic parameters. Thus, future long-term studies should be performed to determine whether the changes in histologic and biochemical parameters are transient or permanent. 7. Keller JN, Hanni KB, Mattson MP, Markesbery WR. Cyclic nucleotides attenuate lipid peroxidation-mediated neuron toxicity. Neuroreport 1998; 9: 3731-4. 8. Guven S, Muci E, Unsal MA, Yulug E, Alver A, Kadioglu Duman M, et al. Peer-review: Externally peer-reviewed. Author Contributions: Surgical and Medical Practices: Ç.Ş., İ.H., O.E.; Concept: Ç.Ş., N.Y., O.E.; Design: Ç.Ş., G.Y., O.E.; Data Collection or Processing: İ.H., A.A.; Analysis or Interpretation: Ç.Ş., S.Ö., O.E.; Literature Search: Ç.Ş., A.A., N.Y.; Writing: Ç.Ş., A.A., O.E. Informed Consent: Patient approval has not been obtained as it is performed on animals. Informed Consent: Patient approval has not been obtained as it is performed on animals. Discussion Scale bars represent 250 µm Table 2. Tissue biochemical parameters in groups Group 1 Group 2 Group 3 Group 4 Group 5 MDA, (nmoL/µg protein) 0.06±0.01 0.75±0.21** 0.41±0.15 1.66±0.20 0.29±0.09## L-Lactate Level (mmoL/L) 6.4±0.80 10.7±1.58* 8.72±1.71 21.3±1.66 13.41±1.07# Plasma cGMP Level (pmol/L) 10.7±1.45 13.7±1.53 29.2±2.7† 17.9±1.6 37.8±5.07# Group 1: Sham operation, Group 2: Torsion, Group 3: Torsion and tadalafil, Group 4: Torsion/detorsion, Group 5: Torsion/detorsion and tadalafil, *: p<0.05, group 1 vs group 2, **: P<0.01, group 1 vs group 2, †: P<0.01, group 2 vs group 3, #: P< 0.01, group 4 vs group 5, ##: P<0.001, group 4 vs group 5 Table 2. Tissue biochemical parameters in groups Table 2. Tissue biochemical parameters in groups Şahin et al. Şahin et al. Tadalafil attenuates ischemic damage as well as reperfusion injury in the rat ovary J Turk Ger Gynecol Assoc 2020; 21: 35-40 39 Conflict of Interest: No conflict of interest is declared by the authors. Financial Disclosure: The authors declared that this study received no financial support. Financial Disclosure: The authors declared that this study received no financial support. Ethics Committee Approval: SPK-HADYEK 34562/2015/24. production is interrupted, which reduces the concentration of ATP in cells. These cells begin anaerobic energy production, increasing the lactic acid content and decreasing the pH level of cells. Arikan et al. (21) previously investigated the effect of tadalafil on IR injury in rat ovaries, and demonstrated decreased MDA concentrations and increased catalase (CAT) and superoxide dismutase (SOD) activities, which are scavenger enzymes for ROS, after using tadalafil (21). Additionally, they observed reduced histopathologic damage in groups that received tadalafil, which led the authors to conclude that tadalafil had a protective effect against IR injury. Similarly, increased MDA and lactate concentrations were ameliorated by tadalafil in our study, and these biochemical findings are supported by the histopathologic results of the tadalafil groups. Another PDE-5 inhibitor, sildenafil, has also been investigated in ovarian IR injury. The authors found changes in MDA, myeloperoxidase (MPO), SOD, glutathione peroxidase (antioxidant enzyme) between treated and untreated groups (22). MPO is secreted by neutrophils and is used as a marker of neutrophil activation in IR injury. These authors observed decreased MDA and MPO concentrations and increased tissue antioxidant enzyme concentrations, as well as an improved histologic appearance, in groups treated with sildenafil (22). Informed Consent: Patient approval has not been obtained as it is performed on animals. References The effects of carbon dioxide pneumoperitoneum on ovarian blood flow, oxidative stress markers, and morphology during laparoscopy: a rabbit model. Fertil Steril 2010; 93: 1327-32. The current management of ovarian torsion includes the protection of ovaries after detorsion, and avoids oophorectomy where possible (23). In this situation, prophylactic measures against injury are implemented, which affect ovarian function (24). Sometimes, the diagnosis of ovarian torsion takes time due to its non-specific symptoms. However, prompt diagnosis of ovarian torsion is important to preserve ovarian function. Therefore, the use of tadalafil in patients with suspected ovarian torsion will provide a greater therapeutic benefit than if treatment is only started following ovary detorsion. This treatment resembles the use of acetylsalicylic acid in patients with suspected myocardial infarction in the ischemic stage. 9. Olson BR, Hoffman GE, Sved AF, Stricker EM, Verbalis JG. Cholecystokinin induces c-fos expression in hypothalamic oxytocinergic neurons projecting to the dorsal vagal complex. Brain Res 1992; 569: 238-48. 10. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein- dye binding. Anal Biochem 1976; 72: 248-54. 11. Akdemir A, Sahin C, Erbas O, Yeniel AO, Sendag F. Is ursodeoxycholic acid crucial for ischemia/reperfusion-induced ovarian injury in rat ovary? Arch Gynecol Obstet 2015; 292: 445-50. 12. Ugurel V, Cicek AC, Cemek M, Demirtas S, Kocaman AT, Karaca T. Antioxidant and antiapoptotic effects of erdosteine in a rat model of ovarian ischemia-reperfusion injury. Iran J Basic Med Sci 2017; 20: 53-8. Şahin et al. Tadalafil attenuates ischemic damage as well as reperfusion injury in the rat ovary Şahin et al. J Turk Ger Gynecol Assoc 2020; 21: 35-40 40 18. Wink DA, Mitchell JB. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med 1998; 25: 434-56. 13. Ahmad N, Wang Y, Ali AK, Ashraf M. Long-acting phosphodiesterase-5 inhibitor, tadalafil, induces sustained cardioprotection against lethal ischemic injury. Am J Physiol Heart Circ Physiol 2009; 297: 387-91. 19. Carden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol 2000; 190: 255-66. 14. Gasanov F, Aytac B, Vuruskan H. The effects of tadalafil on renal ischemia reperfusion injury: an experimental study. Bosn J Basic Med Sci 2011; 11:158-62. 20. Erkanli Senturk G, Erkanli K, Aydin U, Yucel D, Isiksacan N, Ercan F, et al. The protective effect of oxytocin on ischemia/reperfusion injury in rat urinary bladder. References Peptides 2013; 40: 82-8. 15. Lledo-Garcia E, Rodriguez-Martinez D, Cabello-Benavente R, Moncada-Iribarren I, Tejedor-Jorge A, Dulin E, et al. Sildenafil improves immediate posttransplant parameters in warm-ischemic kidney transplants: experimental study. Transplant Proc 2007; 39: 1354-6. 21. Arikan DC, Bakan V, Kurutas EB, Sayar H, Coskun A. Protective effect of tadalafil on ischemia/reperfusion injury of rat ovary. J Pediatr Surg 2010; 45: 2203-9. 22. Celik M, Aksoy AN, Aksoy H, Aksoy Y, Halici Z. Sildenafil reduces ischemia-reperfusion injury in rat ovary: biochemical and histopathological evaluation. Gynecol Obstet Invest 2014; 78: 162-7. 16. Daugan A, Grondin P, Ruault C, Le Monnier de Gouville AC, Coste H, Linget JM, et al. The discovery of tadalafil: a novel and highly selective PDE5 inhibitor. 2: 2,3,6,7,12,12a-hexahydropyrazi no[1’,2’:1,6]pyrido[3,4-b]indole-1,4-dione analogues. J Med Chem 2003; 46: 4533-42. 23. Oelsner G, Shashar D. Adnexal torsion. Clin Obstet Gynecol 2006; 49: 459-63. 24. Özler A, Turgut A, Soydinç HE, Sak ME, Evsen MS, Alabalik U, et al. The biochemical and histologic effects of adnexal torsion and early surgical intervention to unwind detorsion on ovarian reserve: an experimental study. Reprod Sci 2013; 20: 1349-55. 17. Porst H, Padma-Nathan H, Giuliano F, Anglin G, Varanese L, Rosen R. Efficacy of tadalafil for the treatment of erectile dysfunction at 24 and 36 hours after dosing: a randomized controlled trial. Urology 2003; 62: 121-5.
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Monthly Pan Evaporation Modelling using Hybrid machine learning algorithms in a semi-arid environment environment Mustafa Al-Mukhtar  (  mmalmukhtar@gmail.com ) University of Technology https://orcid.org/0000-0002-8850-0899 Ahmed Elbeltagi  Mansoura University Faculty of Agriculture N. L. Kushwaha  Indian Agricultural Research Institute Dinesh Kumar Vishwakarma  G B Pant University of Agriculture and Technology: Govind Ballabh Pant University of Agriculture & Technology Research Article Keywords: Additive regression, Pan Evaporation, machine learning, REPTree Posted Date: July 8th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1832078/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License environment Mustafa Al-Mukhtar  (  mmalmukhtar@gmail.com ) University of Technology https://orcid.org/0000-0002-8850-0899 Ahmed Elbeltagi  Mansoura University Faculty of Agriculture N. L. Kushwaha  Indian Agricultural Research Institute Dinesh Kumar Vishwakarma  G B Pant University of Agriculture and Technology: Govind Ballabh Pant University of Agriculture & Technology Research Article Keywords: Additive regression, Pan Evaporation, machine learning, REPTree Posted Date: July 8th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1832078/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li Monthly Pan Evaporation Modelling using Hybrid machine learning 1 algorithms in a semi-arid environment 2 3 4 Ahmed Elbeltagi1, Mustafa Al-Mukhtar2,*, N. L. Kushwaha3, Dinesh Kumar 5 Vishwakarma4 6 7 1Agricultural Engineering Dept., Faculty of Agriculture, Mansoura University, Mansoura; 35516, 8 Egypt; ahmedelbeltagy81@mans.edu.eg 9 10 2 Civil Engineering Department. University of Technology -Iraq; mmalmukhtar@gmail.com 11 12 3 Division of Agricultural Engineering, ICAR-Indian Agricultural Research Institute, New Delhi- 13 110012, India, nand.kushwaha@icar.gov.in 14 15 4 Department of Irrigation and Drainage Engineering, G.B. Pant University of Agriculture and 16 Technology, Pantnagar, Uttarakhand 263145, India, dinesh.vishwakarma4820@gmail.com 17 18 19 20 Abstract 21 Evaporation is one of the most critical hydrologic processes that have direct impacts on 22 agricultural efficiency, the planning and operation of water resources projects. Hence, an 23 accurate estimation of evaporation is essential for proper management of water resources. 24 However, the non-linearity, non-stationary, and stochastic features of the evaporation process 25 constitute the major problem in developing reliable and robust predictive models. As such, 26 machine learning (ML) algorithms has been identified as efficient alternative methods to 27 overcome such as complexity. The purpose of this study was to explore the applicability of 28 coupling the additive regression model (AR) with four different machine-learning algorithms- 29 Random Subspace (RSS), M5 Pruned (M5P), Reduced Error Pruning Tree (REPTree), and 30 Bagging for predicting pan evaporation rates. The evaluated methods were applied using data 31 from three different agroclimatic stations in Iraq i.e. Baghdad, Mosul, and Basrah. The best-input 32 combinations were determined using the regression model. Results revealed that the best 33 Monthly Pan Evaporation Modelling using Hybrid machine learning 1 algorithms in a semi-arid environment 2 The statistical indices MAE, RMSE, RAE, RRSE, and r from AR-M5P at 37 Baghdad- Mosul during testing were 33.82, 45.05, 24.75, 28.50, and 0.972- 25.82, 35.95, 23.75, 38 29.64, and 0.956, respectively. The findings of this study demonstrated the superior performance 39 of the applied methods which can be employed to address other environmental problems. 40 temperatures. All the ML models performed well in predicting evaporation at the investigated 35 locations. However, the AR-M5P was found to be the best performance among the other 36 evaluated methods. The statistical indices MAE, RMSE, RAE, RRSE, and r from AR-M5P at 37 Baghdad- Mosul during testing were 33.82, 45.05, 24.75, 28.50, and 0.972- 25.82, 35.95, 23.75, 38 29.64, and 0.956, respectively. The findings of this study demonstrated the superior performance 39 of the applied methods which can be employed to address other environmental problems. 40 41 Keywords: Additive regression; Pan Evaporation; machine learning; REPTree 42 43 44 1. Introduction 45 Evaporation is defined as a physical process where the water molecules escaped from the 46 surface once enough energy is absorbed that overcomes the vapour pressure (Malik et al., 2021). 47 Evaporation hence constitutes the majority of losses that might be occurred in any water system 48 which could be thence exacerbated water scarcity. This is especially true in arid to semi-arid 49 areas where the molecules have enough heat energy to escape. Therefore, an accurate estimation 50 of the evaporation losses play a pivotal role in better water resources management, crop water 51 demands and irrigation scheduling (Fan et al., 2016; Gong et al., 2021; Kushwaha et al., 2021). 52 41 Keywords: Additive regression; Pan Evaporation; machine learning; REPTree 42 43 Evaporation is defined as a physical process where the water molecules escaped from the 46 surface once enough energy is absorbed that overcomes the vapour pressure (Malik et al., 2021). 47 Evaporation hence constitutes the majority of losses that might be occurred in any water system 48 which could be thence exacerbated water scarcity. This is especially true in arid to semi-arid 49 areas where the molecules have enough heat energy to escape. Therefore, an accurate estimation 50 of the evaporation losses play a pivotal role in better water resources management, crop water 51 demands and irrigation scheduling (Fan et al., 2016; Gong et al., 2021; Kushwaha et al., 2021). Monthly Pan Evaporation Modelling using Hybrid machine learning 1 algorithms in a semi-arid environment 2 predictions are achieved by incorporating wind speed, relative humidity, the minimum, and mean 34 temperatures. All the ML models performed well in predicting evaporation at the investigated 35 locations. However, the AR-M5P was found to be the best performance among the other 36 evaluated methods. The statistical indices MAE, RMSE, RAE, RRSE, and r from AR-M5P at 37 Baghdad- Mosul during testing were 33.82, 45.05, 24.75, 28.50, and 0.972- 25.82, 35.95, 23.75, 38 29.64, and 0.956, respectively. The findings of this study demonstrated the superior performance 39 of the applied methods which can be employed to address other environmental problems. 40 41 Keywords: Additive regression; Pan Evaporation; machine learning; REPTree 42 43 44 1. Introduction 45 Evaporation is defined as a physical process where the water molecules escaped from the 46 surface once enough energy is absorbed that overcomes the vapour pressure (Malik et al., 2021). 47 Evaporation hence constitutes the majority of losses that might be occurred in any water system 48 which could be thence exacerbated water scarcity. This is especially true in arid to semi-arid 49 areas where the molecules have enough heat energy to escape. Therefore, an accurate estimation 50 of the evaporation losses play a pivotal role in better water resources management, crop water 51 demands and irrigation scheduling (Fan et al., 2016; Gong et al., 2021; Kushwaha et al., 2021). 52 In arid and semi-arid regions, high rates of evaporation are typically be witnessed in the 53 summer season. Thence, water losses into the atmosphere from reservoirs, river basins, and 54 natural lakes might be exacerbated leading to deterioration of water levels (Boers et al., 1986; 55 Sayl et al., 2016, Khan et al., 2019). Therefore, an accurate quantification of water losses from 56 water bodies is crucially important to be considered for proper planning and managing of any 57 water resources project (Moazenzadeh et al., 2018; Vishwakarma et al., 2022). Recently, the 58 impact of climate changes has exacerbated the influence of evaporation on surface water balance 59 predictions are achieved by incorporating wind speed, relative humidity, the minimum, and mean 34 temperatures. All the ML models performed well in predicting evaporation at the investigated 35 locations. However, the AR-M5P was found to be the best performance among the other 36 evaluated methods. Monthly Pan Evaporation Modelling using Hybrid machine learning 1 algorithms in a semi-arid environment 2 52 In arid and semi-arid regions, high rates of evaporation are typically be witnessed in the 53 summer season. Thence, water losses into the atmosphere from reservoirs, river basins, and 54 natural lakes might be exacerbated leading to deterioration of water levels (Boers et al., 1986; 55 Sayl et al., 2016, Khan et al., 2019). Therefore, an accurate quantification of water losses from 56 water bodies is crucially important to be considered for proper planning and managing of any 57 water resources project (Moazenzadeh et al., 2018; Vishwakarma et al., 2022). Recently, the 58 impact of climate changes has exacerbated the influence of evaporation on surface water balance 59 (Sartori, 2000), where the global warming has negative influence on the relationship between 60 evaporation and water management (Eames et al., 1997; Kushwaha et al., 2022). 61 (Sartori, 2000), where the global warming has negative influence on the relationship between 60 evaporation and water management (Eames et al., 1997; Kushwaha et al., 2022). 61 Evaporation can be estimated in two ways; either directly such as the pan evaporation 62 (PE) method or indirectly like mass transfer, water and energy balance, and Penman methods 63 (Lundberg, 1993; Zhao et al., 2013). However, the Class A pan method is globally used for 64 estimating evaporation as it is well adapted to relatively estimate the evaporation levels in 65 different climatic characteristics regions (Masoner et al., 2008). Accordingly, the evaporation 66 amount of 60 cm or 20 cm diameter pans can be converted into that amount of Class A pan. 67 Nonetheless, the costly nature of the class A method represents the essential obstacle of its 68 application in many developing countries (Ashrafzadeh et al., 2019; Wu et al., 2020). In order to 69 be reliably predictive, the evaporation losses models should be accounting for the driving 70 meteorological variables such as relative humidity (RH), sunshine hours (Sh), wind speed (WS), 71 rainfall (RF), minimum (Tmin), maximum (Tmax), and mean (Tmean) temperatures. As such, 72 many of empirical models have been configured for predicting evaporation rates from 73 metrological variables such as Penman-Monteith; Priestley-Taylor; and Thornthwaite equations. Monthly Pan Evaporation Modelling using Hybrid machine learning 1 algorithms in a semi-arid environment 2 (2020), the performance of hybridized extreme learning machine (ELM) model with 96 particle swarm optimization (PSO) was explored to estimate the daily ETo in the arid region of 97 Northwest China. The comparison of the obtained results with those counterparts from the 98 original ELM, artificial neural networks (ANN) and random forests (RF) models as along with 99 six empirical models indicated a superior performance of ELM-PSO models for estimating ETo 100 more accurately than others. A comparative study on the performance of ElasticNet Linear 101 Regression, Extreme Gradient Boosting, Long Short‑Term Memory; in addition to two empirical 102 techniques i.e. Stephens‑Stewart and Thornthwaite was carried out by Abed et al. (2021) in two 103 weather stations in Malaysia. They found that the ML models outperformed the empirical models 104 with the same input configurations. The Bat algorithm (Bat) was coupled with Gradient boosting 105 between predictors and predictands. In the literature, many successful applications of machine 83 learning methods were reported in various topics of hydrology and climatology. For instance, 84 rainfall (Salih et al., 2020; Adnan et al., 2021), streamflow (Parisouj et al., 2020; Feng and Tian, 85 2020), drought (Malik et al., 2020a; Parisouj et al., 2020), surface water quality (Rezaie-Balf et 86 al., 2020; Chen et al., 2020), groundwater (Mosavi et al., 2021; Rahman et al., 2020) and many 87 others (Al-Mukhtar, 2019; Ghaemi et al., 2019; Keshtegar et al., 2019; Majhi et al., 2020; Yang 88 et al., 2020). As a case in point, the applicability of deep neural network architecture with long 89 short-term memory (Deep-LSTM) cells to estimate daily pan evaporation with minimum input 90 features was investigated in a study by Majhi et al. (2020). They proposed to that end number of 91 input combinations to predict the daily evaporation losses in three areas of Chhattisgarh state in 92 India. The investigation suggested that the proposed Deep-LSTM model was able to successfully 93 model the daily evaporation losses with improved accuracy as compared to multilayer artificial 94 neural network and empirical methods (Hargreaves and Blaney–Criddle). In another study by 95 Zhu et al. (2020), the performance of hybridized extreme learning machine (ELM) model with 96 particle swarm optimization (PSO) was explored to estimate the daily ETo in the arid region of 97 Northwest China. Monthly Pan Evaporation Modelling using Hybrid machine learning 1 algorithms in a semi-arid environment 2 74 However, the stochasticity features in addition to the non-linearity and non-stationary of the 75 meteorological variables employed in building a predictive model necessitate developing 76 rigorous and reliable intelligent models that could be capable to eliminate the stochasticity 77 inherited in the evaporation-meteorological variables relationship (Elbeltagi et al., 2022; Kisi et 78 al., 2017a; Salih et al., 2020; Khan et al., 2018; Naganna et al., 2019). 79 Owing to its capacity in tackling the complexity accompanied by highly stochastic 80 Owing to its capacity in tackling the complexity accompanied by highly stochastic 80 features of many environmental problems (Chia et al., 2020), machine learning (ML) methods 81 have been recently identified as paramount method to address various aspects of the association 82 Owing to its capacity in tackling the complexity accompanied by highly stochastic 80 features of many environmental problems (Chia et al., 2020), machine learning (ML) methods 81 have been recently identified as paramount method to address various aspects of the association 82 between predictors and predictands. In the literature, many successful applications of machine 83 learning methods were reported in various topics of hydrology and climatology. For instance, 84 rainfall (Salih et al., 2020; Adnan et al., 2021), streamflow (Parisouj et al., 2020; Feng and Tian, 85 2020), drought (Malik et al., 2020a; Parisouj et al., 2020), surface water quality (Rezaie-Balf et 86 al., 2020; Chen et al., 2020), groundwater (Mosavi et al., 2021; Rahman et al., 2020) and many 87 others (Al-Mukhtar, 2019; Ghaemi et al., 2019; Keshtegar et al., 2019; Majhi et al., 2020; Yang 88 et al., 2020). As a case in point, the applicability of deep neural network architecture with long 89 short-term memory (Deep-LSTM) cells to estimate daily pan evaporation with minimum input 90 features was investigated in a study by Majhi et al. (2020). They proposed to that end number of 91 input combinations to predict the daily evaporation losses in three areas of Chhattisgarh state in 92 India. The investigation suggested that the proposed Deep-LSTM model was able to successfully 93 model the daily evaporation losses with improved accuracy as compared to multilayer artificial 94 neural network and empirical methods (Hargreaves and Blaney–Criddle). In another study by 95 Zhu et al. Monthly Pan Evaporation Modelling using Hybrid machine learning 1 algorithms in a semi-arid environment 2 The comparison of the obtained results with those counterparts from the 98 original ELM, artificial neural networks (ANN) and random forests (RF) models as along with 99 six empirical models indicated a superior performance of ELM-PSO models for estimating ETo 100 more accurately than others. A comparative study on the performance of ElasticNet Linear 101 Regression, Extreme Gradient Boosting, Long Short‑Term Memory; in addition to two empirical 102 techniques i.e. Stephens‑Stewart and Thornthwaite was carried out by Abed et al. (2021) in two 103 weather stations in Malaysia. They found that the ML models outperformed the empirical models 104 with the same input configurations. The Bat algorithm (Bat) was coupled with Gradient boosting 105 in a study by Dong et al. (2021). Its performance was compared with random forest (RF) and 106 original CatBoost (CB) for forecasting daily pan evaporation in arid and semi-arid regions of 107 northwest China. They pointed out that the hybrid model outperformed the other models and 108 presented comprehensive performance results (seasonally and spatially) compared to CatBoost 109 and random forest. Emadi et al. (2021) evaluated the applications of wavelet-hybrids artificial 110 neural networks (WANN), adaptive neuro-fuzzy inference system (WANFIS), and gene 111 expression programming (WGEP) to estimate monthly evaporation in a study area in the 112 Northwest and central part of Iran. They compared their results with those standalone models. It 113 was revealed that the WGEP method has superiority in terms of performance and accuracy in 114 comparison to the others and single models. 115 in a study by Dong et al. (2021). Its performance was compared with random forest (RF) and 106 original CatBoost (CB) for forecasting daily pan evaporation in arid and semi-arid regions of 107 northwest China. They pointed out that the hybrid model outperformed the other models and 108 presented comprehensive performance results (seasonally and spatially) compared to CatBoost 109 and random forest. Emadi et al. (2021) evaluated the applications of wavelet-hybrids artificial 110 neural networks (WANN), adaptive neuro-fuzzy inference system (WANFIS), and gene 111 expression programming (WGEP) to estimate monthly evaporation in a study area in the 112 Northwest and central part of Iran. They compared their results with those standalone models. It 113 was revealed that the WGEP method has superiority in terms of performance and accuracy in 114 comparison to the others and single models. Monthly Pan Evaporation Modelling using Hybrid machine learning 1 algorithms in a semi-arid environment 2 115 From the above-mentioned literature, it is demonstrable the superiority of the ML 116 performances in comparison to other methods. However, the hybrid technique, where two or 117 more models are combined and coupled (Chia et al., 2020), has recently drew more attention in 118 climate and hydrology studies because of its capacity to capture the various patterns in data 119 series by combining multi-technique features in one algorithm (Ghaemi et al., 2019). However, 120 the generalization of the capability of these models over various climatic zones is arguable owing 121 to the fact that each climatic regions is associated with certain characteristics of stochasticity and 122 non-stationarity (Al-Mukhtar 2019). Therefore, it is essential to investigate newly developed 123 models and explore their applicability for specific climatic features. As such, the main purpose of 124 this study was to present new hybrid approaches of bagging and random subspace-based 125 reduced-error pruning tree (REPTree) classifiers for modeling pan evaporation rates. Using these 126 methods in water-related subjects have been rarely reported in the literature. Therefore, this 127 study represents a novel framework to increase the prediction accuracy of applyin 128 learning in solving such complex physical relationships. 129 2. Materials and methods 130 2.1 Study area and data 131 The study area encompasses three different areas in Iraq which are Baghdad, Basrah, a 132 The latitude- longitude- altitude of the above stations are 33°20′26″N- 44°24′03″E- 133 30°31′58″N- 47°47′50″E- 6 m a.s.l, and 36°20′06″N- 43°07′08″E- 228 m a.s.l, re 134 These areas are situated in the middle, south, and north of Iraq, respectively ( 135 Accordingly, the meteorological parameters which include T max., T min., T mean, W 136 to predict the monthly pan evaporation were collected for the study areas. The coll 137 spans over the period of 1990–2013 for Baghdad and Mosul stations, and 1990–2012 f 138 on a monthly time scale. The collected data were subdivided into two datasets i.e 139 training and 25% for testing. The detailed statistical characteristics of the climatic 140 used in modeling configurations are listed in Table 1. As it can be noticed, th 141 parameters ranges from Tmin = 0.7–32.9 °C, Tmax=12.9-47.70 °C, RH%=20-82%, W 142 m/s, and PE=48.70-624.80 mm at Baghdad. Where, Tmin = 4.7–34.60 °C, Tmax=1 143 °C, RH%=17-80%, WS= 1.7-7.7 m/s, and PE=41.40-645.90 mm at Basrah. Monthly Pan Evaporation Modelling using Hybrid machine learning 1 algorithms in a semi-arid environment 2 Lastly, Tm 144 27.40 °C, Tmax=8.30-46.40 °C, RH%=19-88%, WS= 0.2-3.7 m/s, and PE=21.50-464 145 study represents a novel framework to increase the prediction accuracy of applying machine 128 learning in solving such complex physical relationships. 129 study represents a novel framework to increase the prediction accuracy of applying machine 128 learning in solving such complex physical relationships. 129 2. Materials and methods 130 The study area encompasses three different areas in Iraq which are Baghdad, Basrah, and Mosul. 132 The latitude- longitude- altitude of the above stations are 33°20′26″N- 44°24′03″E- 41 m a.s.l, 133 30°31′58″N- 47°47′50″E- 6 m a.s.l, and 36°20′06″N- 43°07′08″E- 228 m a.s.l, respectively. 134 These areas are situated in the middle, south, and north of Iraq, respectively (Figure 1). 135 Accordingly, the meteorological parameters which include T max., T min., T mean, WS, and RH 136 to predict the monthly pan evaporation were collected for the study areas. The collected data 137 spans over the period of 1990–2013 for Baghdad and Mosul stations, and 1990–2012 for Basrah, 138 on a monthly time scale. The collected data were subdivided into two datasets i.e. 75% for 139 training and 25% for testing. The detailed statistical characteristics of the climatic parameters 140 used in modeling configurations are listed in Table 1. As it can be noticed, the climatic 141 parameters ranges from Tmin = 0.7–32.9 °C, Tmax=12.9-47.70 °C, RH%=20-82%, WS= 1.4-2.5 142 m/s, and PE=48.70-624.80 mm at Baghdad. Where, Tmin = 4.7–34.60 °C, Tmax=14.60-48.90 143 °C, RH%=17-80%, WS= 1.7-7.7 m/s, and PE=41.40-645.90 mm at Basrah. Lastly, Tmin = -2.2– 144 27.40 °C, Tmax=8.30-46.40 °C, RH%=19-88%, WS= 0.2-3.7 m/s, and PE=21.50-464.10 mm at 145 Mosul station. 146 Five evolutionary ML methods were evaluated in this study for forecasting PE, which are 148 Additive Regression (AR), Additive Regression- Random Subspace (AR-RSS), AR-Bagging, 149 Additive Regression- Reduced Error Pruning Tree (AR-REPTree), and AR-M5 Pruned models. 150 A brief description on the individual methods are given in the followings. 151 2.2.1 Additive regression AR 152 AR is a non-parametric regression method suggested by Friedman and Stuetzle, (1981). 153 contrast to the ordinary regression, AR uses one smoother function to describe the relationsh 154 between predictors and predictands. As such, it can overcome the issue of curse dimensional 155 inherited in other p-dimensional smoother. The additive model takes the following form: 156 𝐸𝐸ൣ𝑦𝑦𝑖𝑖|𝑥𝑥𝑖𝑖1, … , 𝑥𝑥𝑖𝑖𝑖𝑖൧= 𝛽𝛽𝜊𝜊+ ∑ 𝑓𝑓𝑖𝑖 𝑖𝑖 𝑗𝑗=1 (𝑥𝑥𝑖𝑖𝑗𝑗) 1 157 158 Where ∑ 𝑓𝑓𝑖𝑖 𝑖𝑖 𝑗𝑗=1 (𝑥𝑥𝑖𝑖𝑗𝑗) are the smooth functions fitted from the data, 𝛽𝛽𝜊𝜊 is the regressi 159 coefficient. 160 2.2.2 Random Subspace (RSS) 161 2.2.1 Additive regression AR 152 AR is a non-parametric regression method suggested by Friedman and Stuetzle, (1981). 2. Materials and methods 130 176 One of the attractive features of RSS is being sensibly adapted for high-dimensional 174 problems where the number of features is much larger than the number of training points 175 (Arabameri et al., 2021). 176 2.2.3 M5 Pruned (M5P) 177 M5 model tree method (Quinlan, 1992) is a tree based model used in solving predictor- 178 predictand regression problems of numerical values (Kisi et al., 2017b). In this method, two main 179 steps are adopted in building the M5 model i.e. data splitting into subsets at each node; and 180 constructing a multivariate regression for each node (Malik et al., 2020a). In the first step, the 181 data is split by computing the standard deviation reduction (SDR) at each leaf (Eq. 3). So that the 182 standard deviation of the classes values reaching to a node is treated as an error index at that 183 node. Subsequently, the expected reduction of that error is calculated by testing each attribute at 184 that node. Thence, the subsets data are selected based on maximizing the expected error 185 reduction (Al-Mukhtar, 2021a). 186 𝑠𝑠𝑇 |𝑇𝑇𝑖|𝑇𝑖𝑠𝑠𝑇𝑖 174 2.2.3 M5 Pruned (M5P) 177 M5 model tree method (Quinlan, 1992) is a tree based model used in solving predictor- 178 predictand regression problems of numerical values (Kisi et al., 2017b). In this method, two main 179 steps are adopted in building the M5 model i.e. data splitting into subsets at each node; and 180 constructing a multivariate regression for each node (Malik et al., 2020a). In the first step, the 181 data is split by computing the standard deviation reduction (SDR) at each leaf (Eq. 3). So that the 182 standard deviation of the classes values reaching to a node is treated as an error index at that 183 node. Subsequently, the expected reduction of that error is calculated by testing each attribute at 184 that node. Thence, the subsets data are selected based on maximizing the expected error 185 reduction (Al-Mukhtar, 2021a). 2. Materials and methods 130 In 153 contrast to the ordinary regression, AR uses one smoother function to describe the relationship 154 between predictors and predictands. As such, it can overcome the issue of curse dimensionality 155 inherited in other p-dimensional smoother. The additive model takes the following form: 156 𝐸𝑦𝑖𝑥𝑖𝑥𝑖𝑖𝛽𝜊𝑓𝑖𝑖𝑗𝑥𝑖𝑗 𝐸𝐸ൣ𝑦𝑦𝑖𝑖|𝑥𝑥𝑖𝑖1, … , 𝑥𝑥𝑖𝑖𝑖𝑖൧= 𝛽𝛽𝜊𝜊+ ∑ 𝑓𝑓𝑖𝑖 𝑖𝑖 𝑗𝑗=1 (𝑥𝑥𝑖𝑖𝑗𝑗) 1 157 158𝑓𝑖𝑖𝑗𝑥𝑖𝑗𝛽𝜊 𝐸𝐸ൣ𝑦𝑦𝑖𝑖|𝑥𝑥𝑖𝑖1, … , 𝑥𝑥𝑖𝑖𝑖𝑖൧= 𝛽𝛽𝜊𝜊+ ∑ 𝑓𝑓𝑖𝑖 𝑖𝑖 𝑗𝑗=1 (𝑥𝑥𝑖𝑖𝑗𝑗) 1 157 158𝑓𝑖𝑖𝑗𝑥𝑖𝑗𝛽𝜊 158 Where ∑ 𝑓𝑓𝑖𝑖 𝑖𝑖 𝑗𝑗=1 (𝑥𝑥𝑖𝑖𝑗𝑗) are the smooth functions fitted from the data, 𝛽𝛽𝜊𝜊 is the regression 159 coefficient. 160 Where ∑ 𝑓𝑓𝑖𝑖 𝑖𝑖 𝑗𝑗=1 (𝑥𝑥𝑖𝑖𝑗𝑗) are the smooth functions fitted from the data, 𝛽𝛽𝜊𝜊 is the regression 159 coefficient. 160 2.2.2 Random Subspace (RSS) 161 RSS is one of the machine learning ensemble methods which is used for decision trees. In 162 contrast to other trees-based decision methods, the method uses a systematic construction of 163 decision trees in which each tree is being independently generated based on parallel learning 164 algorithm (Ho, 1998). Additionally, its structure as an ensemble learning method works on 165 reducing the correlation between learners by randomly sampling subset of the training points and 166 features instead of the entire both dataset. Thence, the learners produce different models that can 167 be reliably averaged. The generated trees are randomly clustered into subspaces via a majority 168 voting method. So that, the majority of voting is used in the subspace ensemble system for the 169 sample of X, and is calculated using the following equation: 170 𝛽𝑥𝑎𝑎𝑎𝑎𝑎𝑥𝑦𝑦𝛿𝑠𝑠𝑠𝐶𝑏𝑥𝑦𝑏 2 2 𝛽𝛽(𝑥𝑥) = 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑥𝑥𝑦𝑦𝑦𝑦(−1,1) ∑𝛿𝛿𝑠𝑠𝑠𝑠𝑠𝑠ቀ𝐶𝐶𝑏𝑏(𝑥𝑥)ቁ,𝑦𝑦 𝑏𝑏 2 171 𝛿 Where 𝛿𝛿 is the Kronecker symbol, and y ∈ {-1, 1} is a decision (class label) of the classifier, 172 𝐶𝑏𝑥 𝐶𝐶𝑏𝑏(𝑥𝑥) is the classifier of each subspace b (Skurichina and Duin, 2002). 173 One of the attractive features of RSS is being sensibly adapted for high-dimensional 174 problems where the number of features is much larger than the number of training points 175 (Arabameri et al., 2021). 176 One of the attractive features of RSS is being sensibly adapted for high-dimensional 174 problems where the number of features is much larger than the number of training points 175 (Arabameri et al., 2021). 2. Materials and methods 130 The algorithm is based on the principle of information gain and reducing th 209 variance error (Chen et al., 2019). The data is split based on the information gain reach at eac 210 node and then the subtrees are pruned by the reduced error. The algorithm starts from every non 211 leaf subtree in the test data where the change in misclassification is examined which arises whe 212 the subtree is replaced by the most common classified class at the node (Quinlan, 1987). Th 213 subtree would be replaced by a leaf when the new induced tree has equal or less error tha 214 before. The process is repeated over until any further replacements increases the variance erro 215 of the test data. 216 2.2.5 Bagging 217 Bagging or bootstrap aggregating (Breiman, 1996) is the most common ensemble learnin 218 prediction accuracy (Arabameri et al., 2021). The non-leaf nodes starting from the tree bottom 197 are examined so that the subtree at a node is pruned depending on the least estimated error. If the 198 linear model is opted, the subtree at this node is pruned as a leaf. The prediction accuracy is 199 ultimately enhanced by the smoothing process where the predicted value at the leaf is adjusted 200 by the equation below: 201 𝑃𝑃𝑆𝑠𝑖𝑃𝑃𝑆𝑖𝑘𝑀𝑆 4 4 Where 𝑃𝑃𝑃𝑃(𝑆𝑆𝑖𝑖) is the predicted value at 𝑆𝑆𝑖𝑖, 𝑛𝑛𝑖𝑖number of training cases, 𝑆𝑆𝑖𝑖 is the case follow a 203 branch of subtree 𝑆𝑆, 𝑘𝑘 is the smoothing factor, 𝑀𝑀(𝑆𝑆) is the value given by the model at 𝑆𝑆, 204 2.2.4 Reduced Error Pruning Tree (REPTree) 205 REPTree is one of the pruning algorithm used in machine learning that characterize as simple 206 and computationally speed (Quinlan, 1987). It has been commonly used as a baseline method for 207 the purpose of comparison with other pruning methods (Mohamed et al., 2012; Ganatra and 208 Bhensdadia, 2012). The algorithm is based on the principle of information gain and reducing the 209 variance error (Chen et al., 2019). The data is split based on the information gain reach at each 210 node and then the subtrees are pruned by the reduced error. The algorithm starts from every non- 211 leaf subtree in the test data where the change in misclassification is examined which arises when 212 the subtree is replaced by the most common classified class at the node (Quinlan, 1987). 2. Materials and methods 130 186 𝑠𝑠𝑇𝑇𝑖𝑖𝑠𝑠𝑇𝑖 SDR = 𝑠𝑠𝑠𝑠(𝑇𝑇) −∑ |𝑇𝑇𝑖𝑖| |𝑇𝑇| 𝑖𝑖 × 𝑠𝑠𝑠𝑠(𝑇𝑇𝑖𝑖) 3 187 𝑠𝑠𝑇𝑖 SDR = 𝑠𝑠𝑠𝑠(𝑇𝑇) −∑ |𝑇𝑇𝑖𝑖| |𝑇𝑇| 𝑖𝑖 × 𝑠𝑠𝑠𝑠(𝑇𝑇𝑖𝑖) 3 187 𝑠𝑠𝑇𝑖 SDR = 𝑠𝑠𝑠𝑠(𝑇𝑇) −∑ |𝑇𝑇𝑖𝑖| |𝑇𝑇| 𝑖𝑖 × 𝑠𝑠𝑠𝑠(𝑇𝑇𝑖𝑖) 3 187 𝑠𝑠𝑇𝑖 3 𝑇 Where T is a set of cases in the data that reach a node, 𝑠𝑠𝑠𝑠 is the standard deviation, 𝑇𝑇𝑖𝑖 is the 188 subset of cases that have the ith outcomes of the potential test. 189 Where T is a set of cases in the data that reach a node, 𝑠𝑠𝑠𝑠 is the standard deviation, 𝑇𝑇𝑖𝑖 is the 188 subset of cases that have the ith outcomes of the potential test. 189 In the second step, a multivariate linear regression model is constructed for each node at 190 the model subtree. The M5 model compare the accuracy of the constructed linear model with the 191 subtree at this node to ensure the same level of information. The decision on selection the 192 optimal model is concluded based on the lower estimated error from either the obtained linear 193 model or the model subtree. To minimize the standard error, the linear models are simplified by 194 eliminating parameters using a greedy search method to remove variables which have little 195 contribution to the model. Finally, the pruning and smoothing processes are set up to improve the 196 prediction accuracy (Arabameri et al., 2021). The non-leaf nodes starting from the tree bottom 197 are examined so that the subtree at a node is pruned depending on the least estimated error. If th 198 linear model is opted, the subtree at this node is pruned as a leaf. The prediction accuracy 199 ultimately enhanced by the smoothing process where the predicted value at the leaf is adjuste 200 by the equation below: 201 𝑃𝑃𝑃𝑃(𝑆𝑆) = 𝑠𝑠𝑖𝑖×𝑃𝑃𝑃𝑃(𝑆𝑆𝑖𝑖)+𝑘𝑘×𝑀𝑀(𝑆𝑆) 𝑠𝑠𝑖𝑖+𝑘𝑘 4 202 Where 𝑃𝑃𝑃𝑃(𝑆𝑆𝑖𝑖) is the predicted value at 𝑆𝑆𝑖𝑖, 𝑛𝑛𝑖𝑖number of training cases, 𝑆𝑆𝑖𝑖 is the case follow 203 branch of subtree 𝑆𝑆, 𝑘𝑘 is the smoothing factor, 𝑀𝑀(𝑆𝑆) is the value given by the model at 𝑆𝑆, 204 2.2.4 Reduced Error Pruning Tree (REPTree) 205 REPTree is one of the pruning algorithm used in machine learning that characterize as simp 206 and computationally speed (Quinlan, 1987). It has been commonly used as a baseline method fo 207 the purpose of comparison with other pruning methods (Mohamed et al., 2012; Ganatra an 208 Bhensdadia, 2012). 2. Materials and methods 130 The 213 subtree would be replaced by a leaf when the new induced tree has equal or less error than 214 before. The process is repeated over until any further replacements increases the variance error 215 of the test data. 216 Bagging or bootstrap aggregating (Breiman, 1996) is the most common ensemble learning 218 method which is used to improve the accuracy performance of forecasting models (Li et al., 219 2020) by reducing the variation of a noisy data. In Bagging, multiple learning algorithms are 220 combined to obtain better predictive performance so that a stronger learners are constructed from 221 weak ones (Saha et al., 2016). In contrast to other ensemble learning, a bootstrap replication of 222 the original dataset is used to generate training sets and trains the base learners on these sub 223 datasets (Al-Mukhtar, 2021b). Then, the Bagging averages out the resulting models in regression 224 problems (Tyralis et al., 2019). 225 2020) by reducing the variation of a noisy data. In Bagging, multiple learning algorithms are 220 combined to obtain better predictive performance so that a stronger learners are constructed from 221 weak ones (Saha et al., 2016). In contrast to other ensemble learning, a bootstrap replication of 222 the original dataset is used to generate training sets and trains the base learners on these sub 223 datasets (Al-Mukhtar, 2021b). Then, the Bagging averages out the resulting models in regression 224 problems (Tyralis et al., 2019). 225 3. Results 226 The forecast results for the best data-driven models for each station (Baghdad and Mosul) are 227 shown in the sections below. The projections offered are based on validation data sets for time 228 series of evaporation, which is often used to characterize agricultural meteorological events such 229 as droughts and for irrigation system design. The applied models were set up using data from 230 three different climatic characteristics stations in Iraq. The parameters of the machine learning 231 algorithm used for modeling PE in these three regions were listed as shown in Table 2. 232 3.1 Input selection using best subset model 233 The forecast results for the best data-driven models for each station (Baghdad and Mosul) are 227 shown in the sections below. 2. Materials and methods 130 The projections offered are based on validation data sets for time 228 series of evaporation, which is often used to characterize agricultural meteorological events such 229 as droughts and for irrigation system design. The applied models were set up using data from 230 three different climatic characteristics stations in Iraq. The parameters of the machine learning 231 algorithm used for modeling PE in these three regions were listed as shown in Table 2. 232 3.1 Input selection using best subset model 233 The selection of the optimal input parameters is a critical step in modelling for the best 234 performance of the chosen models. For the optimal input combination, many combinations of 235 meteorological parameters were used. Tables 3 and 4 provide the statistical analysis of the five 236 combinations that were examined in this research. At two stations in Baghdad and Mosul, the 237 optimal input combination was chosen using the seven statistical criteria of MSE, R², Adjusted 238 R², Mallows' Cp, Akaike's AIC, Schwarz's SBC, Amemiya's PC, with the results presented in 239 Tables 3 and 4. The smallest and nearest to zero values of MSE, Mallows' Cp, Akaike's AIC, 240 Schwarz's SBC, Amemiya's PC and the highest and near to 1 values of R², Adjusted R² 241 considered as best input combination in subset linear regression analysis. 242 The bold blue row in Table 3 is observed as the optimum input combination because it 243 contains the lowest values of MSE of 1733.377, Mallows' Cp of 4.229, Akaike's AIC of 244 2151.826, Schwarz's SBC of 2166.478 and Amemiya's PC of 0.103, and the highest values of R2 245 (0.934) and Adj-R2 (0.934) of all input combinations at the Baghdad station. A similar result was 246 seen in Table 4, where the bold blue row was observed as the optimum input combination, with 247 the lowest values of MSE of 979.668, Mallows' Cp of 5.00, Akaike's AIC of 1988.474, 248 Schwarz's SBC of 2006.789 and Amemiya's PC of 0.061, and the highest values of R2 (0.940) 249 and Adj-R2 (0.939) among all the input combinations at the Mosul station. 250 3.2 Sensitivity analysis 251 The inputs combinations predominantly influence on the performance of the models. Some may 252 provide a positive contribution to the accuracy of the chosen model, while others might make a 253 negative contribution. 2. Materials and methods 130 The selection of the most important relevant factors was carried out using 254 sensitivity analysis to capture the optimal performance of daily PE models at two stations in Iraq. 255 Tables 5 and 6, as well as Figures 2 and 3 show the findings of the regression analysis that was 256 conducted. The results of the regression analysis on all input parameters revealed that Tmax, Tmin, 257 T, RH and W, with absolute standard error coefficients of 0.000, 0.000, 0.038, 0.039 and 0.018, 258 were identified as the most influential input parameters for estimation of evaporation at the 259 Baghdad, whereas, absolute standard error coefficients were 0.000, 0.172, 0.213, 0.061 and 260 0.015 at Mosul station, respectively. The standardized coefficients of the input variable for 261 sensitivity analysis are shown in Figures 2 and 3 for Baghdad and Mosul station, respectively. 262 3.3 Modelling of pan evaporation 263 In the present study, five evolutionary machine learning, Additive Regression, AR-RSS, AR- 264 Bagging AR-REPTree and AR-M5P models were applied for forecasting monthly evaporation 265 The bold blue row in Table 3 is observed as the optimum input combination because it 243 contains the lowest values of MSE of 1733.377, Mallows' Cp of 4.229, Akaike's AIC of 244 2151.826, Schwarz's SBC of 2166.478 and Amemiya's PC of 0.103, and the highest values of R2 245 (0.934) and Adj-R2 (0.934) of all input combinations at the Baghdad station. A similar result was 246 seen in Table 4, where the bold blue row was observed as the optimum input combination, with 247 the lowest values of MSE of 979.668, Mallows' Cp of 5.00, Akaike's AIC of 1988.474, 248 Schwarz's SBC of 2006.789 and Amemiya's PC of 0.061, and the highest values of R2 (0.940) 249 and Adj-R2 (0.939) among all the input combinations at the Mosul station. 250 The inputs combinations predominantly influence on the performance of the models. Some may 252 provide a positive contribution to the accuracy of the chosen model, while others might make a 253 negative contribution. The selection of the most important relevant factors was carried out using 254 sensitivity analysis to capture the optimal performance of daily PE models at two stations in Iraq. 255 Tables 5 and 6, as well as Figures 2 and 3 show the findings of the regression analysis that was 256 conducted. 2. Materials and methods 130 The values of MAE, RMSE, RAE, RRSE and r criteria during the training and 271 testing periods for AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P models are presented 272 in Table 7. As evaluated for Baghdad station from Table 7, the AR, AR-RSS, AR-Bagging, AR- 273 REPTree and AR-M5P models provided MAE = 30.34, 31.12, 31.33, 33.313 and 29.65; RMSE 274 = 41.19, 41.50, 40.14, 44.35 and 39.58; RAE = 21.18, 21.73, 21.87, 23.25 and 20.70; RRSE = 275 25.27, 25.47, 24.63, 27.21 and 24.29; r = 0.968, 0.967, 0.970, 0.962 and 0.970 during training 276 period. In addition, the AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P models provided 277 MAE = 35.51, 45.72, 37.74, 37.81 and 33.82; RMSE = 48.68, 59.18, 47.35, 50.13 and 45.05; 278 RAE = 25.98, 33.46, 27.62, 27.67 and 24.75; RRSE = 30.80, 37.44, 29.96, 31.72 and 28.50; r = 279 0.966, 0.949, 0.962, 0.958 and 0.972 during testing period, respectively. Table 7 showing the 280 AR-M5P model attained the most accurate simulation during the testing stage. Therefore, AR- 281 M5P model was the best performed model according to the statistical criteria (i.e., minimum 282 MAE, RMSE, RAE and RRSE values, and maximum r values) in testing stage followed by AR- 283 Bagging model closely. 284 The monthly PE was estimated using AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P 269 models based on MAE, RMSE, RAE, RRSE and r for both training and testing stages at 270 Baghdad station. The values of MAE, RMSE, RAE, RRSE and r criteria during the training and 271 testing periods for AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P models are presented 272 in Table 7. As evaluated for Baghdad station from Table 7, the AR, AR-RSS, AR-Bagging, AR- 273 REPTree and AR-M5P models provided MAE = 30.34, 31.12, 31.33, 33.313 and 29.65; RMSE 274 = 41.19, 41.50, 40.14, 44.35 and 39.58; RAE = 21.18, 21.73, 21.87, 23.25 and 20.70; RRSE = 275 25.27, 25.47, 24.63, 27.21 and 24.29; r = 0.968, 0.967, 0.970, 0.962 and 0.970 during training 276 period. In addition, the AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P models provided 277 MAE = 35.51, 45.72, 37.74, 37.81 and 33.82; RMSE = 48.68, 59.18, 47.35, 50.13 and 45.05; 278 RAE = 25.98, 33.46, 27.62, 27.67 and 24.75; RRSE = 30.80, 37.44, 29.96, 31.72 and 28.50; r = 279 0.966, 0.949, 0.962, 0.958 and 0.972 during testing period, respectively. 2. Materials and methods 130 The results of the regression analysis on all input parameters revealed that Tmax, Tmin, 257 T, RH and W, with absolute standard error coefficients of 0.000, 0.000, 0.038, 0.039 and 0.018, 258 were identified as the most influential input parameters for estimation of evaporation at the 259 Baghdad, whereas, absolute standard error coefficients were 0.000, 0.172, 0.213, 0.061 and 260 0.015 at Mosul station, respectively. The standardized coefficients of the input variable for 261 sensitivity analysis are shown in Figures 2 and 3 for Baghdad and Mosul station, respectively. 262 3.3 Modelling of pan evaporation 263 I h d fi l i hi l i Addi i R i AR RSS AR 264 The inputs combinations predominantly influence on the performance of the models. Some may 252 provide a positive contribution to the accuracy of the chosen model, while others might make a 253 negative contribution. The selection of the most important relevant factors was carried out using 254 sensitivity analysis to capture the optimal performance of daily PE models at two stations in Iraq. 255 Tables 5 and 6, as well as Figures 2 and 3 show the findings of the regression analysis that was 256 conducted. The results of the regression analysis on all input parameters revealed that Tmax, Tmin, 257 T, RH and W, with absolute standard error coefficients of 0.000, 0.000, 0.038, 0.039 and 0.018, 258 were identified as the most influential input parameters for estimation of evaporation at the 259 Baghdad, whereas, absolute standard error coefficients were 0.000, 0.172, 0.213, 0.061 and 260 0.015 at Mosul station, respectively. The standardized coefficients of the input variable for 261 sensitivity analysis are shown in Figures 2 and 3 for Baghdad and Mosul station, respectively. 262 3.3 Modelling of pan evaporation 263 In the present study, five evolutionary machine learning, Additive Regression, AR-RSS, AR- 264 Bagging, AR-REPTree and AR-M5P models, were applied for forecasting monthly evaporation 265 and results were compared with classic AR to see the accuracy improvement of the new 266 methods. For this purpose, the MAE, RMSE, RAE, RRSE and r measures were considered. 267 The monthly PE was estimated using AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P 269 models based on MAE, RMSE, RAE, RRSE and r for both training and testing stages at 270 Baghdad station. 2. Materials and methods 130 Table 7 showing the 280 AR-M5P model attained the most accurate simulation during the testing stage. Therefore, AR- 281 M5P model was the best performed model according to the statistical criteria (i.e., minimum 282 MAE, RMSE, RAE and RRSE values, and maximum r values) in testing stage followed by AR- 283 Bagging model closely. 284 25.27, 25.47, 24.63, 27.21 and 24.29; r = 0.968, 0.967, 0.970, 0.962 and 0.970 during training 276 period. In addition, the AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P models provided 277 MAE = 35.51, 45.72, 37.74, 37.81 and 33.82; RMSE = 48.68, 59.18, 47.35, 50.13 and 45.05; 278 RAE = 25.98, 33.46, 27.62, 27.67 and 24.75; RRSE = 30.80, 37.44, 29.96, 31.72 and 28.50; r = 279 0.966, 0.949, 0.962, 0.958 and 0.972 during testing period, respectively. Table 7 showing the 280 AR-M5P model attained the most accurate simulation during the testing stage. Therefore, AR- 281 M5P model was the best performed model according to the statistical criteria (i.e., minimum 282 MAE, RMSE, RAE and RRSE values, and maximum r values) in testing stage followed by AR- 283 Bagging model closely. 284 The temporal variation along with the scatter plots (right side) of the simulated versus 285 observed monthly evaporation data for the AR, AR-RSS, AR-Bagging, AR-REPTree and AR- 286 M5P models was plotted in Figure 4 (a through e) during the testing stage. In scatter plots, the 287 regression line provided the coefficient of determination (R2) as 0.934 for the Additive 288 Regression (AR) model, 0.902 for the AR-RSS model, 0.925 for AR-Bagging model, 0.918 for 289 AR-REPTree model, and 0.944 for AR-M5P model during the testing stage, respectively. The 290 regression line (RL) and the line of 1:1 were close to each other for all models. The RL was 291 above the best fit (1:1) for AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P models. This 292 means that at Baghdad station, the five models slightly overpredict the monthly PE values. Radar 293 charts display MAE and RSME of AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P 294 models during testing at Baghdad station is plotted in Figure 7. As can be raised from Figure 7, 295 the applied models were very close to each other. In other words, AR-REPTree was seen as the 296 furthest from the observed point which introduces the AR-REPTree model as the worst model. 2. Materials and methods 130 297 On the opposite side, AR-M5P was the closest model to the observed point based on the standard 298 deviation, correlation, and RMSE (Figure 9). This demonstrates the superiority of the AR-M5P 299 model in comparison to the others. 300 Regression (AR) model, 0.902 for the AR-RSS model, 0.925 for AR-Bagging model, 0.918 for 289 AR-REPTree model, and 0.944 for AR-M5P model during the testing stage, respectively. The 290 regression line (RL) and the line of 1:1 were close to each other for all models. The RL was 291 above the best fit (1:1) for AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P models. This 292 means that at Baghdad station, the five models slightly overpredict the monthly PE values. Radar 293 charts display MAE and RSME of AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P 294 models during testing at Baghdad station is plotted in Figure 7. As can be raised from Figure 7, 295 the applied models were very close to each other. In other words, AR-REPTree was seen as the 296 furthest from the observed point which introduces the AR-REPTree model as the worst model. 297 On the opposite side, AR-M5P was the closest model to the observed point based on the standard 298 deviation, correlation, and RMSE (Figure 9). This demonstrates the superiority of the AR-M5P 299 model in comparison to the others. 300 3.3.2 Training and testing the selected models at Mosul station 301 3.3.2 Training and testing the selected models at Mosul station 301 The monthly PE was estimated using AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P 302 models based on MAE, RMSE, RAE, RRSE and r for both training and testing stages at Mosul 303 station. The values of MAE, RMSE, RAE, RRSE and r criteria during the training and testing 304 periods for AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P models are given in Table 8. 305 As evaluated for Mosul station from Table 8, the AR, AR-RSS, AR-Bagging, AR-REPTree and 306 AR-M5P models provided MAE = 23.62, 21.97, 22.46, 26.66 and 22.75; RMSE = 33.96, 29.38, 307 29.97, 38.09 and 29.21; RAE = 20.42, 19.00, 19.42, 23.05 and 19.67; RRSE = 26.24, 22.71, 308 23.16, 29.43 and 22.57; r = 0.965, 0.974, 0.972, 0.957 and 0.974 during training period. In 309 addition, the AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P models provided MAE = 310 29.68, 26.93, 27.37, 27.01 and 25.82; RMSE = 42.34, 37.31, 37.94, 38.62 and 35.95; RAE = 311 27.30, 24.77, 25.17, 24.84 and 23.75; RRSE = 34.92, 30.77, 31.29, 31.85 and 29.64; r = 0.945, 312 0.959, 0.959, 0.962 and 0.956 during testing period, respectively. Table 8 proves that the AR- 313 M5P model outperformed the other models during the testing period according to the statistical 314 criteria (i.e., minimum MAE, RMSE, RAE and RRSE values, and maximum r values) in testing 315 stage followed by AR-RSS model. 316 The temporal variation along with the scatter plots (right side) of the simulated versus 317 observed monthly evaporation data for the AR, AR-RSS, AR-Bagging, AR-REPTree and AR- 318 M5P models was plotted in Figure 5 (a through e) during the testing stage. The coefficient of 319 determination (R2) was 0.894 for the AR model, 0.921 for the AR-RSS model, 0.921 for AR- 320 Bagging model, 0.926 for AR-REPTree model, and 0.915 for AR-M5P model during the testing 321 stage, respectively. It can be revealed that the RL and the line of fit (1:1) were close to each other 322 for all applied models. The RL was above the best fit (1:1) for AR, AR-RSS, AR-Bagging, AR- 323 REPTree and AR-M5P models which implies that at Mosul station, the five models slightly 324 overpredict the monthly PE values. Radar charts display MAE and RSME of AR, AR-RSS, AR- 325 Bagging, AR-REPTree and AR-M5P models during testing at Mosul station is plotted in Figure 326 8. 3.3.2 Training and testing the selected models at Mosul station 301 The use of all of these models in 353 various contexts may only be conceivable after they have been calibrated with fresh data. It was 354 discovered that all of the heuristic models significantly overestimated the pan evaporation 355 values, particularly for the Baghdad, Mosul, and Basrah stations, with the exception of one. One 356 possible explanation for this might be the disparity between the training, test, and validation data 357 The values of MAE, RMSE, RAE, RRSE and r criteria during the validation period AR-M5P 335 models are presented in Table 9. As evaluated for Basrah station from Table 9, the AR-M5P 336 models provided MAE, RMSE, RAE, RRSE and r = 47.23, 67.23, 31.19, 39.30 and 0.942, 337 respectively. 338 The values of MAE, RMSE, RAE, RRSE and r criteria during the validation period AR-M5P 335 models are presented in Table 9. As evaluated for Basrah station from Table 9, the AR-M5P 336 models provided MAE, RMSE, RAE, RRSE and r = 47.23, 67.23, 31.19, 39.30 and 0.942, 337 respectively. 338 The temporal variation along with the scatter plots (right side) of the simulated versus 339 observed monthly evaporation data for the AR, AR-RSS, AR-Bagging, AR-REPTree and AR- 340 M5P models was plotted in Figure 6 (a through e) during the testing stage. In scatter figure, the 341 coefficient of determination (R2) was 0.887. The fitted RL and the perfect line of fit (1:1) were 342 close to each other. The RL was above the best fit (1:1) for AR-M5P models. This means that at 343 Basrah station, the model slightly overpredict the monthly PE values. 344 According to the results of the subset regression analysis, the best input combination for the 346 Baghdad station was selected as T, RH, and W, and the best input combination for the Mosul 347 station was selected as Tmin, T, RH, and W, indicating that all of these variables have an effect 348 on pan evaporation. According to the relevant literature, all of these factors have a physical 349 impact on pan evaporation. This demonstrates that the subset regression analysis was performed 350 correctly. The heuristic models AR-M5P outperform the other algorithm models at both stations 351 when compared to the other algorithms. As a result, the AR-M5P model was employed for the 352 validation of the best candidate model at the Basrah station. 3.3.2 Training and testing the selected models at Mosul station 301 It is noticed that all models were very close to each other, however, AR was the furthest from 327 the observed point while AR-M5P was the closest. Suggesting that AR-REPTree was the worst 328 model and AR-M5P was the best based on the standard deviation, correlation, and RMSE 329 (Figure 10). 330 3.4 Validation best candidate model at Basrah station 331 The best selected model was utilized for validation to predict of monthly evaporation at Basrah 332 station. AR-M5P model was found best algorithm for both stations i.e., Baghdad and Mosul, 333 therefore, AR-M5P model were used for validation of best candidate model at Basrah station. 334 The values of MAE, RMSE, RAE, RRSE and r criteria during the validation period AR-M5P 335 models are presented in Table 9. As evaluated for Basrah station from Table 9, the AR-M5P 336 models provided MAE, RMSE, RAE, RRSE and r = 47.23, 67.23, 31.19, 39.30 and 0.942, 337 respectively. 338 The temporal variation along with the scatter plots (right side) of the simulated versus 339 observed monthly evaporation data for the AR, AR-RSS, AR-Bagging, AR-REPTree and AR- 340 M5P models was plotted in Figure 6 (a through e) during the testing stage. In scatter figure, the 341 coefficient of determination (R2) was 0.887. The fitted RL and the perfect line of fit (1:1) were 342 close to each other. The RL was above the best fit (1:1) for AR-M5P models. This means that at 343 Basrah station, the model slightly overpredict the monthly PE values. 344 4. Discussion 345 According to the results of the subset regression analysis, the best input combination for the 346 Baghdad station was selected as T, RH, and W, and the best input combination for the Mosul 347 station was selected as Tmin, T, RH, and W, indicating that all of these variables have an effect 348 on pan evaporation. According to the relevant literature, all of these factors have a physical 349 impact on pan evaporation. This demonstrates that the subset regression analysis was performed 350 correctly. The heuristic models AR-M5P outperform the other algorithm models at both stations 351 when compared to the other algorithms. As a result, the AR-M5P model was employed for the 352 validation of the best candidate model at the Basrah station. 3.3.2 Training and testing the selected models at Mosul station 301 The use of all of these models in 353 various contexts may only be conceivable after they have been calibrated with fresh data. It was 354 discovered that all of the heuristic models significantly overestimated the pan evaporation 355 values, particularly for the Baghdad, Mosul, and Basrah stations, with the exception of one. One 356 possible explanation for this might be the disparity between the training, test, and validation data 357 ranges at this station. As a result, extrapolating the results of the applicable models becomes 358 challenging. 359 ranges at this station. As a result, extrapolating the results of the applicable models becomes 358 challenging. 359 The results of this study were validated with other recent works ( Chen et al., 2019; 360 Kumar et al., 2021; Kushwaha et al., 2021; Lin et al., 2013; Malik et al., 2020b; Vishwakarma et 361 al., 2022) conducted in different continents of the world. Lin et al. (2013) investigated the 362 performance of two different ML techniques (i.e., SVM and backpropagation network) for 363 estimating daily evaporation values. They demonstrated the superiority of the applied support 364 vector machine to estimate the daily PE values and revealed that it can be used as promising 365 alternative for evaporation prediction. The predictability of five ML methods [i.e., multi-model 366 artificial neural network (MM-ANN), MARS, SVM, multi-gene genetic programming (MGGP), 367 and M5Tree] to predict the monthly PE in India was investigated by Malik et al. (2020a), who 368 made a similar commitment. They reported that the MM-ANN and MGGP algorithms were 369 superior in prediction performance when compared to the MARS and SVM algorithms, as well 370 as the M5Tree method as indicated by lowest RMSE. Kushwaha et al., (2021) evaluated four ML 371 algorithms (i.e., SVM, RT, REPTree, and RSS) under diverse climate conditions in Northern 372 India. They concluded that SVM outperformed over other applied algorithms as it has a high 373 value of correlation coefficient and Willmott index and low value of MAE and RMSE. Similarly, 374 Chen et al. (2019) evaluated the prediction of monthly PE from SVM at 6 different stations, 375 located in the Yangtze River in China. They proved that SVM was better than the traditional 376 methods for estimating PE. 3.3.2 Training and testing the selected models at Mosul station 301 In parallel to the above literature, the findings of this study 377 confirmed that the AR-M5P hybrid algorithm was more accurate than other applied algorithms in 378 terms of predicting the pan evaporation rates at the selected stations. 379 Overall, our findings indicate that hybrid models have a stronger predictive value in real- 380 world situations and maybe employed more effectively in watersheds with little data. In addition 381 to predicting pan evaporation, these types of models may be used to forecast a wide range of 382 hydrological and water resources phenomena, including ETo, suspended and bed sediment loads, 383 rainfall, and groundwater contamination. Especially in developing countries where technical 384 skills and understanding of the processes occurring in the watershed are lacking, these algorithms 385 could be used in data-poor watersheds or for measuring some phenomena that are time- 386 consuming or expensive, such as suspended or bed load in rivers, or nitrate and other heavy 387 metals measurement in groundwater. 388 Overall, our findings indicate that hybrid models have a stronger predictive value in real- 380 world situations and maybe employed more effectively in watersheds with little data. In addition 381 to predicting pan evaporation, these types of models may be used to forecast a wide range of 382 hydrological and water resources phenomena, including ETo, suspended and bed sediment loads, 383 rainfall, and groundwater contamination. Especially in developing countries where technical 384 skills and understanding of the processes occurring in the watershed are lacking, these algorithms 385 could be used in data-poor watersheds or for measuring some phenomena that are time- 386 consuming or expensive, such as suspended or bed load in rivers, or nitrate and other heavy 387 metals measurement in groundwater. 388 380 In this study, five machines learning evolutionary were applied for forecasting monthly 390 evaporation and results were compared with classic AR to see the accuracy improvement of the 391 new methods. The developed models encompass Additive Regression, AR-RSS, AR-Bagging, 392 AR-REPTree and AR-M5P models. Data from three different climatic characteristics regions in 393 Iraq were employed for the sake of models evaluations using several statistical metrics (MAE, 394 RMSE, RAE, RRSE and r). The best input combination was determined based on the regression 395 subset. 3.3.2 Training and testing the selected models at Mosul station 301 As such, the optimal input combinations for the Baghdad station was T, RH, and W, and 396 the best input combination for the Mosul station was Tmin, T, RH, and W, indicating that all of 397 these variables affect pan evaporation. It was concluded that the hybrid models have a stronger 398 predictive capability in real-world situations and maybe employed more effectively in 399 watersheds with little data. However, the AR-M5P was found to be the best performance among 400 the other evaluated methods as it show the least error indices values. 401 Acknowledgment 404 The author would like to express their gratitude to the anonymous reviewer of this manuscript 405 for their valuable comments. 406 407 Contributions 408 Ahmed Elbeltagi, Mustafa Al-Mukhtar: Conceptualization; Data curation; Formal analysis 409 Investigation; Methodology; Software; Supervision; Validation; Visualization; N. L. Kushwaha 410 Dinesh Kumar Vishwakarma: Roles/Writing - original draft; Writing - review & editing. 411 412 Conflict of interest: The authors declare no conflict of interest. 413 Funding: This research did not receive any specific grant from funding agencies in the public, 414 commercial, or not-for-profit sectors. 415 416 417 References 418 Abed, M., Imteaz, M.A., Ahmed, A.N., Huang, Y.F., 2021. Application of long short-term 419 memory neural network technique for predicting monthly pan evaporation. 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Methods to estimate evapotranspiration in humid and subtropical 606 climate conditions. Agricultural Water Management 261, 107378. 607 https://doi.org/10.1016/j.agwat.2021.107378 608 Wu, L., Huang, G., Fan, J., Ma, X., Zhou, H., Zeng, W., 2020. Hybrid extreme learning machine 609 with meta-heuristic algorithms for monthly pan evaporation prediction. Computers and 610 Electronics in Agriculture 168, 105115. https://doi.org/10.1016/j.compag.2019.105115 611 Yang, X., Zhou, J., Fang, W., Wang, Y., 2020. An ensemble flow forecast method based on 612 autoregressive model and hydrological uncertainty processer. Water (Switzerland) 12, 1– 613 15. https://doi.org/10.3390/w12113138 614 autoregressive model and hydrological uncertainty processer. Water (Switzerland) 12, 1– 613 15. https://doi.org/10.3390/w12113138 614 Zhao, L., Xia, J., Xu, C. yu, Wang, Z., Sobkowiak, L., Long, C., 2013. Evapotranspiration 615 estimation methods in hydrological models. Journal of Geographical Sciences 23, 359– 616 369. https://doi.org/10.1007/s11442-013-1015-9 617 Zhu, B., Feng, Y., Gong, D., Jiang, S., Zhao, L., Cui, N., 2020. Hybrid particle swarm 618 optimization with extreme learning machine for daily reference evapotranspiration 619 prediction from limited climatic data. Computers and Electronics in Agriculture 173, 620 105430. https://doi.org/10.1016/j.compag.2020.105430 621 622 623 624 625 626 627 628 629 630 631 Zhao, L., Xia, J., Xu, C. yu, Wang, Z., Sobkowiak, L., Long, C., 2013. Evapotranspiration 615 estimation methods in hydrological models. Journal of Geographical Sciences 23, 359– 616 369. https://doi.org/10.1007/s11442-013-1015-9 617 Zhao, L., Xia, J., Xu, C. yu, Wang, Z., Sobkowiak, L., Long, C., 2013. Evapotranspiration 615 estimation methods in hydrological models. Journal of Geographical Sciences 23, 359– 616 369. https://doi.org/10.1007/s11442-013-1015-9 617 Zhu, B., Feng, Y., Gong, D., Jiang, S., Zhao, L., Cui, N., 2020. Hybrid particle swarm 618 optimization with extreme learning machine for daily reference evapotranspiration 619 prediction from limited climatic data. Computers and Electronics in Agriculture 173, 620 105430. https://doi.org/10.1016/j.compag.2020.105430 621 Zhu, B., Feng, Y., Gong, D., Jiang, S., Zhao, L., Cui, N., 2020. Hybrid particle swarm 618 optimization with extreme learning machine for daily reference evapotranspiration 619 prediction from limited climatic data. Computers and Electronics in Agriculture 173, 620 105430. https://doi.org/10.1007/BF00058655 445 https://doi.org/10.1016/j.compag.2020.105430 621 Table 1 Statistics of the measured meteorological parameters at study areas 632 Parameter Station Min. Max. mean St. Dev CV skewness Max. T (C°) Baghdad 12.90 47.70 31.09 10.32 0.33 -0.13 Basrah 14.60 48.90 33.81 10.47 0.31 -0.17 Mosul 8.30 46.40 28.21 11.05 0.39 -0.01 Min. T (C°) Baghdad 0.70 32.90 15.68 8.05 0.51 -0.06 Basrah 4.70 34.60 19.78 8.09 0.41 -0.17 Mosul -2.20 27.40 13.20 8.21 0.62 0.11 RH (%) Baghdad 20.00 82.00 43.93 16.88 0.38 0.49 Basrah 17.00 80.00 40.50 17.34 0.43 0.45 Mosul 19.00 88.00 51.51 20.33 0.39 0.07 Wind speed (m/sec) Baghdad 1.40 5.20 3.09 0.68 0.22 0.44 Basrah 1.70 7.70 4.14 1.11 0.27 0.67 Mosul 0.20 3.70 1.41 0.56 0.40 0.58 Pan evaporation (mm) Baghdad 48.70 624.80 264.25 161.71 0.61 0.32 Basrah 41.40 645.90 286.75 169.75 0.59 0.22 Mosul 21.50 464.10 177.77 127.14 0.72 0.42 633 Table 2 The tuning parameters of the applied models 634 Model name Description of parameters Random Subspace (RSS) Batch size-100, Classifier = REPTree, random seed- 1, subspace size = 0. 5, numbers of executions slots = 1, number of iteration= 10 Additive Regression (AR) Batch size-100, Classifier = Bagging, shrinkage=1, number of iteration= 30 M5 Pruned (M5P) Batch size-100, Minimum number of instances = 4 Reduced Error Pruning Tree (REPTree) Batch size-100, Initial count=0, number of folds =3, random seed = 1, minimum proportion of the variance = 0.001, minimum number = 2, max depth = 1 Bagging Batch size-100, bag Size percent= 100, Classifier = REPTree, max depth = 0, numbers of executions slots = 1, number of iteration= 10, random seed =1 632 Table 1 Statistics of the measured meteorological parameters at study areas Table 3 Inputs selection using regression analysis for modeling PE at Baghdad station No. https://doi.org/10.1007/BF00058655 445 of variables Variables MSE R² Adjusted R² Mallows' Cp Akaike's AIC Schwarz's SBC Amemiya's PC 1 T 1353.968 0.917 0.916 111.271 2078.702 2086.028 0.084 2 T / W 1073.850 0.934 0.934 30.399 2012.938 2023.927 0.067 3 Tmin / RH / W 979.540 0.940 0.939 3.963 1987.452 2002.104 0.061 4 Tmin / T / RH / W 979.668 0.940 0.939 5.000 1988.474 2006.789 0.061 5 Tmax / Tmin / RH / W 979.668 0.940 0.939 5.000 1988.474 2006.789 0.061 The best model for the selected selection criterion is displayed in green Table 5 Sensitivity analysis of input variables at Baghdad station Source Value Standard error t Pr > |t| Lower bound (95%) Upper bound (95%) Tmax 0.000 0.000 Tmin 0.000 0.000 T 0.693 0.038 18.119 <0.0001 0.618 0.768 RH -0.157 0.039 -3.995 <0.0001 -0.235 -0.080 W 0.206 0.018 11.189 <0.0001 0.170 0.242 Table 6 Sensitivity analysis of input variables at Mosul station Source Value Standard error t Pr > |t| Lower bound (95%) Upper bound (95%) Tmax 0.000 0.000 Tmin 0.412 0.172 2.398 0.017 0.074 0.751 T 0.209 0.213 0.981 0.327 -0.210 0.629 RH -0.320 0.061 -5.247 <0.0001 -0.441 -0.200 W 0.135 0.015 8.836 <0.0001 0.105 0.165 Table 7 MAE, RMSE, RAE, RRSE and r for meta-heuristics algorithms-based models during the training and testing span at Baghdad station Machine learning algorithm Training Testing MAE RMSE RAE RRSE r MAE RMSE RAE RRSE r R 30.34 41.19 21.18 25.27 0.968 35.51 48.68 25.98 30.80 0.966 R-RSS 31.12 41.50 21.73 25.47 0.967 45.72 59.18 33.46 37.44 0.949 R-Bagging 31.33 40.14 21.87 24.63 0.970 37.74 47.35 27.62 29.96 0.962 R-REPTree 33.313 44.35 23.25 27.21 0.962 37.81 50.13 27.67 31.72 0.958 R-M5P 29.65 39.58 20.70 24.29 0.970 33.82 45.05 24.75 28.50 0.972 Table 8 MAE RMSE, RAE, RRSE and r for meta-heuristics algorithms-based models during the training and testing span at Mosul station Machine earning lgorithm Training Testing MAE RMSE RAE RRSE r MAE RMSE RAE RRSE r 23.62 33.96 20.42 26.24 0.965 29.68 42.34 27.30 34.92 0.945 -RSS 21.97 29.38 19.00 22.71 0.974 26.93 37.31 24.77 30.77 0.959 -Bagging 22.46 29.97 19.42 23.16 0.972 27.37 37.94 25.17 31.29 0.959 -REPTree 26.66 38.09 23.05 29.43 0.957 27.01 38.62 24.84 31.85 0.962 -M5P 22.75 29.21 19.67 22.57 0.974 25.82 35.95 23.75 29.64 0.956 Table 9 Validation results (i.e. https://doi.org/10.1007/BF00058655 445 of variables Variables MSE R² Adjusted R² Mallows' Cp Akaike's AIC Schwarz's SBC Amemiya's PC 1 T 2768.697 0.894 0.894 173.192 2284.719 2292.045 0.106 2 Tmax / W 1822.869 0.931 0.930 17.956 2165.336 2176.325 0.070 3 T / RH / W 1733.377 0.934 0.934 4.229 2151.826 2166.478 0.067 4 Tmax / Tmin / RH / W 1731.981 0.935 0.934 5.000 2152.578 2170.893 0.067 5 Tmax / Tmin / RH / W 1731.981 0.935 0.934 5.000 2152.578 2170.893 0.067 The best model for the selected selection criterion is displayed in green Table 4 Inputs selection using regression analysis for modeling PE at Mosul station puts selection using regression analysis for modeling PE at Mosul station No. https://doi.org/10.1007/BF00058655 445 MAE, RMSE, RAE, RRSE and r) for best candidate model at Basrah station Best machine Validation Table 5 Sensitivity analysis of input variables at Baghdad station Table 6 Sensitivity analysis of input variables at Mosul station Table 7 MAE, RMSE, RAE, RRSE and r for meta-heuristics algorithms-based models during the training and testing span at Baghdad station Machine learning algorithm Training Testing MAE RMSE RAE RRSE r MAE RMSE RAE RRSE r AR 30.34 41.19 21.18 25.27 0.968 35.51 48.68 25.98 30.80 0.966 AR-RSS 31.12 41.50 21.73 25.47 0.967 45.72 59.18 33.46 37.44 0.949 AR-Bagging 31.33 40.14 21.87 24.63 0.970 37.74 47.35 27.62 29.96 0.962 AR-REPTree 33.313 44.35 23.25 27.21 0.962 37.81 50.13 27.67 31.72 0.958 AR-M5P 29.65 39.58 20.70 24.29 0.970 33.82 45.05 24.75 28.50 0.972 Table 8 MAE RMSE, RAE, RRSE and r for meta-heuristics algorithms-based models during the training and testing span at Mosul station Machine learning algorithm Training Testing MAE RMSE RAE RRSE r MAE RMSE RAE RRSE r AR 23.62 33.96 20.42 26.24 0.965 29.68 42.34 27.30 34.92 0.945 AR-RSS 21.97 29.38 19.00 22.71 0.974 26.93 37.31 24.77 30.77 0.959 AR-Bagging 22.46 29.97 19.42 23.16 0.972 27.37 37.94 25.17 31.29 0.959 AR-REPTree 26.66 38.09 23.05 29.43 0.957 27.01 38.62 24.84 31.85 0.962 AR-M5P 22.75 29.21 19.67 22.57 0.974 25.82 35.95 23.75 29.64 0.956 Table 9 Validation results (i.e. MAE, RMSE, RAE, RRSE and r) for best candidate model at Basrah station Best machine learning algorithm Validation MAE RMSE RAE RRSE r AR-M5P 47.23 67.23 31.19 39.30 0.942 Figures Figure 1 Figure 3 The standardized coe¨cients of input variable for sensitivity analysis at Mosul station for evaporation The standardized coe¨cients of input variable for sensitivity analysis at Mosul station for evaporation Figure 2 The standardized coe¨cients of input variable for sensitivity analysis at Baghdad station for evaporation The standardized coe¨cients of input variable for sensitivity analysis at Baghdad station for evaporation The standardized coe¨cients of input variable for sensitivity analysis at Baghdad station for evaporation Figure 1 The study area location The study area location Figure 2 Figure 4 Observed vs estimated daily evaporation values by the AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P models during testing at Baghdad station Figure 6 Observed vs estimated daily evaporation values by the best model AR-M5P during validation at Basrah station (a) Temporal variation (b) scatter plot Figure 7 Radar charts display MAE and RSME of AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P models during testing at Baghdad station Figure 5 Observed vs estimated daily evaporation values by the AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P models during testing at Mosul station Radar charts display MAE and RSME of AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P models during testing at Baghdad station Figure 10 Taylor diagrams of AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P during testing span at Mosul station Taylor diagrams of AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P during testing span at Mosul station highlights.docx Figure 8 Radar charts display the best performance indicators of AR, AR-RSS, AR-Bagging, AR-REPTree and AR- M5P models during testing at Mosul station Figure 9 Taylor diagrams of AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P during testing span at Baghdad station Figure 9 Taylor diagrams of AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P during testing span at Baghdad station Taylor diagrams of AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P during testing span at Baghdad station Figure 10 Taylor diagrams of AR, AR-RSS, AR-Bagging, AR-REPTree and AR-M5P during testing span at Mosul station Figure 10 Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. highlights.docx
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English
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INJURIES OF THE NASAL AND NASAL LATERAL CAVITIES
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Bukhara District Medical Association multidisciplinary Central Polyclinic otorhinolaryngologist https://doi.org/10.5281/zenodo.7280055 Bukhara District Medical Association multidisciplinary Central Polyclinic otorhinolaryngologist https://doi.org/10.5281/zenodo.7280055 Annotation: In this article, prevention, treatment, prevention of life- threatening factors of nasal injures, injury of the nasal adjacent cavities are manifested by rupture of the cavity wall, fracture, penetration of bone fragments into the cavity. Key words: Injuries, nasal bones, nasal cavities, symptoms of nasal injury, diagnosis. Injuries of the lateral cavities to the nose and nose occur more often in life, which occur in military and sports activities, in manufacturing enterprises, in blind transport events, during an epilepsy attack. Depending on the strength and nature of the impact of the body that caused the stroke, its direction and how deep it penetrates into the tissue, the injury to the nose can be either open (open wound of the skin covering) or closed (non - breaking of the skin covering). Closed wounds include-eating lat, bleeding into soft tissues, swelling, fracture of the nasal bones, accompanied by the appearance of concomitant cavities in the nose by the walls, the eyelid, cheekbone, the silcification or siljimas of the excretory cavity cages. Open jarring is divided into torn, cut, superficial and chukur, wounds that have penetrated or have not penetrated the nasal cavity. Injuries that enter the nasal cavity are most often caused by a body with an acute tip. Bunda is accompanied by a rupture of the mucous membrane of the nasal cavity, blood flow from the nose, infection into the nasal cavity and adjacent cavities in the nose, the development of nasal obstruction hematoma and abscess. When the direction of the body with an acute tip is directed to the upper wall of the nasal cavity, a fracture of the sieve plate can be observed, in which the patient flows the cerebrospinal fluid. Often, as a result of the fracture of the cartilage of the nasal bones and nasal barrier, or protrusion (figure 93) between the nasal bones and the forehead growth of the upper jaw, the outer nose is smoothed to the side. Sometimes a forehead swelling fracture of the upper jaw is observed. Often, in facial injuries, there is a decrease or complete loss of visual acuity (amnesia), blood flow to the bottom of the eye alma (hyphemas), tuberculosis of the eye alma (enoftalm), paralysis (diplopia) as a result of compression of the eye-moving muscles. INJURIES OF THE NASAL AND NASAL LATERAL CAVITIES Qurbanov Mirvohid Kamolovich Qurbanov Mirvohid Kamolovich ` «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariya va amaliyot» nomli ilmiy, masofaviy, onlayn konferensiya INJURIES OF THE NASAL AND NASAL LATERAL CAVITIES Qurbanov Mirvohid Kamolovich ` «Zamona va amaliy «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariya va amaliyot» nomli ilmiy, masofaviy, onlayn konferensiya Bukhara District Medical Association multidisciplinary Central Polyclinic otorhinolaryngologist https://doi.org/10.5281/zenodo.7280055 Injury of the lateral cavities to the nose is manifested by rupture of the wall of the cavities, fracture, penetration of bone fragments into the cavity. In addition, they are divided into injuries that do not spread or spread to the cranial cavity. 36 36 ` «Zamon va amali ` Zamonaviy dunyoda innovatsion tadqiqotlar: Nazar va amaliyot» nomli ilmiy, masofaviy, onlayn konfere A fracture of the wedge cavity walls is also a fracture of the base of the skull, a relatively threeraydi and vision nerve fiber, accompanied by an injury of the internal sleeping artery, causing a blood flow or aneurysm of the wall of the las sleeping artery from the wound to the cause of death. Symptoms. In the case of a nasal injury, blood flows from the nose, bruises appear on the skin of the nose and on the eyelids, a swelling of soft tissues and subcutaneous emphysema, silcification of bone fragments are observed. As a result of a violation of the breathing activity of the nose or the closure of smel cognition cracks, the patient's ability to know the smell decreases, when the smell cognition nerve fibers are disconnected – the smell cognition activity o the nose is completely lost. Diagnosis. It is put on the basis of patient complaints, Anamnesis, external examination, palpation, sensing, rhinoscopy, endoscopy X-ray, computer tomography. When external examination and palpation, it is determined that the area of the jarohat hurts, there is a swelling of soft tissues, a violation of the external nasal shape. The fact that the nose is shaken to the side, majagled, the joints when the nose is palpated (a sign of a staircase), the pathological mobility of the bones and the fragility of the bone fragments indicate that the bones of the nose are broken, when pressing, the subcutaneous gnashing - the excretory bone is broken and the mucous membrane is torn. In the treatment. The nature of the injury of the lateral cavities to the nose and nostrils, the depth, the general condition of the be-Marie are taken into account. Emergency medical care consists in pain relief, stopping bleeding,giving primary surgery and sending the patient to the hospital immediately. Bukhara District Medical Association multidisciplinary Central Polyclinic otorhinolaryngologist https://doi.org/10.5281/zenodo.7280055 Under general anesthesia, when the outer nose is shaken to the left, broken bone fragments are placed on their place with the thumb of the right hand and the thumb of the left hand when shaking to the right. When the bone fragments fall into their place, a specific creaking sound is heard. Foydalanilgan adabiyotlar: 1. Methods of performing prophylactic otorhinolaryngological examination of the population (method of rec.Kiev 1973). 2. Organization of medical care in children’s kindergarden, schools, boarding schools for children and adolescents.(method.rec.Toshkent.1982). 3. Head of the Department of Otorhinolaryngology B.V.Shevrigin.Moskva.M.1985 y. 4. Handbook of otorhinolaryngology I.B.Soldatov.Moskv.m.1997 y ` «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazar va amaliyot» nomli ilmiy, masofaviy, onlayn konferen external cosmetic defect. It has an important cosmetic ahami-Yat to put bone fragments in their place. The practice of putting the bones in their place (reposition), performed on the first day of the injury, is recognized as the most optimal method, but in severe complications of the skull, the skull can perform it for the last 3 weeks from the injury. In the sitting position of the patient's course, the nasal cavity is anesthetized locally (10% lidocaine spraying on the mucous membrane, 2% dicaine rubbing, 5% cocaine or sending 1% novocaine, 2% lidocaine solutions to the broken area). In children, it is acceptable to perform the act of reposition under general anesthesia. The method of putting a broken piece of bone in its place with the help of a finger. Under general anesthesia, when the outer nose is shaken to the left, broken bone fragments are placed on their place with the thumb of the right hand and the thumb of the left hand when shaking to the right. When the bone fragments fall into their place, a specific creaking sound is heard. Foydalanilgan adabiyotlar: Foydalanilgan adabiyotlar: 1. Methods of performing prophylactic otorhinolaryngological examination of the population (method of rec.Kiev 1973). 1. Methods of performing prophylactic otorhinolaryngological examination of the population (method of rec.Kiev 1973). 2. Organization of medical care in children’s kindergarden, schools, boarding schools for children and adolescents.(method.rec.Toshkent.1982). 3. Head of the Department of Otorhinolaryngology B.V.Shevrigin.Moskva.M.1985 y. 4. Handbook of otorhinolaryngology I.B.Soldatov.Moskv.m.1997 y Bukhara District Medical Association multidisciplinary Central Polyclinic otorhinolaryngologist https://doi.org/10.5281/zenodo.7280055 In the case of an open wound of soft tissues or a lump, primary surgery is performed on the contaminated area of the larynx; the area of the larynx is first washed with an antiseptic solution, then with the help of a special spoon, fragments of broken bones and cartilage are removed, soft tissue is left. The patient is given ana-toxin against tetanus (if vaccinated - 0.5 ml, if not vaccinated - from 1 ml under the skin),blood serum against tetanus (3000 me) on Bezredko after intra-skin test. After the first medical help, when the animal bites the nose area, the patient is vaccinated against rabies. The primary seam is laid after a day after the injury. Usually, after the broken nasal bones are fixed in their place, the blood flow from the nose stops on its own. In most cases, the front nasal congestion, sometimes the back nasal congestion, is performed. The stuffing is replaced every 24-48 hours. Children are usually put on an elastic stuffing. In order to reduce bruises and soft tissue edema on the injured nasal skin, pieces of Ice are placed on the skin of the nose for 5-6 hours. The main method of treatment is the restoration (putting in its place) of the walls of the bones of the nose and nasal adjacent cavities, as well as the practice of inaction of bone fragments from the inside or outside, when the bones of the nose are broken, silenced from their place, as well as the appearance of an 37 37 ` «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariy va amaliyot» nomli ilmiy, masofaviy, onlayn konferens external cosmetic defect. It has an important cosmetic ahami-Yat to put bone fragments in their place. The practice of putting the bones in their place (reposition), performed on the first day of the injury, is recognized as the most optimal method, but in severe complications of the skull, the skull can perform it for the last 3 weeks from the injury. In the sitting position of the patient's course, the nasal cavity is anesthetized locally (10% lidocaine spraying on the mucous membrane, 2% dicaine rubbing, 5% cocaine or sending 1% novocaine, 2% lidocaine solutions to the broken area). In children, it is acceptable to perform the act of reposition under general anesthesia. The method of putting a broken piece of bone in its place with the help of a finger. 38 38
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The Effect of Organizational Changes on the Psychosocial Work Environment: Changes in Psychological and Social Working Conditions Following Organizational Changes
Frontiers in psychology
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ORIGINAL RESEARCH published: 20 December 2019 doi: 10.3389/fpsyg.2019.02845 Edited by: Lise Fløvik*, Stein Knardahl and Jan Olav Christensen* Department of Work Psychology and Physiology, National Institute of Occupational Health (STAMI), Oslo, Norway María del Carmen Pérez-Fuentes, University of Almería, Spain María del Carmen Pérez-Fuentes, University of Almería, Spain Reviewed by: Gabriela Topa, National University of Distance Education (UNED), Spain Artur Victoria, Autonomous University of Lisbon, Portugal Reviewed by: Gabriela Topa, National University of Distance Education (UNED), Spain Artur Victoria, Autonomous University of Lisbon, Portugal Purpose: The present study aimed to clarify the prospective effects of various types and frequencies of organizational changes on aspects in the psychosocial work environment. Methods: The study had a prospective, full-panel, repeated measures design. Data were collected by self-administered, online questionnaires, with a 2-year interval between measurement occasions. Five types of organizational change were assessed – company restructuring, downsizing, layoffs, partial closure, and partial outsourcing. The effects of change on eleven, specific work factors were measured utilizing QPS Nordic. At baseline, 12652 employees participated, while 8965 responded at follow-up. Generalized estimating equations were utilized to estimate the effects of change taking place within the last 12 months or more than 24 months prior. *Correspondence: Lise Fløvik lise.flovik@stami.no; floevik.lise@gmail.com Jan Olav Christensen jan.o.christensen@stami.no Specialty section: This article was submitted to Organizational Psychology, a section of the journal Frontiers in Psychology Results: Cross-sectional analyses, i.e., changes occurring within the last 12 months, showed all 11 work factors to be statistically significantly associated with the organizational changes restructuring, downsizing, and partial closure (coefficients ranging −0.28 to 0.04). In the prospective analyses, i.e., the effects of change taking place more than 24 months prior, associations were no longer significant for a number of work factors, although all types of organizational change remained significantly associated with at least three work factors (coefficients ranging −0.14 to 0.05). Following repeated organizational changes, statistically significant associations were shown for all 11 work factors (coefficients ranging from 0.39 to −0.04). Received: 11 April 2019 Accepted: 02 December 2019 Published: 20 December 2019 Lise Fløvik*, Stein Knardahl and Jan Olav Christensen* Department of Work Psychology and Physiology, National Institute of Occupational Health (STAMI), Oslo, Norway Edited by: María del Carmen Pérez-Fuentes, University of Almería, Spain INTRODUCTION something novel and intrinsically unknown and uncertain for the organization and its members, which may disrupt existing structures and processes. Thus, organizational change can be experienced both as an opportunity to gain and as a risk of losing and may involve redesign of tasks and responsibilities that alter existing work content and – environment in various foreseen and unforeseen ways. Nevertheless, while prior studies have linked organizational change to somatic and mental health (Vahtera et al., 1997; Kivimäki et al., 2001, 2003; Probst, 2003; Moore et al., 2004; Vahtera et al., 2004), less is known about repercussions of organizational change for psychosocial working conditions that are relevant to health. Organizational change has repeatedly been associated with adverse effects on employee health (Oreg et al., 2011). Large- scale organizational changes, such as company restructuring, downsizing and outsourcing have been linked to somatic and mental health complaints, presenteeism and long-term sick leave (Kivimäki et al., 2001; Bamberger et al., 2012). However, a thorough understanding of why organizational changes are associated with adverse health effects is still pending. Clarifying the repercussions of organizational change for workplaces and employees is an essential first step to preventing adverse health effects of and facilitating healthy, successful change. Prior meta-analytic studies have shown a wide range of psychological and social work factors, such as leadership, role conflict and ambiguity, job demands, control and job insecurity to predict employee well-being, health and sick leave (Viswesvaran et al., 1999; Stansfeld and Candy, 2006; Nahrgang et al., 2011; Lang et al., 2012; Schyns and Schilling, 2013; Virtanen et al., 2013; Schmidt et al., 2014; Theorell et al., 2015), as well as the change process and end-result (Schuler and Jackson, 2001; Hoag et al., 2002). In order to elucidate whether extensive company change influence central aspects of the psychosocial work environment the present study aimed to clarify the effects of various types of organizational changes, separately and co-occurring as well as repeated over time, on 11 specific psychological and social work factors. A psychosocial work environment consists of organizational-, social-, and psychological factors which govern and define the content and quality of various aspects of work (Nieuwenhuijsen et al., 2010). Organizational work factors include formal and structural conditions that regulate how work is carried out, e.g., employment contracts and work schedules. Social work factors comprise the relational aspects of a workplace, such as social climate, support from superiors and co-workers. INTRODUCTION Psychological work factors refer to individual-level aspects of work, such as perceived levels of autonomy, job demands and predictability. Prior studies have linked an organization’s psychosocial working conditions to both employee- and organizational outcomes (Stansfeld and Candy, 2006; Holden et al., 2011), such as worker health (Lau and Knardahl, 2008; Väänänen et al., 2008; Bambra et al., 2009; Häusser et al., 2010; Nahrgang et al., 2011; Schmidt et al., 2014; Read and Laschinger, 2015; Schmidt et al., 2018), sick leave (Head et al., 2006) and company productivity (Dollard and Neser, 2013; Dysvik and Kuvaas, 2013; Poulsen et al., 2016; Montano et al., 2017). Despite the aforementioned awareness of the potential of organizational change to upset various organizational systems as well as employee health, few studies seem to have assessed effects of organization change on specific factors in the psychosocial work environment that are known to be associated with health. The present study assessed the effect of various types and frequencies of organizational change on 11 distinct work factors pertaining to job tasks (job control, job demands), job roles (role clarity, role conflict), leadership (fair-, empowering-, supportive leadership), social aspects (support from co-workers, social climate) and predictability (job predictability, future employability). “Organizational change” pertains to the altering of structures, strategies, procedures or cultures of organizations (Quattrone and Hopper, 2001). The term encompasses both the process by which this happens (i.e., “how”) and the content of what is being altered (i.e., “what”). By definition, change implies a shift in the organization from one state to another. This shift may be deliberate, with the aim of gaining or losing specific features of the organization to attain a defined goal, or it may be less deliberate, perhaps occurring as a consequence of developments outside the control of the organization. Moreover, during the change process, additional parts of the organization may be unintentionally affected, particularly when change is experienced as excessive (Stensaker et al., 2001). Such unintended repercussions of organizational change may be both positive and negative (Jian, 2007), and may be more likely when a large number of transactions are required to implement the change decision and many specialized problem-solving capabilities are invoked (Casa and Lodge, 2015). Either way, organizational change represents During change implementation, the organization attends to various change-related tasks in addition to the ordinary, day-to- day activities. Citation: Citation: Fløvik L, Knardahl S and Christensen JO (2019) The Effect of Organizational Changes on the Psychosocial Work Environment: Changes in Psychological and Social Working Conditions Following Organizational Changes. Front. Psychol. 10:2845. doi: 10.3389/fpsyg.2019.02845 Conclusion: Following both separate and repeated organizational change, various psychological and social work factors were altered, with the most pronounced effects following repeated change. These results suggest the implementing organizational December 2019 | Volume 10 | Article 2845 Frontiers in Psychology | www.frontiersin.org 1 Fløvik et al. Organizational Change and the Psychosocial Work Environment change, especially repeated change, may have an adverse effect on various parts of the psychosocial work environment. The negative effects of a company’s psychosocial working conditions may contribute to the adverse health effects often observed following such changes and help explain why many change initiatives fail to reach its intended results. Keywords: organizational change, psychosocial work environment, occupational health, mental distress, sick leave, leadership Keywords: organizational change, psychosocial work environment, occupational health, mental distress, sick leave, leadership December 2019 | Volume 10 | Article 2845 INTRODUCTION The added demands given to managers in this process may also leave fewer resources and room for superiors to provide the support and attentiveness they normally are able to give their employees, which may affect employee’s perception of management as supportive. Hence, organizational change may lead to a decrease in employee perception of leadership as fair (i.e., equal treatment of employees), empowering (i.e., including) and supportive (i.e., attentive and present). The relational aspects of the organizational may also be affected during extensive workplace changes, as the collegial composition may be rearranged or colleagues have to compete over new or remaining positions. As a result, social cohesion within the group may deteriorate and collegial support diminishes (Campbell and Pepper, 2007). Thus, organizational change may be associated with a decrease in perceived support from co-workers and social climate as inclusive and trusting. Employees’ sense of predictability regarding both present and future job prospects could also be affected by exposure to extensive workplace changes (Probst, 2003; Baillien and De Witte, 2009). Change naturally involves some degree of uncertainty regarding the outcome and future. As extensive organizational changes often are management-driven with little employee involvement, uncertainty may be extra prominent. Furthermore, As the rate of organizational change is increasing, a larger part of the workforce is likely to experience multiple changes or repeated organizational changes during their careers, some of which they may deem excessive. To our knowledge, a limited number of studies have examined how exposure to repeated organizational changes influence specific factors in the work environment (Moore et al., 2004). These studies have reported stronger effects following repeated change than separate change, but only on outcomes such as employee health and sick leave (Isaksson et al., 2002; Moore et al., 2004; Wagstaff et al., 2016). Thus, one may speculate whether implementing multiple, repeated changes may also be associated with a stronger effect on psychosocial work factors than single change efforts (Klarner et al., 2011). In developing targeted interventions aimed at reducing the potential adverse effects of organizational change on employee health, identifying the underlying mechanisms in this stressor-strain relationship is an imperative first step. INTRODUCTION Interventions aimed at reducing or alleviating the effect of risk factors in the work environment have shown the potential of such interventions to reduce depressive symptoms and absenteeism and to improve productivity both during and following organizational changes (Bambra et al., 2009; Kelloway and Barling, 2010; Houtman and Lourijsen, 2012). The effect of organizational change on specific factors in the work environment may represent such a mechanism in which the work factors may either moderate or mediate the relationship between change and health. In order to illuminate the effect of various specific types of organizational change as well as repeated change on central aspects of the psychosocial work environment, the current study examined both the cross-sectional and prospective associations of separate and repeated organizational change with 11 specific psychological and social work factors. INTRODUCTION In sum, this may increase the total amount of work and job tasks employees are faced with (Kivimäki et al., 2001). December 2019 | Volume 10 | Article 2845 Frontiers in Psychology | www.frontiersin.org 2 Organizational Change and the Psychosocial Work Environment Fløvik et al. factors such as globalization, market demands or technological innovation, which makes predicting the future jobs even more complex. Hence, organizational changes may be associated with a decrease in employee perception of short-term job predictability and future employability. The need for management to exert control in the planning and implementation process may also leave less room for employees to influence their own job to the same extent as before, and may thus affect employees’ experience of their own job control during the process (Paulsen et al., 2005). Hence, organizational change may be associated with increased job demands (i.e., the amount of work and time demands) and a decrease of job control (i.e., influence over decisions regarding one’s tasks, co-workers and clients). When major shifts take place within an organization, the rearrangement of employee roles and responsibilities are often a central part of the process. Such rearrangement may result in employees facing conflicting demands, lack of resources to complete one’s additional assigned tasks or uncertainty related to the objectives and expectations of one’s new role (Baillien and De Witte, 2009). Thus, large-scaled workplace changes could increase employee’s sense of role conflict (i.e., conflicting demands and lack of resources) and lower the sense of role clarity (i.e., clarity regarding a roles responsibilities and expectations). The need for management to invoke tough and sometimes unpopular decisions, e.g., in a downsizing or layoffprocess, may also affect employees perception of management and superiors following organizational changes (Gilley et al., 2009; Holten and Brenner, 2015; Neves and Schyns, 2018). When the consequences of change involve the potential loss of valued aspects such as specific tasks, collegial relationships or the very existence of one’s job, one may surmise that employee perception of management as just or fair could be affected. Moreover, changes initiated by external forces, e.g., market demands or technological innovation, and invoked by management may also leave less room for including employees in decision making and planning. Exercising an inclusive and empowering leadership style may thus be challenging during change implementation. Frontiers in Psychology | www.frontiersin.org Study Design y g The study was a part of the project “The New Workplace: work, health and participation in working life” initiated and carried out by the Norwegian National Institute of Occupational Health (STAMI). The project was conducted in line with the World Medical Association Declaration of Helsinki, and approved by the Data Inspectorate of Norway and the Norwegian Committee for Medical and Health Research Ethics, Region South East (REC). The study had a prospective, full panel study design, with data collected with a 2-year interval. Baseline data collected between 2004 and 2013, with follow- up 2 years later, respectively. All data were collected by a self-administered, online questionnaire. The participating organizations either contacted STAMI directly as a response December 2019 | Volume 10 | Article 2845 Frontiers in Psychology | www.frontiersin.org 3 Organizational Change and the Psychosocial Work Environment Fløvik et al. Outcome: Psychological and Social Work Factors to an invitation to participate in the study posted on the institute’s webpages, or on requesting assistance in a general work environment survey. The psychological and social work factors were measured by the General Nordic Questionnaire for Psychological and Social Factors at Work (QPSNordic) (Ørhede et al., 2000; Wännström et al., 2009). QPSNordic is a validated questionnaire designed to assess a comprehensive set of social and psychological aspects in the workplace. The effects of organizational changes on 11, specific work factors were assessed. These were six psychological work factors (job control, job demands, job predictability, perceived future employability, role clarity, and role conflict) and five social work factors (empowering leadership, fair leadership, social climate, support from co- worker and support from superior). Each factor was measured by multiple items, ranging from two to five items depending on the factor. Responses on all items were given on a five-point Likert scale, ranging from “1 = very seldom or never” to “5 = very often or always.” For each work factor, a mean score was calculated. For all work factors, Cronbach’s α was calculated at baseline and follow-up and were within the range of 0.71 (“role conflict“) to 0.88 and (“empowering leadership”). Confounders We assessed the effects of five distinct types of organizational changes. These were company restructuring, downsizing, layoffs, partial closure, and partial outsourcing. To clarify, “downsizing” refers to a temporary termination of job contract with the chance of rehiring, while “layoffs” refers to permanent termination of the job contract. Each type of change was assessed by a single item with a dichotomous response (“yes”/”no”) and inquired whether the organization in which the employee worked had implemented a specific type of change within the last 12 months. Examples: “During the last 12 months has your company undergone restructuring?,” and “During the last 12 months has your company undergone downsizing?” All analyses included the variables age, sex, skill level, and place of employment (private vs. public organizations) as potential confounders. Age was divided into three age groups, (i) “<35,” (ii) “35–55,” and (iii) “>55.” Skill level was divided into five categories reflecting years of formal education required in various professions. The categorization was done using the Standard Classification of Occupations (STYRK), which is based on the International Standard Classification of Occupations (ISCO-88) and developed by Statistics Norway (SSB). The five skill level categories were: (i) “<10 years of education,” (ii) “10–12 years of education,” (iii) “13– 15 years of education,” (iv) “>15 years of education,” and (v) “Unspecified,” which included occupations requiring no formal education. Subjects A total of 66 Norwegian organizations participated in the study, representing both public and private sector and a variety of professions, company sizes and sectors. Upon accepting to participate, information regarding the project was initially given at the company level. All current employees were invited to participate in the study and received an information letter by postal mail, containing a unique ID- code for accessing the online questionnaire. Respondents were allotted time during work hours to complete the questionnaire but also had the opportunity to complete the questionnaire at home. Respondents had the opportunity to log in an unlimited number of times to access to complete the questionnaire. Inclusion criteria for both the cross-sectional and prospective sample were completing all items for each individual work factor at both T1 and T2. Statistical Analyses Generalized Estimating Equations The cross-sectional and prospective associations between the separate types and frequencies of organizational changes and the various work factors were estimated utilizing linear regression analyses by the Generalized Estimated Equations method (GEE). The method is based on the generalized linear model and allows for the analyses of correlated observations, such as repeated measures or clustered data. In addition, the method allows for samples to have a non- normal error distribution on the response variable. The GEE approach was chosen as it accounts for the potential correlated responses within sample clusters, which fit the present data well as it was clustered within organizations (Zorn, 2001; Hubbard et al., 2010). The GEE method gives a population parameter estimate based on the average of clusters in the data (Hardin and Hilbe, 2002; Hanley et al., 2003). Hence, the Statistical Analyses To assess the effect of multiple organizational changes occurring simultaneously, a three-category predictor variable was created based on the five change items. The categorical predictor demonstrated whether employees had experienced (i) “No type of organizational change at baseline,” (ii) “One type of organizational change at baseline,” or (iii) “Two or more types of organizational change at baseline.” Cross-Sectional Analyses In the cross-sectional analyses pertaining to specific, separate organizational changes, we ran both uni- and multi-variate regressions separately with each type of change as predictor and each type of work factor as outcome. In the analyses pertaining to the effects of multiple changes, we utilized the aforementioned three-category variable as predictor and ran the analyses for each work factor separately. Multivariate The multivariate analyses showed fewer significant associations, however all work factors were significantly associated with at least one type of specific change each with B-values ranging from 0.20 to −0.17. See Table 5 for further details. Baseline Characteristics The mean age at baseline was 44.34 (SD: 10.5). Of the included subjects 20.9% were under the age of 35, 61.9% were between the age of 35–55, while 17.2% were older than 55. Women constituted 54.7% of the sample. Skill level at baseline was as follows: >15 years of formal education 26.9%, 13–15 years 24.7%, 10–12 years 38.7%, >10 years 1.0%, and Unspecified 8.7%. For further details, see Table 1. Prospective Analyses The analyses of change reported to have occurred during the last 12 months prior to baseline showed company restructuring, downsizing, and partial closure to be statistically significantly associated with all work factors, with b-values ranging from −0.28 to 0.04. Layoffs and partial outsourcing were also statistically significantly associated with most work factors, with the exception of job control, which was not statistically significantly related to partial outsourcing, and role clarity, which was not statistically significantly related to layoffs. See Table 4 for further details. In the prospective analyses, both uni- and multi-variate regressions were run separately with each type of change as predictor and each type of work factor as outcome. The analyses were run in two steps. In the first step, Model I, analyses were adjusted for age, sex, skill level and place of employment, while in step two, Model II, analyses were also adjusted for baseline levels of the work factor in question. In the analyses pertaining to multiple changes, we ran simple regressions with the three-level categorical predictor variable for each work factor separately. As in the analyses pertaining to specific changes, all analyses were conducted in two steps. Lastly, in the analyses pertaining to the effects of repeated change, we ran simple regressions with the aforementioned four-level categorical predictor for each work factor separately. These analyses were also conducted in two-steps. Non-response Analysis GEE method estimates the average response in a population- based on the average of clusters within a sample. The GEE analysis provides the option to predefine the anticipated correlation structure in the data, for instance independent, autoregressive, compound symmetry, or unstructured. In the present analyses, the unstructured option was chosen since no theoretical grounds were present to expect a specific correlation structure in the data. In addition, the unstructured option does not impose any constraints in the correlation structure in the analyses (Hardin and Hilbe, 2002). GEE has previously been widely applied in epidemiological studies where data have been correlated as the method may handle various types of prior, unidentified correlations between measurements (Merlo, 2003; Skrondal and Rabe- Hesketh, 2003; Cui and Qian, 2007). All analyses were run using IBM SPSS Statistics, version 24.0 (IBM, Armonk, NY, United States), with the level of statistical significance set to p < 0.05. Non-response Analysis Women were less likely to be non-respondents (OR 0.72, 95% CI 0.66–0.78), along with employees aged 35–55 years (OR 0.82, 95% CI 0.74–0.91). Respondents employed in private sector companies were also less likely to be non-respondents (OR 0.86, 95% CI 0.78–0.95). As for skill level, respondents employed in jobs requiring 10–12 (OR 1.35, 95% CI 1.22–1.49) and <10 years of formal education (OR 1.84, 95% CI 1.26–2.67) were also more likely to be non-respondents. For further details, see Table 2. Prospective Analyses Univariate The univariate analyses pertaining to the effect of change reported to have taken place more than 24 months prior showed all types of specific changes to be associated with at least three work factors (B-values ranging −0.14 to 0.05 in Model I. In Model II, i.e., also adjusted for baseline levels of each respective work factor, fewer associations remained statistically significant. For further details see Table 4. Sample Attrition Being employed in private sector was linked to dropout at follow- up (OR 1.19, 95% CI 1.07–1.33). Working in an occupation requiring 13–15 years of formal education (OR 1.55, 95% CI 1.36–1.77), 10–12 years (OR 1.65, 95% CI 1.45–1.87) or less than 10 years of formal qualifications (OR 1.86, 95% CI 1.13–3.08) were also associated with not participating at follow-up. On the other hand, employees aged 35–55 were negatively associated with dropout (OR 0.78, 95% CI 0.69–0.87). Gender was not associated with attrition. For further details, see Table 2. Repeated Organizational Change To assess the effects of repeated organizational changes, a four-category predictor variable was created based on the five change items. The predictor demonstrated whether employees had experiences (i) “No type of change at baseline or follow-up,” (ii) “At least one type of change at baseline, but none at follow- up,” (iii) “At least one type of change at follow-up, but none at baseline,” or (iv) “At least one type of change at baseline and at least one type of change at follow-up.” December 2019 | Volume 10 | Article 2845 Frontiers in Psychology | www.frontiersin.org 4 Organizational Change and the Psychosocial Work Environment Fløvik et al. Effects of Separate Organizational Change For a short summary of all associations, see Table 3. Multivariate In the multivariate analyses, most significant associations were no longer statistically significant, although empowering leadership, December 2019 | Volume 10 | Article 2845 Frontiers in Psychology | www.frontiersin.org 5 Organizational Change and the Psychosocial Work Environment Fløvik et al. TABLE 1 | Sample characteristics. Invited subjects Baseline sample Prospective sample N % N % Mean SD N % Mean SD Sex Female 8467 54.7 6478 51.2 4733 52.8 Male 6998 45.3 6174 48.8 4232 47.2 Total 15465 12652 100 8965 Missing 2841 18.3 Age >35 2638 20.9 1740 19.4 35–55 7837 61.9 5742 64.0 >55 2177 17.2 1483 16.5 Total 12652 100 44.34 10.54 8965 100 Skill level >15 3408 26.9 2438 27.2 13–15 3126 24.7 2334 26.0 10–12 4895 38.7 3214 35.9 <10 127 1.0 71 0.8 Unspecified 1096 8.7 908 10.1 Workplace Public sector 11792 76.2 9914 78.4 6995 78.0 Private sector 3673 23.8 2738 21.6 1970 22.0 Organizational change No change 3445 36.1 3780 43.0 One change 3474 36.4 2740 31.2 Two or more changes 2643 27.7 2268 25.8 Organizational change Reorganization 5356 55.0 4448 49.8 Downsizing 2406 15.5 1945 21.8 Layoffs 911 5.9 1081 12.1 Partial closure 1238 8.0 1062 11.9 Partial outsourcing 774 5.0 994 11.2 Characteristics of baseline sample and prospective sample. seen in seven of the work factors (b-values ranging from −0.11 to −0.03). See Table 6 for further details. job demands, role conflict, social climate, job predictability, and future employability remained significantly associated with certain types of specific change. See Table 5 for further details. Cross-Sectional Analyses Separate linear regressions for each work factor as outcome showed exposure to more than one type of change 12 months prior to be statistically significantly associated with all work factors (b-values ranging from 0.27 to −0.26). Effects of Multiple Organizational Changes Separate linear regressions were run with each work factor as the outcome. In Model I, repeated, organizational change was statistically significantly associated with all work factors, with b-values ranging from 0.39 to −0.04. In Model II (controlling for baseline levels of the respective work factor) associations remained statistically significant, with b-values ranging from −19 to 0.18. See Table 7 for further details. DISCUSSION In the prospective analyses most associations were no longer significant, which may indicate that for some work factors, the adverse impact of organizational changes are primarily manifested more proximal to the change. employee’s perception of their superior as fair or empowering is affected proximal to the change, but also remains low 2 years after the layoffhas taken place. An inevitable consequence of a layoffprocess it the termination of job contracts. Both the process and result of deciding who will be let offmay give rise to the feeling of powerlessness, injustice and unfair treatment by superiors and management (Campbell-Jamison et al., 2001). One could surmise that this perception might dwindle over time for the remaining, or “surviving” employees, however, the present results indicate that this may not be the case, as employee perception of fair and empowering leadership continues to be low years after the implementation, even for those who are fortunate to keep their job and remain within the company (Vahtera et al., 2004). These results are in line with prior studies showing survivors of downsizing, layoffs, and outsourcing processes to report a lower sense of job security, productivity, organizational attachment, perceived organizational justice and higher turnover intention (Maertz et al., 2010; Drzensky and Heinz, 2015; van Dick et al., 2016). p g In light of the present results, organizational change seems to have both a short-term and a long-term effect on multiple factors in the work environment. The short-term effect seems to emerge during and be manifest shortly after change implementation, but then diminish over time. For instance, the present results indicate that shortly after a company restructuring process, employees are more likely to report lower role clarity, i.e., more uncertainties regarding their job’s objectives and responsibilities, potentially due to the new job situation or tasks given because of the restructuring process. However, as time passes and employees get more conversant with their new role and responsibilities, the feeling of role clarity seems to increase. In other words, although the present study cannot point to why, the results show that the adverse effect of restructuring on perceived role clarity diminishes over time. In addition to a short-term effect, long-term effects of change on certain work factors were also shown in the present study. These long-term effects may also emerge during or shortly after change has taken place, but then stabilize and last long-term or continue to develop over time. DISCUSSION Separate linear regressions with each work factor as outcome showed exposure to more than one type of change 24 months prior to be statistically significantly associated with all work factors (b-values ranging from 0.27 to −0.07, see Table 2) in Model I. When controlled for baseline levels of the respective work factor, Model II, statistically significant associations were Effects of Separate Organizational Change on the Work Environment The present study demonstrated statistically significant cross- sectional and prospective relationships between various discrete December 2019 | Volume 10 | Article 2845 Frontiers in Psychology | www.frontiersin.org 6 Organizational Change and the Psychosocial Work Environment Fløvik et al. TABLE 2 | Non-response and attrition analyses. Non-response analysis Attrition analysis N % OR 95% CI N % OR 95% CI Sex Female 6478 51.2 4733 52.8 1.09 0.99–1.20 Male 6174 48.8 – – 4232 47.2 – – Total 12652 100.0 8965 100 Age <35 2638 20.9 – – 1740 19.4 – – 35–55 7837 61.9 0.82 0.74–0.91 5742 64.0 0.78 0.69–0.87 >55 2177 17.2 1.10 0.97–1.26 1483 16.5 0.89 0.76–1.04 Skill level >15 3408 26.9 – 2438 27.2 – – 13–15 3126 24.7 0.82 0.73–0.92 2334 26.0 1.55 1.36–1.77 10–12 4895 38.7 1.35 1.22–1.49 3214 35.9 1.65 1.45–1.87 <10 127 1.0 1.84 1.26–2.67 71 0.8 1.86 1.13–3.08 Uspesifisert 7096 8.7 0.53 0.43–0.62 908 10.1 0.80 0.65–0.98 Workplace Public sector 9914 78.4 – – 6995 78.0 – – Private sector 2738 21.6 0.86 0.78–0.95 1790 22.0 1.19 1.07–1.33 Organizational change No change 3184 35.5 – – One change 3310 36.9 1.05 0.94–1.18 Two or more changes 2471 27.6 1.09 0.97–1.23 Non-response analysis and attrition analysis. Non-response defined as not completing work factor items at baseline. Attrition defined as completing work factor items baseline, but not at follow-up. The bold significance values are p < 0.05. TABLE 2 | Non-response and attrition analyses. Non-response analysis and attrition analysis. Non-response defined as not completing work factor items at baseline. Attrition de baseline, but not at follow-up. The bold significance values are p < 0.05. types of organizational change and a number of specific work factors. Hence, changes in multiple work factors were demonstrated when the organizational change had taken place within the last 12 months and more than 24 months prior. Frontiers in Psychology | www.frontiersin.org December 2019 | Volume 10 | Article 2845 Job Tasks (Job Demands and Job Control) Change < 12 months Change > 24 month ago Reorganization Downsizing Layoffs Partial closure Partial outsourcing Reorganization Downsizing Layoffs Partial closure Partial outsourcing powering leadership ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ns ns ∗ ns ns leadership ∗∗∗ ∗∗∗ ∗ ∗ ∗∗ ns ns ∗ ∗∗ ∗ control ∗∗∗ ∗∗∗ ∗∗ ∗∗ ns ns ns ns ns ns demands ∗∗∗ ∗∗∗ ∗∗∗ ∗∗ ∗∗∗ ∗∗ ∗ ns ∗∗ ns e clarity ∗∗∗ ∗∗∗ ns ∗∗∗ ∗ ns ∗ ns ∗∗ ns e conflict ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗ ∗∗∗ ns ∗∗∗ ns ns ial climate ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ns ∗∗ ns ns port superior ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ns ns ns ns ns port co-worker ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ns ns ns ns ns predictability ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗ ∗∗ ∗∗ ∗∗∗ ∗∗∗ ure employability ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗ ∗ ∗ ns ∗∗ mmary of univariate analyses, “adjusted own effect”: cross-sectional and prospective analyses. Separate, linear regressions with separate, organizational change at baseline as predictor and work factor as outcome. nge < 12 months. Adjusted for age, sex, skill level, and place of employment. Change > 24 months (Model II). Adjusted for age, sex, skill level, place of employment, and work factor at baseline. ∗p < 0.001, < 0.01, ∗∗∗p < 0.05. The present results demonstrated how employees perceived job demands to increase both short- and long-term following the implementation of various types of organizational change. In addition, a short-term effect was also observed for job control, with employees reporting less control following all included types of organizational change. These results are in line with prior studies reporting increased demands and lowered control following organizational changes such as restructuring and downsizing (Head et al., 2006; Egan et al., 2007; Tvedt et al., 2009). When implementing large-scaled change, job demands may increase due to change-related tasks, which comes in addition to ordinary tasks and responsibilities. Hence, the total workload may increase both while the process is running and more permanently (Kivimäki et al., 2001). One may speculate that the additional workload associated with change implementation may lower the feeling of job control and leave less resources for co-workers and superiors to be supportive during and following change. DISCUSSION For instance, the present results show that following a layoffprocess, To summarize, one interpretation of the pattern of associations observed in the present study could be that the adverse effects of organizational changes on the psychosocial work factors took place immediately or shortly after the change process. Moreover, while the effect diminished over time for most factors, the adverse effects remained or continued to unfold during the study’s timeframe for others. In the following, a brief discussion of the results pertaining to each, respective work factors is presented. December 2019 | Volume 10 | Article 2845 Frontiers in Psychology | www.frontiersin.org Frontiers in Psychology | www.frontiersin.org 7 Organizational Change and the Psychosocial Work Environment Fløvik et al. Job Tasks (Job Demands and Job Control) The Job Demand-Control (-Support) Model (JDCS) (Karasek and Theorell, 1990; Luchman and González-Morales, 2013) posits that the combination of high demands, low control and lack of support constitutes a high strain work environment, which may influence various employee outcomes, such as mental and somatic health, job satisfaction, turnover intention and productivity (Karasek, 1998; Bordia et al., 2004; Virtanen et al., 2006; Magnusson Hanson et al., 2008; van den Berg et al., 2008; Eller et al., 2009). However, high control and support may buffer the adverse effects of high demands; hence, facilitating a supportive social climate and help employees gain control over the new situation may be particularly important to buffer the adverse effects of the high job demands associated with extensive workplace changes (Van der Doef and Maes, 1999; Levi, 2000; Campbell-Jamison et al., 2001). Frontiers in Psychology | www.frontiersin.org Job Roles (Role Conflict and Role Clarity) Job o es ( o e Co ct a d o e C a ty) The present results also demonstrated reduced role clarity and increased role conflict, both short- and long-term, following various organizational changes. These results are in line with prior studies linking, for instance, exposure to company restructuring to an increase in role conflict and decrease in employees’ experience of clarity regarding their own tasks and responsibilities (Baillien and De Witte, 2009; Oreg et al., 2011). The heightened role conflict following organizational changes may potentially stem from additional or changed job demands without a corresponding adjustment of resource availability during or following changes (Oreg et al., 2011) or difficulties maintaining clearly defined goals and responsibilities for the individual worker at all times during an extensive change process. A restructuring process often involves redefining and rearranging employee tasks and responsibilities, and in the midst of the change-process, it may be challenging to design these explicitly to ensure that new demands are not in conflict with established ones. As December 2019 | Volume 10 | Article 2845 8 Organizational Change and the Psychosocial Work Environment Fløvik et al. TABLE 4 | Separate organizational change. TABLE 4 | Separate organizational change. Job Roles (Role Conflict and Role Clarity) Change <12 months prior Change >24 months prior Model I Model I Model II B p-Value 95% CI B p-Value 95% CI B p-Value 95% CI Empowering leadership Reorganization −0.09 0.000 0.87 to 0.96 −0.08 0.006 −0.13 to −0.02 −0.02 0.537 −0.06 to 0.03 Downsizing −0.15 0.000 0.82 to 0.91 −0.09 0.009 −0.15 to −0.02 −0.02 0.578 −0.07 to 0.04 Layoffs −0.22 0.000 0.74 to 0.87 −0.19 0.000 −0.29 to −0.09 −0.09 0.029 −0.18 to −0.01 Partial closure −0.23 0.000 0.04 to 0.85 −0.14 0.002 −0.22 to −0.05 −0.03 0.447 −0.10 to 0.05 Partial outsourcing −0.12 0.009 0.82 to 0.97 −0.02 0.679 −0.13 to 0.08 0.06 0.197 −0.06 to 0.15 Fair leadership Reorganization −0.08 0.000 0.90 to 0.94 −0.05 0.000 −0.08 to −0.03 −0.02 0.116 −0.04 to 0.01 Downsizing −0.05 0.000 0.92 to 0.97 −0.04 0.009 −0.07 to 0.01 −0.03 0.078 −0.05 to 0.00 Layoffs −0.05 0.013 0.92 to 0.99 −0.07 0.004 −0.12 to −0.02 −0.06 0.010 −0.10 to −0.01 Partial closure −0.08 0.000 0.89 to 0.95 −0.09 0.000 −0.13 to −0.05 −0.05 0.009 −0.09 to −0.01 Partial outsourcing −0.04 0.005 0.92 to 0.99 −0.08 0.002 −0.13 to −0.03 −0.06 0.016 −0.10 to −0.01 Job control decision Reorganization −0.07 0.000 0.90 to 0.97 −0.06 0.006 −0.11 to −0.02 −0.03 0.178 −0.06 to 0.01 Downsizing −0.10 0.000 0.97 to 0.95 −0.07 0.005 −0.13 to −0.02 −0.02 0.425 −0.06 to 0.03 Layoffs −0.11 0.001 0.94 to 0.95 −0.100 0.014 −0.18 to −0.02 −0.04 0.291 −0.11 to 0.03 Partial closure −0.08 0.005 0.97 to 0.98 0.01 0.784 −0.06 to 0.08 0.04 0.168 −0.02 to 0.10 Partial outsourcing −0.05 0.131 0.89 to 1.02 −0.01 0.752 −0.09 to 0.07 0.01 0.816 −0.06 to 0.08 Job demands quantitative Reorganization 0.16 0.000 1.13 to 1.22 0.19 0.000 0.14 to 0.24 0.06 0.019 0.01 to 0.10 Downsizing 0.22 0.000 1.19 to 1.30 0.13 0.002 0.05 to 0.21 0.04 0.289 −0.03 to 0.11 Layoffs 0.15 0.000 1.09 to 1.25 0.14 0.000 0.07 to 0.20 0.08 0.009 0.02 to 0.14 Partial closure 0.09 0.002 1.04 to 1.16 0.12 0.007 0.03 to 0.20 0.05 0.161 −0.02 to 0.12 Partial outsourcing 0.13 0.000 1.06 to 1.22 Role clarity Reorganization −0.09 0.000 0.88 to 0.94 −0.07 0.000 −0.11 to −0.03 −0.03 0.130 −0.06 to 0.01 Downsizing −0.09 0.000 0.87 to 0.95 −0.09 0.000 −0.14 to −0.05 −0.05 0.026 −0.09 to −0.01 Layoffs −0.05 0.075 0.90 to 1.01 −0.07 0.074 −0.14 to 0.01 −0.06 0.085 −0.13 to 0.01 Partial closure −0.12 0.000 0.85 to 0.93 −0.13 0.000 −0.19 to −0.06 −0.07 0.019 −0.12 to −0.01 Partial outsourcing −0.07 0.033 0.88 to 0.99 −0.11 0.007 −0.19 to −0.03 −0.07 0.065 −0.14 to 0.01 Role conflict Reorganization 0.24 0.000 1.23 to 1.33 0.22 0.000 0.18 to 0.26 0.10 0.000 0.06 to 0.13 Downsizing 0.28 0.000 1.27 to 1.39 0.20 0.000 0.14 to 0.25 0.04 0.076 −0.00 to 0.09 Layoffs 0.21 0.000 1.16 to 1.32 0.18 0.000 0.10 to 0.26 0.09 0.009 0.02 to 0.16 Partial closure 0.24 0.000 1.20 to 1.35 0.17 0.000 0.11 to 0.24 0.05 0.090 −0.00 to 0.11 Partial Outsourcing 0.12 0.001 1.05 to 1.21 0.02 0.562 −0.06 to 0.11 −0.04 0.320 −0.11 to 0.04 Social climate Reorganization −0.20 0.000 0.79 to 0.86 −0.20 0.000 −0.24 to −0.15 −0.09 0.000 −0.13 to −0.05 Downsizing −0.13 0.000 0.83 to 0.92 −0.13 0.000 −0.18 to −0.07 −0.03 0.158 −0.08 to 0.01 Layoffs −0.21 0.000 0.75 to 0.87 −0.20 0.000 −0.28 to −0.11 −0.09 0.018 −0.16 to −0.02 Partial closure −0.22 0.000 0.75 to 0.86 −0.21 0.000 −0.28 to −0.14 −0.09 0.005 −0.15 to −0.03 Partial outsourcing −0.23 0.000 0.73 to 0.86 −0.23 0.000 −0.31 to −0.14 −0.14 0.000 −0.22 to −0.06 Support co-worker Reorganization −0.08 0.000 0.89 to 0.95 −0.06 0.002 −0.10 to −0.02 −0.02 0.173 −0.06 to 0.01 Downsizing −0.10 0.000 0.98 to 0.94 −0.09 0.000 −0.13 to −0.04 −0.04 0.072 −0.08 to 0.00 Layoffs −0.11 0.000 0.85 to 0.95 −0.10 0.007 −0.17 to −0.03 −0.05 0.097 −0.12 to 0.01 Partial closure −0.14 0.000 0.83 to 0.91 −0.10 0.001 −0.17 to −0.04 −0.04 0.151 −0.09 to 0.02 Partial outsourcing −0.12 0.000 0.84 to 0.95 −0.09 0.020 −0.17 to −0.02 −0.04 0.251 −0.11 to 0.03 Support superior Reorganization −0.16 0.000 0.82 to 0.89 −0.13 0.000 −0.18 to −0.08 −0.04 0.086 −0.09 to 0.01 Downsizing −0.21 0.000 0.77 to 0.85 −0.14 0.000 −0.20 to −0.08 −0.04 0.181 −0.09 to 0.02 Layoffs −0.25 0.000 0.72 to 0.84 −0.18 0.000 −0.28 to −0.09 −0.07 0.099 −0.15 to 0.01 Partial closure −0.29 0.000 0.70 to 0.80 −0.17 0.000 −0.26 to −0.09 −0.03 0.418 −0.11 to 0.04 Partial outsourcing −0.16 0.000 0.79 to 0.93 −0.11 0.040 −0.21 to −0.01 −0.01 0.799 −0.10 to 0.08 Predictability 1 month Reorganization −0.18 0.000 0.81 to 0.86 −0.13 0.00 −0.17 to −0.09 −0.06 0.001 −0.09 to −0.02 Downsizing −0.15 0.000 0.83 to 0.90 −0.10 0.000 −0.15 to −0.06 −0.05 0.022 −0.10 to −0.01 Layoffs −0.19 0.000 0.78 to 0.88 −0.18 0.000 −0.26 to −0.10 −0.12 0.002 −0.19 to −0.04 Partial closure −0.20 0.000 0.78 to 0.86 −0.21 0.000 −0.28 to −0.15 −0.13 0.000 −0.19 to −0.07 Partial outsourcing −0.21 0.000 0.76 to 0.87 −0.19 0.000 −0.27 to −0.11 −0.12 0.001 −0.20 to −0.05 (Continued) December 2019 | Volume 10 | Article 2845 Frontiers in Psychology | www.frontiersin.org 9 Organizational Change and the Psychosocial Work Environment Fløvik et al. Social Relations (Support From Co-workers and Social Climate) pp p) The present results show that employees perceive their superiors as less fair and empowering following various types of organizational change, both short- and long-term. In addition, support from superior was also perceived to be lower following the included organizational changes, but effects were only present short-term. Various factors may influence employees’ perception of leadership during and following organizational changes. Implementing extensive change may, for instance, put increased pressure and workload on management and superiors, leaving fewer resources to preserve a sense of inclusive, supportive, and fair leadership style (Hoag et al., 2002). The need to make unpopular decisions may also affect how employees perceive management to be fair, empowering or supportive during or following the change process, especially if the process does not follow pre-existing guidelines and expectations (Tyler and De Cremer, 2005). The extent to which management includes employee concerns and perspectives in the process, as well as how management communicates the change have also been reported to affect how superiors and leadership are perceived both prior to, during and following changes (Hoag et al., 2002; Riolli and Savicki, 2006). Prior studies have demonstrated the importance of employee perceptions of organizational justice during organizational changes (Virtanen and Elovainio, 2018). The perception of low fairness from management has been associated with poor social climate and reduced productivity (Schyns and Schilling, 2013; Virtanen and Elovainio, 2018), as well as long-term and reoccurring sick leave, mental distress and somatic health complaints (Tyler and De Cremer, 2005; Riolli and Savicki, 2006; Meierhans et al., 2008; Robbins et al., 2012; Leineweber et al., 2017). On the other hand, employees who perceive leaders to act procedurally fair during organizational changes are more accepting of the change and view management ) Implementing organizational changes may also influence various aspects of an organization’s social and relational environment. The present results show both social climate and perceived support from co-workers to be lower following various types of change, although long-term effects were only shown for social climate. Various aspects of change implementation may affect social relations within the organization. Rearranging collegial composition, i.e., losing and/or being introduced to new co-workers, competing for the same positions during a restructuring process or getting a new superior, may all influence an organization’s social cohesion or employees ability to provide others with the support they normally are able to. Job Roles (Role Conflict and Role Clarity) TABLE 4 | Continued Change <12 months prior Change >24 months prior Model I Model I Model II B p-Value 95% CI B p-Value 95% CI B p-Value 95% CI Predictability 2 years Reorganization −0.13 0.000 0.84 to 0.92 −0.11 0.000 −0.16 to −0.05 −0.06 0.023 −0.11 to 0.01 Downsizing −0.21 0.000 0.77 to 0.86 −0.13 0.000 −0.19 to −0.06 −0.06 0.047 −0.12 to −0.00 Layoffs −0.21 0.000 0.75 to 0.88 −0.15 0.003 −0.25 to −0.05 −0.10 0.021 −0.19 to −0.02 Partial closure −0.28 0.000 0.71 to 0.81 −0.16 0.000 −0.25 to −0.08 −0.06 0.085 −0.14 to 0.01 Partial outsourcing −0.30 0.000 0.68 to 0.81 −0.25 0.000 −0.35 to −0.14 −0.14 0.004 −0.23 to −0.04 Univariate analyses, “adjusted own effect”: cross-sectional and prospective analyses, Separate, linear regressions with separate, organizational change at baseline as predictor and work factor as outcome. Change < 12 months prior. Model I: adjusted for age, sex, skill level, and place of employment. Change > 24 months prior. Model I: adjusted for age, sex, skill level, and place of employment. Model II: adjusted for age, sex, skill level, place of employment, and work factor at baseline. The bold significance values are p < 0.05. Univariate analyses, “adjusted own effect”: cross-sectional and prospective analyses, Separate, linear regressions with separate, organizational change at baseline as predictor and work factor as outcome. Change < 12 months prior. Model I: adjusted for age, sex, skill level, and place of employment. Change > 24 months prior. Model I: adjusted for age, sex, skill level, and place of employment. Model II: adjusted for age, sex, skill level, place of employment, and work factor at baseline. The bold significance values are p < 0.05. and leaders as more competent and trustworthy in handling the change (Tyler and De Cremer, 2005). The present results showing how leaders are rated as less fair and empowering following organizational change processes may be of interest when planning change, as counteracting these effects may improve both the process, consequences and results of extensive workplace changes. prior meta-analyses have shown role conflict and –uncertainty to be related to employee health complaints (Stansfeld and Candy, 2006; Schmidt et al., 2014), it seems crucial to ensure role clarity and prevent role conflict during the process of extensive change. Social Relations (Support From Co-workers and Social Climate) The current results agree with prior studies reporting increased conflict, demoralization and reduced support following organizational change (Campbell and Pepper, 2007). Support and social climate have both been linked to employee health, productivity and (Magnusson Hanson et al., 2008; Ljungblad et al., 2014; Charoensukmongkol et al., 2016; Yang et al., 2016; Geldart et al., 2018) which makes focusing on the effects of change on the organization’s social relations an important aspect to consider in order to secure a healthy and successful change process. Job Predictability (Short-Term Job Predictability and Future Employability) Frontiers in Psychology | www.frontiersin.org Job Predictability (Short-Term Job Predictability and Future Employability) When implementing large-scaled organizational changes, it is naturally challenging to know how both the process and end- result will turn out. Thus, organizational changes are naturally associated with a certain degree of uncertainty. The present December 2019 | Volume 10 | Article 2845 Frontiers in Psychology | www.frontiersin.org 10 Organizational Change and the Psychosocial Work Environment Fløvik et al. TABLE 5 | Separate organizational change. TABLE 5 | Separate organizational change. December 2019 | Volume 10 | Article 2845 Job Predictability (Short-Term Job Predictability and Future Employability) Change <12 months prior Change >24 months prior Model I Model I Model II B p-Value 95% CI B p-Value 95% CI B p-Value 95% CI Empowering leadership Reorganization −0.05 0.108 0.90 to 1.01 −0.05 0.096 0.09 to 1.01 −0.01 0.660 0.94 to 1.039 Downsizing −0.02 0.562 0.91 to 1.05 −0.04 0.355 0.90 to 1.04 −0.00 0.960 −0.94 to 1.06 Layoffs −0.12 0.023 0.79 to 0.98 −0.14 0.010 0.78 to 0.97 −0.09 0.063 0.84 to 1.01 Partial closure −0.11 0.021 0.81 to 0.98 −0.09 0.069 0.83 to 1.01 −0.03 0.547 0.90 to 1.06 Partial outsourcing 0.05 0.328 0.95 to 1.18 0.05 0.357 0.9 to 1.18 0.08 0.092 0.99 to 1.20 Fair leadership Reorganization −0.04 0.009 0.94 to 0.99 −0.04 0.010 0.94 to 0.99 −0.01 0.537 0.97 to 7.02 Downsizing −0.01 0.797 0.96 to 1.03 0.01 0.748 0.96 to 1.03 −0.01 0.707 0.96 to 1.03 Layoffs −0.03 0.291 0.93 to 1.02 −0.04 0.129 0.92 to 1.01 −0.04 0.085 0.92 to 1.01 Partial closure −0.05 0.033 0.91 to 1.00 −0.05 0.024 0.91 to 0.99 −0.02 0.240 0.94 to 1.02 Partial outsourcing −0.04 0.119 0.91 to 1.01 −0.05 0.081 0.91 to 0.1.01 −0.04 0.106 0.92 to 1.01 Job control decision Reorganization −0.05 0.020 0.92 to 0.99 −0.05 0.035 0.91 to 0.99 −0.03 0.199 0.94 to 1.01 Downsizing −0.07 0.009 0.89 to 0.98 −0.07 0.018 0.88 to 0.99 −0.02 0.324 0.93 to 1.02 Layoffs −0.05 0.144 0.88 to 1.02 −0.08 0.057 0.85 to 1.00 −0.04 0.228 0.98 to 1.03 Partial closure −0.02 0.550 0.92 to 1.05 0.06 0.117 0.99 to 1.15 0.06 0.064 1.00 to 1.13 Partial outsourcing 0.01 0.857 0.94 to 1.09 0.01 0.747 0.93 to 1.10 0.01 0.769 0.94 to 1.09 Job demands quantitative Reorganization 0.13 0.000 1.09 to 1.19 0.12 0.000 1.10 to 1.18 0.05 0.008 1.01 to 1.10 Downsizing 0.18 0.000 1.13 to 1.26 0.14 0.000 1.10 to 1.22 0.02 0.355 0.97 to 1.08 Layoffs 0.04 0.333 0.96 to 1.11 0.03 0.453 0.95 to 1.13 0.01 0.830 0.94 to 1.09 Partial closure −0.05 0.141 0.89 to 1.02 0.02 0.544 0.95 to 1.11 0.05 0.165 0.98 to 1.12 Partial outsourcing 0.05 0.210 0.97 to 1.13 0.02 0.634 0.94 to 1.11 0.01 0.742 0.94 to 1.09 Role clarity Reorganization −0.07 0.000 0.90 to 0.96 −0.04 0.038 0.92 to 0.99 −0.01 0.660 0.9 to 1.03 Downsizing −0.06 0.011 0.90 to 0.99 −0.05 0.055 0.91 to 1.00 −0.02 0.365 0.94 to 1.02 Layoffs 0.02 0.525 0.96 to 1.09 −0.02 0.93 to 1.08 −0.03 0.448 0.91 to 1.04 Partial closure −0.06 0.041 0.89 to 0.100 −0.08 0.019 0.96 to 0.99 −0.05 0.149 0.90 to 1.02 Partial outsourcing −0.01 0.820 0.93 to 1.06 −0.05 0.203 0.87 to 1.03 −0.04 0.277 0.89 to 1.03 Role conflict Reorganization 0.19 0.000 1.16 to 1.26 0.19 0.000 1.15 to 1.26 0.09 0.000 1.05 to 1.14 Downsizing 0.20 0.000 1.16 to 1.28 0.12 0.000 1.06 to 1.20 0.00 0.900 0.95 to 1.06 Layoffs 0.07 0.058 1.00 to 1.150 0.09 0.043 1.00 to 1.18 0.08 0.046 1.00 to 1.16 Partial closure 0.09 0.006 1.03 to 1.17 0.08 0.045 1.00 to 1.16 0.03 0.344 0.97 to 1.10 Partial outsourcing −0.04 0.246 0.89 to 1.03 −0.11 0.012 0.82 to 0.98 −0.09 0.022 0.85 to 0.99 Social climate Reorganization −0.17 0.000 0.81 to 0.88 −0.16 0.000 0.82 to 0.89 −0.07 0.000 0.89 to 0.97 Downsizing −0.09 0.001 0.87 to 0.96 −0.02 0.481 0.93 to 1.04 0.02 0.471 0.97 to 1.07 Layoffs −0.13 0.000 0.82 to 0.95 −0.11 0.009 0.82 to 0.97 −0.06 0.122 0.87 to 1.02 Partial closure −0.15 0.000 0.81 to 0.82 −0.11 0.006 0.93 to 0.97 −0.04 0.211 0.89 to 1.03 Partial outsourcing −0.03 0.482 0.91 to 1.05 −0.12 0.013 0.81 to 0.98 −0.10 0.016 0.84 to 0.98 Support co-worker Reorganization −0.05 0.007 0.81 to 0.99 −0.04 0.054 0.92 to 1.00 −0.02 0.328 0.95 to 1.02 Downsizing −0.04 0.102 0.92 to 1.01 −0.04 0.099 0.91 to 1.01 −0.02 0.388 0.94 to 1.03 Layoffs −0.05 0.091 0.89 to 1.01 −0.05 0.208 0.88 to 1.03 −0.03 0.371 0.91 to 1.04 Partial closure −0.07 0.020 0.88 to 0.99 −0.06 0.084 0.88 to 1.01 −0.03 0.416 0.92 to 1.04 Partial outsourcing −0.05 0.132 0.89 to 1.02 −0.04 0.388 0.89 to 1.05 −0.01 0.779 0.92 to 1.06 Support superior Reorganization −0.09 0.000 0.87 to 0.96 −0.09 0.001 0.86 to 0.96 −0.03 0.155 0.92 to 1.01 Downsizing −0.11 0.000 0.85 to 0.95 −0.07 0.037 0.87 to 1.00 −0.02 0.508 0.92 to 1.04 Layoffs −0.14 0.001 0.81 to 0.95 −0.11 0.041 0.83 to 0.99 −0.05 0.251 0.87 to 1.04 Partial closure −0.17 0.000 0.78 to 0.91 −0.10 0.036 0.93 to 0.99 −0.01 0.745 0.91 to 1.07 Partial outsourcing 0.00 0.959 0.92 to 1.09 −0.00 0.983 0.90 to 1.11 0.02 0.687 0.93 to 1.18 Predictability 1 month Reorganization −0.14 0.000 0.83 to 0.90 −0.09 0.000 0.88 to 0.95 −0.04 0.056 0.93 to 1.00 Downsizing −0.07 0.005 0.89 to 0.98 −0.01 0.605 0.93 to 1.04 0.00 0.967 0.95 to 1.05 Layoffs −0.09 0.011 0.86 to 0.98 −0.10 0.021 0.93 to 0.99 −0.07 0.066 0.86 to 1.01 Partial closure −0.08 0.012 0.87 to 0.98 −0.14 0.000 0.81 to 0.94 −0.09 0.009 0.85 to 0.98 Partial outsourcing −0.10 0.004 0.84 to 0.97 −0.09 0.041 0.84 to 0.99 −0.06 0.133 0.87 to 1.02 (Continued) December 2019 | Volume 10 | Article 2845 Frontiers in Psychology | www.frontiersin.org 11 Organizational Change and the Psychosocial Work Environment Fløvik et al. Job Predictability (Short-Term Job Predictability and Future Employability) Reduced job predictability, i.e., attenuated ability to form reasonable expectations about the future, regarding both short-term job characteristics and long-term employment prospects, is intrinsically linked to the concept of job insecurity, which has been linked to employee outcomes such as somatic and mental health complaints (Hellgren et al., 1999; Ferrie, 2001; Ferrie et al., 2002; Staufenbiel and König, 2010; Landsbergis et al., 2014; De Witte et al., 2016), lowered efficiency, reduced organizational citizenship behavior and higher turnover intention (Hellgren et al., 1999; Probst, 2003; Staufenbiel and König, 2010). It may not be surprising that job predictability is temporarily affected by an extensive change process, however, the current results also indicate that the reduction in job predictability persists long after change implementation is completed. Prior studies have also shown that even though the cause of job insecurity was removed, the insecurity did not completely vanish (Ferrie et al., 2002), indicating that the sense of uncertainty may persist for longer periods. Furthermore, this lowered predictability does not only pertain to one’s current job, but also future job prospects, e.g., perceived future employability. Long-term effects on perceived job security have also been reported in prior studies (Ferrie et al., 1998; Probst, 2003). Such long-term effects on perceptions of predictability may be due to reduced trust in management and breaches in the implicit psychological contract in the workplace (Morgan and Zeffane, 2003). Reduced trust in management and perceived breaches in the psychological contract have been linked to various types of organizational change (Turnley and Feldman, 1998; Bellou, 2006). Although reduced predictability may be a common, proximal consequence of organizational change, it does not follow that it is a natural lasting consequence of change. Characteristics of change processes may influence the perception of unpredictability. For instance, prior studies have linked the extent to which employees are involved in the change process, e.g., employee participation in planning and implementing changes, with lower levels of uncertainty and higher levels of perceived control (Bordia et al., 2004). These results have been supported by prior studies reporting employee participation to be linked with higher perceived control, lower levels of job insecurity and reduced mental health complaints and sick leave (Bond and Bunce, 2001; Abildgaard et al., 2018). Results from the present study showed both short- and long-term adverse effects on job predictability and perceived future employability following organizational change. Job Predictability (Short-Term Job Predictability and Future Employability) Characteristics of change processes may influence the perception of unpredictability. For instance, prior studies have linked the extent to which employees are involved in the change process, e.g., employee participation in planning Job Predictability (Short-Term Job Predictability and Future Employability) In light of the aforementioned studies linking both detrimental effects on both health and productivity with such uncertainty (Ferrie, 2001; Ferrie et al., 2002; Hellgren and Sverke, 2003; Staufenbiel and König, 2010; Landsbergis et al., 2014; De Witte et al., 2016), it seems imperative to keep job uncertainty to a minimum during and following change, possibly through involving employees in the process and by promoting a sense of control and support (Bordia et al., 2004). study showed employees’ sense of job predictability and future employability to be lower following various specific company changes both short- and long-term. Similar results have been reported in previous studies (Kivimäki et al., 2001; Probst, 2003; Baillien and De Witte, 2009). Reduced job predictability, i.e., attenuated ability to form reasonable expectations about the future, regarding both short-term job characteristics and long-term employment prospects, is intrinsically linked to the concept of job insecurity, which has been linked to employee outcomes such as somatic and mental health complaints (Hellgren et al., 1999; Ferrie, 2001; Ferrie et al., 2002; Staufenbiel and König, 2010; Landsbergis et al., 2014; De Witte et al., 2016), lowered efficiency, reduced organizational citizenship behavior and higher turnover intention (Hellgren et al., 1999; Probst, 2003; Staufenbiel and König, 2010). It may not be surprising that job predictability is temporarily affected by an extensive change process, however, the current results also indicate that the reduction in job predictability persists long after change implementation is completed. Prior studies have also shown that even though the cause of job insecurity was removed, the insecurity did not completely vanish (Ferrie et al., 2002), indicating that the sense of uncertainty may persist for longer periods. Furthermore, this lowered predictability does not only pertain to one’s current job, but also future job prospects, e.g., perceived future employability. Long-term effects on perceived job security have also been reported in prior studies (Ferrie et al., 1998; Probst, 2003). Such long-term effects on perceptions of predictability may be due to reduced trust in management and breaches in the implicit psychological contract in the workplace (Morgan and Zeffane, 2003). Reduced trust in management and perceived breaches in the psychological contract have been linked to various types of organizational change (Turnley and Feldman, 1998; Bellou, 2006). Although reduced predictability may be a common, proximal consequence of organizational change, it does not follow that it is a natural lasting consequence of change. Frontiers in Psychology | www.frontiersin.org Job Predictability (Short-Term Job Predictability and Future Employability) TABLE 5 | Continued Change <12 months prior Change >24 months prior Model I Model I Model II B p-Value 95% CI B p-Value 95% CI B p-Value 95% CI Predictability 2 years Reorganization −0.07 0.003 0.88 to 0.98 −0.07 0.022 0.88 to 0.99 −0.04 0.179 0.92 to 1.02 Downsizing −0.11 0.000 0.84 to 0.95 −0.05 0.141 0.88 to 1.02 −0.02 0.561 0.92 to 1.05 Layoffs −0.08 0.080 0.85 to 1.01 −0.06 0.230 0.85 to 1.04 −0.06 0.191 0.86 to 1.03 Partial closure −0.16 0.000 0.79 to 0.93 −0.06 0.215 0.96 to 1.04 −0.00 0.981 0.92 to 1.08 Partial outsourcing −0.17 0.000 0.77 to 0.92 −0.18 0.001 0.75 to 0.93 −0.11 0.024 0.81 to 0.99 Multivariate analyses: cross-sectional and prospective analyses. Separate, multivariable linear regressions with work factor as outcome and specific change at baseline as predictor, mutually adjusted for each other. Change < 12 months prior. Model I: adjusted for age, sex, skill level, and place of employment. Change > 24 months prior. Model I: adjusted for age, sex, skill level, and place of employment. Model II: adjusted for age, sex, skill level, place of employment, and work factor at baseline. The bold significance values are p < 0.05. Multivariate analyses: cross-sectional and prospective analyses. Separate, multivariable linear regressions with work factor as outcome and specific change at baseline as predictor, mutually adjusted for each other. Change < 12 months prior. Model I: adjusted for age, sex, skill level, and place of employment. Change > 24 months prior. Model I: adjusted for age, sex, skill level, and place of employment. Model II: adjusted for age, sex, skill level, place of employment, and work factor at baseline. The bold significance values are p < 0.05. study showed employees’ sense of job predictability and future employability to be lower following various specific company changes both short- and long-term. Similar results have been reported in previous studies (Kivimäki et al., 2001; Probst, 2003; Baillien and De Witte, 2009). Effects of Multiple or Repeated Organizational Changes on Factors in the Work Environment The present study demonstrated a stronger adverse effect on the majority of work factors following multiple changes at one time point compared to one specific change only. Furthermore, following repeated organizational changes, the adverse effects were stronger for all included work factors. These results are in line with prior studies reporting stronger effects following exposure to multiple or repeated organizational changes in work factors, such as role conflict and -ambiguity, social support, job insecurity, job demands, trust in management, and turnover intention (Ferrie et al., 2002; Isaksson et al., 2002; Moore et al., 2004; Wagstaffet al., 2016). Prior studies have also reported stronger effects following repeated change on various somatic and mental health complaints (Isaksson et al., 2002; Moore et al., 2004; Oreg et al., 2011). Although it remains uncertain why, these results indicate a cumulative effect of organizational change events on multiple aspects in the work environment, suggesting that organizations and its December 2019 | Volume 10 | Article 2845 Frontiers in Psychology | www.frontiersin.org 12 Organizational Change and the Psychosocial Work Environment Fløvik et al. TABLE 6 | Multiple organizational changes (at baseline). Effects of Multiple or Repeated Organizational Changes on Factors in the Work Environment Change <12 months prior Change >24 months prior Model I Model I Model II B p-Value 95% CI B p-Value 95% CI B p-Value 95% CI Empowering leadership No change at T1 – – – – – – – – – One change at T1 −0.03 0.329 0.91 to 1.03 −0.03 0.362 −0.10 to 0.04 −0.01 0.854 −0.06 to 0.05 Two or more changes at T1 −0.12 0.001 0.83 to 0.95 −0.12 0.001 −0.19 to −0.05 −0.02 0.545 −0.08 to 0.04 Fair leadership No change at T1 – – – – – – – – – One change at T1 −0.02 0.145 0.95 to 1.01 −0.02 0.158 −0.05 to 0.01 −0.00 0.918 −0.03 to 0.03 Two or more changes at T1 −0.07 0.000 0.91 to 0.97 0.07 0.000 −0.10 to−0.04 −0.03 0.036 −0.07 to−0.00 Job demands quantitative No change at T1 – – – – – – – – – One change at T1 0.14 0.000 1.09 to 1.21 0.14 0.000 0.09 to 0.20 0.07 0.003 0.02 to 0.11 Two or more changes at T1 0.25 0.000 1.21 to 1.36 0.25 0.000 0.19 to 0.31 0.10 0.000 0.18 to 0.28 Job control decision No change at T1 – – – – – – – – – One change at T1 −0.05 0.059 0.90 to 1.00 −0.04 0.185 −0.09 to 0.02 −0.03 0.202 −0.07 to 0.02 Two or more changes at T1 −0.09 0.002 0.86 to 0.97 −0.08 0.007 −0.14 to−0.02 −0.02 0.399 −0.07 to 0.03 Role clarity No change at T1 – – – – – – – – – One change at T1 −0.02 0.448 0.94 to 1.03 −0.02 0.403 −0.07 to 0.03 −0.00 0.838 −0.04 to 0.04 Two or more changes at T1 −0.112 0.000 0.84 to 0.94 −0.12 0.000 −0.17 to−0.07 −0.06 0.015 −0.10 to−0.01 Role conflict No change at T1 – – – – – – – – – One change at T1 0.16 0.000 1.11 to 1.24 0.16 0.000 0.11 to 0.22 0.08 0.001 0.03 to 0.12 Two or more changes at T1 0.27 0.000 1.24 to 1.39 0.27 0.000 0.21 to 0.33 0.08 0.003 0.03 to 0.13 Social climate No change at T1 – – – – – – – – – One change at T1 −0.10 0.000 0.86 to 0.95 −0.11 0.000 −0.16 to−0.05 −0.04 0.078 −0.09 to 0.01 Two or more changes at T1 −0.26 0.000 0.73 to 0.82 −0.26 0.000 −0.31 to−0.20 −0.10 0.000 −0.16 to−0.05 Support co-worker No change at T1 – – – – – – – – – One change at T1 −0.01 0.692 0.95 to 1.04 −0.01 0.736 −0.05 to 0.04 0.01 0.764 −0.03 to 0.05 Two or more changes at T1 −0.09 0.000 0.86 to 0.96 −0.10 0.000 −0.15 to 0.04 −0.05 0.052 −0.09 to 0.00 Support superior No change at T1 – – – – – – – – – One change at T1 −0.07 0.019 0.87 to 0.99 −0.07 0.033 −0.13 to−0.01 −0.04 0.207 −0.09 to 0.02 Two or more changes at T1 −0.19 0.000 0.77 to 0.89 −0.19 0.000 −0.26 to−0.12 −0.05 0.096 −0.11 to 0.01 Predictability 1 month No change at T1 – – – – - - - - - One change at T1 −0.07 0.003 0.89 to 0.98 −0.07 0.003 −0.11 to−0.02 −0.03 0.124 −0.07 to 0.01 Two or more changes at T1 −0.21 0.000 0.77 to 0.85 −0.22 0.000 −0.27 to−0.16 −0.11 0.000 −0.16 to−0.06 Predictability 2 years No change at T1 – – – – – – – – – One change at T1 −0.05 0.109 0.89 to 1.01 −0.06 0.098 −0.12 to 0.01 −0.04 0.251 −0.10 to 0.03 Two or more changes at T1 −0.19 0.000 0.77 to 0.89 −0.19 0.000 −0.26 to−0.12 −0.10 0.002 −0.17 to−0.04 Cross-sectional and prospective analyses. Effects of Multiple or Repeated Organizational Changes on Factors in the Work Environment Separate, linear regressions with frequency of change at baseline as predictor and work factor as outcome. Change < 12 months prior. Model I: adjusted for age, sex, skill level, and place of employment. Change > 24 months prior. Model I: adjusted for age, sex, skill level, and place of employment. Model II: adjusted for age, sex, skill level, place of employment, and work factor at baseline. The bold significance values are p < 0.05. TABLE 6 | Multiple organizational changes (at baseline). Cross-sectional and prospective analyses. Separate, linear regressions with frequency of change at baseline as predictor and work factor as outcome. Change < 12 months prior. Model I: adjusted for age, sex, skill level, and place of employment. Change > 24 months prior. Model I: adjusted for age, sex, skill level, and place of employment. Model II: adjusted for age, sex, skill level, place of employment, and work factor at baseline. The bold significance values are p < 0.05. following repeated organizational changes (Zapf et al., 1996). These models posit that repeated exposure to a stressor may wear out an individual’s coping resources, which over time may lead to fatigue and weaken the ability to cope when employees do not adapt to or assimilate to the new situation to the extent that the impact of change dissipates. Stress- vulnerability models may help explain the stronger effects on both perceptions of the work environment and health December 2019 | Volume 10 | Article 2845 Frontiers in Psychology | www.frontiersin.org 13 Organizational Change and the Psychosocial Work Environment Fløvik et al. TABLE 7 | Repeated organizational change. Effects of Multiple or Repeated Organizational Changes on Factors in the Work Environment Model I Model II B p 95% CI B p 95% CI Empowering leadership No change at T1 or T2 – – – – – – At least one change at T1 only −0.08 0.064 −0.17 to 0.01 −0.02 0.536 −0.10 to 0.05 At least one change at T2 only −0.15 0.002 −0.05 to −0.06 −0.13 0.003 −0.21 to −0.04 At least one change at both T1 T2 −0.16 0.000 −0.24 to −0.08 −0.08 0.013 −0.15 to −0.02 Fair leadership No change at T1 or T2 – – – – – – At least one change at T1 only −0.04 0.026 −0.08 to −0.01 −0.02 0.430 −0.05 to 0.02 At least one change at T2 only −0.09 0.000 −0.14 to −0.05 −0.07 0.001 −0.11 to −0.03 At least one change at both T1 T2 −0.11 0.000 −0.14 to −0.07 −0.06 0.000 −0.10 to −0.03 Job demands quantitative No change at T1 or T2 – – – – – – At least one change at T1 only 0.19 0.000 0.12 to 0.26 0.08 0.005 0.23 to 0.14 At least one change at T2 only 0.17 0.000 0.10 to 0.26 0.10 0.001 0.04 to 0.17 At least one change at both T1 T2 0.31 0.000 0.24 to 0.37 0.15 0.000 0.10 to 0.20 Job control decision No change at T1 or T2 – – – – – – At least one change at T1 only −0.03 0.348 −0.10 to 0.04 0.02 0.515 −0.08 to 0.40 At least one change at T2 only −0.10 0.006 −0.19 to 0.03 −0.08 0.019 −0.14 to −0.01 At least one change at both T1 T2 −0.15 0.000 −0.21 to −0.09 −0.10 0.000 −0.15 to −0.04 Role clarity No change at T1 or T2 – – – – – – At least one change at T1 only −0.05 0.087 −0.11 to 0.01 −0.03 0.236 0.08 to 0.02 At least one change at T2 only −0.11 0.002 −0.17 to −0.04 −0.11 0.000 −0.16 to −0.05 At least one change at both T1 T2 −0.13 0.000 −0.19 to −0.08 −0.09 0.000 −0.14 to −0.05 Role conflict No change at T1 or T2 – – – – – – At least one change at T1 only 0.14 0.000 0.07 to 0.21 0.02 0.464 −0.04 to 0.08 At least one change at T2 only 0.20 0.000 0.13 to 0.28 0.10 0.006 0.03 to 0.16 At least one change at both T1 T2 0.39 0.000 −0.33 to 0.45 0.18 0.000 0.12 to 0.24 Social climate No change at T1 or T2 – – – – – – At least one change at T1 only −0.14 0.000 −0.21 to −0.08 −0.06 0.059 −0.11 to 0.00 At least one change at T2 only −0.21 0.000 −0.28 to −0.14 −0.16 0.000 −0.23 to −0.10 At least one change at both T1 T2 −0.34 0.000 −0.40 to −0.28 −0.19 0.000 −0.24 to −0.13 Support co-worker No change at T1 or T2 – – – – – – At least one change at T1 only −0.03 0.343 −0.09 to 0.03 0.01 0.800 −0.05 to 0.06 At least one change at T2 only −0.12 0.001 −0.19 to −0.05 −0.09 0.004 −0.15 to −0.03 At least one change at both T1 T2 −0.13 0.000 −0.18 to −0.08 −0.08 0.001 −0.13 to 0.03 Support superior No change at T1 or T2 – – – – – – At least one change at T1 only −0.11 0.006 −0.19 to −0.03 −0.04 0.322 −0.10 to 0.03 At least one change at T2 only −0.22 0.000 −0.31 to −0.13 −0.17 0.000 −0.24 to −0.09 At least one change at both T1 T2 −0.27 0.000 −0.34 to −0.20 −0.16 0.000 −0.22 to −0.09 Predictability 1 month No change at T1 or T2 – – – – – – At least one change at T1 only −0.06 0.033 −0.12 to −0.01 −0.01 0.741 −0.06 to 0.04 At least one change at T2 only −0.16 0.000 −0.22 to −0.09 −0.11 0.000 −0.17 to −0.06 At least one change at both T1 T2 −0.27 0.000 −0.32 to −0.22 −0.17 0.000 −0.22 to −0.12 Predictability 2 years At least one change at T1 only −0.08 0.063 −0.17 to 0.00 −0.06 0.123 −0.14 to 0.02 At least one change at T2 only −0.12 0.017 −0.22 to −0.02 −0.13 0.004 −0.22 to −0.04 At least one change at both T1 T2 −0.21 0.000 −0.29 to −0.13 −0.16 0.000 −0.23 to −0.08 Separate, linear regressions with repeated change at baseline as predictor and work factor at follow-up as outcome. Frontiers in Psychology | www.frontiersin.org Future Perspectives One may speculate whether employees not holding permanent positions experience extensive company changes as more of a threat to for instance their job security than those permanently employed. Due to the sample composition, the present results may underestimate the impact of organizational change on certain aspects of the work environment. p All data were collected by questionnaires; hence, both self- report bias and common-method bias could influence responses (Moorman and Podsakoff, 1992; Donaldson and Grant-Vallone, 2002). Precautions were taken in order to minimize such effects, e.g., a temporal separation of measurements, forced-choice items (Nederhof, 1985), baseline adjustments for all outcome variables and differences in wording and response options for predictor and outcome may all reduce the risk of common- method variance (Podsakoffet al., 2003). However, one cannot rule of the potential effects, the study had a prospective design with a 2-year interval between baseline and follow-up. This may not be the optimal interval to measure the effects of changes on the psychosocial work environment, with short- term effects possibly being present, but diminishing in the years between baseline and follow-up (Oreg et al., 2011). For this reason, the present study included both cross-sectional and prospective analyses. Keeping in mind the limitations in cross- sectional design regarding inference of causation (Levin, 2006), we chose to include these analyses due to items pertaining to change inquired into change taking place up to 12 months Effects of Multiple or Repeated Organizational Changes on Factors in the Work Environment Model I: adjusted for age, sex, skill level, and place of employment. Model II: adjusted for age, sex, skill level, place of employment, and work factor at baseline. The bold significance values are p < 0.05. TABLE 7 | Repeated organizational change. to be associated with adverse effects on multiple factors in the psychosocial work environment known to influence both employee health and productivity, it seems imperative for organizations to prevent these unfavorable effects when planning and implementing change in order to secure both employee health and company sustainability. As the rate of change is re-exposed to the stressor. Hence, the stronger effects on employee perception of various aspect of the work environment following repeated company changes could be a result of fatigue and reduced resources to cope with change as exposure to prior changes have worn-out coping resources. As both prior and the present results show organizational changes December 2019 | Volume 10 | Article 2845 Frontiers in Psychology | www.frontiersin.org 14 Organizational Change and the Psychosocial Work Environment Fløvik et al. increasing in contemporary work life a larger proportion of the workforce is likely to be exposed to organizational changes more than once during their career. As more employees will be facing multiple, large-scaled workplace changes, a focus on the prevention of the adverse effects associated with such changes seems vital. prior, while items related to work factors considered the perception of the employee’s current work environment. In addition, the risk of reverse causation in the relationship between organizational change and work factors in the study is considered small as extensive company changes are likely to be events whose occurrence and frequency to a lesser degree is affected by employee’s perception of specific psychological and social work factors. DATA AVAILABILITY STATEMENT The datasets generated for this study are available on request to the corresponding author. Methodological Considerations Certain methodological limitations may have affected the generalizability of the present results. Regarding attrition, response rate at baseline was 82%, while 58% participated at follow up. Dropout was associated with being employed in the private sector, working in professions requiring 13– 15, 10–12, and less than 10 years of formal education respectively. Hence, selection bias may have affected the external validity and by that compromised the generalizability of the present results. Future Perspectives p The present study elucidates the negative effects of exposure to separate and repeated organizational changes on employee’s perception of multiple aspects of the organization’s psychosocial work environment. In order to implement organizational changes in a healthy and successful manner, securing a healthy and productive work environment is crucial. Prior studies have indicated that participation in decision-making process (Egan et al., 2007), adhering to pre-existing guidelines (Korsgaard et al., 2002) and provide adequate and effective information and communication flow (Allen et al., 2007; Rehman, 2011) may influence employee health, attitudes and behaviors during and following organizational changes. Hence, organizations may potentially alleviate the effects of change by following pre-defined procedural and ethical guidelines and include employees in the change process. The present results highlight the work factors most susceptible to adverse effects following extensive workplace changes. An assessment of the underlying mechanisms in these relationships, explaining why and how different types of organizational changes affect the various psychosocial work factors differently, were outside the scope of the present study. To gain a further understanding of why and how implementing organizational change influence the various aspects of the work environment, further studies are needed to elucidate the specific mechanisms in order to obtain a more thorough understanding of these unique relationships. Preventing negative effects in psychosocial work factors should be a pivotal part of both change planning and implementation, as an unfavorable psychosocial work environment has been associated with adverse effects on both employee and company outcomes, such as health, turnover intention, productivity and profitability. p The participating companies primarily contacted STAMI after an invitation to participate in the project was posted on the institute’s web pages, hence sampling was not random. Management was not asked about their reasons for participation. It may be that the organizations who contacted STAMI in order to participate, constitute a subset of Norwegian companies especially focused on the subject, perhaps more so than the average Norwegian firm. Furthermore, a larger part of the respondents was holding permanent positions and were employed in public sector compared to the general working population in Norway (Nergaard, 2016). 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Emberland, Bjørn Lau, Anne Lene Andersen, and Margrethe Schøning for their help in the survey administration. The authors also thank all organizations and companies participating in the study. ETHICS STATEMENT This study was carried out in accordance with the recommendations of the Data Inspectorate of Norway and the Regional Committee for Medical and Health Research Ethics (REK), with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol December 2019 | Volume 10 | Article 2845 Frontiers in Psychology | www.frontiersin.org Frontiers in Psychology | www.frontiersin.org 15 Organizational Change and the Psychosocial Work Environment Fløvik et al. participated in the idea development, contributed to structure and content, and read all versions of the manuscript. was approved by the Regional Committee for Medical and Health Research Ethics (REK). 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Hydrogen-induced transgranular to intergranular fracture transition in bi-crystalline nickel
Scripta materialia
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a r t i c l e i n f o Article history: Received 31 May 2021 Revised 28 June 2021 Accepted 28 June 2021 Available online 14 July 2021 Keywords: Hydrogen embrittlement Fracture Grain boundary Molecular dynamics (MD) Article history: Received 31 May 2021 Revised 28 June 2021 Accepted 28 June 2021 Available online 14 July 2021 Keywords: Hydrogen embrittlement Fracture Grain boundary Molecular dynamics (MD) Article history: Received 31 May 2021 Revised 28 June 2021 Accepted 28 June 2021 Available online 14 July 2021 Keywords: Hydrogen embrittlement Fracture Grain boundary Molecular dynamics (MD) It is known that hydrogen can influence the dislocation plasticity and fracture mode transition of metallic materials, however, the nanoscale interaction mechanism between hydrogen and grain boundary largely remains illusive. By uniaxial straining of bi-crystalline Ni with a 5(210)[001] grain boundary, a trans- granular to intergranular fracture transition facilitated by hydrogen is elucidated by atomistic modeling, and a specific hydrogen-controlled plasticity mechanism is revealed. Hydrogen is found to form a local atmosphere in the vicinity of grain boundary, which induces a local stress concentration and inhibits the subsequent stress relaxation at the grain boundary during deformation. It is this local stress concentra- tion that promotes earlier dislocation emission, twinning evolution, and generation of more vacancies that facilitate nanovoiding. The nucleation and growth of nanovoids finally leads to intergranular fracture at the grain boundary, in contrast to the transgranular fracture of hydrogen-free sample. © 2021 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) H at crack tips could contribute to the weakening of metal bonds resulting in fracture, but it does not make an explanation for the enhanced plasticity. HESIV [ 28 –32 ] assumes that the vacancy clus- ters generated during plastic deformation are stabilized by forming H-vacancy complexes, which will further interact with the dislo- cations. However, the connection of those stabilized vacancies to embrittlement remains unexplained. All these mechanisms can be viable at the GBs, which is a material interface with intensive dis- location activity and high H trapping capacity [33] . The hydrogen- enhanced plasticity mediated failure mechanism [3] has been pro- posed as a connection between HELP and HEDE, trying to make a universal explanation to HE phenomena. It should be mentioned that the whole framework was established as a posteriori inter- pretation of evolved microstructures. ∗Corresponding author. E-mail address: zhiliang.zhang@ntnu.no (Z. Zhang). Hydrogen-induced transgranular to intergranular fracture transition in bi-crystalline nickel Yu Ding a , Haiyang Yu b , Kai Zhao c , Meichao Lin a , Senbo Xiao a , Michael Ortiz d Jianying He a , Zhiliang Zhang a , ∗ a Department of Structural Engineering, Norwegian University of Science and Technology (NTNU), Trondheim 7491, Norway b Division of Applied Mechanics, Department of Materials Science and Engineering, Uppsala University, Uppsala SE-75121, Sweden c Jiangsu Key Laboratory of Advanced Food Manufacturing Equipment and Technology, Jiangnan University, Wuxi 214122, China d Graduate Aerospace Laboratories, California Institute of Technology, 1200 E. California Blvd., Pasadena, CA 91125, United States Scripta Materialia 204 (2021) 114122 Scripta Materialia 204 (2021) 114122 https://doi.org/10.1016/j.scriptamat.2021.114122 1359-6462/© 2021 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) a r t i c l e i n f o During the H charging process, each step of GCMC runs (insertion or dele- tion of one randomly chosen H atom) was followed by 50 steps of MD runs in canonical (NVT) ensemble until the H concentra- tion fluctuates in a narrow range ( ±1% atomic ratio). The simula- tions were first performed for the perfect FCC Ni lattice without GB, to obtain the relationship between c 0 and μ ( Fig. 1 d). Then μ = −2.31 eV was chosen as the charging parameters for all the subsequent study corresponding to c 0 = 0.001 in the bulk grain. The equilibrium hydrogenated GB after charging was regarded as the 100% saturation case with the 0, 13, 25, 40, 60% saturation cases as comparison. After charging, the system was relaxed in the NPT ensemble for 100 ps. To obtain a realistic stress state in front of a crack tip and avoid unphysical elongation, uniaxial straining in the NVT ensemble was carried out by moving the upper boundary layer at a constant velocity of 2 m/s along Y direction while the lower layer is kept stationary. The atomic stress during deforma- tion was analyzed by Viral theory [42] , illustrations of all simula- tion snapshots were achieved by the coordination number centro- symmetry parameter [43] and common neighbor analysis (CNA) [44] , and dislocations were identified by Ovito [45] . The H distribution during charging is shown in Fig. 1 e. The H concentration in the grains and GB reaches c 0 = 0.001 in the first due to the high trapping energy and excess volumes in the sub- sequent MC steps. However, the H concentration inside the grains is slightly reduced and kept around c = 0.0 0 08, which is mainly caused by the attractive interaction between the formed H atmo- sphere around the GB and newly inserted H. Finally, the GB struc- ture reaches an equilibrium at c = 0.25 after 15,0 0 0,0 0 0 MC steps in Fig. 1 b. The locally high H concentration agrees well with the experimental results [33] and theoretical calculation [46] , indicat- ing that this type of 5 GB could be a preferred gathering site for H. For comparison, a 3 coherent twin boundary (CTB) is charged under the same conditions. The evenly distributed H in Fig. a r t i c l e i n f o 1 c im- plies that CTB has an inconsequential effect to trap H because of the compact structure and few trapping sites, indicating that CTBs have good resistance to HE [ 16 , 47 ]. g [ , ] Fig. 2 a shows the stress (Syy)-strain curve in the Y direction with varying H concentrations during deformation. Fig. 2 c–h are the snapshots of the elastic stage, plastic stage, and final frac- ture for the cases without H and with H in the 100% saturation case, respectively. Without H , the sample fractured ( ε = 0.208) with nanovoid nucleation in the grains near the upper boundary layer due to the accumulation of high-density dislocations. With H in the 100% saturation case, the sample fractured ( ε = 0.173) with nanovoid nucleation on the GB. This shift of nanovoid nu- cleation site from grain interior to the GB caused by H is mani- fested as H-induced transgranular to intergranular fracture transi- tion. The sample deforms mainly through the nucleation and glid- ing of 1/6 < 112 > Shockley dislocations (green pipelines) and leaves the twins (red stacking fault) on the path in which they have glided. At the first drop of stress (critical stress), the twins start to grow and soon get pinned by the top and bottom boundary. The inserted H could facilitate earlier twinning nucleation ( ε = 0.08 without H and ε = 0.062 with H) and decrease the critical stress (Syy = 11.06 GPa without H and ε = 9.55 GPa with H ) during this stage. Higher H concentration could amplify those effects. To re- lease the stress caused by subsequent deformation, more disloca- tions nucleate, multiplicate and exit on the GB, which generates more twins. These twins interact with each other in the junctions, further increasing the dislocation density. As shown in Fig. 2 b, the twins volume fraction evolution displays a zig-zag pattern until fracture, which is the main plasticity activity during deformation. It should be noted that hydrogen promoted deformation twinning, revealed in the MD simulation, has not been experimentally ob- served in typical Ni microstructures [ 39 , 48 –50 ]. This is probably because the MD simulation assumes no pre-existing dislocations before deformation, so continued plasticity dominated by dislo- cation gliding was source-controlled and twinning was facilitated, this can be the case for nanocrystalline Ni [ 51 , 52 ]. a r t i c l e i n f o (b, c) H distribution map for 5 GB and 3 CTB, respectively. (d) The relationship between Equilibrium bulk H concentration c 0 in perfect FCC Ni and chemical potential μ used in the charging process. (e) H concentration in the GB region (1 nm around GB), inside the grains, and in the whole box as a function of MC charging time. has not been observed. Recent experimental studies [ 39 –41 ] indi- cate that mobile H-deformation interaction is not an intrinsic re- quirement for H-induced intergranular fracture, implying that the initial H segregation on the GB is the key. But it is still unclear how the segregated H interacts with the GB. The present work zooms into the GB region with segregated H and probes the mechanisms behind H-induced intergranular fracture. A 5(210)[001] GB was created by constructing two separate crystals with desired crystal- lographic orientations and joining them along a plane normal to the Y-direction ( Fig. 1 a). The initial configuration was then modi- fied by shifting the upper grain in the X-Z plane, deleting overlap- ping atoms, and applying the conjugate gradient method to equi- librate the system. The simulation box was divided into three re- gions: The vicinity ±1 nm to the GB as GB region, 1 nm thick re- gion on the top of grain1 and bottom of grain2 as boundary layers to apply displacement controlled loading, and rest part as grains. Periodic boundary conditions were imposed along the X and Z di- rections while a free boundary condition was employed along the Y direction. After equilibrium in the isothermal–isobaric (NPT) en- semble at 300 K for 100 ps, the mixed grand canonical Monte Carlo (GCMC)/molecular dynamic (MD) method was utilized to ob- tain the dynamic trapping map around the GB. For the GCMC im- plementation, the system was kept at constant chemical potential μ, volume V and temperature T . There is a relationship between chemical potential μ and equilibrium bulk H concentration c 0 in perfect lattice: μ = kT ln c 0 + E , where E is the segregation en- ergy of one H atom and k is the Boltzmann constant. a r t i c l e i n f o The transition process of the fracture mode has not been directly demonstrated by in-situ exper- iment or simulation. Verification of this mechanism is one of the outstanding issues in HE research. As indicated by its name, a key feature of hydrogen embrit- tlement (HE) [ 1 –3 ] is the transition from ductile to “brittle” frac- ture in the presence of hydrogen. For polycrystalline materials, this transition is usually attributed to hydrogen-induced transgranular to intergranular fracture at the microscopic scale, observed in a large number of ex-situ experiments [ 4 –7 ]. All the studies [ 8 –16 ] show that grain boundary (GB) plays an important role in HE and the hydrogen-GB interactions hold the key to understanding the transgranular to intergranular fracture transition. Nowadays, it is well understood that the transition process may involve the syn- ergistic action of several important HE mechanisms [ 11 , 17 ]. Three widely accepted mechanisms are hydrogen enhanced local plas- ticity (HELP), hydrogen enhanced decohesion (HEDE), and hydro- gen enhanced strain-induced vacancy formation (HESIV). The the- ory of HELP [ 18 –22 ] is based on the experimental evidence of enhanced dislocation mobility and well-evolved dislocation struc- tures beneath the fracture surfaces of hydrogen-embrittled sam- ples. However, there is still a large gap in the understanding of how this locally ductile behavior could lead to the eventual “brit- tle” fracture. HEDE [ 23 –27 ] postulates that local accumulation of The interaction between hydrogen and grain boundary/material interface has been simulated both using the continuum approach [34] and the atomistic method [35] . Hydrogen segregation around GBs [ 36 , 37 ] and the influence of hydrogen on the propagation of an existing crack [38] have been elaborated. However, the direct transgranular to intergranular transition without an initial crack Y. Ding, H. Yu, K. Zhao et al. Scripta Materialia 204 (2021) 114122 Fig. 1. (a) Equilibrium structures of perfect 5(210)[001] GB. (b, c) H distribution map for 5 GB and 3 CTB, respectively. (d) The relationship between Equilibrium bulk H concentration c 0 in perfect FCC Ni and chemical potential μ used in the charging process. (e) H concentration in the GB region (1 nm around GB), inside the grains, and in the whole box as a function of MC charging time. Fig. 1. (a) Equilibrium structures of perfect 5(210)[001] GB. a r t i c l e i n f o Therefore, cau- tion should be taken when applying hydrogen-induced twinning mechanism in Ni, since this is likely applicable to highly special- ized cases instead of the typical coarse-grained microstructures. The H distribution during charging is shown in Fig. 1 e. The H concentration in the grains and GB reaches c 0 = 0.001 in the first 10 0,0 0 0 MC steps. H atoms continue to pump into the GB region 2 Y. Ding, H. Yu, K. Zhao et al. Scripta Materialia 204 (2021) 114122 Fig. 2. (a) stress-strain curve in the Y direction for varying H concentrations. (b) Twins volume fraction as a function of strain with varying H concentration. (c–e) atomic structure without H at different strains. (f–h) atomic structure with 100% H at different strains. Only atoms in defect structure are colored, HCP atoms are marked red, white atoms indicate other type atoms, and green pipelines outline the 1/6 < 112 > Shockley dislocations. Fig. 2. (a) stress-strain curve in the Y direction for varying H concentrations. (b) Twins volume fraction as a function of strain with varying H concentration. (c–e) atomic structure without H at different strains. (f–h) atomic structure with 100% H at different strains. Only atoms in defect structure are colored, HCP atoms are marked red, white atoms indicate other type atoms, and green pipelines outline the 1/6 < 112 > Shockley dislocations. Fig. 3. (a) Average Von Mises stress-strain curve in different regions. (b, c) Average Von Mises stress distribution along the Y direction without or with H , respectively. Fig. 3. (a) Average Von Mises stress-strain curve in different regions. (b, c) Average Von Mises stress distribution along the Y direction without or with H , respectively. ess-strain curve in different regions. (b, c) Average Von Mises stress distribution along the Y direction without or with H , respective Fig. 3. (a) Average Von Mises stress-strain curve in different regions. (b, c) Average Von Mises stress distribution along the Y dire Nevertheless, this can still be a viable mechanism in a number of FCC alloys that undergo deformation twinning, such as in TWIP steels [ 53 , 54 ] or those with nanocrystalline grain structures. To better understand how H influences local plasticity, the average von Mises stress distribution along the Y direction in different re- gions is plotted in Fig. 3 . It is interesting to note that in Fig. a r t i c l e i n f o 3 b the 5(210)[001] GB could release the initial stress and always keep its stress level lower than in the grains during deformation without H. This might be a good explanation for the huge amount of point defects in the grains without H ( Fig. 2 c) and the eventual trans- granular fracture. The saturated H can increase the initial stress concentration in the GB region and more importantly inhibit the stress-releasing ability of the GB during deformation. The GB thus stays at an activated state [11] with a more disordered atomistic structure due to the higher stress level maintained at the GB than the grain interior, which induces the earlier twinning nucleation and void formation on the GB. Increased H concentration could in- crease the local stress concentration which induces more serious plastic deformation and earlier fracture. process of vacancy formation, which could be the embryo of the fi- nal void [31] , is analyzed in the GB region. The extremely distorted atoms are identified as vacancy cluster and the vacancy volume fraction-strain curve is plotted in Fig. 4 a. It is found that there are few vacancies formed around the GB before twinning nucleation without H , but the fraction of vacancy volume comes to grow after more plasticity occurs around the GB. With H , the vacancy volume fraction comes to grow in the elastic stage which shows that the GB has entered a plasticity-activated state [33] , and the vacancy volume fraction rises with increasing pre-charged H concentration. It indicates that H accelerates vacancy formation at early plastic- ity stage on the GB, possibly through lattice dislocation interaction and jog formation. Previous studies also show that vacancies could be stabilized by forming Va-H complexes [ 31 , 32 ], consistent with the high vacancy volume fraction observed in the H charged cases. Those Va-H complexes could be the reason for the earlier void for- mation with H in Fig. 4 g. 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Robertson , Acta Mater. 60 (13–14) (2012) 5182–5189 . a r t i c l e i n f o (a) Vacancy volume fraction as a function of strain with varying H concentration. (b,–g) Snapshots of vacancy surface atoms at strain = 0.18 with varying H concen- tration. Only highly distorted atoms with coordination numbers less than 8 are colored. Fig. 4. (a) Vacancy volume fraction as a function of strain with varying H concentration. (b,–g) Snapshots of vacancy surface atoms at strain = 0.18 with varying H concen- tration. Only highly distorted atoms with coordination numbers less than 8 are colored. plasticity. Compared with the 3 coherent twin GB which traps nearly no H atoms, H will form an atmosphere at the 5 GB due to the high trapping energy and excess volume and induce a higher initial local stress. In the case without H , less stress is built up at the 5 GB region during deformation, which leads to transgran- ular fracture. 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Combinational Immunotherapy for Hepatocellular Carcinoma: Radiotherapy, Immune Checkpoint Blockade and Beyond
Frontiers in immunology
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Edited by: Marco Carbone, University of Milano-Bicocca, Italy Reviewed by: Raghavan Chinnadurai, Mercer University, United States Cristina Maccalli, Sidra Medicine, Qatar *Correspondence: Valerie Chew Valerie.chew.s.p@singhealth.com.sg Edited by: Marco Carbone, University of Milano-Bicocca, Italy Reviewed by: Raghavan Chinnadurai, Mercer University, United States Cristina Maccalli, Sidra Medicine, Qatar Specialty section: This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology Received: 02 June 2020 Accepted: 14 September 2020 Published: 30 September 2020 Combinational Immunotherapy for Hepatocellular Carcinoma: Radiotherapy, Immune Checkpoint Blockade and Beyond The systemic treatment landscape for advanced hepatocellular carcinoma (HCC) has experienced tremendous paradigm shift towards targeting tumor microenvironment (TME) following recent trials utilizing immune checkpoint blockade (ICB). However, limited success of ICB as monotherapy mandates the evaluation of combination strategies incorporating immunotherapy for improved clinical efficacy. Radiotherapy (RT) is an integral component in treatment of solid cancers, including HCC. Radiation mediates localized tumor killing and TME modification, thereby potentiating the action of ICB. Several preclinical and clinical studies have explored the efficacy of combining RT and ICB in HCC with promising outcomes. Greater efforts are required in discovery and understanding of novel combination strategies to maximize clinical benefit with tolerable adverse effects. This current review provides a comprehensive assessment of RT and ICB in HCC, their respective impact on TME, the rationale for their synergistic combination, as well as the current potential biomarkers available to predict clinical response. We also speculate on novel future strategies to further enhance the efficacy of this combination. REVIEW published: 30 September 2020 doi: 10.3389/fimmu.2020.568759 INTRODUCTION AND BACKGROUND ON CURRENT TREATMENT LANDSCAPE FOR HCC Liver cancer ranks second as the leading cause of cancer deaths in men and fourth highest cancer mortality in both genders globally with an estimate of 781,631 deaths in 2018 (1). Hepatocellular carcinoma (HCC), which comprises 75–85% of liver cancer cases, is a complex cancer with various etiologies such as hepatitis viral infection, alcohol abuse, and obesity (2). Chronic viral infection remains the primary contributing factor of liver cirrhosis, a chronic liver disease that precedes HCC, characterized by the irreversible scarring and hardening of the liver tissue with decreased hepatocyte proliferation (3, 4).The chronic liver inflammation causes fibrous scar tissues to replace healthy tissues overtime and eventually occlude blood flow through the liver, resulting in the onset of portal hypertension and creates a hypoxic environment that favors tumor growth (5–7). Cirrhosis also leads Keywords: radiotherapy, immunotherapy, immune checkpoint blockade (ICB), combination therapy, hepatocellular carcinoma (HCC) Edited by: Marco Carbone, University of Milano-Bicocca, Italy Reviewed by: Raghavan Chinnadurai, Mercer University, United States Cristina Maccalli, Sidra Medicine, Qatar *Correspondence: Valerie Chew Valerie.chew.s.p@singhealth.com.sg Citation: Tumor-associated cytokines and chemokines are able to recruit and polarize immune subsets into a pro-tumor phenotype and fuel tumorigenesis. One of such immune subsets is the tumor-associated macrophages (TAMs) which are polarized into M2 macrophage phenotype by IL-10, TGF-b, IL-4, or IL-13 present in the TME, and drive tumor progression by supporting angiogenesis and recruitment of CD4+FoxP3+ T regulatory cells (Tregs) (Figure 1) (35, 36). The accumulation of TAMs in the tumor region is frequently associated with poor prognosis across cancer types, including HCC (35, 37). In addition, Tregs which regulate or dampen CD8 T cells’ activation and cytotoxic capacity, also play a critical role in promoting tumorigenesis (38, 39). We previously reported that Tregs from HBV-positive HCC tumors exhibited higher expression of PD-1 and displayed superior suppressive capacity against CD8 T cells (30). Higher intra-tumoral interleukin-17- producing Tregs have also been consistently reported in HCC and correlated with poorer prognosis and reduced survival in HCC patients (30, 37, 38, 40, 41). Likewise, TGF-b-rich TME of HCC favours the differentiation of Th17, a CD4 T helper subset that also produces IL-17 (42). High intra-tumoral frequencies of Th17 were positively correlated with microvascular invasion in HCC patients and were implicated in shorter OS and disease free survival (DFS) of patients (43). More recently, immune checkpoint blockade (ICB) has emerged as a promising therapeutic option for advanced HCC patients (17). Both phase II and phase III ICB trials in advanced HCC using antibodies against programmed cell death protein 1 (PD-1) have demonstrated clinical benefit and fewer incidences of serious treatment-related adverse events (17–20). The recent IMbrave150 trial, which investigated the combination of atezolizumab (anti-PD- L1) and bevacizumab (anti-VEGF-A; anti-angiogenesis agent) in patients with advanced HCC, showed survival benefit over sorafenib in the first-line setting and highlighted the potential of combining ICB with a tumor microenvironment (TME)-modifying agent (21). In March 2020, FDA also approved the use of ipilimumab (anti-CTLA-4) combined with nivolumab (anti-PD-1) in sorafenib-experienced patients based on the high objective response rates (RR) and durability of responses (19). On the other hand, CD4 T helper 1 cells (Th1) that are capable of producing IFN-g can activate dendritic cells which leads to enhanced priming and maturation of CD8 T cells (44, 45). Th1 can also trigger DC-mediated tumor-killing activities via IFN-g- dependent mechanisms which will further fuel downstream anti- tumor immune responses (Figure 1) (46). Citation: Lee YH, Tai D, Yip C, Choo SP and Chew V (2020) Combinational Immunotherapy for Hepatocellular Carcinoma: Radiotherapy, Immune Checkpoint Blockade and Beyond. Front. Immunol. 11:568759. doi: 10.3389/fimmu.2020.568759 September 2020 | Volume 11 | Article 568759 Frontiers in Immunology | www.frontiersin.org Combination Therapy in Liver Cancer Lee et al. to impairment of the immune surveillance within the liver through the reduced synthesis of innate immunity proteins and pattern- recognition receptors (PRRs). This in turn damages the antigen- presenting and phagocytic capacity of Kupffer cells and sinusoidal endothelial cells which creates an immuno-deficient environment in patients and contributes to hepatocarcinogenesis (8, 9). confers protection against pathogens and repair tissue damage, however, sustained liver inflammation can perturb the microenvironment and tip the scales in favor of carcinogenesis (28–30). For instance, cytokines and chemokines secreted within the liver can promote angiogenesis, immune evasion and anti-apoptotic responses as well as to recruit immune cells with the capacity to create a niche microenvironment that facilitates tumor growth (31, 32). In particular, anti-inflammatory cytokine interleukin-10 (IL-10) was shown to be highly enriched in HCC tumors as compared to adjacent non-tumor or healthy liver tissues (33). High levels of transforming growth factor beta (TGF-b) was also associated with increased tumor invasiveness in advanced HCC (Figure 1) (34). The management of early stage HCC with curative intent includes surgical resection or liver transplantation with expected 5-year survival rate between 60 and 80% (10). However, less than 20% of HCC patients qualify for these curative treatments (11). Loco-regional therapies such as radiofrequency ablation, chemo, or radio-embolization are recommended for local disease control when curative treatments are contraindicated (12). For unresectable advanced HCC, systemic therapies such as tyrosine kinase inhibitors (TKI) (e.g. Sorafenib) have been used (13). Sorafenib was approved by the Food and Drug Administration (FDA) as first- line therapy for inoperable HCC a decade ago and remains the current standard of care despite its relatively modest activity (14, 15). Lenvatinib, another first-line treatment option for advanced HCC, demonstrated non-inferiority in terms of median overall survival (OS), but superiority in progression-free survival (PFS) and overall response rate (ORR) compared to sorafenib (16). Regorafenib, cabozantinib, and ramucirumab have also attained FDA approval in recent years as second-line treatments for patients with advanced HCC who exhibited progressive disease after one or more systemic therapies (12). Citation: An increase in Th1 response correlated with favorable outcomes in HCC patients following treatment with transarterial chemoembolization (TACE) (47). While Th1 demonstrated anti-tumor capabilities, type 2 CD4 T helper (Th2) were found to be enriched in HCC tissues as compared to normal healthy livers and were inversely associated with OS of HCC patients (48). Th2 cytokines such as IL-4, IL-10, and IL-13 are able to induce M2 macrophages which have reduced cytotoxic activity and dampens CD8 T cell-mediated anti-tumor activity (Figure 1) (49, 50). Furthermore, upregulation of Th2 cytokine production was linked to increased likelihood of HCV- induced HCC (51). Besides combinations of multiple ICBs or ICB with targeted therapies, growing evidence indicates the role of radiotherapy (RT) in potentiating tumor immunity (22, 23). For instance, preclinical studies have shown that combination of RT and ICB can synergistically augment the anti-tumor responses induced by both agents (24–26). In this systemic review, we will discuss the impact of RT and ICB on the TME and how this combinational therapy, and potentially along with another therapeutic agent, could bring about a synergistic control of HCC progression. IMMUNE LANDSCAPE OF HCC Presence of CD4+ T helper 1 (Th1) cells correlated with favorable outcomes in HCC as they are able to enhance activation of CD8+ T cells via dendritic cells (DC) and trigger DC-mediated tumor-killing. Despite the capability to kill tumor cells and association with good prognosis in HCC, CD8+ T cells showed upregulation of exhaustion markers (e.g. PD-1, CTLA-4, and LAG-3), which dampens its killing capacity in chronic conditions leading to tumor progression. play a pivotal role in anti-tumor immunity (37, 40). However, it was reported that NK cells found within the tumor regions of HCC tumors exhibited inferior cytolytic capacity and production of IFN-g as compared to NK cells derived from non-tumor regions (52). The authors also observed that the addition of HCC patient-derived Tregs impaired the tumor-killing ability of autologous NK cells in vitro (Figure 1). While the presence of Trm and Tem cells was associated with good prognosis in HCC (30), they often express exhaustion markers such as PD-1, lymphocyte-activation gene 3 (LAG-3), and cytotoxic T-lymphocyte-associated antigen 4 (CTLA- 4), which correlated negatively with their functional competency (Figure 1) (37, 53). Thus, these exhaustion markers have been the prime targets of ICB to reinvigorate and restore the cytotoxic capacity of CD8 T cells (39). conform radiation doses to tumors (54). However, recent improved techniques such as stereotactic body radiation therapy (SBRT) and radioembolization (RE), allow for high doses of radiation to be delivered to the tumor while limiting the damage to surrounding healthy tissues. SBRT delivers highly conformal hypo-fractionated external beam radiation in relatively fewer fractions, enabled by technological advances in on-board imaging, radiation planning, and delivery systems (12). In contrast, RE is an internal radiation technique that utilizes predominantly beta-emissions with limited range of tissue penetration from radio-labeled (e.g. Y-90) micro-embolic particles that are directly introduced to HCC via hepatic artery (10). These advancements have broadened the range of RT applications for HCC treatment. SBRT has demonstrated good tumor control with 2-year local control rates between 84 and 95% (55, 56). However, the overall survial (OS) is limited by “out-of-field” intra- and extra-hepatic disease progression (57), highlighting the need for concurrent systemic disease control. SBRT is also generally well tolerated particularly in those with Child-Pugh (CP) scores less than 7. IMMUNE LANDSCAPE OF HCC Chronic hepatitis infection or long-standing liver injury often place the liver in a chronic inflammatory state (27). Modest inflammation Cytotoxic immune populations such as CD8 T resident memory (Trm), CD8 T effector memory (Tem), and natural killer (NK) cells, September 2020 | Volume 11 | Article 568759 Frontiers in Immunology | www.frontiersin.org Combination Therapy in Liver Cancer Lee et al. Lee et al. FIGURE 1 | The immunological events contributing to good versus bad prognosis in HCC. Enrichment of CD4+ T helper 2 (Th2) cells are associated with poor overall survival of HCC patients. TGF-b as well as cytokines secreted by Th2 such as IL-4, IL-10, and IL-13, drives the polarization of tumor-associated macrophages (TAMs) into immunosuppressive M2 macrophage phenotype which further recruits T regulatory cells (Tregs). Tregs in turn further enhances the immunosuppressive tumor microenvironment through inhibition of CD8+ T memory/effector cells’ and natural killer (NK) cells’ tumor-killing capacity, leading to poor prognosis in HCC. TGF-b also drives differentiation of CD4+ T helper 17 (Th17) cells and high frequencies of Th17 have been correlated with increased microvascular invasion and shorter OS and DFS of HCC patients. Presence of CD4+ T helper 1 (Th1) cells correlated with favorable outcomes in HCC as they are able to enhance activation of CD8+ T cells via dendritic cells (DC) and trigger DC-mediated tumor-killing. Despite the capability to kill tumor cells and association with good prognosis in HCC, CD8+ T cells showed upregulation of exhaustion markers (e.g. PD-1, CTLA-4, and LAG-3), which dampens its killing capacity in chronic conditions leading to tumor progression. FIGURE 1 | The immunological events contributing to good versus bad prognosis in HCC. Enrichment of CD4+ T helper 2 (Th2) cells are associated with poor overall survival of HCC patients. TGF-b as well as cytokines secreted by Th2 such as IL-4, IL-10, and IL-13, drives the polarization of tumor-associated macrophages (TAMs) into immunosuppressive M2 macrophage phenotype which further recruits T regulatory cells (Tregs). Tregs in turn further enhances the immunosuppressive tumor microenvironment through inhibition of CD8+ T memory/effector cells’ and natural killer (NK) cells’ tumor-killing capacity, leading to poor prognosis in HCC. TGF-b also drives differentiation of CD4+ T helper 17 (Th17) cells and high frequencies of Th17 have been correlated with increased microvascular invasion and shorter OS and DFS of HCC patients. IMMUNE CHECKPOINT BLOCKADES IN HCC In the past decade, the importance of immune system in tumor progression, and concept of immune-surveillance and evasion by cancer cells have been widely accepted as one of the key hallmarks of cancer (29). Immunotherapies have since gained recognition as a promising alternative cancer treatment with ICB in the forefront of clinically approved immune-modulating agents across cancer types (75). The benefits of immunotherapy in HCC have also been discussed substantially in several reviews (76–79). In particular with ICB, inhibitors against PD-1, PD-L1, and CTLA-4 have also been tested extensively in clinical trials for HCC. p Traditionally, the rationale behind RT for cancer treatment is to induce lethal DNA damage to tumor cells with high-energy particles leading to subsequent cell death (64). However, the ability of RT to elicit an immune-mediated anti-tumor response, a phenomenon known as “abscopal effect” denoted by the downsizing of non-targeted distant tumors following ionizing radiation treatment, has gained increased prominence in the recent decade (65). RT causes immunogenic cell death and cellular stress, which increases the pool of tumor-associated antigens and damage-associated molecular patterns (DAMPs) (66). These in turn activate dendritic cells, a professional antigen- presenting cell (APC) that primes tumor-specific CD8+ T cells to further enhance the anti-tumor responses and promote immune cell infiltration into the TME (Figure 2) (23). Indeed, studies revealed increased antigen presentation activity following Y-90- RE in HCC and SBRT across cancer types (22, 64). Additionally, DAMPs are taken up by PRRs which activates stimulator of interferon genes (STING) pathway that mediates the production of type I interferons (IFN-a and IFN-b) involved in the activation of downstream immune responses (67). Further evidences of radiation-induced immune responses were reported by both pre-clinical and clinical studies where heightened activation and recruitment of anti-tumor immune subsets such as CD4+, CD8+ T cells, cytotoxic NK, and CD8 +CD56+ natural killer T (NKT) cells to the TME were observed (22, 64, 68). Altogether, these findings are concordant with the notion that RT can convert an otherwise “cold” tumor that has low immunogenicity and poorly infiltrated with immune cells to an immune-reactive “hot” tumor, which is well infiltrated by the immune cells (Figure 2). Upregulation of PD-L1 expression on tumor cells, which binds particularly to PD-1 expressed by tumor-infiltrating activated T cells, induces exhaustion and dampens the anti-tumor immune activities of these effector cells hence, allowing immune evasion by tumor cells (Figure 3) (80). RADIOTHERAPY (RT) IN HCC RT for HCC was traditionally linked to suboptimal results due to limited tolerance of whole liver irradiation and the inability to September 2020 | Volume 11 | Article 568759 Frontiers in Immunology | www.frontiersin.org 3 Combination Therapy in Liver Cancer Lee et al. Lee et al. However, in those with CP score >7, there is a higher risk of radiation-induced liver disease (RILD) and worsening of CP scores (58, 59). In this group of patients, the risk of liver toxicities can be as high as 31–35%. Furthermore, SBRT may also result in gastrointestinal perforation or hemorrhage, particularly in lesions adjacent to luminal organs or those with a history of gastrointestinal ulcers (56, 60). The selective use of RE in the treatment of Barcelona Clinic Liver Cancer (BCLC) B and C HCCs have also demonstrated clinical benefit and/or local tumor control (12, 61), and less than 9% of patients who underwent RE experienced adverse events greater than grade 3 (62). While it was reported across various studies that the 20–70% of patients experienced post-RE syndrome (PRS), which included fatigue, nausea, and abdominal pain, these symptoms could be usually treated with over-the-counter drugs (62). Due to their overall efficacy and safety profile, these radiation therapies are now more widely adopted for local tumor control and bridging/ downstaging treatment for future curative treatments such as transplant/resection (54, 61, 63). the expression of Tim-3 and PD-1, following treatment with Y-90- RE in HCC patients (22). Other studies have also observed that RT resulted in upregulation of PD-L1 expression by the tumor cells, which can attenuate anti-tumor responses (Figure 2) (73, 74). Hence, tumor cells are able to subvert immunosurveillance, where immunosuppressive responses overwhelm the anti-tumor immune response and eventually result in radio-resistance. Therefore, the strategic combinational use of immunotherapy would be necessary to circumvent such resistance and to enhance clinical benefits of RT. Frontiers in Immunology | www.frontiersin.org IMMUNE CHECKPOINT BLOCKADES IN HCC (Left panel) Immune responses induced by radiotherapy (RT): Initial (1°C) anti-tumor immune response includes (A) increased pool of tumor antigens and DAMPs which results in antigen presentation activity, (B) subsequent activation of T cells and enhanced infiltration of anti-tumor immune cells into the TME to facilitate tumor-killing; Secondary (2°C) wave of immunosuppressive response is denoted by (C) recruitment of immunosuppressive immune subsets, Tregs and TAMs and (D) upregulated expression of immune checkpoint molecules by tumor cells (PD-L1) and cytotoxic CD8+ T cells (PD-1 and CTLA-4) which dampens anti-tumor activity. (Right panel) Immune responses mediated by immune checkpoint blockade (ICB): (A) Anti-PD-1 and anti-PD-L1 interferes with PD-1/PD-L1 interaction and relieves suppression of CD8+ T cells by tumor cells. (B) Anti- CTLA-4 blocks inhibitory signaling by inhibiting B7/CTLA-4 binding and allows for the activation of APCs and T cells. (Bottom panel) Potential synergistic effects of combining RT and ICB include enhanced infiltration of anti-tumor into TME post-RT and reversion of radiation-induced exhaustion and immunosuppression by ICB. FIGURE 2 | Immune modifying effects of radiotherapy (RT) and immune-checkpoint blockade (ICB). (Left panel) Immune responses induced by radiotherapy (RT): Initial (1°C) anti-tumor immune response includes (A) increased pool of tumor antigens and DAMPs which results in antigen presentation activity, (B) subsequent activation of T cells and enhanced infiltration of anti-tumor immune cells into the TME to facilitate tumor-killing; Secondary (2°C) wave of immunosuppressive response is denoted by (C) recruitment of immunosuppressive immune subsets, Tregs and TAMs and (D) upregulated expression of immune checkpoint molecules by tumor cells (PD-L1) and cytotoxic CD8+ T cells (PD-1 and CTLA-4) which dampens anti-tumor activity. (Right panel) Immune responses mediated by immune checkpoint blockade (ICB): (A) Anti-PD-1 and anti-PD-L1 interferes with PD-1/PD-L1 interaction and relieves suppression of CD8+ T cells by tumor cells. (B) Anti- CTLA-4 blocks inhibitory signaling by inhibiting B7/CTLA-4 binding and allows for the activation of APCs and T cells. (Bottom panel) Potential synergistic effects of combining RT and ICB include enhanced infiltration of anti-tumor into TME post-RT and reversion of radiation-induced exhaustion and immunosuppression by ICB. CTLA-4) and nivolumab (anti-PD-1) in sorafenib-experienced advanced HCC patients (19). response rate between viral versus uninfected HCC towards ICBs to date. Patients from various cancer types with higher tumor PD-1 and PD-L1 expression were evidenced to have improved OS and response as compared to those with lower expression levels (86). Frontiers in Immunology | www.frontiersin.org IMMUNE CHECKPOINT BLOCKADES IN HCC Inhibition of PD-1/PD-L1 interaction can reverse the exhaustive state of these cytotoxic immune cells and reinvigorate their anti-tumor activities (Figure 2) (81). Initial promising results were demonstrated by successful phase II ICB trials in HCC (CheckMate 040 and KEYNOTE-224) using antibodies against PD-1 (19, 20). However, subsequent phase III clinical trials (CheckMate 459 and KEYNOTE-240) which compared anti-PD-1 antibodies to sorafenib in HCC as first-line and second-line therapy, respectively, did not meet the pre-defined statistical significance improvement for OS (17, 18). Despite that, the clinical benefit of the ICB in advanced HCC is notable with superior overall response rate (ORR) and fewer incidences of grade 3/4 treatment-related adverse events in both trials (17, 18). Another target of ICB is CTLA-4, an inhibitory receptor found on surface of T cells, which negatively modulates T cell activation and proliferation upon binding with B7 costimulatory molecule on APCs (Figure 2) (82). Unlike PD-1/PD-L1 pathway, CTLA-4 act primarily upstream at T cells priming stage and is also known to have a wider adverse events profile due to this (83). Both pre-clinical and clinical studies have shown that administration of anti-CTLA-4 led to the direct induction of CD4+ and CD8+ effector T cells by alleviating the immunosuppressive effect of Tregs (81, 84). Multiple phase III clinical trials investigating the use of anti-CTLA drugs, such as tremelimumab and ipilimumab, as monotherapy or in combination with other ICBs are currently underway (19, 85), with a recent FDA’s approval for the combination of ipilimumab (anti- Despite the initial immune activation, RT can indirectly lead to subsequent immunosuppressive effects such as the second wave of recruitment of Tregs and TAMs to the TME (69, 70). Radiation- induced interferon activity could also cause upregulation of exhaustion molecules or signaling pathways on tumor- infiltrating cytotoxic T cells (71, 72). Our previous findings demonstrated an increase in exhausted CD8 T cells, denoted by September 2020 | Volume 11 | Article 568759 Frontiers in Immunology | www.frontiersin.org Combination Therapy in Liver Cancer Lee et al. Lee et al. FIGURE 2 | Immune modifying effects of radiotherapy (RT) and immune-checkpoint blockade (ICB). IMMUNE CHECKPOINT BLOCKADES IN HCC However, PD-L1 expression was not a significant biomarker for response to anti-PD-1 in HCC as evidenced by the findings from ICB trials CheckMate 040 and KEYNOTE-224 (20, 91). Studies have also attributed the innate variability of each patient’s pre- existing immunity to the differential response observed towards Despite initial success observed with ICB therapies across a broad range of tumors, a decrease in their efficacy and acquired resistance were reported following initial response to ICB (86, 87). This varying response rate in HCC patients have been intensively reviewed previously (88–90). Despite a previous study reporting the distinct immune landscape between viral versus non-viral HCC (30), there is no concrete evidence that suggests a difference in September 2020 | Volume 11 | Article 568759 Frontiers in Immunology | www.frontiersin.org 5 Combination Therapy in Liver Cancer Lee et al. FIGURE 3 | Rationale of combination therapy with radiotherapy, immune checkpoint blockade, and anti-angiogenesis agents. Diagram illustrates the key immune modifying effects by each therapeutic agent and the potential synergetic effects of their combination in HCC. RT enhances immune infiltrates into TME but induces upregulation of immune checkpoint molecules (e.g. PD-1, PD-L1, and CTLA-4) and VEGF. Anti-VEGF promotes normalization of vessel formation, which improves efficacy of RT and/or further boosts infiltration of cytotoxic cells into TME but increases PD-1 expression by CD4+ T cells. Synergistic combination with ICB restores and further enhances anti-tumor immune responses to improve efficacy of RT and anti-VEGF therapies. FIGURE 3 | Rationale of combination therapy with radiotherapy, immune checkpoint blockade, and anti-angiogenesis agents. Diagram illustrates the key immune modifying effects by each therapeutic agent and the potential synergetic effects of their combination in HCC. RT enhances immune infiltrates into TME but induces upregulation of immune checkpoint molecules (e.g. PD-1, PD-L1, and CTLA-4) and VEGF. Anti-VEGF promotes normalization of vessel formation, which improves efficacy of RT and/or further boosts infiltration of cytotoxic cells into TME but increases PD-1 expression by CD4+ T cells. Synergistic combination with ICB restores and further enhances anti-tumor immune responses to improve efficacy of RT and anti-VEGF therapies. various ICB therapies across cancer types (92–94). Specifically in HCC, tumors with higher transcriptomic diversity were associated with worse OS in patients treated with ICB and these tumor cells also expressed a significantly higher level of vascular endothelial growth factor A (VEGF-A) (95). Concordant to this finding, Chen et al. IMMUNE CHECKPOINT BLOCKADES IN HCC showed in a separate study that responders to anti-PD-1 treatment have decreased expression levels of VEGF-A while the non-responders have increased VEFG-A expression (94). This in turn suggests that the VEGF pathway is an important mechanism for resistance to the anti-PD-1 therapy, where it could hamper tumor infiltration and functions of T effector cells (96, 97). Therefore, the degree of transcriptomic diversity of HCC tumors, VEGF expression level, and pre-existing immunity of each individual could provide a rationale for the observed differential responses of HCC patients towards immune checkpoint blockade therapies. system and produce a synergistic anti-tumor immunity for durable disease control when combined with immunotherapy (Figure 2, bottom). For instance, RT enhances inflammatory immune response and intra-tumoral infiltration by cytotoxic immune cells while ICB could overcome the radiation-induced exhaustion in CD8 T cells and restore their anti-tumor immune responses. Kim et al. demonstrated the dose-dependent upregulation of PD-L1 expression following irradiation of various HCC cell lines, which was found to be mediated predominantly through the IFN-g-STAT3 signaling pathway (25). Likewise, clinical studies have reported post-RT upregulated expressions of PD-1 and PD-L1 by CD8 T cells and tumor cells, respectively (22, 73, 74). These studies provided a sound rationale for the combination of RT with ICB, which should be considered against the major concerns for a successful treatment strategy in HCC as outlined in Table 1. Preclinical data have shown that the combination of RT and ICB exhibited therapeutic synergism as well as superior tumor control (24–26, 73). Significant tumor growth suppression and improved OS were observed in HCC mice treated with single 10Gy RT and anti-PD-L1 compared to either therapy alone (25). Anti-PD-L1 and 12Gy RT exerted abscopal tumor control and COMBINATIONAL STRATEGIES: RT AND ICB The interest in combining radiotherapy and immunotherapy stems from the rationale that radiation primes the immune September 2020 | Volume 11 | Article 568759 Frontiers in Immunology | www.frontiersin.org 6 Combination Therapy in Liver Cancer Lee et al. TABLE 1 | Major considerations for the combination of RT and ICB. Major concerns of intervention(s) Potential solution How can we subvert RT-induced exhaustion? ICB that targets exhaustion pathways can help reinvigorate the exhausted cytotoxic immune cells (24, 26, 73, 74). How to overcome the ineffectiveness of ICB against cold tumors? RT can trigger immune activity and switch a “cold” tumor to a “hot” tumor with enhanced inflammation and tumor infiltration by the immune cells (22, 25, 64, 68). Will there be severe toxicities in the combined therapy of RT and ICB? Preliminary findings from early phase trials for combined use of RT and ICB have showed tolerable safety profile (98–100). How can we predict the differential responses by patients towards treatments? Discovery of predictive biomarkers for response towards various cancer treatments (i.e. RT and ICB) and in combination is essential to select the most appropriate therapeutic agent and therapy for the patients (22, 95, 101). TABLE 1 | Major considerations for the combination of RT and ICB. Potential solution Preliminary findings from early phase trials for combined use of RT and ICB have showed tolerable safety profile (98–100). ? Discovery of predictive biomarkers for response towards various cancer treatments (i.e. RT and ICB) and in combination is essential to select the most appropriate therapeutic agent and therapy for the patients (22, 95, 101). Considerations for the radio-sensitivity of the surrounding vasculature, toxicity profile and identification of pro- immunogenic signatures following RT would also be essential to optimize protocols for combining RT and ICB (106). Therefore, it is imperative to understand the key immune checkpoint molecules modified by RT in the TME, the sequence and mechanisms of their modification and subsequent role in tumor immunity to determine the type of ICB to be used and RT/ICB dosing sequence. superior local control of irradiated tumors in a mammary cancer murine xenograft model compared to monotherapy (24). Similar findings were replicated in CT26 murine colon carcinoma xenografts treated with fractionated RT (2Gy x 5) and/or anti- PD-1 (73). Synergism between RT and anti-CTLA-4 were also demonstrated in several murine tumor models (26, 102). COMBINATIONAL STRATEGIES: RT AND ICB More importantly, these effects were durable as shown by the rejection of tumor re-challenge that was dependent on CD8 T-cells (24, 73). Despite encouraging preclinical findings, there is no published prospective clinical data to our knowledge on combined RT and ICB therapy in HCC, except a few small series that have shown promising clinical activity (98, 99). Chiang et al. reported 100% ORR in 5 patients treated with SBRT followed by nivolumab for large unresectable HCC and another case report showed complete pathological response following Y-90-RE and nivolumab bridging therapy prior to partial hepatectomy (98, 99). Tai et al. also showed that combination of Y-90-RE with nivolumab had an optimistic ORR of 31%, and the combination therapy was safe and tolerable with only 11% of treated advanced HCC patients experienced grade 3/4 treatment-related adverse events (100). Phase I trial (NCT02239900) that evaluated liver/lung SBRT with ipilimumab reported that 23% of patients experienced clinical benefit which corresponded to increase in CD8+ T cells and CD8 +/CD4+ ratios (101). Grade 3 toxicities were found in 34% of patients with no grade 4/5 toxicities. A separate phase I basket trial evaluated the safety of multi-site SBRT followed by pembrolizumab in patients with advanced solid tumors (NCT02608385), including one case of HCC. Similar levels of toxicity were observed as compared to monotherapy-treated patients with a dose-limiting toxicity rate of 9.7% (103). Several other prospective clinical trials are currently on-going to evaluate the combined approach of RT and immunotherapy in HCC (Table 2). Frontiers in Immunology | www.frontiersin.org FUTURE DEVELOPMENTS IN COMBINATIONAL STRATEGIES FOR HCC Beyond the combination of RT and ICB, there have been remarkable advancement in the use of anti-angiogenesis agent in treatment for HCC reported by the phase III IMbrave150 trial, which demonstrated superiority in terms of ORR, OS, and PFS in HCC patients treated with atezolizumab (anti-PD-L1) and bevacizumab (anti-VEGF-A) versus patients treated with sorafenib (21). As one of the hallmarks of cancer, angiogenesis plays critical role in tumor formation and growth and have long been a promising drug target in HCC (29, 31). Dysregulated VEGF expression was shown to be responsible for the vascular abnormalities observed in HCC tumors (31, 107). Various other studies have also consistently showed that elevated circulating VEGF expression following surgery or RFA correlated to poor prognosis in HCC patients (31, 108, 109). Shigeta et al. revealed the mechanism behind the anti-tumorigenic effects of this anti- PD-L1 and anti-VEGF-A dual therapy using orthotopic murine HCC model. They reported that VEGFR2-blockade alone increased PD-1 expression on CD4+ cells in the tumor but when combined with anti-PD-1 therapy, CD4+ cells’ functions were restored and aided in normalization of vessel formation. The combination therapy also increased tumor infiltration and activation of cytotoxic CD8+ T cells, while reducing infiltration of Tregs and monocytes (110). As this field is in its preliminary stages, most studies have showed encouraging efficacies and tolerable toxicity profile in patients treated with combination of RT and ICB but did not specify an optimal RT dose, fractionation scheme and RT/ICB sequencing. There is consensus that these parameters are highly dependent on cancer type, choice of ICB, as well as tumor histology and mutational burden (104). In addition, hypo- fractionation appears to be favored over conventional fractionation as it appears to elicit a more effective anti-tumor response and a dose of 8–10Gy RT with one to three fractions was suggested to induce abscopal effect (104, 105). Importantly, VEGF expression was found to be elevated in post-RT treated HCC tumors (109). Based on this, we propose that it would be meaningful to examine the efficacy of treating HCC patients with anti-angiogenesis and ICB following RT due to their ability to fuel efficient tumor-killing activities and limit the tumor cells’ ability to re-stablish themselves (Figure 3). September 2020 | Volume 11 | Article 568759 FUTURE DEVELOPMENTS IN COMBINATIONAL STRATEGIES FOR HCC However, such treatment regimens have to be carefully evaluated as anti-angiogenics are associated with increased risk of severe RT-related gastrointestinal luminal toxicities (112). Other potentially more tolerable combination such as dual ICBs with RT could also be considered. effects (111). ICB could be administered following this combination treatment to further sustain anti-tumor immunity (Figure 3). However, such treatment regimens have to be carefully evaluated as anti-angiogenics are associated with increased risk of severe RT-related gastrointestinal luminal toxicities (112). Other potentially more tolerable combination such as dual ICBs with RT could also be considered. are being explored as novel immunotherapeutic targets in other cancer types (119). In HCC, both independent or combined expression of B7-H2 and B7-H3 have correlated to recurrence and poorer survival rate of the patients (120). Although further understanding of the tumor-promoting mechanisms by B7 ligands in the context of HCC is required, they could become promising combinatorial agents in treating HCC in the future. Similarly, chimeric antigen receptor T (CAR-T) cell therapy have shown promising results in lymphoma patients and solid tumors with strong driver mutations that could be used as specific targets for CAR-T cell (121, 122). However, HCC is a largely transcriptomically heterogeneous solid tumor (95) and attempts to target HCC-specific antigens, such as AFP, had been evaluated previously with disappointing outcomes (NCT03349255). Nonetheless, findings from a recently concluded phase I trial (NCT02395250) which investigated the safety profile of glypican-3 (GPC3) CAR-T cell therapy in HCC patients showed that it was well tolerated and demonstrated indications of anti-tumor activity (123). GPC3 is involved in the regulation of cell division and growth and its expression in tumor cells was previously associated with poor prognosis for HCC (124). The rapid development in the safety and efficacy of CAR-T cell therapies for solid tumors is optimistic and are highly anticipated as the future of novel immunotherapeutic strategies in cancer. Apart from the current immune checkpoint inhibitors that have been heavily evaluated in HCC, we can also consider other novel targets such as indoleamine 2,3 dioxygenase (IDO). IDO is an enzyme that is involved in immune homeostasis and also escape from immunosurvelliance by tumor cells (113). Overexpression of IDO in HCC tumors was associated to poor prognosis where dendritic cells suppress T cells via IDO and contribute to progression in HCC (114). However, Ishio et al. FUTURE DEVELOPMENTS IN COMBINATIONAL STRATEGIES FOR HCC An alternative strategy would be to administer anti-angiogenic agent prior to RT, which could “normalize” tumor vasculature and in turn, potentially promote greater efficacy of the radiotherapy September 2020 | Volume 11 | Article 568759 7 Combination Therapy in Liver Cancer Lee et al. TABLE 2 | Ongoing clinical trials investigating combined use of RT and ICB in HCC/liver cancer. Clinical trial identification (Study Name) Phase Disease Type of radiative intervention Type of ICB Treatment design Est. enrolment Primary endpoint Secondary endpoints NCT03033446 II HCC Y-90 RE Nivolumab Y-90 RE -> Nivolumab 40 RR TTR, DoR, TTP, PFS, OS, QOL, AEs NCT03482102 II HCC and biliary tract cancer Radiotherapy (not specified) Durvalumab(anti-PD- L1) and Tremelimumab (anti- CTLA_4) Durvalumab + tremelimumab -> RT 70 Best ORR AEs, OS, DCR, PFS, DoR, TTP NCT02239900 II Liver and lung cancer SBRT Ipilimumab(anti-CTLA- 4) Ipilimumab + SBRT orIpilimumab -> SBRT 143 MTD RR NCT02608385 I Solid tumors SBRT Pembrolizumab(anti- PD-1) SBRT -> pembrolizumab 130 Recommended SBRT dose AEs, long-term AEs, RR, PFS, OS, LC, radiation-induced changes in TME, DCR NCT03203304 I HCC SBRT Nivolumab or Ipilimumab SBRT -> Nivolumab or Nivolumab + ipilimumab 50 AEs ORR, long-term AEs, PFS, OS, DCR, LC NCT03817736 (START-FIT) II HCC TACE + SBRT Not specified TACE + SBRT -> ICB 33 No. of patients amendable to curative surgical intervention RR, TTP, PFS, OS, QOL, AEs, PR RE, radioembolization; SBRT, stereotactic body radiation therapy; TACE, transcatheter arterial chemoembolization; RR, response rate; ORR, overall response rate; MTD, maximum tolerated dose; AEs, adverse events; TTR, time to response (TTR); DoR, duration of response; TTP, time to progression; PFS, progression-free survival; OS, overall survival; QOL, quality of life; DCR, disease control rate; LC, local control; PR, pathological response; ->, followed by. TABLE 2 | Ongoing clinical trials investigating combined use of RT and ICB in HCC/liver cancer. RE, radioembolization; SBRT, stereotactic body radiation therapy; TACE, transcatheter arterial chemoembolization; RR, response rate; ORR, overall response rate; MTD, maximum tolerated dose; AEs, adverse events; TTR, time to response (TTR); DoR, duration of response; TTP, time to progression; PFS, progression-free survival; OS, overall survival; QOL, quality of life; DCR, disease control rate; LC, local control; PR, pathological response; ->, followed by. effects (111). ICB could be administered following this combination treatment to further sustain anti-tumor immunity (Figure 3). Frontiers in Immunology | www.frontiersin.org FUTURE DEVELOPMENTS IN COMBINATIONAL STRATEGIES FOR HCC found that IDO could also have anti-tumor properties and its expression level was correlated with gene expressions of IFN-g, tumor necrosis factor alpha (TNFa), and interleukin 1 beta (IL-1b) in HCC (115). Another study found that increased tumor expression of IDO (T- IDO) correlated with increased CD8+ T cells infiltration and favorable outcome in HCC (116). Contrary to above findings, a study conducted in patients of NSCLC found that low activity of IDO following chemoradiation was associated favorably with survival but the effect of radiation on the activity level of IDO was heterogenous (117). A preclinical study carried out with Lewis lung mouse model also showed that treatment with IDO inhibitor and RT synergistically reduced Tregs and expression of exhaustion molecules such as PD-1, PD-L1, and TIM-3 by dendritic cells and T cells (118). Taken altogether, the potential of IDO as a combination therapy with RT in HCC remains to be determined. A crucial aspect to ensure a successful treatment outcome lies in the selection of patients who would benefit most from any given treatment strategies. Mounting evidence shows that variability in pre-existing immunity of each patient reflects their respective clinical response to immunotherapies (93, 94). Crittenden et al. also demonstrated that in vivo blockade of pre-existing immune responses in mice rendered the combination of RT and ICB Currently, several other B7 family ligands such as B7-H2 (a.k.a. inducible T cell costimulator ligand; ICOSL) and B7-H3 September 2020 | Volume 11 | Article 568759 Frontiers in Immunology | www.frontiersin.org 8 Combination Therapy in Liver Cancer Lee et al. ineffective (125). However, there are limited studies on predictive biomarkers for response in various HCC treatments to date. Initially, tumor tissue expression of PD-L1 was used to select patients for enrolment into various ICB trials, however, it was later proven not a strong predictor of response due to heterogeneity in its expression in tumor tissues and various technical issues with detection of its expression (126, 127). Instead, a recent study in a small cohort of liver cancer patients showed that higher cytolytic T cell infiltrates is associated with response towards ICB in HCC (95). Our group constructed a predictive model based on pre-treatment PBMCs of HCC patients and concluded that existence of pre- activated PD-1+ and Tim-3+CD8+ T cells with homing capability (CCR5+ and CXCR6+) was key in predicting response to Y-90-RE (22). REFERENCES 8. Albillos A, Lario M, Alvarez-Mon M. Cirrhosis-associated immune dysfunction: distinctive features and clinical relevance. J Hepatol (2014) 61(6):1385–96. doi: 10.1016/j.jhep.2014.08.010 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin (2018) 68 (6):394–424. doi: 10.3322/caac.21492 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. 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From cirrhosis to hepatocellular carcinoma: new molecular insights on inflammation and cellular senescence. Liver Cancer (2013) 2(3-4):367–83. doi: 10.1159/000343852 12. Vogel A, Cervantes A, Chau I, Daniele B, Llovet J, Meyer T, et al. Hepatocellular carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol (2018) 29(Suppl 4):iv238– 55. doi: 10.1093/annonc/mdy308 5. Schuppan D, Afdhal NH. Liver cirrhosis. Lancet (2008) 371(9615):838–51. doi: 10.1016/S0140-6736(08)60383-9 13. Barbier L, Muscari F, Le Guellec S, Pariente A, Otal P, Suc B. ACKNOWLEDGMENTS We would like to thank the medical oncology team from National Cancer Centre, Singapore (NCCS) and research scientists from TII for the insightful immunological discussion when constructing the manuscript. FUTURE DEVELOPMENTS IN COMBINATIONAL STRATEGIES FOR HCC Similarly, peripheral immune phenotypes may predict for therapeutic efficacy of SBRT and concurrent SBRT and ICB therapy (101, 128). The use of radio-sensitivity index and radiosensitive gene signatures have also been heavily investigated but fell short in their value as predictive biomarkers (129). Taken altogether, the lack of reliable predictive biomarker(s) for various treatments in HCC indicates that greater effort and emphasis is required in this area in order to more accurately select for therapy that provides the maximum clinical benefit and minimal adverse effect for patients. otherwise “cold” TME to an immune-reactive “hot” TME, which synergistically improves the effectiveness of ICB. Beyond RT and ICB, combinational use of anti-angiogenesis agent, which normalizes tumor vasculature and induce local immune response in TME, can also be explored. Most importantly, the sharp increase in the variety of combinational immunotherapeutic options in recent years demands for the discovery of predictive biomarkers in order to determine the most appropriate therapeutic strategy for optimal clinical benefit. FUNDING This work was supported by the National Medical Research Council (NMRC), Singapore (ref numbers: TCR15Jun006, CIRG16may048, CSAS16Nov006, CSASI17may003, and LCG17MAY003). CONCLUDING REMARKS The immunosuppressive landscape of HCC, as evidenced by the presence of pro-tumoral and exhausted immune cells, renders ICB a promising treatment option for HCC patients. Improvements in radiation techniques over the past two decades have boosted the efficacies of RT in HCC. In combination, ICB could overcome the upregulation of immune checkpoint molecules/pathways induced by RT and restore the anti-tumor immunity. In turn, RT can also re-model an AUTHOR CONTRIBUTIONS YL and VC contributed in design, drafting, revising, and approving the final version of the manuscript. DT, CY, and SC assisted in editing the manuscript. All authors contributed to the article and approved the submitted version. 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Outcomes of Observation vs Stereotactic Ablative Radiation for Oligometastatic Prostate September 2020 | Volume 11 | Article 568759 Frontiers in Immunology | www.frontiersin.org 13
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The Use of Bright and Dark Types of Humour is Rooted in the Brain
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Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2017 the Use of Bright and Dark types of Humour is Rooted in the Brain Ilona papousek1, Willibald Ruch2, Christian Rominger1, elisabeth Kindermann1, Katharina scheidl1, Günter schulter1, Andreas Fink1 & elisabeth M. Weiss1 received: 11 October 2016 Accepted: 17 January 2017 Published: 17 February 2017 the ways in which humour can be used are related to the manifold interpersonal functions humour can serve, some of which are positive, and some negative. In the present study, phasic changes in the functional coupling of prefrontal and posterior cortex (eeG coherence) during other people’s auditory displays of happy and sad mood were recorded to predict people’s typical use of humour in social interactions. Greater use of benevolent humour, the intentions of which are in keeping with the characteristics of “laughing-with” humour, was associated with greater decreases of prefrontal- posterior coupling during the processing of happy laughter. More loose prefrontal-posterior coupling indicates loosening of control of the prefrontal cortex over the incoming perceptual information, thereby opening up the perceptual gate and allowing the brain to become more affected by the social- emotional signals. Greater use of humour styles linked to malicious intentions of “laughing-at” humour was associated with responses indicating a wider opened perceptual gate during the processing of other people’s crying. The findings are consistent with the idea that typical humour styles develop in line with the rewarding values of their outcomes (e.g., interaction partners are happy or hurt), which in turn are defined through the individuals’ latent interpersonal goals. The use of humour is a common component in social interaction, where it can serve different purposes. A com- mon distinction, also confirmed by psychometric analysis, is that between using humour with the intention to laugh with others versus the intention to laugh at others1. The ways in which humour can be used are related to the manifold interpersonal functions humour can serve, some of which are positive, and some negative2. Shared humour (laughing with somebody else) is an important social bonding mechanism, it aids the formation, enhancement, and maintenance of social relationships, and enhances feelings of connectedness and closeness3–11. Interpersonal functions of laughing at others include manipulative control, status enhancement or maintenance, ostracism of out-group members, and enforcement of conformity12–14. g p y If the interpersonal intentions coupled with the ways in which humour can be used are not limited to the moment but are manifested as a trait and, thus, are mirrored in the typical use of certain types of humour, the latter may be rooted in relevant social-emotional brain functions. 1University of Graz, Department of Psychology, Biological Psychology Unit, Graz, Austria. 2University of Zurich, Department of Psychology, Zurich, Switzerland. correspondence and requests for materials should be addressed to i.P. (email: ilona.papousek@uni-graz.at) The use of bright and dark types of humour is rooted in the brain Papousek, Ilona ; Ruch, Willibald ; Rominger, Christian ; Kindermann, Elisabeth ; Scheidl, Katharina ; Schulter, Günter ; Fink, Andreas ; Weiss, Elisabeth M DOI: https://doi.org/10.1038/srep42967 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-136109 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 Interna Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-136109 Journal Article Published Version ollowing work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Papousek, Ilona; Ruch, Willibald; Rominger, Christian; Kindermann, Elisabeth; Scheidl, Katharina; Schulter, Gün- ter; Fink, Andreas; Weiss, Elisabeth M (2017). The use of bright and dark types of humour is rooted in the brain. Scientific Reports, 7:42967. DOI: https://doi.org/10.1038/srep42967 Originally published at: Papousek, Ilona; Ruch, Willibald; Rominger, Christian; Kindermann, Elisabeth; Scheidl, Katharina; Schulter, Gün- ter; Fink, Andreas; Weiss, Elisabeth M (2017). The use of bright and dark types of humour is rooted in the brain. Scientific Reports, 7:42967. DOI: https://doi.org/10.1038/srep42967 Originally published at: Papousek, Ilona; Ruch, Willibald; Rominger, Christian; Kindermann, Elisabeth; Scheidl, Katharina; Schulter, Gün- ter; Fink, Andreas; Weiss, Elisabeth M (2017). The use of bright and dark types of humour is rooted in the brain. Scientific Reports, 7:42967. DOI: https://doi.org/10.1038/srep42967 www.nature.com/scientificreports www.nature.com/scientificreports Scientific RepoRts | 7:42967 | DOI: 10.1038/srep42967 received: 11 October 2016 Accepted: 17 January 2017 Published: 17 February 2017 Results S d d Standard multiple regression analyses were performed to examine whether individual differences in changes of prefrontal-posterior coupling during the perception of other people’s laughter or crying may predict the typical use of each of the eight types of humour captured by the 8SHCS. Change-of-coherence scores (∆ coh) during lis- tening to the laughter and crying stimuli were used as the predictors. Because of the wide age range in the sample, and because brain connectivity33,34 as well as humour preferences35,36 may change with aging, age was included as an additional predictor to control for its potential influence. Results of the effects of the change-of-coherence scores (∆ coh) in the right hemisphere during listening to other people’s laughter and crying are summarised in Table 1. The regression models were significant for cyn- icism (F(3,48) = 2.8, p = 0.049, R2 = 0.15), sarcasm (F(3,48) = 3.3, p = 0.027, R2 = 0.17), irony (F(3,48) = 4.0, p = 0.013, R2 = 0.20), wit (F(3,48) = 2.9, p = 0.045, R2 = 0.15), nonsense (F(3,48) = 3.8, p = 0.017, R2 = 0.19), and benevolent humour (F(3,48) = 3.3, p = 0.029, R2 = 0.17), and were not significant for satire (F(3,48) = 2.0, p = 0.132, R2 = 0.11) and fun (F(3,48) = 0.8, p = 0.494, R2 = .05; α = 0.05). With the use of a p < 0.005 criterion for Mahalanobis distance no outliers among the cases were found. The detailed results of the regression analyses depicted in Table 1 show that greater decreases of ∆ coh during the perception of other people’s crying, that indicate a relatively more opened perceptual gate during this stim- ulus, were associated with a greater propensity to use cynical, sarcastic, and ironic humour, and wit. In contrast, greater decreases of ∆ coh during the perception of other people’s laughter were associated with a greater pro- pensity to use benevolent humour. Changes in prefrontal-posterior coupling during the laughter or the crying stimulus did not correlate with the use of satire, fun (clowning around), and nonsense humour. The semi-partial correlations denote the correlations between ∆ coh during one social-emotional stimulus and use of a humour style, adjusted for ∆ coh during the other stimulus and age. Table 1 shows that the semi-partial correlations remained virtually unchanged compared to the respective zero-order correlations. www.nature.com/scientificreports/ to make others laugh about them are goals of the benevolent humour style. Schmidt-Hidding did not attribute significant social goals to the styles nonsense and fun. The set of humour styles overlaps with previous ones (e.g., 8) but is more extensive15. ) Broadly defined, goals refer to internal representations of desired states19. The goals attributed to the humour styles most probably refer to “latent” goals19, which can motivate action and direct behaviour outside of people’s awareness20 and which play key roles in many aspects of social life (e.g., moral behaviour, social discrimination21). It is believed that positive reward signals are attached to the outcomes of goals via the mesolimbic dopamine sys- tem20,22,23. Whether a goal is pursued depends on its rewarding value, that is, on the extent to which the outcome is desired and rewarding19. g Considering an interpersonal view of the desired outcomes, the goals attached to the humour styles may be summarised as either to make the targets happy and laugh (“laughing-with” type of humour), or to hurt the targets, make them cry (“laughing-at” styles). Taken together, this means that the respective outcomes (social interaction partners are happy or hurt) and, as a consequence thereof, the respective displays of happy/exhilarated (laughter) or depressed/despaired (crying) emotional states have a desired, rewarding value. Pursuing this train of thought, one may expect different brain responses in individuals with lesser versus greater use of bright (“laughing-with”) or dark (“laughing-at”) types of humour when they are exposed to other people’s laughter and crying, depending on the supposed rewarding value of the stimulus. Specifically, this con- cerns phasic changes in the functional coupling of prefrontal and posterior association cortex, measured by changes of EEG coherence, which signify modulation of incoming affectively laden social information. More loose prefrontal-posterior coupling during social-emotional processing, especially in the right hemisphere, indi- cates loosening of control of the prefrontal cortex over the incoming perceptual information, thereby opening up the perceptual gate and allowing the brain to become more affected by the social-emotional signals. By contrast, functional coupling increases during exposure to aversive information, protecting the individual from being unduly affected by the aversive input24–29. yf y p It may be expected, therefore, that typical use of “laughing-with” humour will be correlated with decreases of prefrontal-posterior coupling (EEG coherence) during the processing of other people’s happy laughter, allowing the brain to become more affected by this rewarding social-emotional signal. www.nature.com/scientificreports/ On the other hand, typical use of dark, “laughing-at” humour may be correlated with relative decreases of prefrontal-posterior coupling during the processing of other people’s crying. p g p p y g However, there is at least one other possible outcome. Dark humour styles were associated with low interper- sonal competence, particularly with poor ability to perceive other people’s emotions30–32. Therefore, it is possible that individuals poor in the perception of emotions tend to use humour in compromising ways, because they do not (appropriately) interprete the target’s emotional feedback. In regard to the brain responses, during the expo- sure to social-emotional signals, prefrontal-posterior coupling was also higher (gate more closed) in individuals with a generally lower propensity for perceiving the emotional states of other persons26. Consequently, if poorer emotion perception is the more crucial process underlying strong tendencies to use dark humour, use of dark humour styles may be expected to be associated with increases, rather than decreases, of prefrontal-posterior coupling in response to other people’s crying. Scientific RepoRts | 7:42967 | DOI: 10.1038/srep42967 the Use of Bright and Dark types of Humour is Rooted in the Brain Ilona papousek1, Willibald Ruch2, Christian Rominger1, elisabeth Kindermann1, Katharina scheidl1, Günter schulter1, Andreas Fink1 & elisabeth M. Weiss1 For the following reasons, the brain’s auto- matic responses to the perception of other people’s displays of happy and depressed or despaired emotional states seemed to be a promising candidate for finding neurological roots of bright and dark humour styles. Humour styles refer to the ways in which humour is typically used in social interactions, and there are mul- tiple ways to categorise them15. We used a classification in the present study that specifically focuses on the use/ production of humour (as opposed to the appreciation of humour produced by others) and includes the attempt to define the goals and intentions of the use of humour paired with the attitudes towards the targets. The classifi- cation goes back to Schmidt-Hidding16, who initially used a lexical approach and analysed humour in literature. The eight humour styles identified by Schmidt-Hidding were picked up by Ruch who analyzed them from a psy- chological perspective and cast them in a psychometrically sound self-report instrument15,17,18. Factor analysis of the structure of these eight humour styles suggested a clear cluster referring to “laughing-at” styles comprising cynicism, sarcasm, and irony, one factor comprising only benevolent humour (“laughing with”), and one separate factor comprising fun (clowning around) and nonsense humour. Wit and satire had double loadings and, thus, can apparently be used in negative as well as positive ways17,18. The empirical clusters largely match the social goals that Schmidt-Hidding had attributed to the humour styles: Malicious, mean-spirited goals and attitudes, intentions of hurting other persons and demonstrating superiority are attributes of cynicism, sarcasm, irony, in part also of satire and wit. To brighten others up and point up funny sides of adversities or short-comings in order Scientific RepoRts | 7:42967 | DOI: 10.1038/srep42967 1 www.nature.com/scientificreports/ Results S d d ∆coh (laughter) ∆coh (crying) Age r (p) sr (p) r (p) sr (p) r (p) sr (p) Cynicism* − 0.05 (0.712) − 0.07 (0.600) −0.33 (0.018) −0.31 (0.024) − 0.23 (0.104) − 0.19 (0.156) Sarcasm* 0.05 (0.719) 0.04 (0.774) −0.31 (0.027) −0.27 (0.044) −0.31 (0.027) −0.28 (0.040) Irony* − 0.10 (0.483) − 0.11 (0.384) −0.32 (0.021) −0.30 (0.027) −0.32 (0.020) −0.29 (0.032) Satire − 0.20 (0.157) − 0.22 (0.113) − 0.23 (0.098) − 0.26 (0.067) 0.06 (0.672) 0.09 (0.499) Wit* − 0.08 (0.561) − 0.10 (0.445) −0.34 (0.014) −0.33 (0.018) − 0.20 (0.152) − 0.16 (0.226) Fun 0.04 (0.781) 0.03 (0.839) − 0.19 (0.171) − 0.18 (0.212) − 0.12 (0.393) − 0.10 (0.472) Nonsense* 0.08 (0.575) 0.09 (0.539) − 0.18 (0.216) − 0.13 (0.338) −0.41 (0.003) −0.39 (0.004) Benevol. humour* −0.30 (0.029) −0.31 (0.021) − 0.21 (0.136) − 0.21 (0.110) − 0.19 (0.186) − 0.15 (0.247) Table 1. Prediction of use of types of humour by brain responses to other people’s laughter and crying Table 1. Prediction of use of types of humour by brain responses to other people’s laughter and crying (changes of prefrontal-posterior EEG coherences, ∆coh). Note: *Statistically significant regression models (F-test). r = zero-order correlation, sr = semipartial correlation, p = p-value (two-tailed). Coherence changes in the right hemisphere (beta frequency range) relative to neutral stimulation. Negative scores of ∆ coh denote a relative decrease of prefrontal-posterior coherence (more opened perceptual gate), positive scores denote an increase (gate more closed). Significant zero-order and semi-partial correlations are highlighted in bold font (α = 0.05). N = 52. Table 1. Prediction of use of types of humour by brain responses to other people’s laughter and crying (changes of prefrontal-posterior EEG coherences, ∆coh). Note: *Statistically significant regression models (F-test). r = zero-order correlation, sr = semipartial correlation, p = p-value (two-tailed). Coherence changes in the right hemisphere (beta frequency range) relative to neutral stimulation. Negative scores of ∆ coh denote a relative decrease of prefrontal-posterior coherence (more opened perceptual gate), positive scores denote an increase (gate more closed). Significant zero-order and semi-partial correlations are highlighted in bold font (α = 0.05). N = 52. “laughing-at” “laughing-with” r (p) sr (p) r (p) sr (p) ∆ coh (laughter) − 0.21 (0.136) − 0.07 (0.584) −0.30 (0.029) −0.32 (0.024) ∆ coh (crying) −0.35 (0.010) −0.29 (0.033) − 0.03 (0.815) 0.10 (0.476) Table 2. Results S d d Correlations of brain responses to other people’s laughter and crying (changes of prefrontal- posterior EEG coherences, ∆coh) with unique variance of the typical use of “laughing-at” and “laughing- with” humour. Note: r = zero-order correlation, sr = semipartial correlation, p = p-value (two-tailed). Coherence changes in the right hemisphere (beta frequency range) relative to neutral stimulation. Negative scores of ∆ coh denote a relative decrease of prefrontal-posterior coherence (more opened perceptual gate), positive scores denote an increase (gate more closed). Significant zero-order and semi-partial correlations are highlighted in bold font (α = 0.05). These correlations are considered to be of medium size according to the common conventions of Cohen60. N = 52. Table 2. Correlations of brain responses to other people’s laughter and crying (changes of prefrontal- posterior EEG coherences, ∆coh) with unique variance of the typical use of “laughing-at” and “laughing- with” humour. Note: r = zero-order correlation, sr = semipartial correlation, p = p-value (two-tailed). Coherence changes in the right hemisphere (beta frequency range) relative to neutral stimulation. Negative scores of ∆ coh denote a relative decrease of prefrontal-posterior coherence (more opened perceptual gate), positive scores denote an increase (gate more closed). Significant zero-order and semi-partial correlations are highlighted in bold font (α = 0.05). These correlations are considered to be of medium size according to the common conventions of Cohen60. N = 52. between the use of cynical, sarcastic, and ironic humour with the brain responses to other people’s crying are due to a common underlying characteristic of these humour styles. Th l d h h d d l ’ f “l h ” “l h h” h The regression analyses demonstrate that the individuals’ use of “laughing-at” or “laughing-with” humour were specifically predicted by the changes of prefrontal-posterior coupling in response to other people’s crying or laughter, respectively. Additionally, two supplemental regression analyses were conducted to assess the cor- relations between the use of dark humour styles (i.e. averaged across cynicism, sarcasm, and irony) and benev- olent humour, and ∆ coh during the crying or the laughter stimulus. Results of these analyses are summarised in Table 2. The semipartial correlations show that decreases in prefrontal-posterior coupling during the crying stimulus were specifically correlated with the use of “laughing-at” humour but not with the use of “laughing-with” humour. Results S d d Conversely, decreases in prefrontal-posterior coupling during the laughter stimulus were specifically correlated with the use of “laughing-with” humour but not with the use of “laughing-at” humour. Figures 1 and 2 show scatter plots of these correlations. The correlation between the use of “laughing-at” and “laughing-with” humour was r = 0.40 (p = 0.003, two-tailed), reflecting an additional superordinate factor of humourousness17. (p )l g p Analogous regression analyses for the left hemisphere did not reveal significant correlations between prefrontal-posterior coherence changes and humour styles (highest sr = − 0.12, p = 0.406). Use of the humour styles did not differ between men and women (in independent t-Tests t-values ranged from t(50) = 0.2, p = 0.866 to t(50) = 1.7, p = 0.105), and did not depend on the educational level of the participants (less then high school vs high school graduate or university; t-values ranged from t(50) = 0.5, p = 0.655 to t(50) = 1.8, p = 0.076), except for nonsense humour (greater use with higher educational levels, M = 4.2, SD = 1.2; M = 3.4, SD = 0.9; t(50) = 2.9, p = .006, η2 = 0.14). Results S d d This suggests that the rela- tionships between changes of prefrontal-posterior coupling and the use of humour were present independently from eventual correlations with age, and also that the responses to the two stimuli were largely independent. Additionally, the analyses revealed that, independently from the brain responses to the social-emotional stimuli, older participants indicated less use of sarcastic, ironic, and nonsense humour. The pattern observed in these results corroborates the findings of the factor analysis in a larger sample which clearly yielded a factor comprising cynicism, sarcasm, and irony, and another factor comprising only benevolent humour17. Wit did not unequivocally load on either of these factors. It therefore seems that the correlations Scientific RepoRts | 7:42967 | DOI: 10.1038/srep42967 2 www.nature.com/scientificreports/ ∆coh (laughter) ∆coh (crying) Age r (p) sr (p) r (p) sr (p) r (p) sr (p) Cynicism* − 0.05 (0.712) − 0.07 (0.600) −0.33 (0.018) −0.31 (0.024) − 0.23 (0.104) − 0.19 (0.156) Sarcasm* 0.05 (0.719) 0.04 (0.774) −0.31 (0.027) −0.27 (0.044) −0.31 (0.027) −0.28 (0.040) Irony* − 0.10 (0.483) − 0.11 (0.384) −0.32 (0.021) −0.30 (0.027) −0.32 (0.020) −0.29 (0.032) Satire − 0.20 (0.157) − 0.22 (0.113) − 0.23 (0.098) − 0.26 (0.067) 0.06 (0.672) 0.09 (0.499) Wit* − 0.08 (0.561) − 0.10 (0.445) −0.34 (0.014) −0.33 (0.018) − 0.20 (0.152) − 0.16 (0.226) Fun 0.04 (0.781) 0.03 (0.839) − 0.19 (0.171) − 0.18 (0.212) − 0.12 (0.393) − 0.10 (0.472) Nonsense* 0.08 (0.575) 0.09 (0.539) − 0.18 (0.216) − 0.13 (0.338) −0.41 (0.003) −0.39 (0.004) Benevol. humour* −0.30 (0.029) −0.31 (0.021) − 0.21 (0.136) − 0.21 (0.110) − 0.19 (0.186) − 0.15 (0.247) Table 1. Prediction of use of types of humour by brain responses to other people’s laughter and crying (changes of prefrontal-posterior EEG coherences, ∆coh). Note: *Statistically significant regression models (F-test). r = zero-order correlation, sr = semipartial correlation, p = p-value (two-tailed). Coherence changes in the right hemisphere (beta frequency range) relative to neutral stimulation. Negative scores of ∆ coh denote a relative decrease of prefrontal-posterior coherence (more opened perceptual gate), positive scores denote an increase (gate more closed). Significant zero-order and semi-partial correlations are highlighted in bold font (α = 0.05). N = 52. Scientific RepoRts | 7:42967 | DOI: 10.1038/srep42967 Discussion The study showed that an individual’s typical use of bright (“laughing-with”) and dark (“laughing-at”) humour is linked to the brain’s automatic responses to incoming social-emotional information. Greater typical use of benevolent humour, the goals and intentions of which are in keeping with the characteristics of “laughing-with” humour, was associated with greater relative decreases of prefrontal-posterior coupling during the process- ing of other people’s happy laughter, allowing the brain to become more affected by this apparently rewarding social-emotional signal24–29. Greater typical use of the three humour styles that were consistent with malicious intentions of “laughing-at” humour (cynicism, sarcasm, irony17) was associated with greater relative decreases of prefrontal-posterior coupling (i.e., a wider opened perceptual gate) during the processing of other people’s crying. Scientific RepoRts | 7:42967 | DOI: 10.1038/srep42967 3 www.nature.com/scientificreports/ Figure 1. Correlation between changes of prefrontal-posterior EEG coherence in response to the perception of other people’s crying and use of dark (“laughing-at”) humour. Coherence changes in the right hemisphere (beta frequency range) relative to neutral stimulation. The plot shows standardised residuals (∆ coh; see Methods section). Negative scores of ∆ coh denote a relative decrease of prefrontal-posterior coherence (more opened perceptual gate), positive scores denote an increase (gate more closed). “Laughing-at” humour comprises the styles cynicism, sarcasm, and irony. Figure 1. Correlation between changes of prefrontal-posterior EEG coherence in response to the perception of other people’s crying and use of dark (“laughing-at”) humour. Coherence changes in the right hemisphere (beta frequency range) relative to neutral stimulation. The plot shows standardised residuals (∆ coh; see Methods section). Negative scores of ∆ coh denote a relative decrease of prefrontal-posterior coherence (more opened perceptual gate), positive scores denote an increase (gate more closed). “Laughing-at” humour comprises the styles cynicism, sarcasm, and irony. Figure 2. Correlation between changes of prefrontal-posterior EEG coherence in response to the perception of other people’s laughter and use of benevolent (“laughing-with”) humour. Coherence changes in the right hemisphere (beta frequency range) relative to neutral stimulation. The plot shows standardised residuals (∆ coh; see Methods section). Negative scores of ∆ coh denote a relative decrease of prefrontal- posterior coherence (more opened perceptual gate), positive scores denote an increase (gate more closed). Figure 2. Correlation between changes of prefrontal-posterior EEG coherence in response to the perception of other people’s laughter and use of benevolent (“laughing-with”) humour. Coherence changes in the right hemisphere (beta frequency range) relative to neutral stimulation. Methods p ti i participants. A total of n = 52 participants (21 men, 31 women) completed the experiment (age range 20 to 71 years, M = 36.7, SD = 14.4). Levels of education were: less than high school (29), high school graduate (19), university degree (4). All participants were right-handed as confirmed by a standardised hand skill test. Individuals who reported having a neuropsychiatric disease or using psychoactive medication were not included in the study. Participants were requested to refrain from alcohol for twelve hours and from coffee and other stim- ulating beverages for two hours prior to their lab appointment, and to come to the session well rested. The study was performed in accordance with the American Psychological Association’s Ethics Code and the Declaration of Helsinki and was approved by the ethics committee of the University of Graz. Informed consent was obtained from all participants. Assessment of Humour styles. In the 8SHCS (8 Schmidt-Hidding Comic Styles18), participants are asked to rate the extent to which 48 statements apply to the way they typically express humour on a seven-point Likert scale (from 0 – “strongly disagree” to 6 – “strongly agree”). The questionnaire comprises eight subscales (6 items each) capturing the propensity to use humour in the form of sarcasm (e.g., “Biting mockery suits me”, test reliabil- ity was α = 0.87 in the present study, M = 2.22, SD = 1.31), cynicism (e.g., “I tend to show no reverence for certain moral concepts and ideals, but only scorn and derision”, α = 0.89, M = 2.52, SD = 1.31), irony (e.g., “My irony unveils who is smart enough and understands something and who does not”, α = 0.80, M = 3.25, SD = 1.11), sat- ire (“I like to ridicule moral badness to induce or increase a critical attitude in other people”, α = 0.81, M = 2.89, SD = 1.15), wit (e.g. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Taken together, the findings do not support the alternative possibility that poor perception of other people’s emotions may be the most decisive factor predicting a high propensity for using “laughing-at” types of humour, despite the fact that dark humour styles were associated with low interpersonal competence30–32. If poor percep- tion of other’s emotions had been more important, greater tendencies to use “laughing-at” humour would have been correlated with increased prefrontal-posterior coupling during the confrontation with other people’s crying (i.e., a more closed perceptual gate26). p p g It has been clearly recognized that the social effect of joking and laughing with somebody else, that is, the happy laughter of interacting partners, is an immediately pleasurable and rewarding experience for most people who produce humour6–11. The current findings and their theoretical foundation suggest that in some people (i.e., in those with a high propensity to use “laughing-at” humour), expressions of hurt, depressed or despaired feel- ings may have a rewarding value. Laughing at or ridiculing another person is an expression of disapproval that induces strong negative feelings in that person such as shame, feelings of inferiority and devaluation, which are very painful emotions39 and, therefore, have marked effects on the person’s outward emotional expression. There is, in fact, evidence that observing or causing other people’s adversity can be a source of pleasure40, a feature that is associated with psychopathic personality traits41. In line with that, several studies have shown relationships between the propensity of laughing at others and psychopathic personality traits such as antagonism, manipula- tion and callousness14,42–44. Previous research indicated that the use of dark types of humour may be one factor perpetuating maladaptive cognitive schemas that implicate the belief that one is superior to others and that others should be controlled and dominated45. These maladaptive cognitive schemas are also associated with vulnerability to development of externalising/aggressive psychopathology46–48. p g gg p y p gy Apart from potentially maladaptive features and developments in those with high tendencies to use humour in dark ways, using humour with a view to laughing at other people can have drastic negative social conse- quences. www.nature.com/scientificreports/ For instance, in line with the interpersonal functions, goals, and desired outcomes that are attached to “laughing-at” types of humour, it was shown that, from a very early age on (6 years) a high propensity to enjoy laughing at others was related to bullying behaviour49, which can entail devastating effects on the victims and their social behaviour50,51. On the other hand, there is evidence that the use of benevolent humour targeted to laugh with somebody else may help to protect against the development of depression52. g y y p p g p p Collectively, the pattern of the present findings is largely consistent with the idea that an individual’s typical humour style develops in line with the rewarding values of its outcomes (e.g., social interaction partners are happy or hurt), which in turn are defined through the – latent – interpersonal goals of the individual. That way, the typical use of bright and dark types of humour seems to be rooted in the brain, and hence may provide indi- cations of biologically anchored clinically and socially relevant personality features. Discussion The plot shows standardised residuals (∆ coh; see Methods section). Negative scores of ∆ coh denote a relative decrease of prefrontal- posterior coherence (more opened perceptual gate), positive scores denote an increase (gate more closed). This finding is congruent with the assumed rewarding value of this social-emotional signal in people with a high propensity to use “laughing-at” kinds of humour. Use of nonsense humour and fun (clowning around), to which Schmidt-Hidding16 had not attributed significant social goals, and which constituted a separate factor (i.e., sep- arate from typical “laughing-at” and “laughing-with” kinds of humour) in the large-sample study by Ruch17, was not related to the brain responses to other people’s affect expressions. It seems noteworthy, in this context, that benevolent humour may involve moral goodness or virtue37, which is lacking in sheer fun. This finding is congruent with the assumed rewarding value of this social-emotional signal in people with a high propensity to use “laughing-at” kinds of humour. Use of nonsense humour and fun (clowning around), to which Schmidt-Hidding16 had not attributed significant social goals, and which constituted a separate factor (i.e., sep- arate from typical “laughing-at” and “laughing-with” kinds of humour) in the large-sample study by Ruch17, was not related to the brain responses to other people’s affect expressions. It seems noteworthy, in this context, that benevolent humour may involve moral goodness or virtue37, which is lacking in sheer fun. y g g However, also one of the two more ambivalent types of humour (wit)17 correlated with decreases of prefrontal-posterior coupling during the crying stimulus. This might be explained by the nature of typical jokes, which almost always are made at someone else’s expense38. Thus, some malicious intentions are included in this type of humour, which in the current sample perhaps might have outweighed the benevolent parts. Scientific RepoRts | 7:42967 | DOI: 10.1038/srep42967 4 www.nature.com/scientificreports/ offline to a mathematically averaged ears refs 53, 54. All data were inspected visually, in order to eliminate inter- vals in which ocular or muscle artefacts occurred. At least 30 s of artefact free data was obtained for each par- ticipants for each of the recording periods and for each of the electrode positions of interest. Artefact-free EEG data were submitted to Fast Fourier Analysis using a Hanning window (epoch length 1 s, overlapping 50%), and spectral coherence (Fisher’s z-transformed) was obtained. Previous research on EEG coherence in the context of affective processing indicated that connectivity changes during evoked emotions occurred primarily in the beta frequency range24–29,55–57. Consequently, we focused on coherence in the beta range (13–30 Hz). Following previous relevant research24–29,56, coherence pairs were grouped into anatomically valid clusters corresponding to the left and right, prefrontal and posterior association cortex regions. Coherence scores of nine electrode pairs each were averaged to summarise interaction within the left and the right hemisphere, respectively (left: Fp1-T3, Fp1-P3, Fp1-T5, F3-T3, F3-P3, F3-T5, F7-T3, F7-P3, F7-T5; right: Fp2-T4, Fp2-P4, Fp2-T6, F4-T4, F4-P4, F4-T6, F8-T4, F8-P4, F8-T6). , , , , ) Linear regressions were conducted using the EEG beta coherence during the neutral (reference) stimulus preceding the emotional stimulus to predict the coherence during listening to each of the emotional sound clips, in order to calculate residualised change scores (cf.25–29,56). These were used as indexes of state-dependent rela- tive decreases or increases of intra-hemispheric coherence in response to the social-emotional stimulation. This was done to ensure that the analysed residual variability was due to the experimental manipulation, and not to individual differences in baseline levels, and to control for measurement error inherent in the use of repeated measures of the same kind58,59. The abbreviation “∆ coh” is used for these change-of-coherence scores. Negative scores indicate a relative decrease in prefrontal-posterior coherence, positive scores indicate a relative increase. p p p Since previous research indicated strong right-hemisphere dominated effects of coherence changes in the context of emotional processing, the analysis focused on prefrontal-posterior coherence changes in the right hemisphere, and a separate (supplemental) set of regression analyses tested for potential effects in the left hemi- sphere (cf.25–29). procedure. After completing the handedness test, participants were seated in an acoustically and electrically shielded examination room, and electrodes were attached. www.nature.com/scientificreports/ They were instructed to close their eyes, to direct their whole attention to the sound recordings, and to imagine that they were amidst the happenings. A two-minutes resting period preceded the stimulation. The order of emotional sounds was counterbalanced, and emotional and neutral sound clips were presented in alternating order, so that each emotional sound was preceded by the neutral sound (i.e., N-S-N-C or N-C-N-S). Before each sound clip, the instructions were briefly repeated. The 8SHCS was completed in a separate test session. References The effects of power on laughter Pers psychological well-being: Development of the humor styles questionnaire. J. Res. Pers. 37, 48–75 (2003). 9. Stillman, T. F., Baumeister, R. F. & DeWall, C. N. What’s so funny about not having money? The effects of power on laughter. Pers. S P h l B ll 33 1547 1558 (2007) 9. Stillman, T. F., Baumeister, R. F. & DeWall, C. N. What’s so funny about not having money? The effects of power on laughter. Soc. Psychol. Bull. 33, 1547–1558 (2007). y 10. Vinton, K. L. Humor in the work place: Is it more than telling jokes. Small Group Behav. 20, 151–166 (1989). p g j p 11. Weisfeld, G. E. The adaptive value of humor and laughter. Ethol. Sociobiol. 14, 141–169 (1993). mor theory and the fear of being laughed at. Humor: Int. J. Humor R 13. Martin, R. A. The psychology of humor: An integrative approach (Elsevier, 2007). h p y gy f g pp 14. Proyer, R. T., Flisch, R., Tschupp, S., Platt, T. & Ruch, W. How does psychopathy relate to humor and laughter. Dispositions towards ridicule and being laughed at, the sense of humor, and psychopathic personality traits. Int. J. Law Psychiatry 35, 263–268 (2012). 4. Proyer, R. T., Flisch, R., Tschupp, S., Platt, T. & Ruch, W. How does psychopathy relate to humor and laughter. Dispositions toward idi l d b i l h d h f h d h hi li i I J L P hi 35 263 268 (2012) ridicule and being laughed at, the sense of humor, and psychopathic personality traits. Int. J. Law Psychiatry 35, 263–268 (2012). 15. Ruch, W. & Heintz, S. The German version of the Humor Styles Questionnaire: Psychometric properties and overlap with other styles of humor. Eur. J. Psychol. 12, 434–455 (2016). 15. Ruch, W. & Heintz, S. The German version of the Humor Styles Questionnaire: Psychometric properties and overlap with styles of humor. Eur. J. Psychol. 12, 434–455 (2016). y J y , ( ) 16. Schmidt-Hidding, W. Humor und WitzHumour and Wit (Huebner, 1963). y J y ( ) 16. Schmidt-Hidding, W. Humor und WitzHumour and Wit (Huebner, 1963). 17. Ruch, W. Towards a new structural model of the sense of humor: Preliminary findings. AAAI Techn. Rep. Methods p ti i “I surprise others with funny remarks and accurate judgements of current issues, which occur to me spontaneously”, α = 0.88, M = 3.48, SD = 1.08), benevolent humour (e.g., “When my humour is aimed at human weaknesses, I include both myself and others”, α = 0.69, M = 3.99, SD = 0.77), fun (e.g., “I like to make jests and to be silly”, α = 0.89, M = 3.53, SD = 1.30), and nonsense (e.g., “Humour doesn’t have to make sense; the opposite holds true for me: the more absurd, the funnier”, α = 0.83, M = 3.76, SD = 1.11). Self-peer correlations were between r = 0.40 and r = 0.56 with a median of r = 0.4918. social-emotional stimulation. Three sound recordings in which a small mixed-gender group of people audibly expressed the respective affect without using language (words or parts of words) were used (90 s each): Laughter (good-natured, hearty laughter), Crying (bitter crying and sobbing), and a neutral recording (soft mur- murs and trivial everyday sounds without understandable language), serving as the reference condition. The clips were matched for peak sound intensity and sound level range, and were presented over headphones. They have been used in several previous studies25–29. The displayed emotions are unambiguous and intense; healthy partici- pants have no difficulties identifying and differentiating the expressed affective states25,29. EEG Recording and Quantification. 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Reiser, E. M. et al. Prefrontal-posterior coupling while observing the suffering of other people, and the development of intrusive memories. Psychophysiology 51, 546–555 (2014). y p y gy 57. Schellberg, D., Besthorn, C., Klos, T. & Gasser, T. EEG power and coherence while male adults watch emotional video films. Int. J. Psychophysiol. 9, 279–291 (1990). y p y , ( ) 58. Linden, W., Earle, L., Gerin, W. & Christenfeld, N. Physiological stress reactivity and recovery: Conceptual siblings separated at birth? J. Psychosom. Res. 42, 117–135 (1997). y 9. Steketee, G. S. & Chambless, D. L. Methodological issues in the prediction of treatment outcome. Clin. Psychol. Rev. 12, 387–400 (1992) 0. Cohen, J. Statistical power analysis for the behavioral sciences, 2nd ed. (Lawrence Erlbaum, 1988). g p y 60. Scientific RepoRts | 7:42967 | DOI: 10.1038/srep42967 Additional Information Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Papousek, I. et al. The Use of Bright and Dark Types of Humour is Rooted in the Brain. ci. Rep. 7, 42967; doi: 10.1038/srep42967 (2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Scientific RepoRts | 7:42967 | DOI: 10.1038/srep42967 7 www.nature.com/scientificreports/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. 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Prominent Heart Organ-Level Performance Deficits in a Genetic Model of Targeted Severe and Progressive SERCA2 Deficiency
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Received May 14, 2013; Accepted September 24, 2013; Published November 4, 2013 nis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright:  2013 Heinis et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health (NIH) 5R01HL059301-14 to JMM. FIH was supported by NIA training grant 5T32AG029796-05. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: metzgerj@umn.edu * E-mail: metzgerj@umn.edu * E-mail: metzgerj@umn.edu Prominent Heart Organ-Level Performance Deficits in a Genetic Model of Targeted Severe and Progressive SERCA2 Deficiency Frazer I. Heinis1,2, Kristin B. Andersson3,4, Geir Christensen3,4, Joseph M. Metzger1* Frazer I. Heinis1,2, Kristin B. Andersson3,4, Geir Christensen3,4, Joseph M. Metzger1* 1 Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America, 2 Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America, 3 Institute for Experimental Medical Research, Oslo University Hospital Ullevaal and University of Oslo, Oslo, Norway, 4 Center for Heart Failure Research, University of Oslo, Oslo, Norway 1 Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America, 2 Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America, 3 Institute for Experimental Medical Research, Oslo University Hospital Ullevaal and University of Oslo, Oslo, Norway, 4 Center for Heart Failure Research, University of Oslo, Oslo, Norway November 2013 | Volume 8 | Issue 11 | e79609 Abstract The cardiac SERCA2 Ca2+ pump is critical for maintaining normal Ca2+ handling in the heart. Reduced SERCA2a content and blunted Ca2+ reuptake are frequently observed in failing hearts and evidence implicates poor cardiac Ca2+ handling in the progression of heart failure. To gain insight into mechanism we investigated a novel genetic mouse model of inducible severe and progressive SERCA2 deficiency (inducible Serca2 knockout, SERCA2 KO). These mice eventually die from overt heart failure 7-10 weeks after knockout but as yet there have been no reports on intrinsic mechanical performance at the isolated whole heart organ level. Thus we studied whole-organ ex vivo function of hearts isolated from SERCA2 KO mice at one and four weeks post-knockout in adult animals. We found that isolated KO heart function was only modestly impaired one week post-knockout, when SERCA2a protein was 32% of normal. At four weeks post-knockout, function was severely impaired with near non-detectable levels of SERCA2. During perfusion with 10 mM caffeine, LV developed pressures were similar between 4-week KO and control hearts, and end-diastolic pressures were lower in KO. When hearts were subjected to ischemia-reperfusion injury, recovery was not different between control and KO hearts at either one or four weeks post- knockout. Our findings indicate that ex vivo function of isolated SERCA2 KO hearts is severely impaired long before symptoms appear in vivo, suggesting that physiologically relevant heart function in vivo can be sustained for weeks in the absence of robust SR Ca2+ flux. Citation: Heinis FI, Andersson KB, Christensen G, Metzger JM (2013) Prominent Heart Organ-Level Performance Deficits in a Genetic Model of Targeted Severe and Progressive SERCA2 Deficiency. PLoS ONE 8(11): e79609. doi:10.1371/journal.pone.0079609 Editor: Rajesh Gopalrao Katare, University of Otago, New Zealand Editor: Rajesh Gopalrao Katare, University of Otago, New Zealand Editor: Rajesh Gopalrao Katare, University of Otago, New Zealand Received May 14, 2013; Accepted September 24, 2013; Published November 4, 2013 Received May 14, 2013; Accepted September 24, 2013; Published November 4, 2013 Introduction Experimental timeline and SERCA2 protein determination in FL and KO hearts. A, Experimental Timeline. All animals were injected with tamoxifen then sacrificed either 1 or 4 weeks later for heart isolation. KO mice expressed the aMHC-MerCreMer transgene, which efficiently excised LoxP-flanked exons of the Serca2 locus in response to tamoxifen. FL mice received tamoxifen, but retained normal SERCA2 levels due to lack of MerCreMer. B-C, at 1 week post-tamoxifen, SERCA2 content in KO hearts was diminished to 32% of FL. D-E, at 4 weeks post-tamoxifen, faint SERCA2 bands were observed in 20 mg of heart lysate. ***: P,0.001 for FL vs. KO at either 1 week or 4 weeks post-tamoxifen injection, as determined by unpaired two-tailed t test. doi:10.1371/journal.pone.0079609.g001 Figure 1. Experimental timeline and SERCA2 protein determination in FL and KO hearts. A, Experimental Timeline. All animals were injected with tamoxifen then sacrificed either 1 or 4 weeks later for heart isolation. KO mice expressed the aMHC-MerCreMer transgene, which efficiently excised LoxP-flanked exons of the Serca2 locus in response to tamoxifen. FL mice received tamoxifen, but retained normal SERCA2 levels due to lack of MerCreMer. B-C, at 1 week post-tamoxifen, SERCA2 content in KO hearts was diminished to 32% of FL. D-E, at 4 weeks post-tamoxifen, faint SERCA2 bands were observed in 20 mg of heart lysate. ***: P,0.001 for FL vs. KO at either 1 week or 4 weeks post-tamoxifen injection, as determined by unpaired two-tailed t test. doi:10.1371/journal.pone.0079609.g001 for much longer than predicted based on current knowledge of cardiac Ca2+ handling and SR function (reviewed in [15]). dysfunction ex vivo at times when in vivo pathology is not yet manifest. We studied Serca2 KO and FL control hearts at 1 and 4 weeks post-knockout, time points well prior to the onset of overt pump failure, but where severe dysfunction of isolated cardiac- myocytes is manifest, to establish an organ-level biological SERCA2 dose-response relationship. Since sarcolemmal Ca2+ currents are increased following Serca2 knockout[11,13,14], we perfused isolated 4-week Serca2 FL and KO hearts with caffeine to reveal SR-independent contractile function. In addition, we used this model to test whether altered SR Ca2+ handling could improve the functional recovery of isolated hearts from ischemia- reperfusion injury. Introduction allows for selective disruption of the Serca2 gene in the hearts of adult mice[10,11]. In this model, exons 2 and 3 of Serca2 gene locus are flanked by loxP sites, thereby allowing selective disruption of Serca2 in adult cardiac myocytes conferred by a tamoxifen-sensitive, cardiac myocyte-expressed MerCreMer trans- gene[12]. Normal heart pump function requires the highly regulated, cyclical release and reuptake of Ca2+ in the myoplasm of cardiac myocytes. Reuptake of Ca2+ into the sarcoplasmic reticulum (SR), accomplished by the cardiac SR Ca2+ ATPase (SERCA2), plays a major role in cardiac muscle relaxation and also is critical in determining SR Ca2+ load and subsequent systolic Ca2+ release. Defects in Ca2+ handling are clearly associated with cardiac dysfunction and heart failure[1–6]. Although diminished Ca2+ flux and reduced SERCA2a expression are frequently observed in the failing heart, the exact relationship between these observations is not fully known. It is not clearly established whether loss of SERCA2a is a driving primary cause of severe cardiac dysfunction or whether this is a secondary consequence of the emerging cardiac pathology. g [ ] This model of genetic SERCA2 manipulation has been characterized in vitro in isolated cardiac myocyte studies, as well as in vivo by survival, echocardiography and invasive micromano- metry studies[11,13,14]. Following Serca2 gene disruption, SER- CA2a protein is rapidly lost from the heart (t1/2 ,3 days). Despite the emergence of severe cardiac SERCA2a deficiency within weeks, the SERCA2 KO mice survive for up to 10 weeks post- knockout before finally succumbing to end-stage heart failure and pulmonary congestion[13]. Myocytes isolated from SERCA2 KO hearts at 4 and 7 weeks post-knockout reveal severe impairments in contractility, intracellular Ca2+ transient magnitude, and Ca2+ transient decay rates[11,13]. The in vivo phenotype at these time- points as assessed by echocardiography, however, is surprisingly mild. This suggests that SERCA2 KO mice are capable of compensating, at least temporarily, for the loss of SERCA2 activity Serca2+/2 mice, which express approximately 60% of normal SERCA2a protein content, have only mildly impaired func- tion[7],[8],[9]. Given that Serca2 heterozygous mice represent only one state of diminished SERCA2a activity, a more refined approach to study the relationship between SERCA2 expression and heart function in adult mice is necessary. A recently developed model of inducible SERCA2 knockout, the SERCA2 KO mouse, November 2013 | Volume 8 | Issue 11 | e79609 1 PLOS ONE | www.plosone.org SERCA2 Deficiency and Whole Heart Performance Figure 1. Introduction Because there are reports that increasing SR Ca2+ load can severely impair recovery from ischemic injury ex vivo[17], we hypothesized that decreasing SR Ca2+ content by Serca2 disruption would improve the recovery of KO hearts relative to controls. The disconnect between severely impaired in vitro function of isolated myocytes with the apparent preserved in vivo function after Serca2 disruption is surprising and warrants more detailed study. Interestingly, a recent parallel study using isolated rabbit hearts and the potent SERCA2a inhibitor thapsigargin showed that heart pump function can be sustained, at least in the short term, in absence of any appreciable myocyte SR Ca2+ handling capabil- ities[16]. These findings raise important new questions regarding the precise relationship between SERCA2a deficiency and the progression of heart failure. Although the contractile function and electrophysiology of isolated myocytes from inducible Serca2 KO have been studied, and in vivo function has been assessed by echocardiography and invasive micromanometry, no studies thus far have reported the contractile function of whole hearts isolated from these mice. It therefore remains unclear whether the whole-organ function of KO hearts will most closely resemble the severely impaired isolated myocyte phenotype or the largely preserved in vivo hemodynamic phenotype. In order to address this gap in knowledge, we isolated hearts at specific time-points after cardiac Serca2 gene disruption and directly examined whole heart performance. We hypothesized that, similar to isolated myocytes, isolated Serca2 KO hearts would exhibit severe contractile Animal Handling and Ethics Statement All experiments were approved by the University of Minnesota Institutional Animal Care and Use Committee (NIH Animal Welfare Assurance #A3456-01). Mice were housed on a 12- 12 hour light-dark cycle and provided rodent chow and tap water ad libitum. All mice were homozygous for loxP sites in introns 1 and November 2013 | Volume 8 | Issue 11 | e79609 PLOS ONE | www.plosone.org 2 SERCA2 Deficiency and Whole Heart Performance Figure 2. Langendorff protocol and individual LV pressure traces. A, Langendorff Protocol. After initial equilibration, pacing frequency w stepped between 3 and 12 Hz in 1-Hz intervals. After pacing at 12 Hz, hearts were re-equilibrated for 10 minutes in KHB lacking pyruvate and th subjected to 20 minutes no-flow ischemia and 60 minutes reperfusion. B, Representative traces of LV pressure from 1-week FL (solid line), 1-week (dashed line), and 4-week KO (dotted line) hearts sampled over 0.6 seconds. C, Individual peaks normalized to developed pressure. Note t differences in time scale for each normalized peak. i, ii, and iii: 3, 7, and 12 Hz traces, respectively, for either absolute LV pressure (B) or LV pressu normalized to own developed pressure (C). doi:10 1371/journal pone 0079609 g002 SERCA2 Deficiency and Whole Heart Performan Figure 2. Langendorff protocol and individual LV pressure traces. A, Langendorff Protocol. After initial equilibration, pacing frequency wa stepped between 3 and 12 Hz in 1-Hz intervals. After pacing at 12 Hz, hearts were re-equilibrated for 10 minutes in KHB lacking pyruvate and the subjected to 20 minutes no-flow ischemia and 60 minutes reperfusion. B, Representative traces of LV pressure from 1-week FL (solid line), 1-week KO (dashed line), and 4-week KO (dotted line) hearts sampled over 0.6 seconds. C, Individual peaks normalized to developed pressure. Note th differences in time scale for each normalized peak. i, ii, and iii: 3, 7, and 12 Hz traces, respectively, for either absolute LV pressure (B) or LV pressur normalized to own developed pressure (C). Figure 2. Langendorff protocol and individual LV pressure traces. A, Langendorff Protocol. After initial equilibration, pacing frequency was stepped between 3 and 12 Hz in 1-Hz intervals. After pacing at 12 Hz, hearts were re-equilibrated for 10 minutes in KHB lacking pyruvate and then subjected to 20 minutes no-flow ischemia and 60 minutes reperfusion. Langendorff protocol Mice were anesthetized with sodium pentobarbital (100 mg/kg i.p.) and heparinized (250 IU i.p.). Upon loss of toe pinch reflex, the ribcage was opened and the heart was removed to a dish of ice- cold Krebs-Henseleit solution (KHB: In mmol/L, 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 0.5 NaEDTA, 2.5 CaCl2, 15 Glucose, 25 NaHCO3, 0.5 NaPyruvate). The aorta was trimmed and cannulated, and the heart was mounted on a Langendorff apparatus (Radnoti, Inc) and retrogradely perfused with KHB bubbled with 95% O2 / 5% CO2 and maintained near 37 uC with SERCA2 Deficiency and Whole Heart Performance Serca2fl/fl; TG:aMHC-MerCreMer+/o mice ef- ficiently deleted Serca2 in response to tamoxifen and became Serca20 (KO) in the heart, while Serca2fl/fl; NTG animals retained the floxed Serca2 gene and normal SERCA2 expression (FL controls). FL and KO groups were sacrificed by sodium pentobarbital injection (100 mg/kg i.p.) and exsanguination at 1 and 4 weeks post-tamoxifen injection and their hearts were removed for ex vivo functional analysis (Figure 1A). All mice were 8–12 weeks of age at the time of sacrifice, and were of both sexes in roughly equal proportion. FL and KO animals were sacrificed 7 days post-tamoxifen for the 1-week time point, and 29–30 days post-tamoxifen for the 4-week time point. 3 of the Serca2 gene (Serca2fl/fl), and either contained (TG) or did not contain (NTG) the aMHC-MerCreMer+/o transgene. Mice were genotyped as described [11]. All mice were injected with tamoxifen dissolved at 10 mg/ml in peanut oil (1640 mg/kg intraperitoneally). Serca2fl/fl; TG:aMHC-MerCreMer+/o mice ef- ficiently deleted Serca2 in response to tamoxifen and became Serca20 (KO) in the heart, while Serca2fl/fl; NTG animals retained the floxed Serca2 gene and normal SERCA2 expression (FL controls). FL and KO groups were sacrificed by sodium pentobarbital injection (100 mg/kg i.p.) and exsanguination at 1 and 4 weeks post-tamoxifen injection and their hearts were removed for ex vivo functional analysis (Figure 1A). All mice were 8–12 weeks of age at the time of sacrifice, and were of both sexes in roughly equal proportion. FL and KO animals were sacrificed 7 days post-tamoxifen for the 1-week time point, and 29–30 days post-tamoxifen for the 4-week time point. After re-equilibration, pacing was ceased and hearts were subjected to 20 minutes of no-flow ischemia, followed by 60 minutes of reperfusion. 7 Hz pacing was resumed after 8 minutes of reperfusion and continued until the end of the experiment. Caffeine perfusion Serca2 knockout was performed and 4-week FL and KO hearts were mounted in the Langendorff mode as described above. KHB composition was as above, except glucose was 10 mM and pyruvate was not used. Hearts were allowed to equilibrate in normal KHB for 10 minutes. After equilibration, 5 minutes of baseline performance were recorded and the perfusate was switched to a reservoir containing KHB +10 mM caffeine. Hearts were perfused with KHB + caffeine for 10 minutes, followed by 20 minutes washout in normal KHB. Pacing frequency was 7 Hz throughout. Arrhythmic behavior during and after caffeine perfusion was defined as the time from start of caffeine perfusion until the first 15-second interval in which contractile frequency deviated outside 760.5 Hz (420630 BPM). SERCA2 Deficiency and Whole Heart Performance a water jacket. The atria were removed, and a balloon catheter was inserted into the left ventricle (LV) to measure isovolumic LV pressure. An electrode placed at the base of the heart controlled pacing frequency, which was set at 7 Hz (pacing cycle length of 1000/7 = 143 milliseconds) for baseline and equilibration. Table 1. Animal Characteristics. Table 1. Animal Characteristics. Group 1wk FL 1wk KO 4wk FL 4wk KO Heart Weight (mg) 11768.6 107611.0 11564.3 10465.2 Body Weight (g) 22.662.3 21.662.2 22.61.2 20.762.2 HW:BW Ratio (mg/g) 5.260.2 5.060.3 5.460.2 5.160.3 Age (wks) 8.760.4 8.960.5 11.660.2 12.060.3 Time Post-Tamoxifen (days) 760 760.3 2960.6 3060.9 No significant differences in heart weight, body weight, HW:BW ratio, or age were found between FL and KO hearts at 1 and 4 weeks post-tamoxifen injection. Unpaired two-tailed t tests were used to compare FL to KO hearts at each time point. Values are mean 6 SEM. doi:10.1371/journal.pone.0079609.t001 The Langendorff protocol is presented in Figure 2A. Hearts were equilibrated at 7 Hz for 5 minutes, followed by stepped changes in pacing frequency from 3 to 12 Hz: frequency was first reduced stepwise from 7 Hz to 3 Hz, then returned to 7 Hz and increased stepwise to 12 Hz in 1-Hz increments. Each frequency step was maintained until an equilibrium developed, at which point the frequency was again increased (usually ,1 minute per step). Parameters from 5 to 10 beats were averaged at this equilibrium state of each pacing step. After pacing steps to 12 Hz were completed, frequency was set at 7 Hz, perfusion was switched to KHB lacking pyruvate, and hearts were re-equilibrat- ed at 7 Hz for 10 minutes to wash out pyruvate from the pacing protocol. No significant differences in heart weight, body weight, HW:BW ratio, or age were found between FL and KO hearts at 1 and 4 weeks post-tamoxifen injection. Unpaired two-tailed t tests were used to compare FL to KO hearts at each time point. Values are mean 6 SEM. doi:10.1371/journal.pone.0079609.t001 3 of the Serca2 gene (Serca2fl/fl), and either contained (TG) or did not contain (NTG) the aMHC-MerCreMer+/o transgene. Mice were genotyped as described [11]. All mice were injected with tamoxifen dissolved at 10 mg/ml in peanut oil (1640 mg/kg intraperitoneally). Animal Handling and Ethics Statement B, Representative traces of LV pressure from 1-week FL (solid line), 1-week KO (dashed line), and 4-week KO (dotted line) hearts sampled over 0.6 seconds. C, Individual peaks normalized to developed pressure. Note the differences in time scale for each normalized peak. i, ii, and iii: 3, 7, and 12 Hz traces, respectively, for either absolute LV pressure (B) or LV pressure normalized to own developed pressure (C). doi:10.1371/journal.pone.0079609.g002 November 2013 | Volume 8 | Issue 11 | e79609 PLOS ONE | www.plosone.org 3 SERCA2 Deficiency and Whole Heart Performance Tissue handling Some FL hearts could not be paced at very low frequencies: for 1-week FL, the 3 and 4 Hz data points represent N = 3 observations, not N = 4 for all other pacing frequencies. For 4-week FL, 3 Hz represents N = 3 observations, and 4 Hz represents N = 4 observations, rather than N = 5 for all other pacing frequencies. *: P,0.05; **: P,0.01; ***: P,0.001 for FL vs. KO at either 1 week or 4 weeks post-tamoxifen injection, as determined by two-way ANOVA with Bonferroni post-test. Symbols are mean 6 SEM. doi:10.1371/journal.pone.0079609.g003 Data Acquisition and Statistical Analysis Data was acquired using LabChart 6 software (AD Instruments) and analyzed using Prism 5.02 (GraphPad). Significance was tested by unpaired two-tailed t test or two-way analysis of variance with Bonferroni post-test, where appropriate. Significance was set at P , 0.05. Data is presented as mean 6 SEM unless SEM was smaller than figure icon. RIPA (in mmol/L, 50 Tris, 150 NaCl, 1 EDTA, plus 0.5% w/v SDS and protease inhibitors [1 mM PMSF and 0.001 mg/ml each of aprotinin, leupeptin, and pepstatin, all added fresh]), briefly sonicated, and centrifuged at 14,000xg for 2 minutes. Supernatant protein concentration was determined using bicin- chroninic acid. Results Serca2 FL and Serca2 KO hearts were studied at one and four weeks after tamoxifen injection. Following tamoxifen injection, SERCA2 protein was rapidly lost from the heart (Figure 1). One week post-tamoxifen, SERCA2 protein in KO hearts was 32% of FL control (Figure 1B,C). By four weeks post-tamoxifen, only faint SERCA2 bands were found in heavily loaded samples (Figure 1D). Despite weeks of severe SERCA2a depletion in these mice, there were no overt, readily observable differences in animal appearance and no differences in heart weight, body weight, or heart weight- body weight ratio between FL and KO groups at either the one or four week time points (Table 1). Tissue handling For 4-week FL, 3 Hz represents N = 3 observations, and 4 Hz represents N = 4 observations, rather than N = 5 for all other pacing frequencies. *: P,0.05; **: P,0.01; ***: P,0.001 for FL vs. KO at either 1 week or 4 weeks post-tamoxifen injection, as determined by two-way ANOVA with Bonferroni post-test. Symbols are mean 6 SEM. doi:10.1371/journal.pone.0079609.g003 Developed Pressure (LVDP) in arts. A, 1-week KO vs. 1-week FL equency range (3–12 Hz). B, 4-week 1-week and 4-week FL hearts force-frequency response as pacing aseline. 1-week KO vs. 4-week KO: ome FL hearts could not be paced at k FL, the 3 and 4 Hz data points N = 4 for all other pacing frequencies. 3 b ti d 4 H t Figure 4. Summary of LV End-Diastolic Pressures (LVEDP) in isolated Serca2fl/fl and KO hearts. A, LVEDP of 1-week FL and KO hearts from 3–12 Hz stimulation frequency. B, LVEDP of 4-week FL and KO hearts from 3–12 Hz stimulation frequency. KO hearts at both 1 and 4 weeks post-tamoxifen injection underwent a pronounced increase in LVEDP as stimulation frequency increased. *: P,0.05; **: P,0.01; *** P,0.001 for FL vs. KO at either 1 week or 4 weeks post-tamoxifen injection, as determined by two-way ANOVA with Bonferroni post-test doi:10.1371/journal.pone.0079609.g004 Figure 4. Summary of LV End-Diastolic Pressures (LVEDP) in isolated Serca2fl/fl and KO hearts. A, LVEDP of 1-week FL and KO hearts from 3–12 Hz stimulation frequency. B, LVEDP of 4-week FL and KO hearts from 3–12 Hz stimulation frequency. KO hearts at both 1 and 4 weeks post-tamoxifen injection underwent a pronounced increase in LVEDP as stimulation frequency increased. *: P,0.05; **: P,0.01; ***: P,0.001 for FL vs. KO at either 1 week or 4 weeks post-tamoxifen injection, as determined by two-way ANOVA with Bonferroni post-test. doi:10.1371/journal.pone.0079609.g004 Figure 3. Summary of LV Developed Pressure (LVDP) in isolated Serca2fl/fl and KO hearts. A, 1-week KO vs. 1-week FL LVDP across a wide stimulation frequency range (3–12 Hz). B, 4-week KO vs. 4-week FL LVDP. Both 1-week and 4-week FL hearts demonstrated a negative staircase force-frequency response as pacing frequency increased from 7 Hz baseline. 1-week KO vs. 4-week KO: P,0.05 at all pacing frequencies. Tissue handling After the Langendorff protocol was complete, hearts were removed from the cannula, quickly blotted and weighed, and frozen using liquid nitrogen. Frozen hearts were pulverized in a liquid nitrogen-cooled steel deadblow, resuspended in 750 ml Table 2. Baseline Isolated Heart Function. Group 1wk FL 1wk KO 4wk FL 4wk KO # Hearts 4 4 5 4 Developed Pressure (mm Hg) 99.268.5 72.667.8 (n.s.) 90.465.4 20.464.9 (***) End-Diastolic Pressure (mm Hg) 5.962.1 7.560.8 (n.s.) 5.860.4 18.862.6 (***) dP/dt Max (mm Hg s21) 30346268 22326307 (n.s.) 25126189 4876120 (***) dP/dt Min (mm Hg s21) 224416292 213596148 (*) 222316190 2401688 (***) T50% Rise (ms) 25.5260.63 30.5161.29 (*) 27.4660.54 33.9561.09 (***) T50% Relaxation (ms) 31.1661.21 41.1961.26 (**) 30.4261.09 41.2460.78 (***) FDHM (ms) 56.6860.87 71.6962.42 (**) 57.8860.94 75.1961.55 (***) Isolated heart performance at baseline 7 Hz pacing frequency. Values shown, other than time to 50% rise, are also presented with other pacing frequencies in Figures 3, 4, and 5. Values represent mean and SEM of each group’s isolated heart performance; values for each heart were determined by averaging 5–10 pressure peaks at each pacing frequency after the heart’s performance at that step had stabilized. *: P,0.05; **:P,0.01; ***: P,0.001 for FL vs. KO at either 1 week or 4 weeks post-tamoxifen injection, as determined by unpaired two-tailed t test. doi:10.1371/journal.pone.0079609.t002 Table 2. Baseline Isolated Heart Function. Table 2. Baseline Isolated Heart Function. Isolated heart performance at baseline 7 Hz pacing frequency. Values shown, other than time to 50% rise, are also presented with other pacing frequencies in Figures 3, 4, and 5. Values represent mean and SEM of each group’s isolated heart performance; values for each heart were determined by averaging 5–10 pressure peaks at each pacing frequency after the heart’s performance at that step had stabilized. *: P,0.05; **:P,0.01; ***: P,0.001 for FL vs. KO at either 1 week or 4 weeks post-tamoxifen injection, as determined by unpaired two-tailed t test. doi:10.1371/journal.pone.0079609.t002 November 2013 | Volume 8 | Issue 11 | e79609 PLOS ONE | www.plosone.org 4 SERCA2 Deficiency and Whole Heart Performance Data Acquisition and Statistical Analysis D t i d i L bCh t 6 ft (AD I t t ) Figure 3. Summary of LV Developed Pressure (LVDP) in isolated Serca2fl/fl and KO hearts. A, 1-week KO vs. 1-week FL LVDP across a wide stimulation frequency range (3–12 Hz). B, 4-week KO vs. 4-week FL LVDP. Tissue handling Both 1-week and 4-week FL hearts demonstrated a negative staircase force-frequency response as pacing frequency increased from 7 Hz baseline. 1-week KO vs. 4-week KO: P,0.05 at all pacing frequencies. Some FL hearts could not be paced at very low frequencies: for 1-week FL, the 3 and 4 Hz data points represent N = 3 observations, not N = 4 for all other pacing frequencies. For 4-week FL, 3 Hz represents N = 3 observations, and 4 Hz represents N = 4 observations, rather than N = 5 for all other pacing frequencies. *: P,0.05; **: P,0.01; ***: P,0.001 for FL vs. KO at either 1 week or 4 Figure 4. Summary of LV End-Diastolic Pressures (LVEDP) in isolated Serca2fl/fl and KO hearts. A, LVEDP of 1-week FL and KO hearts from 3–12 Hz stimulation frequency. B, LVEDP of 4-week FL and KO hearts from 3–12 Hz stimulation frequency. KO hearts at both 1 and 4 weeks post-tamoxifen injection underwent a pronounced increase in LVEDP as stimulation frequency increased. *: P,0.05; **: P,0.01; ***: P,0.001 for FL vs. KO at either 1 week or 4 weeks post-tamoxifen injection, as determined by two-way ANOVA with Bonferroni post-test. doi:10.1371/journal.pone.0079609.g004 Figure 4. Summary of LV End-Diastolic Pressures (LVEDP) in isolated Serca2fl/fl and KO hearts. A, LVEDP of 1-week FL and KO hearts from 3–12 Hz stimulation frequency. B, LVEDP of 4-week FL and KO hearts from 3–12 Hz stimulation frequency. KO hearts at both 1 and 4 weeks post-tamoxifen injection underwent a pronounced increase in LVEDP as stimulation frequency increased. *: P,0.05; **: P,0.01; ***: P,0.001 for FL vs. KO at either 1 week or 4 weeks post-tamoxifen injection, as determined by two-way ANOVA with Bonferroni post-test. doi:10.1371/journal.pone.0079609.g004 Figure 3. Summary of LV Developed Pressure (LVDP) in isolated Serca2fl/fl and KO hearts. A, 1-week KO vs. 1-week FL LVDP across a wide stimulation frequency range (3–12 Hz). B, 4-week KO vs. 4-week FL LVDP. Both 1-week and 4-week FL hearts demonstrated a negative staircase force-frequency response as pacing frequency increased from 7 Hz baseline. 1-week KO vs. 4-week KO: P,0.05 at all pacing frequencies. Some FL hearts could not be paced at very low frequencies: for 1-week FL, the 3 and 4 Hz data points represent N = 3 observations, not N = 4 for all other pacing frequencies. Western Blotting C, dashed line indicates the calculated pacing cycle length in milliseconds (PCL = 1000 ms s21 / Pacing Frequency s21) to compare the contractile cycle length to the maximum possible peak width at each pacing frequency. Rest Time (D) is the difference between calculated PCL and Peak Width. Symbols indicate mean 6 SEM. #: P,0.05 FL vs KO (1-week). 1: P,0.05 FL vs KO (4-week). {: P,0.05 1-week KO vs 4-week KO. doi:10.1371/journal.pone.0079609.g005 Figure 3 summarizes one-week and four-week control and KO heart systolic function (LVDP) across a broad range of pacing frequencies. FL hearts at both time points exhibited a negative staircase force-frequency response as pacing frequency increased above 7 Hz baseline. At one week post-tamoxifen (Figure 3A), KO systolic function was diminished at low and high, but not moderate pacing frequencies (P , 0.05 between KO and FL at 3–5 and 8– 11 Hz). At four weeks, however, KO hearts had severe systolic impairment at all pacing frequencies (Figure 3B). Isolated heart contractile parameters at baseline are detailed in Table 2. One week post-tamoxifen, KO hearts exhibited mild systolic and diastolic dysfunction. The minimal first derivative of LV pressure (dP/dtmin, the fastest rate of pressure decay each beat) was significantly reduced in KO hearts compared to FL. In addition, times to 50% pressure rise (T50% Rise) and fall (T50% Relaxation or T50R) were significantly increased in KO hearts relative to FL at 1 week post-tamoxifen. In comparison, at four weeks post-tamoxifen, KO hearts had severe systolic and diastolic dysfunction relative to FL. In KO hearts LV developed pressure and maximal and minimal dP/dt were markedly decreased, and end-diastolic pressure (LVEDP) and times to 50% rise and 50% relaxation were significantly increased (P,0.05). Representative traces of LV pressure (Figure 2B, LV pressures; 2C, normalized to magnitude) from one-week FL, one-week KO, and four-week KO reveal severe, progressive decline in isolated heart contractile performance as SERCA2 protein is lost from the myocardium. At low to moderate pacing frequencies (3 and 7 Hz, Figure 2Bi-ii and 2Ci-ii), KO hearts contracted more weakly and relaxed more slowly than FL hearts. At higher pacing frequencies, these differences in relaxation performance were diminished. At four- weeks KO heart diastolic function was severely impaired at all pacing frequencies (Figure 2B). Western Blotting Heart lysates were diluted to 1 mg/ml with RIPA and Laemmli buffer, and then 20 mg protein per lane was fractionated on 12.5% Tris-Glycine gels (BioRad Criterion) and transferred to PVDF membranes. SERCA2a and Actin were detected by immunoblot (primary antibodies: SERCA2a, 2A7-A1 (Sigma) at 1:1000; Actin, A-2103 (Sigma) at 1:10,000) using an Odyssey imaging system (LiCor, Inc). PLOS ONE | www.plosone.org November 2013 | Volume 8 | Issue 11 | e79609 5 SERCA2 Deficiency and Whole Heart Performance Figure 5. Time to 50% Relaxation (T50R), Full-Duration at Half-Maximum (FDHM), Peak Width, and Rest Time in isolated Serca2fl/fl and KO hearts. A, T50R; B, FDHM; C, Peak Width; and D, Rest Time in 1-week FL, 1-week KO, 4-week FL, and 4-week KO hearts across 3–12 Hz stimulation frequencies. T50R (A) is the time (in ms) required for pressure to decay from peak to 50% of peak. FDHM (B) is the sum of the time to 50% pressure rise and the time to 50% pressure relaxation and indicates time spent in the contracted state. Peak Width (C) is the time duration between initiation of contraction and return to baseline. C, dashed line indicates the calculated pacing cycle length in milliseconds (PCL = 1000 ms s21 / Pacing Frequency s21) to compare the contractile cycle length to the maximum possible peak width at each pacing frequency. Rest Time (D) is the difference between calculated PCL and Peak Width. Symbols indicate mean 6 SEM. #: P,0.05 FL vs KO (1-week). 1: P,0.05 FL vs KO (4-week). {: P,0.05 1-week KO vs 4-week KO. doi:10.1371/journal.pone.0079609.g005 Figure 5. Time to 50% Relaxation (T50R), Full-Duration at Half-Maximum (FDHM), Peak Width, and Rest Time in isolated Serca2fl/fl and KO hearts. A, T50R; B, FDHM; C, Peak Width; and D, Rest Time in 1-week FL, 1-week KO, 4-week FL, and 4-week KO hearts across 3–12 Hz stimulation frequencies. T50R (A) is the time (in ms) required for pressure to decay from peak to 50% of peak. FDHM (B) is the sum of the time to 50% pressure rise and the time to 50% pressure relaxation and indicates time spent in the contracted state. Peak Width (C) is the time duration between initiation of contraction and return to baseline. November 2013 | Volume 8 | Issue 11 | e79609 Western Blotting p p g q ( g ) In both one-week and four-week KO hearts, there was a substantial rise in end-diastolic pressure (LVEDP) as pacing frequency increased (Figures 4A and 4B). At four weeks, KO end- diastolic pressures were significantly elevated at lower frequencies compared to one-week KO or FL, but at the maximal pacing frequency of 12 Hz both KO LVEDPs were similar (one-week KO: 31.561.5 mm Hg; four-week KO: 36.664.2 mm Hg). Further underscoring the relaxation deficit of KO hearts, times to 50% relaxation (T50R) were significantly prolonged in one- and four-week KO hearts relative to FL, particularly at low pacing frequencies (Figure 5A). KO hearts had also a significantly prolonged contracted state (Figure 5B: full-duration at half maximum, FDHM) compared to FL hearts at both time points. Likewise, the peak width (time duration of each pressure peak) progressively increased in one- and four-week KO hearts November 2013 | Volume 8 | Issue 11 | e79609 PLOS ONE | www.plosone.org 6 SERCA2 Deficiency and Whole Heart Performance Figure 6. LV Developed Pressures (LVDP) and LVDP normalized to baseline performance during ischemia-reperfusion injury in Serca2fl/fl and KO hearts. A, LVDP of 1-week FL and 1-week KO hearts at baseline, during ischemia (black bar, minutes 1–20), and during reperfusion (gray bar, minutes 30–80). B, as in (A) except all values normalized to each heart’s baseline LVDP. C and D, as with (A) and (B) for 4-week FL and 4-week KO hearts. Baseline values collected immediately prior to the onset of ischemia. One 1-week FL heart encountered a bubble in the perfusion line between pacing and ischemia-reperfusion and became infarcted, so 1-week FL N = 3 for ischemia-reperfusion (Figures 6 and 7). Symbols indicate mean 6 SEM. No significant differences between FL and KO at 1 week or 4 weeks post-tamoxifen injection were found by two-way ANOVA. doi:10 1371/journal pone 0079609 g006 Figure 6. LV Developed Pressures (LVDP) and LVDP normalized to baseline performance during ischemia-reperfusion injury in Serca2fl/fl and KO hearts. A, LVDP of 1-week FL and 1-week KO hearts at baseline, during ischemia (black bar, minutes 1–20), and during reperfusion (gray bar, minutes 30–80). B, as in (A) except all values normalized to each heart’s baseline LVDP. C and D, as with (A) and (B) for 4-week FL and 4-week KO hearts. Baseline values collected immediately prior to the onset of ischemia. Western Blotting One 1-week FL heart encountered a bubble in the perfusion line between pacing and ischemia-reperfusion and became infarcted, so 1-week FL N = 3 for ischemia-reperfusion (Figures 6 and 7). Symbols indicate mean 6 SEM. No significant differences between FL and KO at 1 week or 4 weeks post-tamoxifen injection were found by two-way ANOVA. d i 10 1371/j l 0079609 006 doi:10.1371/journal.pone.0079609.g006 In an additional set of experiments, we sought to further evaluate Ca2+ handling in FL and KO hearts by using caffeine to modulate excitation-contraction coupling. Isolated 4-week FL and KO hearts were perfused with 10 mM caffeine, a ryanodine receptor (RyR) activator, to modulate SR Ca2+ handling and reveal non-SR dependent contractile function. Caffeine perfusion releases SR Ca2+ stores, thus forcing hearts to depend on SR- independent Ca2+ handling pathways for contractility. Represen- tative traces of 4-week FL and KO hearts during caffeine perfusion are shown in Figure 8A. Upon perfusion with 10 mM caffeine, isolated hearts initially underwent a brief period of increased contractile force (Figure 8B). Following this initial increase, LV developed pressure diminished in both FL and KO and remained low until removal of caffeine during washout. Upon returning to normal KHB perfusion, FL hearts recovered developed pressure more rapidly than KO hearts. LV end-diastolic pressure increased during caffeine perfusion in both groups, but to a greater extent in FL hearts than KO (Figure 8C). In FL hearts, LV end-systolic pressure (LVESP) decreased during caffeine perfusion, whereas in KO hearts LVESP increased (Figure 8D). During caffeine perfusion, KO hearts were more susceptible than FL hearts to pacing irregularities, defined as a 15-second period in which a heart’s contractile frequency deviated outside 760.5 Hz. Each (Figure 5C). The rest time, defined as the difference between the calculated pacing cycle length and the peak width (PCL = 1000 ms s21/ pacing frequency s21), thus representing the time interval between the end of one peak and the beginning of the next, was elevated in KO hearts at low pacing frequencies (Figure 5D). These measures differed between KO and FL groups at low pacing frequencies and converged as pacing rate increased (7 Hz and above: no significant differences). (Figure 5C). SERCA2 Deficiency and Whole Heart Performance heart’s time from the start of caffeine perfusion to the first of th events was plotted in Figure 8E. Here, 9 of 12 KO hearts, and Figure 7. LV End-Diastolic Pressures (LVEDP) during ischem reperfusion injury in Serca2fl/fl and KO hearts. A, LVEDP of 1-w FL and KO hearts at baseline, during ischemia (black bars, minute 20), and during reperfusion (gray bar, minutes 30–80). B, as (A) fo week FL and KO hearts. FL and KO groups were not significa different (P.0.05) at 1 or 4 weeks post-KO, by two-way ANOVA. doi:10.1371/journal.pone.0079609.g007 previously in isolated myocytes from SERCA2a KO mice[11,13] and are in contrast to in vivo function where heart morphology and whole behavior appear relatively normal. This later point is in keeping with findings of apparent normal echocardiography assessment, at least in the early weeks after severe SERCA2a depletion in vivo[11]. Based on whole heart function results reported here it is surprising that SERCA2a KO mice survive well beyond time points where intrinsic whole heart performance is severely compromised. Our results from the inducible SERCA2a knockout mouse are interesting in the context of previous findings in Serca2+/2 mice, which constitutively express about 60% of normal SERCA2 protein. Serca2 heterozygous mice have slightly impaired cardiac function, but this does not appear to negatively impact the overall health of these mice[7,9]. The inducible SERCA2 KO mice[10] offer greater experimental latitude by permitting study of animals at multiple time points between initiation of knockout and the onset of in vivo pathology. In this way it is possible to perform detailed study of heart function as SERCA2 protein is specifically and progressively lost from the myocardium. In this context, severe SERCA2a deficiency from 32% of control levels down to near zero levels markedly disrupts heart organ function and yet at , 1,000,000 cardiac pump cycles/day the animal can survive for many weeks before overt cardio-respiratory failure ensues. Figure 7. LV End-Diastolic Pressures (LVEDP) during ischemia- reperfusion injury in Serca2fl/fl and KO hearts. A, LVEDP of 1-week FL and KO hearts at baseline, during ischemia (black bars, minutes 1– 20), and during reperfusion (gray bar, minutes 30–80). B, as (A) for 4- week FL and KO hearts. FL and KO groups were not significantly different (P.0.05) at 1 or 4 weeks post-KO, by two-way ANOVA. SERCA2 Deficiency and Whole Heart Performance doi:10.1371/journal.pone.0079609.g007 The physiological disconnect between the ex vivo heart performance and in vivo function is evidence that KO mice are capable of compensating for the loss of cardiac SERCA2 protein for up to two months before ultimately failing. Along these lines, recent reports on isolated rabbit hearts exposed to the SERCA2 inhibitor, thapsigargin, also show a surprising extended time of whole heart function that too appears to uncouple isolated myocyte SR Ca2+ flux and whole-heart contractility[16]. y y y[ ] As of yet the mechanism allowing for this compensation is not clear, although there are several changes shown to occur following Serca2 disruption that may support heart function in the absence of robust SR Ca2+ flux. For example, there are modest increases in the expression and activity of the L-type Ca2+ channel, the Na+- Ca+ exchanger, and the plasma membrane Ca2+ ATPase[13]. In addition, Na-K ATPase expression and activity is decreased[13], and serum norepinephrine is elevated in KO mice[11]. Collec- tively, these changes prompt the hypothesis that Serca2 KO cardiac performance in vivo is maintained by enhancing trans-sarcolemmal Ca2+ transport while SR Ca2+ handling is reduced. KO myocytes exhibit greater Ca2+ transients than FL when stimulated during caffeine perfusion[18]. The contractile performance of isolated KO hearts perfused with caffeine, however, suggests that trans- sarcolemmal Ca2+ flux is unlikely to account for preserved systolic function after Serca2 disruption. Systolic performance in FL and KO hearts is nearly identical during caffeine perfusion. End- diastolic pressures are lower in KO hearts during caffeine perfusion, suggesting that increased NCX and PMCA levels found 4 weeks post-KO can help sustain diastolic performance. KO hearts tend to deviate from the programmed stimulation frequency, 7 Hz, soon after the application of caffeine, indicating that this increased dependence on trans-sarcolemmal Ca2+ fluxes can sensitize KO hearts to arrhythmic behavior[19]. This finding is notable because ,1 week KO mice exhibit fewer ventricular extrasystoles than FL mice in telemetry studies[14]. This difference is likely accounted for by the progressive nature of this Serca2 KO model, as the 4-week KO hearts used in this study have diminished SR function and greater NCX activity[13] than ,1 week KO hearts[14]. Therefore, compensatory changes in Ca2+ handling mechanisms of Serca2 KO hearts may contribute to comparatively preserved end-diastolic pressures, but not systolic Figure 7. LV End-Diastolic Pressures (LVEDP) during ischemia- reperfusion injury in Serca2fl/fl and KO hearts. SERCA2 Deficiency and Whole Heart Performance A, LVEDP of 1-week FL and KO hearts at baseline, during ischemia (black bars, minutes 1– 20), and during reperfusion (gray bar, minutes 30–80). B, as (A) for 4- week FL and KO hearts. FL and KO groups were not significantly different (P.0.05) at 1 or 4 weeks post-KO, by two-way ANOVA. doi:10.1371/journal.pone.0079609.g007 heart’s time from the start of caffeine perfusion to the first of these events was plotted in Figure 8E. Here, 9 of 12 KO hearts, and 0 of 6 FL hearts, experienced arrhythmias during caffeine perfusion, and 2 of 6 FL hearts experienced arrhythmias shortly after returning to normal KHB perfusion. Western Blotting The rest time, defined as the difference between the calculated pacing cycle length and the peak width (PCL = 1000 ms s21/ pacing frequency s21), thus representing the time interval between the end of one peak and the beginning of the next, was elevated in KO hearts at low pacing frequencies (Figure 5D). These measures differed between KO and FL groups at low pacing frequencies and converged as pacing rate increased (7 Hz and above: no significant differences). Following force-frequency response determination, whole hearts were subjected to ischemia-reperfusion (I/R) challenge to test the hypothesis that the magnitude of I/R-mediated LV pressure dysfunction is modulated by progressive SERCA2a deficiency. At one-week, KO hearts had reduced pre-ischemic baseline LVDP compared to FL hearts, however both groups produced similar absolute developed pressures during reperfusion (Figure 6A). In contrast, at four weeks, KO hearts recovered much lower developed pressures than control hearts during reperfusion. When the developed pressure of each heart was normalized to pre- ischemic baseline values (Figures 6B and 6D), the recovery of KO hearts was not significantly different from FL controls. Also KO and FL hearts developed similar rigor pressure during ischemia, and recovery of LV end-diastolic pressure was not different between groups (Figure 7). November 2013 | Volume 8 | Issue 11 | e79609 PLOS ONE | www.plosone.org 7 SERCA2 Deficiency and Whole Heart Performance Discussion This study reports new findings of left ventricular whole heart function after targeted Serca2 gene disruption with the goal to interrogate organ level performance in the context of progressive and severe SERCA2a protein depletion. Under standard condi- tions at one week after Serca2 gene disruption, where cardiac SERCA2a protein content is 32% of control values, isolated whole heart performance is surprisingly near normal. In comparison, under the same standard testing conditions of whole heart function four weeks after Serca2 gene disruption, where cardiac SERCA2a protein is at near non-detectable levels, there are severe deficiencies in both systolic and diastolic LV function at the level of the isolated intact heart. Cardiac stress testing by increasing pacing rates further and dramatically unmasks relaxation perfor- mance deficits at early and late time points after gene disruption and establishes the SERCA2a dose-diastolic performance response relationship. Overall these new findings parallel those obtained November 2013 | Volume 8 | Issue 11 | e79609 PLOS ONE | www.plosone.org 8 performance, during caffeine exposure. Increased dependence on Ca2+ current, however, may render KO hearts susceptible to However, in this context it is interesting to note that patients w Darier’s disease, which owing to Serca2 loss-of-function mutatio Figure 8. Caffeine perfusion of isolated 4-week FL and KO hearts. A, Representative traces of LV pressure from FL and KO hearts spann the final minute of baseline recording and the first nine minutes of caffeine perfusion. B, LVDP of 4-week FL (N = 6) and KO (N = 12) hearts up perfusion with 10 mM caffeine. Washout with normal KHB began after 10 minutes of caffeine perfusion and proceeded for 20 minutes. Baseline (‘‘B indicates heart performance at the fifth minute of baseline recording, just prior to caffeine perfusion. Dead space in perfusion line took abou minute to clear at both switch timepoints. C, LVEDP of 4-week FL and KO hearts upon perfusion with 10 mM caffeine. D, LV End-Systolic Press (LVESP) of 4-week FL and KO hearts upon perfusion with 10 mM caffeine, normalized to baseline level. E, Time of survival for 4-week FL and KO hea from beginning of caffeine perfusion until first 15-second window in which contractile frequency was outside 760.5 Hz. Log-rank test: P,0 between FL and KO groups. Icons indicate mean, and error bars indicate SEM unless smaller than icon. Panels B, C, and D analyzed by two-w ANOVA. Brackets indicate results of Bonferroni post-tests. References 11. Andersson KB, Birkeland JAK, Finsen AV, Louch WE, Sjaastad I, et al. (2009) Moderate heart dysfunction in mice with inducible cardiomyocyte-specific excision of the Serca2 gene. J Mol Cell Cardiol 47: 180–187. 1. Gwathmey JK, Copelas L, MacKinnon R, Schoen FJ, Feldman MD, et al. (1987) Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ Res 61: 70–76. g 2. Engelhardt S, Hein L, Dyachenkow V, Kranias EG, Isenberg G, et al. (2004) Altered calcium handling is critically involved in the cardiotoxic effects of chronic beta-adrenergic stimulation. Circulation 109: 1154–1160. 12. Sohal DS, Nghiem M, Crackower MA, Witt SA, Kimball TR, et al. (2001) Temporally Regulated and Tissue-Specific Gene Manipulations in the Adult and Embryonic Heart Using a Tamoxifen-Inducible Cre Protein. Circ Res 89: 20–25. 3. Louch WE, Mørk HK, Sexton J, Strømme TA, Laake P, et al. (2006) T-tubule disorganization and reduced synchrony of Ca2+ release in murine cardiomy- ocytes following myocardial infarction. J Physiol 574: 519–533. 13. Louch WE, Hougen K, Mørk HK, Swift F, Aronsen JM, et al. (2010) Sodium accumulation promotes diastolic dysfunction in end-stage heart failure following Serca2 knockout. J Physiol 588: 465–478. 4. Schmidt U, Hajjar RJ, Helm PA, Kim CS, Doye AA, et al. (1998) Contribution of abnormal sarcoplasmic reticulum ATPase activity to systolic and diastolic dysfunction in human heart failure. J Mol Cell Cardiol 30: 1929–1937. 14. Stokke MK, Hougen K, Sjaastad I, Louch WE, Briston SJ, et al. (2010) Reduced SERCA2 abundance decreases the propensity for Ca2+ wave development in ventricular myocytes. Cardiovasc Res 86: 63–71. y J 5. Wei S, Guo A, Chen B, Kutschke W, Xie YP, et al. (2010) T-tubule remodeling during transition from hypertrophy to heart failure. Circ Res 107: 520–531. 15. Bers D (2002) Cardiac excitation–contraction coupling. Nature 415: 198–205. 6. Lompre´ AM, Lambert F, Lakatta EG, Schwartz K (1991) Expression of sarcoplasmic reticulum Ca(2+)-ATPase and calsequestrin genes in rat heart during ontogenic development and aging. Circ Res 69: 1380–1388. 16. Elliott EB, Kelly A, Smith GL, Loughrey CM (2012) Isolated rabbit working heart function during progressive inhibition of myocardial SERCA activity. Circ Res 110: 1618–1627. 7. Ji Y, Lalli MJ, Babu GJ, Xu Y, Kirkpatrick DL, et al. (2000) Disruption of a single copy of the SERCA2 gene results in altered Ca2+ homeostasis and cardiomyocyte function. J Biol Chem 275: 38073–38080. 17. Author Contributions Conceived and designed the experiments: FIH JMM. Performed the experiments: FIH. Analyzed the data: FIH JMM. Contributed reagents/ materials/analysis tools: KBA GC. Wrote the paper: FIH JMM. Conceived and designed the experiments: FIH JMM. Performed the experiments: FIH. Analyzed the data: FIH JMM. Contributed reagents/ materials/analysis tools: KBA GC. Wrote the paper: FIH JMM. In conclusion, these findings show that progressive loss of SERCA2 protein from heart following inducible knockout initiates SERCA2 Deficiency and Whole Heart Performance Another motivation for studying Serca2 KO mice was to assess the relationship between SR function and ischemia-reperfusion injury. Earlier reports of altered SR function using phospholam- ban deficient mice reported much more severe I/R-mediated heart performance deficits than in controls[17]. We speculated that a converse model whereby SR Ca2+ uptake and load are reduced would have opposite effects and paradoxically could be beneficial in terms of relative LV functional deficits in I/R. In 1- week KO hearts with SERCA2a reduced to 32% of control, mean values for LVDP tended to be higher than controls but this did not reach statistical significance by ANOVA analysis. Thus, although increasing SR Ca2+ load by phospholamban ablation severely impairs recovery from ischemic injury[17], decreasing SERCA2a content to 32% or less appears not to confer any benefit to recovery. rapid, severe decline in the contractile performance of isolated whole hearts. Although the mechanisms allowing for sustained in vivo cardiac function and survival are currently unclear, identifying the manner in which hearts compensate for severely diminished SR Ca2+ flux could prove highly beneficial to our understanding of the interrelationship between SR Ca2+ derangements and cardiac disease. Acknowledgments We thank the Lillehei Heart Institute for their continued support. Discussion doi:10.1371/journal.pone.0079609.g008 SERCA2 Deficiency and Whole Heart Performan SERCA2 Deficiency and Whole Heart Performance Figure 8. Caffeine perfusion of isolated 4-week FL and KO hearts. A, Representative traces of LV pressure from FL and KO hearts spanning the final minute of baseline recording and the first nine minutes of caffeine perfusion. B, LVDP of 4-week FL (N = 6) and KO (N = 12) hearts upon perfusion with 10 mM caffeine. Washout with normal KHB began after 10 minutes of caffeine perfusion and proceeded for 20 minutes. Baseline (‘‘BL’’) indicates heart performance at the fifth minute of baseline recording, just prior to caffeine perfusion. Dead space in perfusion line took about 1 minute to clear at both switch timepoints. C, LVEDP of 4-week FL and KO hearts upon perfusion with 10 mM caffeine. D, LV End-Systolic Pressure (LVESP) of 4-week FL and KO hearts upon perfusion with 10 mM caffeine, normalized to baseline level. E, Time of survival for 4-week FL and KO hearts from beginning of caffeine perfusion until first 15-second window in which contractile frequency was outside 760.5 Hz. Log-rank test: P,0.05 between FL and KO groups. Icons indicate mean, and error bars indicate SEM unless smaller than icon. Panels B, C, and D analyzed by two-way ANOVA. Brackets indicate results of Bonferroni post-tests. doi:10.1371/journal.pone.0079609.g008 However, in this context it is interesting to note that patients with Darier’s disease, which owing to Serca2 loss-of-function mutations are functional Serca2+/2 heterozygotes, show no sign of cardiac disease or dysfunction[20]. This observation supports the apparent disconnection between in vivo cardiac function, cardiac SERCA2 content/activity, and isolated organ and cell function. performance, during caffeine exposure. Increased dependence on Ca2+ current, however, may render KO hearts susceptible to arrhythmias. Additional studies will be required to further specify how KO hearts maintain systolic performance in vivo as SR function is gradually abolished. Loss of SERCA2 expression and activity is well documented within a wide variety of cardiac pathologies in human patients. November 2013 | Volume 8 | Issue 11 | e79609 PLOS ONE | www.plosone.org 9 SERCA2 Deficiency and Whole Heart Performance References Cross HR, Kranias EG, Murphy E, Steenbergen C (2003) Ablation of PLB exacerbates ischemic injury to a lesser extent in female than male mice: protective role of NO. Am J Physiol Heart Circ Physiol 284: H683–90. 8. Hiranandani N, Raman S, Kalyanasundaram A, Periasamy M, Janssen PML (2007) Frequency-dependent contractile strength in mice over- and under- expressing the sarco(endo)plasmic reticulum calcium-ATPase. Am J Physiol Regul Integr Comp Physiol 293: R30–6. 18. Swift F, Franzini-Armstrong C, Øyehaug L, Enger UH, Andersson KB, et al. (2012) Extreme sarcoplasmic reticulum volume loss and compensatory T-tubule remodeling after Serca2 knockout. Proc Natl Acad Sci U S A 109: 3997–4001. g 19. Pogwizd SM, Qi M, Yuan W, Samarel AM, Bers DM (1999) Upregulation of Na+/Ca2+ Exchanger Expression and Function in an Arrhythmogenic Rabbit Model of Heart Failure. Circ Res 85: 1009–1019. 9. Periasamy M, Reed TD, Liu LH, Ji Y, Loukianov E, et al. (1999) Impaired cardiac performance in heterozygous mice with a null mutation in the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) gene. J Biol Chem 274: 2556–2562. 20. Mayosi BM, Kardos A, Davies CH, Gumedze F, Hovnanian a, et al. (2006) Heterozygous disruption of SERCA2a is not associated with impairment of cardiac performance in humans: implications for SERCA2a as a therapeutic target in heart failure. Heart 92: 105–109. 10. Andersson KB, Finsen AV, Sja˚land C, Winer LH, Sjaastad I, et al. (2009) Mice carrying a conditional Serca2(flox) allele for the generation of Ca(2+) handling- deficient mouse models. Cell Calcium 46: 219–225. November 2013 | Volume 8 | Issue 11 | e79609 PLOS ONE | www.plosone.org 10
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A randomized cross over trial of tolerability and compliance of a micronutrient supplement with low iron separated from calcium vs high iron combined with calcium in pregnant women [ISRCTN56071145]
BMC pregnancy and childbirth
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Received: 17 October 2005 Accepted: 04 April 2006 Received: 17 October 2005 Accepted: 04 April 2006 Received: 17 October 2005 Accepted: 04 April 2006 This article is available from: http://www.biomedcentral.com/1471-2393/6/10 This article is available from: http://www.biomedcentral.com/1471-2393/6/10 ; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BioMed Central BioMed Central BioMed Central BMC Pregnancy and Childbirth Open Access Open Acc Research article A randomized cross over trial of tolerability and compliance of a micronutrient supplement with low iron separated from calcium vs high iron combined with calcium in pregnant women [ISRCTN56071145] Address: 1The University of Toronto, Clinique de Gynécologie-Obstétrique Pierre Boucher, Longueuil, Québec, Ca Pharmacology/Toxicology, The Hospital for Sick Children, North York General Hospital, Toronto, Canada Email: Eric Ahn - eric.ahn@utoronto.ca; Nicholas Pairaudeau - npairaudeau@hotmail.com; Nicholas Pairaudeau - npairaudeau@hotmail.com; Yves Cérat - yvescerat@videotron.ca; Bernard Couturier - bcouturier@videotron.ca; Andre Fortier - jandrej.fortier@videotron.ca; Éric Paradis - ericparadis65@hotmail.ca; Gideon Koren* - gkoren@sickkids.ca * Corresponding author Published: 04 April 2006 BMC Pregnancy and Childbirth 2006, 6:10 doi:10.1186/1471-2393-6-10 Received: 17 October 2005 Accepted: 04 April 2006 This article is available from: http://www.biomedcentral.com/1471-2393/6/10 © 2006 Ahn et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Methods g Prenatal micronutrient supplementation is indicated for all pregnant women, because the recommended daily allowances (RDA) for many vitamins and minerals are not met during pregnancy with dietary sources alone [1,2]. Prior to 1990's, physicians typically commenced prenatal micronutrients at the time of diagnosis of pregnancy. However, proving the ability of folic acid supplementa- tion to prevent neural tube defects, has led to the recom- mendation to start prenatal micronutrient supplementation when planning pregnancy [3,4]. A recent analysis has further documented the importance of micronutrient supplementation during pregnancy in pre- venting malformations other than neural tube defects [5]. The findings are consistent with the notion that pericon- ceptional multivitamin use, containing folic acid, reduces the overall occurrence of birth defects by 15–33%, in addition to the demonstrated reduction in neural tube defects [6]. Approval of the study protocol was obtained from the local ethics committee at The Hospital for Sick Children, in Toronto, Ontario, the North York General Hospital (NYGH) in Toronto, Ontario and the Clinique de Gyné- cologie-Obstétrique Pierre Boucher (CGOPB) in Quebec. Appropriate sample size was determined to be one hun- dred participants based on a desired effect size of 20% fewer adverse effects with PregVit® versus Materna®. Participants were eligible to enroll in the study if they were pregnant and between 18 and 45 years of age. This was a prospective, randomized, open labeled, cross over study. Between June 2003 and November 2003, women attending out patients obstetric clinics at NYGH and CGOPB were invited to enroll in the study. During their regular first obstetric appointment, participants reviewed the protocol and signed an informed consent form. Partic- ipating women were asked to complete a standardized questionnaire recording demographic data, pregnancy and, medical history, drug and vitamin use and informa- tion on their normal bowel movements. Based on the existence of NVP, participants were block-randomized to receive either Materna® or PregVit® to be taken for one month. The block randomization aimed at ensuring sim- ilar numbers of women with NVP/no NVP receiving each of the products first as NVP may affect compliance. They were asked to keep a diary of any adverse event (e.g. con- stipation, nausea and headache). The severity of their NVP was monitored by the Pregnancy-Unique Questionnaire of Emesis and nausea (PUQE) scores (Table 2) [13]. Abstract Background: Prenatal micronutrient combinations with high iron content are associated with high rates of gastrointestinal symptoms. This coupled with nausea and vomiting of pregnancy results in women often discontinuing their multivitamins. A new prescription supplement (PregVit®) that separates iron from calcium in two tablets – morning and evening, has lower elemental iron content (35 mg), but results in similar extent of iron absorption when compared to another supplement containing (60 mg) of elemental iron (Materna®). The objectives of this study were to compare tolerability and compliance with PregVit® vs. a supplement with high iron content (Materna®), in pregnant women. Methods: Randomized, crossover open labeled study in 135 pregnant women attending outpatient clinics in Ontario and Quebec. Results: Use of PregVit® was associated with a 30% reduction in constipation rate as compared to Materna®. Both products demonstrated similar compliance rates. Results: Use of PregVit® was associated with a 30% reduction in constipation rate as compared to Materna®. Both products demonstrated similar compliance rates. Compliance of Materna® was negatively associated with the severity of nausea and vomiting of pregnancy. No such correlation was found for PregVvit®. Compliance of Materna® was negatively associated with the severity of nausea and vomiting of pregnancy. No such correlation was found for PregVvit®. Conclusion: PregVit®, a supplement with lower iron content (35 mg), has significantly decreased constipation rates as compared to 60 mg iron- Materna and has similar compliance rates. High iron content in multivitamin supplements is associated with adverse effects in pregnancy. Page 1 of 6 (page number not for citation purposes) Page 1 of 6 (page number not for citation purposes) (page number not for citation purposes) BMC Pregnancy and Childbirth 2006, 6:10 http://www.biomedcentral.com/1471-2393/6/10 http://www.biomedcentral.com/1471-2393/6/10 Methods In addition, changes in diet as well as the use of medications and adherence to the study drug were recorded in the diary. At the end of the month a pill count was conducted to corroborate their diary reports. One month later, upon their regular second follow-up visit, patients returned their diaries and pill bottles. The women were then given the alternative product and were asked to record the same information in the diary for the following month. Upon completion of the two month cross over, participants were asked to return their diaries and supplement bottles for a pill count. Typically, many prenatal supplements contain, among other minerals, elemental iron at 50–60 mg, to prevent pregnancy-induced anemia secondary to increasing fetal needs. However, this level of iron content has been asso- ciated adverse gastrointestinal effects, including nausea, vomiting and constipation. The common occurrence of nausea and vomiting of pregnancy (NVP) has further led women to discontinue prenatal supplementation con- taining these levels of iron [7]. A new prescription prenatal micronutrient supplement PregVit® has recently been introduced into the Canadian Market aiming to address these issues: PregVit®, taken twice daily, contains less elemental iron (35 mg) than many other prenatal supplements. Iron is incorporated in the morning dose (Table 1). The evening dose of PregVit® contains calcium, which is known to inhibit iron absorp- tion from the gastrointestinal tract [8-11]. By separating the two elements it was hypothesized that despite a lower total iron dose – a similar systemic exposure to iron would occur as with the other high iron prenatal supplements. Moreover, PregVit® contains higher doses of vitamin C, which is also known to facilitate iron absorption (Table 1). Before starting the study, a pilot sample of 10 adult volun- teers was tested for the feasibility of completing the diary. They took PregVit® for one week followed by Materna® for one week and were asked to fill out the diary. Minor changes were made to the diary based on comments col- lected during the pilot phase. A recent pharmacokinetic study has shown that the extent of iron absorption with PregVit® (in terms of area under the concentration-time curve) was similar to that of Materna®, despite PregVit®containing only about half of the iron dose [12]. Sample size and statistical analysis D (cholecalciferol)ε 250 IU 250 IU 5 µg* Calcium 300 mg 250 mg 1000–1300 mg* Biotin 0 30 µg - Chromium 0 25 µg 29–30 µg* Manganese 0 5 mg 2 mg* Molybdenum 0 25 µg 50 µg Selenium 0 25 µg 60 µg Pill Size Small large * able 1: Composition of two prenatal supplements and Recommended Dietary Allowance (RDA) values12 q ** Applies only to PregVit® ** Applies only to PregVit® ψVIT A – 1 International Unit (IU) of retinol is equivalent to 0.3 µg retinol (750 µg of retinol is 2,500 IU of retinol) 2 µg of supplemental β-carotene is equivalent to 1 µg retinol 3 (page 92 & 565) ξVIT E – 1 International Unite (IU) is equivalent to 0.67 µg of α-tocopherol (15 mg is equivalent to 22,388 IU) However, different conversion factors are used for different forms of vitamin E εVIT D – 1 IU is equivalent to 0.025 µg of cholecalciferol (5 µg is equivalent to 200 IU) ***Please note: Since the completion of this study a new formulation of Materna was introduced containing 27 mg of elemental iron. in rate of constipation (from 30% to 20%) at power of 80% and alpha of 5%. in rate of constipation (from 30% to 20%) at power of 80% and alpha of 5%. arms by paired Student's t test or signed rank test, as appropriate. Other variables such as mean days of experi- encing adverse drug event (ADE), nausea, constipation and other ADE, excluding nausea and constipation, (labeled as other), were also compared using the same sta- tistical tests, as appropriate. The incidence of nausea and noncompliance due to constipation were compared between the two groups using chi-square. All statistical tests were performed using Sigmastat® statistical software, Version 2.03 (SPSS Inc, Chicago, IL). Subsequently, mul- tilinear regression analysis was conducted to elucidate determinants that affected compliance rate with the study drugs. Determinants of compliance were chosen based on factors showing significant in recent literature [14-17]. arms by paired Student's t test or signed rank test, as appropriate. Other variables such as mean days of experi- encing adverse drug event (ADE), nausea, constipation and other ADE, excluding nausea and constipation, (labeled as other), were also compared using the same sta- tistical tests, as appropriate. The incidence of nausea and noncompliance due to constipation were compared between the two groups using chi-square. Sample size and statistical analysis All statistical tests were performed using Sigmastat® statistical software, Version 2.03 (SPSS Inc, Chicago, IL). Subsequently, mul- tilinear regression analysis was conducted to elucidate determinants that affected compliance rate with the study drugs. Determinants of compliance were chosen based on factors showing significant in recent literature [14-17]. Sample size and statistical analysis The objective of the present study was to compare tolera- bility and compliance with PregVit® and Materna® among pregnant women. The primary end point of interest was the mean rate of adverse events in the two study arms, including decreased compliance. The mean compliance based on pill counts and diary reports were compared between the two study Page 2 of 6 (page number not for citation purposes) (page number not for citation purposes) BMC Pregnancy and Childbirth 2006, 6:10 http://www.biomedcentral.com/1471-2393/6/10 http://www.biomedcentral.com/1471-2393/6/10 Table 1: Composition of two prenatal supplements and Recommended Dietary Allowance (RDA) values12 Component PregVit® Materna® *** Recommended dietary allowance (RDA) a.m. tablet** Vit. Aψ 2700 IU (β-Carotene) 1500 IU (β-Carotene) 1500 IU (acetate) 750–770 µg retinol Vit. Eξ 30 IU 30 IU 15 mg Vit. C 120 mg 100 mg 80–85 mg Vit. B1 (thiamin) 3 mg 3 mg 1.4 mg Vit. B2 (riboflavin) 3.4 mg 3.4 mg 1.4 mg Niacinamide 20 mg 20 mg 18 mg Vit. B6 10 mg 10 mg 1.9 mg Pantothenic acid (calcium pantothenate) 5 mg 10 mg 6 mg* Magnesium 50 mg 50 mg 350–400 mg Iodine 0.15 mg 0.15 mg 0.22 mg Iron 35 mg (as fumarate) 60 mg (as fumarate) 27 mg Copper 2 mg 2 mg 1 mg Zinc 15 mg 25 mg 11–12 mg p.m. tablet** Folic Acid 1.1 mg 1 mg 0.6 mg Vit. B12 (cyanocaobalamin) 12 µg 12 µg 2.6 µg Vit. Page 3 of 6 (page number not for citation purposes) Results A total of 109 patients from NYGH consented to enroll in the study. Of these 109 patients, 37 patients with NVP and 45 Non NVP patients completed both arms of the study, totaling 82 patients (75.2%). Of 100 patients agreed to enroll in the study at the CGOPB (35 NVP and 65 Non NVP), 18 NVP patients and 38 Non NVP patients, for a total of 56 patients, completed the study (56%). Hence, a total of 138 patients completed the study and were included in the data analysis. The mean gestational age at the start was 8 ± 2 weeks. There was identical gestational age at start in the two arms. The women who dropped out from the study almost entirely due to the fact that they did not return for follow-up or did not fill out diaries on returning the pill bottles. They did not differ from those who completed the study in any characteristic collected Rates of constipation were the primary end point of inter- est, as high iron content is known to induce constipation. Sample size of 100 was calculated to show a 33% decrease Page 3 of 6 (page number not for citation purposes) Page 3 of 6 (page number not for citation purposes) BMC Pregnancy and Childbirth 2006, 6:10 http://www.biomedcentral.com/1471-2393/6/10 Table 2: The PUQE Score (12). Patients were asked to circle the appropriate box next to the three symptoms of nausea and vomiting of pregnancy (NVP). The score of each box is represented by the top row. The total score is calculated and determines the severity of NVP14. A score of 3 to 6 represents Mild NVP. A score of 7 to 12 indicates Moderate NVP. Finally a score of 13 and greater represents Severe NVP. 1 2 3 4 5 QUESTION 1In the last 12 hours, how long have you felt nauseated or sick at your stomach? Not at all 1 hour or less 2–3 hours 4–6 hours More than 6 hours (Please specify number of hours) QUESTION 2In the last 12 hours, have you vomited or thrown up? I did not throw up 1–2 times 3–4 times 5–6 times 7 or more times (Please specify number of times) QUESTION 3In the last 12 hours, how many times have you had retching or dry heaves without bringing anything up? Results No Time 1–2 times 3–4 times 5–6 times 7 or more (Please specify number of times) and reported by us. Among those who did not complete the study, there was no higher prevalence to any of the two arms (e.g., PregVit® vs Materna®). Finally, multiple linear regression with patients and their reported compliance while taking Materna® revealed that marital status and NVP severity predicted their reported compliance; single motherhood predicted higher compli- ance rates whereas more severe NVP predicted a lower compliance rate (Table 5). No such correlation was detected for PregVit®. Finally, multiple linear regression with patients and their reported compliance while taking Materna® revealed that marital status and NVP severity predicted their reported compliance; single motherhood predicted higher compli- ance rates whereas more severe NVP predicted a lower compliance rate (Table 5). No such correlation was detected for PregVit®. There was a significantly higher incidence of reported con- stipation, as well as average duration of constipation when taking Materna® as compared to PregVit® (Table 3). n represents the number of patients (out of 138) who completed any particular item. Discussion h l Independent Variable Coefficient Standard Error p value Age 0.00 0.002 0.43 Education -0.01 0.011 0.58 Province 0.04 0.026 0.17 Marital Status -0.02 0.028 0.43 Weight 0.00 0.000 0.14 NVP Severity -0.02 0.012 0.08 Substance use* 0.00 0.036 0.79 Multivitamin Supplement -0.01 0.019 0.75 * alcohol, tobacco or illicit drugs Table 4: Multiple linear regression of factors affecting compliance in both treatment arms. * alcohol, tobacco or illicit drugs related to the severity of nausea and vomiting of preg- nancy. This agrees with a recent study documenting that the severity of morning sickness adversely affects use of Materna® (6). This may be explained also by a substan- tially larger pill size as compared to PregVit®, in addition to the direct effect of iron. These findings suggest that PregVit® may confer an advantage to women suffering from NVP due to both higher tolerability because of lower iron content and a smaller tablet size. Because NVP can very from slight food aversion to hyperemesis gravi- darum, we used a validated tool that quantifies the sever- ity of NVP (Table 2). content than PregVit®, it is possible that the systemic absorption of iron of Materna® may be subtherapeutic14. Moreover, studies have suggested an antagonistic relation- ship between iron and zinc and manganese, in which zinc and manganese reduce the positive effects of iron supple- mentation and vice versa [19,21]. It is likely that the higher dose of zinc, found in Materna® versus PregVit® plus manganese, (which is not included in PregVit®) all interfere with the absorption of iron. A 1993 study(16) found that supplementation with a low daily dose (18 mg) of iron given in a prenatal supplement together with inhibitive minerals was not sufficient to cover the iron needs of most pregnant women. Impor- tantly, many prenatal supplements currently available still contain 50–60 mg of elemental iron. Potential weaknesses of this study are that the participa- tion in this cross over protocol may act to improve com- pliance among participant, and that, similar to other randomized controlled studies, the demonstrated high compliance figures do not reflect those of naturalistic "real life" drug administration. We experienced a rela- tively high drop out rate. This did not reflect failure to take the two products but rather lack of motivation of healthy pregnant women to participate in this type of intensive protocol including daily diaries and returning pills. Discussion h l Multivariate linear regression was conducted to elucidate factors that independently affected compliance. The fac- tors tested included: age, education, province, marital sta- tus, weight, NVP severity, any substance used such as illicit drugs and multivitamin supplement (which multivitamin they started the trial with). These factors have been shown in previous studies to affect compliance [18]. The depend- ent variable used was reported compliance. Including all 138 patients, using both arms of the study, no variable sig- nificantly predicted compliance rate (Table 4). The results of this study demonstrate that pregnant women may experience less constipation when taking PregVit®. These results are likely attributed to the lower iron dose contained in PregVit® relative to Materna® (35 mg and 60 mg, respectively) (Table 1). The other differ- ences in micronutrient content between the two prepara- tions are not known to affect constipation rate. It should be noted that since the completion of this study the dose of iron in Materna® has been reduced to 27 mg. The refor- mulated Materna® has the same levels of calcium, manga- nese and zinc as before. Thus, with lower vitamin C Page 4 of 6 Table 3: Overall study results, comparing Pregvit® (low iron) to Materna® (high iron) over a month administration in a cross-over design. (n)* PregVit® (SD) Materna® (SD) p value Pill Count (% of 100%) 78 87.7 ± 20 90.9 ± 17 0.11 Reported Compliance (%) 138 90.4 ± 18 92.4 ± 15 0.42 Adverse events (%) 138 17.6 ± .24 20.3 ± 24 0.14 Nausea (%) 138 9.3 ± 19 10.1 ± 18 0.71 Constipation time length (%) 138 3.1 ± 8 4.7 ± 11 0.05 Other adverse events 138 (n) 10.4 ± 21 99.9 ± 20 0.95 Nausea rate (%) 138 41.3 (57/81) 45.7 (63/75) 0.54 Constipation Rate (%) 138 22.5% (31/107) 34.8% (48/90) 0.03 n represents the number of patients (out of 138) who completed any particular item. ring Pregvit® (low iron) to Materna® (high iron) over a month administration in a cross-over Table 3: Overall study results, comparing Pregvit® (low iron) to Materna® (high iron) over a m design. Table 3: Overall study results, comparing Pregvit® (low iron) to Materna® (high iron) over a month administration in a cross-over BMC Pregnancy and Childbirth 2006, 6:10 http://www.biomedcentral.com/1471-2393/6/10 Table 4: Multiple linear regression of factors affecting compliance in both treatment arms. ** alcohol, tobacco or illicit drugs * More severe NVP was associated with significantly lower compliance. No such effect was found with Pregvit®, wh tablet, containing much lower iron content. Competing interests p g y J y ( pp ) 14. Leopold NA, Plansky M, Hurka MR: Drug adherence in Parkin- son's disease. Mar Disord 2004, 19:513-7. G. Koren has been a paid consultant for Duchesnay Inc. All other authors declare they have no competing inter- ests'. 15. Yavuz A, Tuncer M, Erdogran O, Gurkan A, Cetinkaya R, Akbas SH, et al.: Is there any effect of compliance on clinical parameters of renal transplant recipients? Transplant Proc 2004, 36:120-1. p p p 16. Reimherr FW, Strong RE, Merchant BK, Hedges DW, Wender PH: Factors affecting return of symptoms 1 year after treatment in major depression. J Clin Psychiatry 2001, 6(Suppl 22):16-23. Pre-publication history p The pre-publication history for this paper can be accessed here: The pre-publication history for this paper can be accessed here: Authors' contributions Eric Ahn collected all data in Toronto, analyzed the data and wrote the draft manuscript. j p J y y ( pp ) 17. Smetana GW, Davis RB, Phillis RS: Factors that influence patient response to requests to change to a unified restrictive for- mulary. J Gen Intern Med 2004, 19:1212-9. Nicholas Pairaudeau oversaw the study in the Toronto site. Nicholas Pairaudeau oversaw the study in the Toronto site. y J 18. Ekenved G, Halvorsen L, Solvell L: Influence of a liquid antacid on the absorption of different iron salts. Scand J Haematol Suppl 1976, 28:65-77. 19. Crofton RW, Gvozdanovic D, Gvozdanovic S, Khin CC, Brunt PW, Mowat NA, Aggett PJ: Inorganic zinc and the intestinal absorp- tion offerrous iron. Am J Clin Nutr 1989, 50:141-4. Nicholas Pairaudeau Jr. participated in collection and analysis of data. J 20. Rossander-Hulten L, Brune M, Sandstrom B, Lonnerdal B, Hallberg L: Competitive inhibition of iron absorption by manganese and zinc in humans. Am J Clin Nutr 1991, 54:152-6. Yves Cerat, B. Couturier, A. Fortier and E. Paradis oversaw patients and collected the data in the Quebec Site. Yves Cerat, B. Couturier, A. Fortier and E. Paradis oversaw patients and collected the data in the Quebec Site. J 21. Thomsen JK, Prien-Larsen JC, Devantier A, Fogh-Andersen N: Low dose iron supplementation does not cover the need for iron during pregnancy. Acta Obstet Gynecol Scand 1993, 72:93-8. Gideon Koren wrote the protocol and oversaw all stages of collection, analysis and writing. Gideon Koren wrote the protocol and oversaw all stages of collection, analysis and writing. Conclusion p J 10. Zijp IM, Korver O, Tijburg LB: Effect of tea and other dietary fac- tors on iron absorption. Crit Rev Food Sci Nutr 2000, 40:371-98. PregVit®, a supplement with lower iron content (35 mg), has significantly decreased constipation rates as compared to 60 mg iron- Materna and has similar compliance rates. High iron content in multivitamin supplements is associ- ated with adverse effects in pregnancy. p 11. Bothwell TH: Iron requirements in pregnancy and strategies to meet them. Am J Clin Nutr 2000, 72:257S-64S. 2 G i i i i i i J 12. Ahn E, Kapur B, Koren G: Iron bioavailability in prenatal multi- vitamin supplements with separated and combined iron and calcium. J Soc Obst Gynecol Can 2004, 26:809-815. J y 13. Koren G, Boscovic R, Hard M, Maltepe C, Navioz Y, Einarson A: Motherisk – PUQE scoring system for nausea and vomiting of pregnancy. Am J Obstetr Gynecol 2002, 186(55 Suppl):228-31. 13. Koren G, Boscovic R, Hard M, Maltepe C, Navioz Y, Einarson A: Motherisk – PUQE scoring system for nausea and vomiting of pregnancy. Am J Obstetr Gynecol 2002, 186(55 Suppl):228-31. 14. Leopold NA, Plansky M, Hurka MR: Drug adherence in Parkin- son's disease. Mar Disord 2004, 19:513-7. References National Research Council (U.S.): Subcommittee on the Tenth Edition of the RDAs. Recommended dietary allowances/Sub- committee on the Tenth Edition of the RDAs, Food and Nutrition Board, Commission on Life Sciences, National Research Council. Washington, D.C: National Academy Press; 1989. 2. Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Food and Nutrition Board, Institute of Medicine: Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington, D.C: National Academy Press; 2002:770-73. Yet, this study, comparing tolerability and compliance rates between two prenatal micronutrient supplements different in their iron content and administration sched- ule, has identified several determinants of potential clini- cal significance, which may affect pregnant women's adherence to micronutrient supplementation. y 3. Czeizel AE, Dudas I: Prevention of the first occurrence of neu- ral-tube defects by periconceptional vitamin supplementa- tion. N Engl J Med 1992, 327:1832-35. g J 4. Wald N, Hack Shaw A: Folic acid and prevention of neural-tube defects. Lancet 1992, 350:605. 5. Botto LD, Olney RS, Erickson JD: Vitamin supplements and the risk for congenital anomalies other than neural tube defects. Am J Med Genet 2004, 125C:12-21. A major problem in primary prevention of congenital anomalies by folic acid or folic acid containing micronu- trients is the need to commence supplementation precon- ceptionally. There is a serious and urgent need to educate potential parents to start such supplementation before pregnancy is confirmed. 6. Botto LD, Olney RS, Erickson JD: Vitamin supplements and the risk for congenital anomalies other than neural tube defects. Am J Med Genet C Semin Med Genet 2004, 125:12-21. J 7. Koren G, Piwko C, Ahn E, Boskovic R, et al.: Validation studies of the pregnancy unique quantification of emesis (PUQE) scores. J Obstet Gynecol in press. 8. Bendich A: Calcium supplementation and iron status offe- males. Nutr 2001, 17:46-51. 9. Cook JD, Dassenko SA, Whittaker P: Calcium supplementation: effect on iron absorption. Am J Clin Nutr 1991, 53:106-11. Discussion h l How- ever, because each woman served as her own control, the attrition does not bias the results. The women who dropped out were not different in their characteristics from those who completed the protocol. Importantly, Two methods were used to estimate compliance: Patients' reported compliance and pill count. Using these meas- ures, compliance was found to be similar for both prod- ucts. The similar compliance may be a reflection of better tolerability with PregVit®, being partially offset by the need to take this supplement twice daily, as compared to once daily with Materna®. Of potential clinical importance, the multivariate analysis revealed that compliance with Materna® was negatively Table 5: Multiple linear regression of compliance in patients while taking Materna® Independent Variable Coefficient Standard Error p value Age 0.00 0.002 0.73 Education -0.02 0.014 0.09 Province 0.03 0.031 0.37 Race 0.02 0.010 0.11 Marital Status -0.08 0.034 0.02 Weight 0.00 0.000 0.14 NVP Severity* -0.05 0.014 0.002 Substance** -0.06 0.044 0.20 * More severe NVP was associated with significantly lower compliance. No such effect was found with Pregvit®, which is a substantially smaller tablet, containing much lower iron content. ** alcohol, tobacco or illicit drugs Table 5: Multiple linear regression of compliance in patients while taking Materna® iple linear regression of compliance in patients while taking Materna® * More severe NVP was associated with significantly lower compliance. No such effect was found with Pregvit®, which is a substantially smaller tablet, containing much lower iron content. ** alcohol, tobacco or illicit drugs BMC Pregnancy and Childbirth 2006, 6:10 http://www.biomedcentral.com/1471-2393/6/10 because compliance rate was an important endpoint we refrained from contacting the women to remind them of their participation, an act that could artificially affect com- pliance, with the medications. Previous studies also reported on diarrhea occurring with micronutrients, how- ever, in our study the occurrence of diarrhea was very rare and not different between the two study interventions. because compliance rate was an important endpoint we refrained from contacting the women to remind them of their participation, an act that could artificially affect com- pliance, with the medications. Previous studies also reported on diarrhea occurring with micronutrients, how- ever, in our study the occurrence of diarrhea was very rare and not different between the two study interventions. Acknowledgements g The study was sponsored by Duchesnay Inc., Laval, Québec, Canada. GK is a Senior Scientist of the Canadian Institutes for Health Research and holds the Research Leadership for Better Pharmacotherapy during Pregnancy and Lactation; and the Ivey Chair in Molecular Toxicology, The University of Western Ontario. http://www.biomedcentral.com/1471-2393/6/10/prepub Page 6 of 6 (page number not for citation purposes) Page 6 of 6 (page number not for citation purposes)
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Genome-wide analysis of health-related biomarkers in the UK Household Longitudinal Study reveals novel associations
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Genome-wide analysis of health- related biomarkers in the UK Household Longitudinal Study reveals novel associations Received: 10 May 2017 Accepted: 8 August 2017 Published: xx xx xxxx Bram P. Prins1, Karoline B. Kuchenbaecker1, Yanchun Bao2, Melissa Smart2, Delilah Zabaneh3, Ghazaleh Fatemifar4, Jian’an Luan5, Nick J. Wareham5, Robert A. Scott5, John R. B. Perry5, Claudia Langenberg5, Michaela Benzeval2, Meena Kumari2 & Eleftheria Zeggini   1 Serum biomarker levels are associated with the risk of complex diseases. Here, we aimed to gain insights into the genetic architecture of biomarker traits which can reflect health status. We performed genome-wide association analyses for twenty serum biomarkers involved in organ function and reproductive health. 9,961 individuals from the UK Household Longitudinal Study were genotyped using the Illumina HumanCoreExome array and variants imputed to the 1000 Genomes Project and UK10K haplotypes. We establish a polygenic heritability for all biomarkers, confirm associations of fifty-four established loci, and identify five novel, replicating associations at genome-wide significance. A low-frequency variant, rs28929474, (beta = 0.04, P = 2 × 10−10) was associated with levels of alanine transaminase, an indicator of liver damage. The variant is located in the gene encoding serine protease inhibitor, low levels of which are associated with alpha-1 antitrypsin deficiency which leads to liver disease. We identified novel associations (rs78900934, beta = 0.05, P = 6 × 10−12; rs2911280, beta = 0.09, P = 6 × 10−10) for dihydroepiandrosterone sulphate, a precursor to major sex-hormones, and for glycated haemoglobin (rs12819124, beta = −0.03, P = 4 × 10−9; rs761772, beta = 0.05, P = 5 × 10−9). rs12819124 is nominally associated with risk of type 2 diabetes. Our study offers insights into the genetic architecture of well-known and less well-studied biomarkers. Serum biomarker levels are associated with the risk of complex diseases and are therefore increasingly used in clinical practice to assist with diagnosis, status monitoring and disease management. Well-known examples include the measurement of lipid levels in the context of cardiovascular disease or liver enzymes and albumin to assess liver function. Serum biomarker levels have a polygenic basis. As demonstrated in the case of lipids, identifying genetic associations can provide new insights into disease aetiology which can in turn guide drug discovery and be use- ful for diagnosis and risk stratification1–3. However, the genetic architecture of most health-related biomarkers has not been studied as extensively as for lipids. Alleles identified to be associated with protein biomarkers to date are predominantly common (minor allele frequency (MAF) >5%). www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | 7: 11008 | DOI:10.1038/s41598-017-10812-1 Received: 10 May 2017 Accepted: 8 August 2017 Published: xx xx xxxx Genome-wide analysis of health- related biomarkers in the UK Household Longitudinal Study reveals novel associations This is primarily driven by genotyping technology and composition of arrays or imputation reference panels used to date4–6. Systematically evaluating the association of low frequency and rare variants can provide new insights regarding the genetic architecture of protein biomarkers.h The importance of studying the joint impact of genetic and non-genetic factors on health has been recog- nised by the UK Household Longitudinal Study (UKHLS, www.understandingsociety.ac.uk), also known as Understanding Society. Involving a total of 40,000 households representative of the UK population, UKHLS is the largest panel survey in the world to support social research. A wide range of social, economic, environment, 1Wellcome Trust Sanger Institute, Hinxton, UK. 2Institute for Social and Economic Research, University of Essex, Wivenhoe Park, Colchester, Essex, UK. 3MRC Social, Genetic & Developmental Psychiatry Centre, IoPPN, KCL, London, UK. 4Institute for Health Informatics, UCL and the Farr Institute of Health Informatics, London, UK. 5MRC Epidemiology Unit, University of Cambridge School of Clinical Medicine, Box 285 Institute of Metabolic Science, Cambridge Biomedical Campus, Cambridge, UK. Bram P. Prins and Karoline B. Kuchenbaecker contributed equally to this work. Correspondence and requests for materials should be addressed to E.Z. Genome-wide analysis of health- related biomarkers in the UK Household Longitudinal Study reveals novel associations (email: eleftheria.zeggini@sanger.ac.uk) Scientific Reports | 7: 11008 | DOI:10.1038/s41598-017-10812-1 1 www.nature.com/scientificreports/ Variable units N missing Female Male N mean IQR* min max N mean IQR* min max Age years 0 5574 52.1 25 16 99 4387 52.82 25 16 97 BMI 285 5416 28.02 7.4 14.5 75.7 4260 28.09 5.6 15.8 66.5 Albumin G/L 137 5501 46.24 4 36 57 4323 47.48 4 36 57 Alkaline Phosphatase lu/L 228 5451 70.99 26 22 191 4282 71.86 24 22 217 Alanine Transaminase lu/L 230 5458 23.66 10 5 152 4273 32.23 16 5 150 Aspartate Transaminase lu/L 498 5321 28.09 8 13 84 4142 32.12 9 12 82 Fibrinogen G/L 199 5468 2.87 0.7 1.5 5.2 4294 2.76 0.7 1.5 5.2 Total Cholesterol Mmol/L 144 5495 5.49 1.5 2.2 10 4322 5.29 1.6 2 10 Dihydroepiandrosterone Sulphate Umol/L 239 5414 3.76 3.3 0.4 19 4308 5.67 4.9 0.4 25.3 Creatinine Umol/L 158 5497 68.27 14 33 173 4306 85.9 17 44 178 Gamma Glutamyl Transferase lu/L 214 5467 27.33 16 5 382 4280 39.75 25 5 368 Glycated haemoglobin Mmol/mol 525 5288 36.05 6 15 57 4148 36.56 6 18 57 HDL cholesterol Mmol/L 165 5482 1.68 0.5 0.5 3.4 4314 1.37 0.5 0.4 3.4 Haemoglobin G/L 294 5392 130.49 13 82 174 4275 145.62 14 84 185 C-Reactive Protein (hs assay) Mg/L 420 5350 3.53 3 0.2 115.5 4191 3 2.2 0.2 104.9 Insulin-like growth factor 1 Nmol/L 229 5455 17.74 8 2 47 4277 18.43 8 3 47 Ferritin G/L 143 5499 92.93 82 3 1292 4319 189.11 143 7 3044 Testosterone (for males only) Nmol/L 5702 NA NA NA NA NA 4259 15.59 7.3 2.9 40.1 Triglycerides Mmol/L 216 5482 1.58 1 0.3 6.3 4263 1.99 1.3 0.3 6.3 Urea Mmol/L 143 5498 5.94 2 2.2 16.5 4320 6.53 2 2.1 16.5 Table 1. Descriptive statistics for the sample and the measured biomarkers. *IQR = inter quartile range. Table 1. Descriptive statistics for the sample and the measured biomarkers. *IQR = inter quartile range. behavioural, attitudinal, physiological and biomedical variables, including a large panel of the most commonly used clinical biomarkers, have been measured for a representative selection of the sample. This study represents a large sample with very homogenous biomarker measurements, in which recruitment and processing have been carried out consistently and following strict protocols. Genome-wide analysis of health- related biomarkers in the UK Household Longitudinal Study reveals novel associations behavioural, attitudinal, physiological and biomedical variables, including a large panel of the most commonly used clinical biomarkers, have been measured for a representative selection of the sample. This study represents a large sample with very homogenous biomarker measurements, in which recruitment and processing have been carried out consistently and following strict protocols. Here we describe genome-wide investigation of associations with 20 biomarkers relevant to blood clot forma- tion (fibrinogen), diabetic status (glycated haemoglobin [HbA1c]), insulin-like growth factor 1 [IGF-1]), inflam- mation (C-reactive protein [CRP]), iron homeostasis (ferritin, haemoglobin), lipid metabolism (HDL-, LDL- and total cholesterol, triglycerides), liver function (alanine and aspartate transaminase, alkaline phosphatase, gamma glutamyl transferase [GGT]), liver and kidney function (albumin, creatinine, eGFR, urea), and reproductive health (dihydroepiandrosterone sulphate [DHEAS], testosterone) in 9,961 individuals from UKHLS. We also leverage the homogeneity of the sample and its size to estimate the narrow sense heritability which has not yet been quantified for many of these biomarkers. www.nature.com/scientificreports/ www.nature.com/scientificreports/ biomarker name h2 standard error p-value Albumin 0.15 0.04 8.9 × 10−6 Alkaline Phosphatase 0.28 0.04 2.2 × 10−13 Alanine Transaminase 0.09 0.04 6.8 × 10−3 Aspartate Transaminase 0.09 0.04 2.9 × 10−3 Fibrinogen 0.17 0.04 6.5 × 10−6 Total Cholesterol 0.07 0.04 0.023 Dihydroepiandrosterone Sulphate 0.17 0.04 4.7 × 10−6 in men 0.14 0.08 0.045 in women 0.20 0.07 1.6 × 10−3 Creatinine 0.21 0.04 5.2 × 10−9 eGFR 0.12 0.04 9.0 × 10−4 Gamma Glutamyl Transferase 0.22 0.04 2.4 × 10−9 Glycated haemoglobin 0.22 0.04 2.8 × 10−9 HDL cholesterol 0.23 0.04 5.9 × 10−10 LDL cholesterol 0.08 0.04 0.013 Haemoglobin 0.17 0.04 5.5 × 10−7 C-Reactive Protein (hs assay) 0.16 0.04 1.1 × 10−5 Insulin-like growth factor 1 0.20 0.04 4.6 × 10−9 Ferritin 0.06 0.04 0.043 Testosterone (for males only) 0.27 0.08 4.8 × 10−4 Triglycerides 0.23 0.04 3.6 × 10−10 Urea 0.14 0.04 2.1 × 10−4 Table 2. Array heritability (h2) estimates and standard errors for 20 biomarkers. biomarker name h2 standard error p-value Albumin 0.15 0.04 8.9 × 10−6 Alkaline Phosphatase 0.28 0.04 2.2 × 10−13 Alanine Transaminase 0.09 0.04 6.8 × 10−3 Aspartate Transaminase 0.09 0.04 2.9 × 10−3 Fibrinogen 0.17 0.04 6.5 × 10−6 Total Cholesterol 0.07 0.04 0.023 Dihydroepiandrosterone Sulphate 0.17 0.04 4.7 × 10−6 in men 0.14 0.08 0.045 in women 0.20 0.07 1.6 × 10−3 Creatinine 0.21 0.04 5.2 × 10−9 eGFR 0.12 0.04 9.0 × 10−4 Gamma Glutamyl Transferase 0.22 0.04 2.4 × 10−9 Glycated haemoglobin 0.22 0.04 2.8 × 10−9 HDL cholesterol 0.23 0.04 5.9 × 10−10 LDL cholesterol 0.08 0.04 0.013 Haemoglobin 0.17 0.04 5.5 × 10−7 C-Reactive Protein (hs assay) 0.16 0.04 1.1 × 10−5 Insulin-like growth factor 1 0.20 0.04 4.6 × 10−9 Ferritin 0.06 0.04 0.043 Testosterone (for males only) 0.27 0.08 4.8 × 10−4 Triglycerides 0.23 0.04 3.6 × 10−10 Urea 0.14 0.04 2.1 × 10−4 Table 2 Array heritability (h2) estimates and standard errors for Table 2. Array heritability (h2) estimates and standard errors for 20 biomarkers. Figure 1. Genetic correlations between different biomarker levels. Colour-coding indicates the strength of the correlations. The lower triangle uses only the red color-coding to make it easier to compare the strength of all correlations. Stars indicate statistically significant associations. Table 2.  Array heritability (h2) estimates and standard errors for 20 biomarkers. Results i Imputation and genomic coverage. After quality control, genotype data for 525,314 variants were avail- able for 9,961 individuals (Table 1). Following imputation based on the combined reference panel of UK10K and 1000 Genomes Project phase 3, we analysed 23,756,480 variants with imputation accuracy >0.4. Of those, 14,364,872 were rare (MAF <1%, minor allele count (MAC) >10) (2,237,400 of which had imputation accuracy >0.9), 2,732,394 low-frequency (1%≤ MAF <5%) and 6,659,214 common (MAF ≥5%). Heritability and genetic overlap analyses. For all biomarkers except overall and LDL-cholesterol, ala- nine transaminase and ferritin there was significant (p < 3.6 × 10−3) evidence for a heritable polygenic compo- nent (Table 2). Alkaline phosphatase and testosterone had the highest array heritability estimates with h2 = 27.7% (standard error (SE): 0.040) and h2 = 27.1% (SE: 0.084), respectively. Creatinine, GGT, HbA1c, HDL, IGF1, and triglycerides all had estimates larger than 0.20 while the lowest estimate was observed for ferritin (h2 = 6.1%, SE: 0.037). We found statistically significant (p < 5.5 × 10−4) evidence of genome-wide pleiotropy between different biomarkers (Fig. 1). There was genetic correlation between lipid biomarkers: triglyceride and HDL-cholesterol levels (genetic correlation rg = −0.67, p = 9.9 × 10−18). Triglyceride levels were also inversely genetically linked with DHEAS (rg = −0.53 p = 4.0 × 10−4). The genetic correlation between two markers of inflammation, C-reactive protein and fibrinogen, was also significant (rg = 0.60 p = 3.2 × 10−8). Finally, the genetic factors for creatinine and urea were positively correlated (rg = 0.56 p = 1.2 × 10−5). Genome-wide association analyses. The genome-wide significance threshold of P < 3.56 × 10−9 for this study was derived by taking the conventional genome-wide significance threshold (P < 5 × 10−8) divided by the effective number of independent traits analysed (N = 14.05, details in Methods). Across fifteen biomarkers, we observed associations of 54 previously reported loci at this threshold (Fig. 2). This includes a low frequency vari- ant, rs148685782 at 4q31 in the fibrinogen gamma chain gene (weighted effect allele frequency [WEAF] = 0.4%, beta[SE] = −0.18[0.02], P = 4.0 × 10−21), associated with levels of fibrinogen, a glycoprotein that assists in the Scientific Reports | 7: 11008 | DOI:10.1038/s41598-017-10812-1 2 www.nature.com/scientificreports/ Albumin: alb, Alkaline Phosphatase: alkp, Alanine Transaminase: alt, Aspartate Transaminase: ast, Fibrinogen: cfib, Total Cholesterol: chol, LDL cholesterol: ldl, Dihydroepiandrosterone Sulphate: dheas, Creatinine: ecre, Gamma Glutamyl Transferase: ggt, Glycated haemoglobin: hba1c, HDL cholesterol: hdl, Haemoglobin: hgb, C-Reactive Protein: hscrp, Insulin-like growth factor 1: igfi, Ferritin: rtin, Testosterone: testo, Triglycerides: trig, Urea: ure. Figure 1. Genetic correlations between different biomarker levels. Colour-coding indicates the strength of the correlations. The lower triangle uses only the red color-coding to make it easier to compare the strength of all correlations. Stars indicate statistically significant associations. Albumin: alb, Alkaline Phosphatase: alkp, Alanine Transaminase: alt, Aspartate Transaminase: ast, Fibrinogen: cfib, Total Cholesterol: chol, LDL cholesterol: ldl, Dihydroepiandrosterone Sulphate: dheas, Creatinine: ecre, Gamma Glutamyl Transferase: ggt, Glycated haemoglobin: hba1c, HDL cholesterol: hdl, Haemoglobin: hgb, C-Reactive Protein: hscrp, Insulin-like growth factor 1: igfi, Ferritin: rtin, Testosterone: testo, Triglycerides: trig, Urea: ure. blood clot formation. This variant is a missense mutation and has been previously reported to be associated with fibrinogen levels7 as well as with hypofibrinogenemia and haemorrhage8–10. F h di h i d f d 573 i d d ( i i 2 0 01) i h i From the discovery phase we carried forward 573 independent (pairwise r2 < 0.01) variants that were associ- ated with biomarker levels at P < 1 × 10−5 and were located more than 500 kb away from any known index variant for the respective biomarker. Using data from up to 25,897 samples from 4 independent studies (Supplementary Table S1), five loci provided evidence of replication and reached P < 3.6 × 10−9 for the combined analysis of dis- covery and replication data (Table 3). y p rs28929474 at 14q32 (WEAF = 2%, beta[SE] = 0.04[0.01], P = 1.7 × 10−10), a low-frequency variant associated with alanine transaminase (ALT), resides in the serpin family A member 1 (SERPINA1) gene (Figs 3A and 4). Scientific Reports | 7: 11008 | DOI:10.1038/s41598-017-10812-1 3 www.nature.com/scientificreports/ p Figure 2. Scatter plot of effect size by frequency of genome-wide significant variants. Effect sizes and 95% confidence intervals (absolute value of beta, expressed in standard deviation units) as a function of minor allele frequencies (MAF), based on the discovery stage of this study. Novel variants (Table 1) are displayed as diamonds, whilst known variants that reach genome-wide significance (P<3.56 × 10-9, two-sided) in the discovery stages are display as circles. www.nature.com/scientificreports/ Alkaline Phosphatase: alkp, Alanine Transaminase: alt, Fibrinogen: cfib, Total Cholesterol: chol, LDL cholesterol: ldl, Dihydroepiandrosterone Sulphate: dheas, Gamma Glutamyl Transferase: ggt, Glycated haemoglobin: hba1c, HDL cholesterol: hdl, Haemoglobin: hgb, C-Reactive Protein: hscrp, Insulin-like growth factor 1: igfi, Ferritin: rtin, Testosterone: testo_m, Triglycerides: trig. Figure 2. Scatter plot of effect size by frequency of genome-wide significant variants. Effect sizes and 95% confidence intervals (absolute value of beta, expressed in standard deviation units) as a function of minor allele frequencies (MAF), based on the discovery stage of this study. Novel variants (Table 1) are displayed as diamonds, whilst known variants that reach genome-wide significance (P<3.56 × 10-9, two-sided) in the discovery stages are display as circles. Alkaline Phosphatase: alkp, Alanine Transaminase: alt, Fibrinogen: cfib, Total Cholesterol: chol, LDL cholesterol: ldl, Dihydroepiandrosterone Sulphate: dheas, Gamma Glutamyl Transferase: ggt, Glycated haemoglobin: hba1c, HDL cholesterol: hdl, Haemoglobin: hgb, C-Reactive Protein: hscrp, Insulin-like growth factor 1: igfi, Ferritin: rtin, Testosterone: testo_m, Triglycerides: trig. SERPINA1 encodes alpha-1-antitrypsin (AAT), which is a serine protease inhibitor produced in the liver11. Low levels of this protein are the hallmark of a genetic disorder called alpha-1 antitrypsin deficiency (A1AD), which leads to liver disease12.i We identified two novel replicating associations for DHEAS (Fig. 3B,C). DHEAS is the sulphated form of DHEA, a precursor to major sex-hormones such as testosterone and oestrogen, and is synthesized in the adrenal glands. It is an important marker of adrenal gland function. rs78900934 at chromosome 1p21 (WEAF = 30.9%, beta[SE] = 0.05[0.01], P = 5.9 × 10−12) is located 1 kb upstream of a pseudogene, peptidylprolyl isomerase A pseudogene 7 (PPIAP7). This gene shows a high degree of similarity to cyclophilin A (PPIA), the product of which is involved in a number of biological processes including signal transduction13, inflammation14 and apop- tosis15. At the second novel locus associated with DHEAS the index variant, rs2911280 at 16q13 (WEAF = 7.5%, beta[SE] = 0.09[0.01], P = 6.0 × 10−10), is located in an intron of the gene encoding c-Maf inducing protein (CMIP), thought to play a role in the T-cell signalling pathway.16i g p y g g p y Two novel replicating associations with HbA1c levels were identified (Fig. 3D,E). HbA1c represents the three-month average plasma glucose concentration and is used to diagnose as well as manage type 2 diabetes. www.nature.com/scientificreports/ The index variant at 12q13, rs12819124 (WEAF = 46.7%, beta[SE] = −0.03[0.01], P = 4.2 × 10−9) lies in an intron of RP1-228P16.4, a long non-coding RNA. The index variant of the second novel locus, rs761772 at 17q25 (WEAF = 12.4%, beta[SE] = 0.05[0.01], P = 4.9 × 10−9), lies within a non-coding exon in the transmembrane channel-like 6 (TMC6) gene and has been shown to affect the expression of TMC6, as well as TNRC6C antisense RNA 1 (TNRC6C-AS1) and transmembrane channel like 8 (TMC8), in cardiac, thyroid, and vascular tissue, as well as whole blood in the GTEx database17. Discussioni and highly biologically plausible finding as both markers are increased in blood when glomerular filtration rate declines, reflecting impaired kidney function. Characterising the genetic architecture of health-related biomark- ers in this way is informative with respect to their biology as well as the design of future association studies. While each known locus individually explains only a small proportion of the variance in biomarker levels, these analyses demonstrate that the joint effect of many variants can be much larger. f We examined less-well studied health-related biomarkers in addition to routine blood measures used in clin- ical practice. This made it possible to identify novel associations of common and low frequency variants with DHEAS, HbA1c and ALT. These associations could provide novel biological insights. rs2911280, which we found to be associated with DHEAS, is located in an intron of the gene encoding c-Maf inducing protein (CMIP). CMIP is a highly pleiotropic gene, and is associated with several metabolic traits such as adiponectin and HDL choles- terol levels. Cholesterol is a precursor of DHEA in its synthesis process22. p y p rs28929474 at 14q32 is associated with levels of ALT, which is used in clinical practice to assess liver damage. This variant is located in SERPINA1, encoding the serine protease inhibitor alpha-1-antitrypsin (AAT), which is largely produced in the liver. Associations of variants in this gene were previously found for cortisol23 and height24. Mutations of this gene can cause alpha-1 antitrypsin deficiency (A1AD) which can lead to an accumula- tion of aberrant proteins in hepatocytes causing liver damage25. This in turn may elevate levels of ALT, warranting future assessment of the association between this signal and liver-related clinical endpoints. g p We identify two novel associations with HbA1c levels. In a lookup using published data from an independ- ent large-scale meta-analysis by the MAGIC consortium26, rs12819124 was associated with HbA1c levels with P = 1.8 × 10−6. The direction of effect was consistent with our findings. rs12819124 was also nominally asso- ciated with risk of type 2 diabetes at P = 0.025 using data from the DIAGRAM study27. Moreover, association results from published cohorts suggest a possible pleiotropic association with mental disorders and wellbeing (P = 9.0 × 10−6 for bipolar disorder and schizophrenia28, P = 6.4 × 10−5 for subjective wellbeing29). No HbA1c association results were available for rs761772 in MAGIC. Discussioni We identify five new biomarker loci, across common and low frequency variants, associated with DHEAS, HbA1c and ALT. We demonstrate polygenic heritability of the majority of biomarkers included in this study and observe large differences in their polygenic component. To our knowledge this is the first report of SNP-based heritability estimates for DHEAS, insulin-like growth factor 1, testosterone and urea. The large sample set with homogeneous biomarker measurements afforded by UKHLS enables reliable estimation for this population. We also identify genetic correlations between several of the biomarkers. Genetic correlation between two traits is an indicator of shared genetic factors and consequently genome-wide pleiotropy. The patterns of heritability and genetic corre- lations we observe for lipid biomarkers are consistent with previous reports in independent samples18. For total and LDL cholesterol, the SNP-based heritability is less than 10% whilst for HDL it is higher at 23.2%. All these estimates represent a lower bound for the narrow sense heritability. Our estimate of the negative genetic correla- tion between levels of HDL-cholesterol with triglycerides of rg = −0.67 is similar to the estimate derived from an independent study (rg = −0.61)19. High levels of triglycerides are mechanistically related to low levels of HDL20, 21, which could explain the reverse influence of the shared genetic factors on the biomarkers. We show for the first time that polygenic factors for triglyceride are also negatively correlated with DHEAS. There is a statistically significant genetic correlation between CRP and fibrinogen levels, which could be due to shared inflammation pathways. Discussioni Finally, the genetic correlation we observe between creatinine and urea is a previously unreported Scientific Reports | 7: 11008 | DOI:10.1038/s41598-017-10812-1 4 www.nature.com/scientificreports/ biomarker rs-id function nearest gene cytoband EA/NEA discovery replication combined EAF beta (SE), p-value N r2 imputed EAF beta (SE), p-value N EAF beta (SE), p-value N Alanine Transaminase rs28929474 missense SERPINA1 14q32 T/C 0.02 0.04 (0.01), 2.61 × 10−6 9731 1.00 no 0.02 0.04 (0.01), 1.47 × 10−5 9881 0.02 0.04 (0.01), 1.72 × 10−10 19612 Dihydroepiandrosterone Sulphate rs78900934 upstream gene PPIAP7 1p21 A/C 0.31 0.05 (0.01), 7.95 × 10−8 9722 1.00 yes 0.31 0.08 (0.02), 4.32 × 10−6 3630 0.31 0.05 (0.01), 5.88 × 10−12 13352 Dihydroepiandrosterone Sulphate rs2911280 intron CMIP 16q23 A/G 0.08 0.09 (0.02), 2.25 × 10−8 9722 0.97 yes 0.07 0.08 (0.03), 8.63 × 10−3 3630 0.08 0.09 (0.01), 5.97 × 10−10 13352 Glycated haemoglobin rs12819124 intron RP1- 228P16.4 12q13 A/C 0.47 −0.04 (0.01), 5.94 × 10−8 9436 0.99 yes 0.47 −0.02 (0.01), 1.12 × 10−3 7970 0.47 −0.03 (0.01), 4.20 × 10−9 17406 Glycated haemoglobin rs761772 non-coding exonic TMC6 17q25 C/T 0.13 0.06 (0.01), 5.94 × 10−8 9436 0.92 yes 0.12 0.03 (0.01), 3.83 × 10−3 5190 0.12 0.05 (0.01), 4.86 × 10−9 14626 Table 3. Association results of replicating novel signals. function: variant functional consequence; nearest gene: gene nearest to lead variant with 500Kb from either side; chr: chromosome; EA/NEA: effect allele/non-effect allele; EAF; effect allele frequency; beta(SE), p-value: effect size (standard error) and p-value; N: total number of individuals analysed for this variant; r2: imputation accuracy. Table 3. Association results of replicating novel signals. function: variant functional consequence; nearest gene: gene nearest to lead variant with 500Kb from either side; chr: chromosome; EA/NEA: effect allele/non-effect allele; EAF; effect allele frequency; beta(SE), p-value: effect size (standard error) and p-value; N: total number of individuals analysed for this variant; r2: imputation accuracy. and highly biologically plausible finding as both markers are increased in blood when glomerular filtration rate declines, reflecting impaired kidney function. Characterising the genetic architecture of health-related biomark- ers in this way is informative with respect to their biology as well as the design of future association studies. While each known locus individually explains only a small proportion of the variance in biomarker levels, these analyses demonstrate that the joint effect of many variants can be much larger. Discussioni For a proxy SNP, rs429216 (r2 = 0.75), the p-value for the association with HbA1c was in the same direction and reached P = 2.7 × 10−3.hf The UKHLS sample size is modest compared to some of the previous large-scale GWAS meta-analysis efforts (e.g., >45,000 individuals for HbA1c levels26). The relative gain in power leading to novel locus identification in this study can be attributed to several factors. Two of the newly reported signals have relatively low allele fre- quency (2% and 7.5%, respectively). These were captured here through use of the Illumina HumanCoreExome array and imputation to a comprehensive reference panel consisting of 1000 Genomes combined with the UK10K haplotypes30. A further power advantage was afforded by the homogeneous measurement of biomarkers in UKHLS and in two of the replication studies. Each biomarker was measured using the same assay for each sample, and processed on the same machine, avoiding loss of information due to diverse biomarker assays with different sensitivity, dynamic range and detection limit, potentially leading to power reductions31. y y g p y g p Larger-scale homogeneous studies and synthesis in massive-scale meta-analyses will help further elucidate the genetic architecture of medically-relevant biomarker traits. Insights into the genetic determinants of population variation in biomarker levels can help us to understand basic processes involved in maintaining health. Methods Ethi P Ethics. Participants gave informed written consent for their blood to be taken and stored for future scientific analysis. The UKHLS has been approved by the University of Essex Ethics Committee and the nurse data collec- tion by the National Research Ethics Service (10/H0604/2). Study population. The United Kingdom Household Longitudinal Study, also known as Understanding Society (https://www.understandingsociety.ac.uk) is a longitudinal panel survey of 40,000 UK households from England, Scotland, Wales and Northern Ireland). Participants are surveyed annually since 2009 and contribute information relating to their socioeconomic circumstances, attitudes, and behaviours via a computer assisted Scientific Reports | 7: 11008 | DOI:10.1038/s41598-017-10812-1 5 www.nature.com/scientificreports/ Figure 3. Regional association plots of novel genome-wide significant loci. Panel A–E : Regional association plots for replicating lead variants for alanine transaminase (A), DHEAS (B,C), HbA1c (D,E) respectively. Pairwise LD (r2) with the index variant is indicated following a color-coded scale. Both the p-values for the discovery as well as the combined discovery + replication are plotted for the index variant, results for all other variants were based on discovery-only data. Figure 3. Regional association plots of novel genome-wide significant loci. Panel A–E : Regional association plots for replicating lead variants for alanine transaminase (A), DHEAS (B,C), HbA1c (D,E) respectively. Pairwise LD (r2) with the index variant is indicated following a color-coded scale. Both the p-values for the discovery as well as the combined discovery + replication are plotted for the index variant, results for all other variants were based on discovery-only data. interview. As recruitment was household based, the study contains related individuals. The study includes phe- notypical data for a representative sample of participants for a wide range of social and economic indicators as well as a biological sample collection encompassing biometric, physiological, biochemical, and haematolog- ical measurements and self-reported medical history and medication use (https://www.understandingsociety. ac.uk/d/100/7251_User_Guide_Health_Assmt_w2_w3.pdf?1392855567). For each participant non-fasting blood samples were collected through venepuncture, were centrifuged to separate plasma and serum, aliquoted and frozen at −80 °C. DNA has been extracted and stored for genetic analyses. g y For replication, data were available for 5533 individuals from ELSA32, 9888 from Fenland33 (Supplemental Table 1), 7621 from HRS (http://hrsonline.isr.umich.edu)34, 2859 from NCDS35. These studies have been described in detail elsewhere. Sample collection were carried out consistently and analysed by the same laborato- ries for UKHLS, ELSA and NCDS. Biomarker measurements. www.nature.com/scientificreports/ www.nature.com/scientificreports/ tificreports/ Figure 4. Power calculations. Power calculations for individual variants selected for replication per trait, number of samples needed to reach 80% power to reach genome-wide significance (P<3.56 × 10−9, two-sided). The size of the circles represents the relative effect size (standardised) compared amongst all traits. Figure 4. Power calculations. Power calculations for individual variants selected for replication per trait, number of samples needed to reach 80% power to reach genome-wide significance (P<3.56 × 10−9, two-sided). The size of the circles represents the relative effect size (standardised) compared amongst all traits. Figure 4. Power calculations. Power calculations for individual variants selected for replication per trait, number of samples needed to reach 80% power to reach genome-wide significance (P<3.56 × 10−9, two-side The size of the circles represents the relative effect size (standardised) compared amongst all traits. Figure 4. Power calculations. Power calculations for individual variants selected for replication per trait, number of samples needed to reach 80% power to reach genome-wide significance (P<3.56 × 10−9, two-sided). The size of the circles represents the relative effect size (standardised) compared amongst all traits. Phenotype transformations and exclusions. The measurements for biomarkers used in the associa- tion analyses were prepared according to protocols from the largest genetic association study published for each specific trait at the time when analyses commenced, details for which are available in Supplementary Table S2. Genotyping. In total, 10,484 UKHLS samples have been typed using the Illumina Infinium HumanCoreExome BeadChip Kit® (12v1-0). This array contains a set of >250,000 highly informative genome-wide tagging single nucleotide polymorphisms as well as a panel of functional (protein-altering) exonic markers, including a large proportion of low-frequency (MAF 1–5%) and rare (MAF <1%) variants. Genotype calling was performed with the gencall algorithm using GenomeStudio (Illumina Inc.). For quality control (QC) we excluded individuals based on the following criteria: sample call rate <98%, autosomal heterozygosity outliers (>3SD), gender mismatches, duplicates as established by identity by descent (IBD) analysis (PI_HAT > 0.9). Individuals with non-European ancestry were also excluded. For this we estimated the genomic kinship between all pairs of individuals along with 1000 Genomes Project data. These were converted to distances and subjected to multidimensional scaling. Prior to variant QC, we first mapped all 538,448 variants to the human reference genome build 37. www.nature.com/scientificreports/ Variants with Hardy-Weinberg equilibrium p-value < 1 × 10−4, call rate below 98% or poor genotype clustering values (<0.4) were excluded, leaving 525,314 variants passing QC. For typed variants in our GWAS analyses that were brought forward for replication we inspected cluster plots manually using Scattershot 0.75 beta (Supplementary Fig. S1). All QC procedures were carried out using PLINK (v1.07) and R. Imputation. We imputed our genotype data using a combined reference panel consisting of 7,562 haplotypes from the UK10K project and 2,184 haplotypes from 1000 Genomes phase 3. Details regarding the creation of this combined imputation panel are described elsewhere37. Prior to imputation, we first pre-phased using SHAPEIT (v2.r). Data were then imputed using IMPUTE2 (v2.3.0), resulting in an initial set of 38,310,212 variants. Variants with an IMPUTE info score <0.4, and variants with a Hardy-Weinberg p-value < 1 × 10−4 were excluded, leaving 26,851,013 variants for analysis. Data availability. The UKHLS EGA accession number is EGAD00010000918. ELSA EGA accession number is EGAC00001000270. NCDS accession number is EGAC00000000001. HRS is available through dbGAP, Study Accession number phs000428.v1.p1. Genotype-phenotype data access for UKHLS, ELSA and NCDS is available by application to Metadac (www.metadac.ac.uk). Statistical analysesh. Heritability analyses and genetic correlations. The proportion of trait variance explained by the genotyped and imputed variants was estimated using the GREML method as implemented in the GCTA software38, 39 (v1.26). We included all variants with minor allele frequency (MAF) > 0.01. We excluded variants with imputation accuracy less than 0.4. We computed the genetic relationship matrix (GRM) for each autosome and then used GCTA to combine them into one matrix. We excluded relatives from the estimation by filtering based on the GRM using a threshold of 0.1 after inspecting the distribution. This led to the exclusion of 672 individuals for this analysis. We also performed bivariate REML analysis in order to estimate genetic correla- tions between different biomarkers40. We applied a Bonferroni adjusted significance threshold using the effective number of traits for the heritability analyses and using the number of pairs based on the effective number of traits for the genetic correlation analyses. Association analyses. The association analyses were carried out using a multivariate linear mixed model to account for relatedness as implemented in GEMMA (v0.95). QQ plots and genomic inflation factors, as well as Manhattan plots for traits where we identified novel associations are displayed in Supplementary Fig. S2. Methods Ethi P In total, biomarker data were successfully obtained from 13,107 eligible indi- viduals who gave consent to give blood samples to be stored for future analysis (https://www.understanding- society.ac.uk/d/154/7251-UnderstandingSociety-Biomarker-UserGuide-2014.pdf?1418057881). All biomarkers were measured from serum (non-fasting), using a variety of suitable assays, and the majority analysed on a single Roche P module analyser36. On each machine Internal Quality Controls (IQC) were at regular intervals per day. External Quality Assurance (EQA) systems were in place to monitor all tests. Scientific Reports | 7: 11008 | DOI:10.1038/s41598-017-10812-1 6 References 1. Teslovich, T. M. et al. Biological, clinical and population relevance of 95 loci for blood lipids. Nature 466, 707–713 (2010). 1. Teslovich, T. M. et al. Biological, clinical and population relevance of 95 loci for blood lipids. Nature 466, 707–713 (2010). 2. Cohen, J. C., Boerwinkle, E., Mosley, T. H. & Hobbs, H. H. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N. Engl. J. Med. 354, 1264–1272 (2006). , g , p p p , ( ) 2. Cohen, J. C., Boerwinkle, E., Mosley, T. H. & Hobbs, H. H. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N. Engl. J. Med. 354, 1264–1272 (2006). g 3. Timpson, N. J. et al. A rare variant in APOC3 is associated with plasma triglyceride and VLDL levels in Europeans. Nat. Com 5, 4871 (2014).i , ( ) 4. Manolio, T. A. Bringing genome-wide association findings into clinical use. Nat. Rev. Genet. 14, 549–558 (2013).h i F. et al. The metabochip, a custom genotyping array for genetic studies of metabolic, cardiovascular, and anthropometric S Genet. 8, e1002793 (2012).h 5. Voight, B. F. et al. The metabochip, a custom genotyping array for genetic studies of metabolic, cardiovascular, an traits. PLoS Genet. 8, e1002793 (2012).h 6. Cortes, A. & Brown, M. A. Promise and pitfalls of the Immunochip. Arthritis Res. Ther. 13, 101 (2011).fi p ph 7. Huffman, J. E. et al. Rare and low-frequency variants and their association with plasma levels of fibrinogen, FVII, FVIII, and vWF. Blood 126, e19–29 (2015).i ( ) 8. Brennan, S. O., Fellowes, A. P., Faed, J. M. & George, P. M. Hypofibrinogenemia in an individual with 2 coding (gamma82 A– > G and Bbeta235 P– > L) and 2 noncoding mutations. Blood 95, 1709–1713 (2000).i 9. Wyatt, J., Brennan, S. O., May, S. & George, P. M. Hypofibrinogenaemia with compound heterozygosity for two gamma chain mutations - gamma 82 Ala– > Gly and an intron two GT– > AT splice site mutation. Thromb. Haemost. 84, 449–452 (2000). h 10. Ivaskevicius, V. et al. gammaAla82Gly represents a common fibrinogen gamma-chain variant in Caucasians. Blood Coagul. Fibrinolysis Int. J. Haemost. Thromb. 16, 205–208 (2005).i y Jh , ( ) 11. Stoller, J. K. & Aboussouan, L. S. A review of α1-antitrypsin deficiency. Am. J. Respir. Crit. Care Med. 185, 246–259 (2012).i yi y p 12. de Serres, F. www.nature.com/scientificreports/ g g All methods were performed in accordance with the relevant guidelines and regulations. All methods were performed in accordance with the relevant guidelines and regula www.nature.com/scientificreports/ www.nature.com/scientificreports/ implemented in METAL (v2011-03-25). We calculated an adjusted genome-wide significance threshold, for the effective number of traits, as several of our biomarkers have correlated levels. The effective number of traits was derived by computing the eigenvalues for the correlation matrix of the 20 biomarkers (effective number: 14.05). The routinely used GWAS threshold of p < 5 × 10−8 was then adjusted for this using the Bonferroni approach: 5 × 10−8/14.05 = 3.56 × 10−9. implemented in METAL (v2011-03-25). We calculated an adjusted genome-wide significance threshold, for the effective number of traits, as several of our biomarkers have correlated levels. The effective number of traits was derived by computing the eigenvalues for the correlation matrix of the 20 biomarkers (effective number: 14.05). The routinely used GWAS threshold of p < 5 × 10−8 was then adjusted for this using the Bonferroni approach: 5 × 10−8/14.05 = 3.56 × 10−9. Power calculations. We carried out power calculations using Quanto (v1.2.4), for discrete per-variant frequency and (standardised) effect sizes combinations, representative of variants identified in the discovery. Per-trait and per selected variant power analyses showed that we would minimally need 5,000 to 15,000 samples to replicate our variants with P < 3.56 × 10−9, two-sided, for testosterone levels, whereas the largest replication sample of 25,000 to 60,000 would be needed for eGFR (Fig. 4). Selection of replication SNPs, and criteria for novel loci. For replication we selected independent SNPs (LD r2 < 0.1), with MAF > 0.01 and a discovery p-value of P < 1 × 10−5 and at least > 500 Kb away from the nearest known reported index SNP for a given trait. We also took forward independent rare variants with a MAF ≤ 0.01 that were typed and reached P < 1 × 10−5, regardless whether they represented known associations for a given trait. Known index SNPs for all biomarkers analysed in this study were obtained through the GWAS catalog41 (accessed August 4, 2016) > , supplemented by manual searches in PubMed. Annotation. For annotation of our lead variants we used an in-house annotation script that automatically retrieves variant annotations from ENSEMBL42, including variant function, the nearest gene IDs within < 500Kb from a given variant, transcript and protein IDs for these genes, as well as conservation scores. It also calculates GWAVA43 scores for non-genic variants amongst other annotations. References J., Blanco, I. & Fernández-Bustillo, E. Genetic epidemiology of alpha-1 antitrypsin deficiency in North Americ Australia/New Zealand: Australia, Canada, New Zealand and the United States of America. Clin. Genet. 64, 382–397 (2003). 13. Walsh, C. T., Zydowsky, L. D. & McKeon, F. D. Cyclosporin A, the cyclophilin class of peptidylprolyl isomerases, and blockade cell signal transduction. J. Biol. Chem. 267, 13115–13118 (1992).lh cell signal transduction. J. Biol. Chem. 267, 13115–13118 (1992).l g 4. Jin, Z.-G. et al. Cyclophilin A is a proinflammatory cytokine that activates endothelial cells. Arterioscler. Thromb. Vasc. Biol. 24 1186–1191 (2004). 15. Yazdanbakhsh, K., Choi, J. W., Li, Y., Lau, L. F. & Choi, Y. Cyclosporin A blocks apoptosis by inhibiting the DNA binding activity of the transcription factor Nur77. Proc. Natl. Acad. Sci. USA 92, 437–441 (1995).hh 6. Grimbert, P. et al. The Filamin-A is a partner of Tc-mip, a new adapter protein involved in c-maf-dependent Th2 signaling pathway Mol. Immunol. 40, 1257–1261 (2004).h 17. Carithers, L. J. et al. A Novel Approach to High-Quality Postmortem Tissue Procurement: The GTEx Project. Biopreservation Biobanking 13, 311–319 (2015).hhi g 18. The UK10K Consortium. The UK10K project identifies rare va hh p ji ( ) 19. Bulik-Sullivan, B. et al. An atlas of genetic correlations across human diseases and traits. Nat. Genet. 47, 1236–1241 (2015). 19. Bulik-Sullivan, B. et al. An atlas of genetic correlations across human diseases and traits. Nat. Genet. 47, 1236–1241 (2015). g 20. Pownall, H. J. et al. Correlation of serum triglyceride and its reduction by omega-3 fatty acids with lipid transfer ac neutral lipid compositions of high-density and low-density lipoproteins. Atherosclerosis 143, 285–297 (1999). all, H. J. et al. Correlation of serum triglyceride and its reduction by omega-3 fatty acids with lipid transfer activity and the l lipid compositions of high-density and low-density lipoproteins Atherosclerosis 143 285–297 (1999) g y y 21. Tall, A. R. Plasma cholesteryl ester transfer protein. J. Lipid Res. 34, 1255–1274 (1993). y p p 22. Miller, W. L. Androgen biosynthesis from cholesterol to DHEA. Mol. Cell. Endocrinol. 198, 7–14 (2002).i Androgen biosynthesis from cholesterol to DHEA. Mol. Cell. Endoc 23. Bolton, J. L. et al. Genome Wide Association Identifies Common Variants at the SERPINA6/SERPINA1 Locus Influencing P Cortisol and Corticosteroid Binding Globulin. PLOS Genet. 10, e1004474 (2014). 23. Bolton, J. L. et al. Genome Wide Association Identifies Common Variants at the SER Cortisol and Corticosteroid Binding Globulin. PLOS Genet. Scientific Reports | 7: 11008 | DOI:10.1038/s41598-017-10812-1 www.nature.com/scientificreports/ Replication analyses were carried out in R and PLINK, following the same trait preparation protocols as used in the discovery stage. The association summary statistics from the replication analyses, as well as the combined discovery and replication stage were meta-analysed using a fixed-effects inverse variance weighted approach Scientific Reports | 7: 11008 | DOI:10.1038/s41598-017-10812-1 7 www.nature.com/scientificreports/ & Flicek, P. Functional annotation of noncoding sequence variants. Nat. Methods 11, 294–296 (2014). Acknowledgements g We would like to acknowledge the Understanding Society Scientific Group which includes the following members: Michaela Benzeval, Jonathan Burton, Nicholas Buck, Annette Jäckle, Meena Kumari, Heather Laurie, Peter Lynn, Stephen Pudney, Birgitta Rabe, Dieter Wolke; The UK Household Longitudinal Study is led by the Institute for Social and Economic Research at the University of Essex. The UK Household Longitudinal Study is funded by the Economic and Social Research Council (ES/H029745/1). The survey was conducted by NatCen and the genome- wide scan data were analysed and deposited by the Wellcome Trust Sanger Institute (WT098051). Information on how to access the data can be found on the Understanding Society website https://www.understandingsociety.ac.uk/. Genetic analysis in The English Longitudinal Study of Ageing is sponsored by the Economic and Social Research council (ES/K005774/1). The Health and Retirement Study genetic data project was conducted by the University of Michigan and sponsored by the National Institute on Aging (grant numbers U01AG009740, RC2AG036495, and RC4AG039029). The Fenland Study is funded by the Wellcome Trust and the Medical Research Council (MC_ U106179471). We further acknowledge support from the Medical research council (MC_UU_12015/1). We are grateful to all the volunteers for their time and help, and to the General Practitioners and practice staff for assistance with recruitment. Biochemical assays were performed by the National Institute for Health Research, Cambridge Biomedical Research Centre, Core Biochemistry Assay Laboratory and the Cambridge University Hospitals NHS Foundation Trust, Department of Clinical Biochemistry. www.nature.com/scientificreports/ 0. Kim, Y. J. et al. Large-scale genome-wide association studies in east Asians identify new genetic loci influencing metabolic traits. Nat Genet. 43, 990–995 (2011). Genet. 43, 990–995 (2011). 31. Sluis, S., van der, Verhage, M., Posthuma, D. & Dolan, C. V. Phenotypic Complexity, Measurement Bias, and Poor Phenotypic Resolution Contribute to the Missing Heritability Problem in Genetic Association Studies PLOS ONE 5 e13929 (2010) ( ) 1. Sluis, S., van der, Verhage, M., Posthuma, D. & Dolan, C. V. Phenotypic Complexity, Measurement Bias, and Poor Phenotypic Resolution Contribute to the Missing Heritability Problem in Genetic Association Studies. PLOS ONE 5, e13929 (2010).i 32. Steptoe, A., Breeze, E., Banks, J. & Nazroo, J. Cohort profile: the English longitudinal study of ageing. Int. J. Epidemiol. 42, 1640–1648 (2013). 33. Lotta, L. A. et al. Integrative genomic analysis implicates limited peripheral adipose storage capacity in the pathogenesis of hu insulin resistance. Nat. Genet., doi:10.1038/ng.3714 (2016).i g ( ) 34. Sonnega, A. et al. Cohort Profile: the Health and Retirement Study (HRS). Int. J. Epidemiol. 43, 576–585 (2014).i i 35. Power, C. & Elliott, J. Cohort profile: 1958 British birth cohort (National Child Development Study). Int. J. Epidemiol. 35, 3 (2006). 36. M Benzeval, A Davillas, M Kumari, P Lynn Understanding Society: the UK Household Longitudinal Study Biomarker User Guide and Glossary, ISER, University of Essex - Google Search. Available at: https://www.google.co.uk/search?q=M+Benzeval, +A+Davillas, +M+Kumari, +P+Lynn+ (2014)+Understanding+Society: +the+UK+Household+­Longitudinal+Study+Biomarker+User+Gui de+ and +Glossary, +ISER,+University+of+Essex&ie=utf-8&oe=utf-8&gws_rd=cr&ei=XfRSWOjVGdetgAa3qIKoDg. (Accessed: 15th December 2016) (2014). 37. Huang, J. et al. Improved imputation of low-frequency and rare variants using the UK10K haplotype reference panel. Nat. Com 6, 8111 (2015). ( ) 38. Yang, J. et al. Common SNPs explain a large proportion of the heritability for human height. Nat. Genet. 42, 565–569 (2010). 39. Yang, J., Lee, S. H., Goddard, M. E. & Visscher, P. M. GCTA: a tool for genome-wide complex trait analysis. Am. J. Hum. Gene 76–82 (2011). 40. Lee, S. H., Yang, J., Goddard, M. E., Visscher, P. M. & Wray, N. R. Estimation of pleiotropy between complex diseases using single- nucleotide polymorphism-derived genomic relationships and restricted maximum likelihood. Bioinforma. Oxf. Engl. 28, 2540–2542 (2012).h 1. Welter, D. et al. The NHGRI GWAS Catalog, a curated resource of SNP-trait associations. Nucleic Acids Res. 42, D1001–1006 (2014) 2. Yates, A. et al. Ensembl 2016. Nucleic Acids Res. 44, D710–716 (2016). 43. Ritchie, G. R. S., Dunham, I., Zeggini, E. Author Contributionst B.P.P., K.B.K. and E.Z. drafted the initial manuscript. B.P.P. and K.B.K. performed the statistical analyses. E.Z. designed and supervised the study. Y.B., M.S., D.Z., G.F., J.L. carried out the replication analyses. N.J.W., R.A.S., J.R.B.P. and C.L. contributed data for the replication. M.B. is the principal investigator of UKHLS. M.K. is the genetics lead of UKHLS. All authors read and approved the final manuscript. References 10, e1004474 (2014). g 4. North, T.-L. et al. A study of common Mendelian disease carriers across ageing British cohorts: meta-analyses reveal heterozygosity for alpha 1-antitrypsin deficiency increases respiratory capacity and height. J. Med. Genet. 53, 280–288 (2016).h yi y y y g 5. Lomas, D. A., Evans, D. L., Finch, J. T. & Carrell, R. W. The mechanism of Z alpha 1-antitrypsin accumulation in the liver. Nature 357, 605–607 (1992).l 6. Soranzo, N. et al. Common variants at 10 genomic loci influence hemoglobin A1(C) levels via glycemic and nonglycemic pathways Diabetes 59, 3229–3239 (2010).h 27. Fuchsberger, C. et al. The genetic architecture of type 2 diabetes. Nature 536, 41–47 (2016).i 27. Fuchsberger, C. et al. The genetic architecture of type 2 diabetes. Nature 536, 41–47 (2016).i h 28. Wang, K.-S., Liu, X.-F. & Aragam, N. A genome-wide meta-analysis identifies novel loci associated with schizophrenia and bi disorder. Schizophr. Res. 124, 192–199 (2010).i 29. Okbay, A. et al. Genetic variants associated with subjective well-being, depressive symptoms, and neuroticism identified through genome-wide analyses. Nat. Genet. 48, 624–633 (2016). Scientific Reports | 7: 11008 | DOI:10.1038/s41598-017-10812-1 Scientific Reports | 7: 11008 | DOI:10.1038/s41598-017-10812-1 8 www.nature.com/scientificreports/ Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-10812-1h Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2017 Scientific Reports | 7: 11008 | DOI:10.1038/s41598-017-10812-1 9
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Comprehensive Care Response and Systematic Management of COVID-19 in Querétaro, Mexico
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A. Aguilar Galarza envelope symbol · S. Celada Martínez University Health Service, Universidad Autonoma de Queretaro, Queretaro, Mexico e-mail: adrianaag.ga@gmail.com A. Aguilar Galarza envelope symbol · S. Celada Martínez S. Celada Martínez e-mail: ln.sandra.celada@gmail.com Introduction In this chapter, the impact of the pandemic on health in Mexico, in particular, in the state of Querétaro, is presented. Epidemiological trends regarding incidence, prevalence, and mortality are examined. And health policies are reviewed. On December 31, 2019, Wuhan, China, reported a conglomerate of cases of acute respiratory syndrome of unknown origin. Some of these cases were vendors in the Wuhan seafood market which was closed on January 1, 2020 [1]. On January 7, 2021, the Chinese authorities reported that a new coronavirus (nCoV) had been identified. On January 30, the World Health Organization (WHO) declared that the outbreak of 2019-nCoV was a public health emergency of international concern [2]. p g y The first confirmed case of COVID-19 was detected on February 27, 2020, in Mexico City. The patient was a Mexican who had traveled to Italy; he had mild symptoms [3]. On March 11, 2020, the WHO declared COVID-19 a pandemic. A few days later on March 18, the first death from COVID-19 was registered in Mexico. Mexico featured among the countries with the highest fatality rates. It ranked second in terms of the fatality rate (7.6 deaths per 100 infections). Peru ranked first [4]. y ( p ) According to official data from the National Epidemiological Surveillance System (SS), close to 4.0 million confirmed cases and 306,062 deaths from COVID-19 have been reported in Mexico. The incidence rate of COVID-19 is 2,9860 per 100,000 inhabitants. Figure 1 shows Mexico´s epidemic curve with the number of confirmed cases and deaths according to SS. The epidemic curve presents two infection peaks. The first one was in mid-January 2021. The second, in early August 2021, exceeded the first peak in the number of cases. Sex disaggregated data shows that there is a gender difference worldwide [5]. In Mexico, at the beginning, the distribution by sex in confirmed cases showed a higher prevalence in men (58%). Currently, the number of confirmed cases is similar in men and women (49% vs. 51%) but men have higher rates of hospitalization, intensive care admissions, and deaths (Fig. 2). The median age of COVID-19 cases in Mexico is 44 years. g y There were 395,000 accumulated deaths by October 2021 (Fig. 1). Mortality was higher in people with pre-existing comorbidities. Comorbidities associated with higher mortality were hypertension, diabetes, obesity, and smoking [6]. Comprehensive Care Response and Systematic Management of COVID-19 in Querétaro, Mexico Adriana Aguilar Galarza, Sandra Celada Martínez, Oscar San Roman Orozco, Isidro Amadeo Gutiérrez Álvarez, Izarelly Rosillo Pantoja, and Nuri G. Villaseñor Cuspinera Abstract The health impact of the pandemic in Queretaro, México is assessed. The socioeconomic conditions of the population and health policies implemented at the federal and state level as well as in the Universidad Autonoma de Queretaro are examined. The work is presented in three parts: In the first part, epidemiological data related to the incidence, prevalence, and mortality from COVID-19 in Mexico and Querétaro is presented. In the second part, the epidemiological panorama of Queretaro is presented as an analysis tool to assess the epidemiological behavior of the population and the social, economic, and health conditions in the state. Finally, in the third part, health policies implemented by the state university and the experi- ence of an integrative care model, implemented in the ‘Clinica de Atencion Integral COVID’, which provides multidisciplinary assessment and treatment for COVID-19, is presented. A. Aguilar Galarza envelope symbol · S. Celada Martínez University Health Service, Universidad Autonoma de Queretaro, Queretaro, Mexico e-mail: adrianaag.ga@gmail.com S. Celada Martínez e-mail: ln.sandra.celada@gmail.com O. San Roman Orozco The BORN Project, Universidad Autonoma de Queretaro, Queretaro, Mexico e-mail: oscarsanroman@gmail.com I. A. Gutiérrez Álvarez Applied Global Public Health Initiatives, Universidad Autonoma de Queretaro, Queretaro, Mexico I. Rosillo Pantoja School of Law, Universidad Autonoma de Queretaro, Queretaro, Mexico e-mail: izarellyrosillo@gmail.com N. G. Villaseñor Cuspinera School of Medicine, Universidad Autónoma de Queretaro, Queretaro, Mexico e-mail: nuguvicu@gmail.com © The Author(s) 2023 S. Pachauri and A. Pachauri (eds.), Global Perspectives of COVID-19 Pandemic on Health, Education, and Role of Media, https://doi.org/10.1007/978-981-99-1106-6_3 45 A. Aguilar Galarza envelope symbol · S. Celada Martínez University Health Service, Universidad Autonoma de Queretaro, Queretaro, Mexico e-mail: adrianaag.ga@gmail.com S. Celada Martínez e-mail: ln.sandra.celada@gmail.com O. San Roman Orozco The BORN Project, Universidad Autonoma de Queretaro, Queretaro, Mexico e-mail: oscarsanroman@gmail.com I. A. Gutiérrez Álvarez Applied Global Public Health Initiatives, Universidad Autonoma de Queretaro, Queretaro, Mexico I. Rosillo Pantoja School of Law, Universidad Autonoma de Queretaro, Queretaro, Mexico e-mail: izarellyrosillo@gmail.com N. G. Villaseñor Cuspinera School of Medicine, Universidad Autónoma de Queretaro, Queretaro, Mexico e-mail: nuguvicu@gmail.com © The Author(s) 2023 S. Pachauri and A. Pachauri (eds.), Global Perspectives of COVID-19 Pandemic on Health, Education, and Role of Media, https://doi.org/10.1007/978-981-99-1106-6_3 45 45 A. Aguilar Galarza et al. 46 Introduction 3 Population distribution of the state of Querétaro in relation to the national territory. Source Instituto Nacional de Estadística y Geografía (INEGI), 2011 laces, respectively, according to the ranking of the United Nations Development rogramme (UNDP, 2015). places, respectively, according to the ranking of the United Nations Development Programme (UNDP, 2015). The state of Querétaro is organized into four health jurisdictions (Queretaro, San Juan del Rio, Cadereyta, and Jalpan). There are more than 250 public health insti- tutions in Queretaro and San Juan del Rio. There are five second level hospitals (2 in Queretaro, 1 in San Juan del Rio, 1 in Cadereyta, and 1 in Jalpan), and approxi- mately 200 first level healthcare centers [10]. In addition, healthcare is provided by a voluntary public program Seguro Popular which is financed by the Federal Govern- ment and private insurance (IMSS, ISSSTE). This program covers about 85% of the Mexican population [11]. The main causes of death in the state are non-communicable diseases including ischemic heart disease, diabetes mellitus, and cancer. Influenza and pneumonia are among the top 10 causes of death in the state [12]. Excessive weight is linked to several health problems [13]. Epidemiological studies suggest that obesity could have an adverse impact on COVID-19, especially in severe cases, and could increase mortality [14]. In Mexico, obesity is the strongest predictor of COVID-19 followed by diabetes and hypertension [15]. On March 11, 2020, the first confirmed case of COVID-19 was reported in Quere- taro [16]. By October 30, 2021, there were more than 90,000 confirmed cases. The highest peak was in January with a second peak in August (Fig. 4). The incidence of COVID-19 was similar in women (45%) and men (55%). Although not considered a risk group, there were more confirmed cases in persons 25–34 years of age (27%) [17]. Higher mortality was reported in men than in women 63% and 37% respectively Higher mortality was reported in men than in women, 63% and 37%, respectively. Mortality was highest in persons over 60 years (57%). Comorbidities associated with deaths from COVID (n = 5,929) were hypertension (40%), diabetes (28%), obesity (21%), and chronic kidney disease (6%) [18]. Queretaro showed the lowest case fatality rate for COVID-19 in Mexico (Fig. 5). Despite having a larger number of confirmed cases in the capital of Queretaro, the fatality rate was two percentage points below the national average (Table 1). Introduction Until November 12, 2021, 38.2% of the deaths were reported in women and 61.7% in men (Fig. 2). A study of COVID-19 in eight countries in Latin America (Brazil, Peru, Mexico, Argentina, Colombia, Venezuela, Ecuador, and Bolivia) showed that hyper- tension (12.1%) was the most common comorbidity followed by diabetes (8.3%), and obesity (4.5%) [7]. According to the National Survey on Health and Nutrition (ENSANUT), Mexico has a high prevalence of obesity and diabetes. The prevalence of overweight and obesity in Mexico has increased in recent years. From 1980 to date, the prevalence of these diseases tripled. Currently, the weight of over 70.0% of the adult population in Mexico is above the recommended level [8]. Excess weight is one of the main risk factors in the development of chronic non-communicable diseases such as diabetes Comprehensive Care Response and Systematic Management … 47 Fig. 1 Daily confirmed COVID-19 cases and deaths in Mexico. Source Authors’ elaborated based on the General Directorate of Epidemiology (GDE) database Fig. 1 Daily confirmed COVID-19 cases and deaths in Mexico. Source Authors’ elaborated based on the General Directorate of Epidemiology (GDE) database Fig. 2 Distribution of COVID-19 cases and deaths by gender in Mexico. Source Authors’ elaborated based on the General Directorate of Epidemiology (GDE) database Fig. 2 Distribution of COVID-19 cases and deaths by gender in Mexico. Source Authors elaborated based on the General Directorate of Epidemiology (GDE) database mellitus, hypertension, and cardiovascular diseases. And these diseases increase the severity of COVID-19. With a population of 2,038,372, Querétaro ranks 22 in the number of inhabitants among the states of the Federation. Queretaro represents 0.6% of the territorial exten- sion of the Mexican Republic (Fig. 3); 175,000 of its inhabitants are over 60 years of age and so are at risk of contracting COVID-19 [9]. Regarding the Human Develop- ment Index (HDI) and health, the state of Querétaro occupies the seventh and twelfth 48 A. Aguilar Galarza et al. Fig. 3 Population distribution of the state of Querétaro in relation to the national territory. Source Instituto Nacional de Estadística y Geografía (INEGI), 2011 places, respectively, according to the ranking of the United Nations Development Programme (UNDP, 2015). Th t t f Q ét i i d i t f h lth j i di ti (Q t S 48 A. Aguilar Galarza et al. Fig. Introduction According to the State Development Plan 2016–2020, Queretaro did not have the number of beds recommended by the World Health Organization (1 bed per 1,000 population) [19]. To avoid overburdening the healthcare system, hospitalization was Comprehensive Care Response and Systematic Management … 49 Fig. 4 Daily confirmed COVID-19 cases and deaths in Queretaro. Source Authors’ elaborated based on the General Directorate of Epidemiology (GDE) database Fig 4 Daily confirmed COVID 19 cases and deaths in Queretaro Source Authors’ elaborate Fig. 4 Daily confirmed COVID-19 cases and deaths in Queretaro. Source Authors’ elaborated based on the General Directorate of Epidemiology (GDE) database Fig. 5 Case fatality ratio in Mexico. Source Authors’ elaborated based on the General Directorate of Epidemiology (GDE) database Fig. 5 Case fatality ratio in Mexico. Source Authors’ elaborated based on the General Directorate of Epidemiology (GDE) database reserved for severely ill patients. In Queretaro, 12% of confirmed cases required hospitalization which was three percentage points below the national average [17]. 50 A. Aguilar Galarza et al. Municipality Confirmed cases Deaths % Fatality rate Tequisquiapan 1084 134 12.4 Huimilpan 316 35 11.1 Amealco de Bonfil 655 58 8.9 Ezequiel Montes 795 60 7.5 Cadereyta de Montes 1616 119 7.4 Colon 942 68 7.2 San Juan del Rio 9170 658 7.2 Peñamiller 234 16 6.8 Pedro Escobedo 2149 135 6.3 Queretaro 65,380 3644 5.6 El Marques 5440 295 5.4 Pinal de Amoles 356 19 5.3 Landa de Matamoros 255 13 5.1 Toliman 482 24 5.0 Corregidora 6270 226 3.6 San Joaquin 311 10 3.2 Jalpan de Serra 1590 38 2.4 Arroyo Seco 356 7 2.0 Source General Directorate of Epidemiology (GDE) database. Table 1 Confirmed cases and deaths in Queretaro Source General Directorate of Epidemiology (GDE) database. Table 1 Confirmed cases and deaths in Queretaro Health Policies in Mexico and Querétaro When the first death was registered in Mexico, the first, second, and third level Epidemiological/Hospital Surveillance Units (UVEH) in the country, members of the National Network of Public Health Laboratories, and staff of the National Health System issued a report through the National Committee for Epidemiological Surveil- lance (CONAVE) wherein parameters were established to identify suspected and confirmed cases of COVID-19. A suspected case was a person of any age who presented acute, mild, or severe respiratory disease and who had any of the following antecedents up to 14 days before the onset of symptoms: (1) had been in contact with a confirmed case or had been under investigation for COVID-19 and (2) had made a trip or stayed in countries with local transmission of COVID-19. A confirmed case was a person who met the operational definition of a suspected case and had Comprehensive Care Response and Systematic Management … 51 a confirmed diagnosis by the National Institute for Epidemiological Diagnosis and Reference (InDRE) [20]. On March 23, 2020, 367 cases and four deaths from COVID-19 were confirmed in Mexico: two in Mexico City, one in Durango and one in Jalisco. The Federal Government closed all schools. A program called ‘Sana Distancia’ was launched by the Mexican Ministry of Health. Recommendations were made regarding basic prevention measures such as frequent hand washing, respiratory etiquette, greeting from a distance, and staying home if symptoms were present. There was a temporary suspension of non-essential activities. There was a rescheduling of mass events. And measures to protect and care for the elderly were implemented. To emphasize the importance of physical distance, a communication campaign was designed using cartoons that featured a heroine called ‘Susana Distancia’ [21]. On March 30, 2020, the General Health Council declared COVID-19 a health emergency, and all non-essential activities were suspended till April 30, 2020, to mitigate the spread of COVID-19 in the community and to reduce the burden of disease [22]. Although there was a 44% increase in the number of cases [2,527 new confirmed cases], in order to reactivate economic and social activities, on May 13, 2020, the plan to reopen non-essential activities called ‘New normality’ was announced [23]. An epidemiological traffic light was used for monitoring. The traffic light was defined weekly according to the risk by region. Its color indicated what activities could be carried out. Health Policies in Mexico and Querétaro The regions identified with the red color represented the maximum epidemiological risk. Orange represented a high epidemiological risk. The colors yellow and green represented intermediate and low epidemiological risk, respectively. This traffic light system began on June 1, 2020, at the state level [24]. In regions identified with red color, only essential activities were allowed. In regions identified with the orange color, in addition to essential activities, non-essential activ- ities could be reactivated although they were to be carried out at a capacity of 30% and with strict safe distance measures. In regions identified with the yellow and green colors, essential and non-essential economic activities could be conducted at full capacity as long as measures were taken to protect the health of workers [25]. On July 24, 2020, the Ministry of Health of the Government of Mexico, through the Institute of Health for Wellbeing (INSABI) and the Undersecretariat for Preven- tion and Health Promotion (SPPS), in collaboration with the Pan American Health Organization (PAHO), Mexico and the Secretariat of Welfare designed a strategy for promotion, prevention, care, and mitigation of COVID-19 and for monitoring essential public health actions at the community and the first levels of care within the framework of primary healthcare (PHC). The aim was to strengthen the response of the local health system to the pandemic. The following three action groups with specific action objectives were established [26]: (1) The Community Health Promotion Brigade: Its function was to request information from the Director of the Health Unit about people with risk factors for COVID, generate data updates, apply general questionnaires and notify, provide information to reduce the risk of contagion, identify persons within the patient’s home for follow-up, strengthen health promotion actions, and develop a directory 52 A. Aguilar Galarza et al. of public health officials according to the health problem and characteristics of the identified population. (2) The Specialized Brigades: Their function was to follow-up suspected cases that belonged to the risk groups described above. Daily follow-up was conducted by telephone or in-person with people in the community with ARI (acute respi- ratory infection). The status of the identified persons with any health problems was evaluated. Patients suspected of COVID-19 were monitored. Blood pres- sure of all adults 20 years of age and older was taken. Cases that required care in remote consultation units (UCID) were identified. Health Policies in Mexico and Querétaro Patients considered in risk groups were clinically evaluated to determine if they should be isolated. Preven- tive actions were implemented. A ‘brief mental health screening’ questionnaire was completed. Patients were provided information on self-care. They were also provided First Psychological Aid if needed and were given timely information on specialized mental health services. Pregnant women were also reviewed. (3) The Clinical Care Team: Its function was to evaluate and treat patients referred by the brigades, provide clear information on the evolution of the disease and its consequences, communicate with the isolated persons, provide psycho- education, provide information on the lines of support, provide psychological support, and refer in a timely manner to Specialized Mental Health Services. On March 11, 2020, the Secretary of Health of the State of Querétaro (SESEQ) confirmed the first case in the city. This was a 43-year-old man from Spain who was isolated at his home. He was kept under medical observation. His situation was stable. Coordination with the federal authorities was maintained in order to provide timely follow-up using protocols and guidelines and to provide adequate care. The staff of the Ministry of Health had the necessary supplies and were trained to address this situation [27]. The public was exhorted to participate in containing the disease by implementing preventive measures and visiting the doctor when symptoms of an acute respiratory disease appeared (fever, cough, respiratory distress or chest pain). Two operational criteria had to be present: (1) the patient had been contacted 14 days prior to the appearance of symptoms with a person confirmed with COVID-19 and (2) the patient had traveled to a city that had community transmission [28]. Preventive measures included: frequent hand washing with soap and water or 70% alcohol gel solution, covering the nose and mouth with a disposable tissue when sneezing or coughing or using the internal angle of the arm, avoiding spitting, but if necessary, spitting in a disposable tissue which would be put in a plastic bag and thrown away, cleaning and disinfecting surfaces and objects commonly used at home and in schools, offices, closed places, transport, meeting centers, etc. Health Policies in Mexico and Querétaro Avoiding touching the face, especially the nose, mouth, and eyes, avoiding direct contact with people with symptoms of cold or flu, visiting the doctor when there were respiratory ailments (fever greater than 38 degrees, headache, sore throat, runny nose, etc.), avoiding self-medication, staying at home when there were respiratory symptoms, avoiding going to crowded places, keeping workspaces and housing units well ventilated, and drinking plenty of fluids. Finally, the use of face masks was Comprehensive Care Response and Systematic Management … 53 recommended for patients with respiratory symptoms and people were urged to follow information provided through institutional channels [29]. The SESEQ included within its website, a COVID section on the Health Risks Directorate section and incorporated a tab called COVID-19 legislation in documents that were made available to citizens. At least 15 agreements were issued during the COVID-19 pandemic [27]. In these agreements, general recommendations were made to undertake health security measures. These were shown to the general popu- lation and to the health personnel. Other agreements focused on the suspension of economic, productive, and social activities. During the emergency period, a Call Center for Medical and Emotional Attention was set up to inform people about physical and psychological symptoms related to COVID-19, resolve their doubts, and make appropriate referrals [30]. The Mexican Institute of Social Security (IMSS) implemented a hospital recon- version strategy in eight entities to address bed requirements which meant increasing the number of beds by 39% to reach 6,116 by the end of January 2021 in the states of Querétaro, Hidalgo, Puebla, Morelos, Guanajuato, Nuevo León, Jalisco, and Michoacán. In Querétaro, there were 380 beds and reconversion meant adding 104 to reach a total of 484 beds which was the number needed [31]. The Querétaro General Hospital shelter and the Children’s and Women’s Specialties Hospital were upgraded to provide medical care to COVID-19 cases that needed hospitalization. In the second half of April 2020, the Congress Center became a Medical and Isolation Unit (UMA) to serve patients who were positive for COVID-19 but did not require hospitalization. UMA served 250 patients—125 women and 125 men. This number was later increased to 700 patients [32]. On November 28, 2020, the Secretariat of Health of the Executive Power of the State of Querétaro through the official gazette, envisioned future scenarios. Health Policies in Mexico and Querétaro In accor- dance with the criteria of the Technical Committee for Attention to COVID-19 and the Specialized Multisectorial Group for Epidemiological Surveillance of COVID- 19, three scenarios were envisioned: Scenario A remission: hospital occupancy at 39%. Scenario B prevention: hospital occupancy between 40 and 70%. And Scenario C containment: hospital occupancy greater than 70% [33]. With a total of 30,215 accumulated cases and 2,049 deaths, on December 19, 2020, Scenario C was established in the State of Querétaro [34]. The positivity index was close to 38% and the increase in hospital occupancy in the absolute number of beds occupied by patients without the use of assisted ventilation was 48%. The percentage of patients with ventilator support was 49%. A total of 471 hospitalized patients reached 60% hospital occupancy [35]. According to the number of active cases of SARS-CoV-2 virus infection, the positivity index, and the increase in hospital occupancy, Scenario C remained in force until February 14, 2021. By then, the total number of accumulated cases was 51,162 and there were 3,371 deaths [36]. On February 12, 2021, Scenario B was established in the State of Querétaro. Sanitary security measures were implemented and remained in force until April 22, 2021. On February 10, 2021, hospital occupancy was reduced by 12% with 37% occupancy of beds with ventilator support and 50% occupancy of beds without ventilator support [37]. 54 A. Aguilar Galarza et al. A. Aguilar Galarza et al. Local Strategies to Respond to COVID-19 The COVID-19 pandemic dramatically changed health systems around the world. It changed the way in which outpatient care was delivered to decrease the risk of transmitting the virus to patients and to healthcare workers. Vaccines and treatments were also developed [38]. On the other hand, the lockdown period imposed drastic changes in the behaviors and lifestyles of the people in terms of physical activity and quality of diet both of which are known to play an important role in disease management [39, 40]. The management of large numbers of COVID-19 patients over a short period of time disrupted the healthcare system. High demand for hospitalization beds over- whelmed the healthcare system [41]. As a response to the pandemic, the Mexican government promoted several strategies like physical distancing to limit the spread of COVID-19. However, it is important to note that very few strategies were imple- mented to ensure the continuity of essential health services [42]. The public and private health sectors repurposed multiple hospitals, reallocated health personnel, and diverted medical equipment and supplies to treat COVID-19 patients. There has been a lack of investment in the health sector in Mexico historically. Health expenditure in Mexico is only 5.5% of the Gross Domestic Product (GDP) [43]. Through the University Health System, the Autonomous University of Queretaro organized an integrative care clinic ‘Clinica de Atencion Integral COVID’ dedicated to providing COVID-19 services and diagnosing and monitoring patients at home. The goal of this clinical service was to provide multidisciplinary assessment and treatment by a system that included virtual phone-based assessment and clinical home monitoring. The Clinica de Atencion Integral COVID offered a multidisci- plinary care model in which physicians, nutritionists, physical therapists, pharmaco- logical chemists, psychologists, and other staff members played a role in delivering comprehensive care. A description of the activities in each of the clinic’s care areas is provided below. Detection Area At the beginning of the pandemic, a group of scientists from the Autonomous Univer- sity of Queretaro offered SARS-CoV-2 virus detection services to the people through the use of the university’s own resources. Detection was carried out by means of a molecular screening test. More than 2,000 free tests were offered to the population at risk. It was found that approximately 80% of infected people did not have symptoms or had mild symptoms. This was an important finding because it made it possible to detect and isolate carriers to prevent the spread of the virus, particularly for those most susceptible. Comprehensive Care Response and Systematic Management … 55 Clinical Household Follow-Up Program Carriers of SARS-CoV-2 were invited for clinical follow-up which included a symp- toms questionnaire to be administered by telephone screening to find out if the patient was a candidate for follow-up at home. Only patients with low and medium risk were accepted into this program. Patients with high risk were referred to a specialized care service. A series of visits were made to the patient’s home to record signs and symp- toms such as temperature, oxygenation, an olfactory test, and an antibody test. The first home visit was made by medical personnel. In subsequent visits, a pharmaco- logical chemist obtained a blood sample to measure antibodies and a nutritionist performed a nutritional assessment. Nutrition plays an important role in the management of COVID-19 [44]. Under- nutrition, micronutrient deficiencies, and overnutrition increase the risk of devel- oping serious complications [45]. The aim of the nutritional assessment was to iden- tify nutritional risk in COVID-19 patients and assess its association with disease outcomes. In the beginning, the assessments were focused on identifying the risks of undernutrition. Nutritional assessment was later modified to assess metabolic risk factors. In patients included in this program risk factors like hypertension (30%), obesity BMI > 30 (27%), and diabetes (10%) were also assessed. Rehabilitation: Post-COVID-19 Program COVID-19 resulted in several medical, social, and psychological consequences like multi-organ failure of the heart and kidneys and vascular damage [46]. Specific reha- bilitation needs to be undertaken for post-COVID-19 patients to achieve respiratory improvement and functional and cognitive recovery, decrease disability, and improve the quality of life [47]. Persistent symptoms were present in patients who had recovered from COVID-19 in Mexico [48]. There was no rehabilitation clinic in the private or the public sector where comprehensive multidisciplinary services could be offered for the treatment sequelae of COVID-19. A multidisciplinary team participated to address cardio- pulmonary, nutritional, and psychological sequelae. The assessment included a clinical history to obtain the patient’s sociodemo- graphic data, data related to diabetes, cardiovascular risk factors, and other comor- bidities. Information on smoking and alcohol consumption and dietary intake was obtained by using a food frequency questionnaire. A battery of tests were conducted including laboratory testing, testing for respiratory conditions, functional status, quality of life, psychiatric conditions, and nutritional status. The goal of the nutrition service was to assess the nutritional sequelae of COVID- 19 and then to give personalized advice to the patients. Body composition analyses were performed by bioimpedance with a Body Composition Analyzer mBCA 514/ 56 A. Aguilar Galarza et al. A. Aguilar Galarza et al. 515 Seca GmbH & Co. KG, Hamburg. This breaks down weight into body compart- ments (muscle mass, body fat, and visceral fat). As would be expected, the principal nutritional risks found in patients post-COVID-19 were obesity, loss of muscle mass, and a decline in overall physical functioning. However, in our experience, after six weeks, muscle mass and nutritional status improved in these patients. It is important to note that medical and physiotherapists participated in the program. The assessment also included circadian rhythmic features. It has been repeatedly acknowledged that the adequate functioning of the rhythmical system is essential for maintaining the homeostasis of an organism [49]. Studies focusing on the effects of lockdown as a result of the pandemic showed that human performance and health, especially quality and quantity of sleep [50], nutrition, and physical activity were affected [51]. A recent study shows that the complex pathogenesis of severe acute respiratory syndrome by SARS-CoV-2 infection is related to circadian disruption [52]. Rehabilitation: Post-COVID-19 Program Thus, in considering the disturbances of the biological timing for nutritional assessment according to light–dark cycles, timing of food intake, light at night, nocturnal feeding, physical activity, jet lag, and shift work are of prime importance [53]. In conclusion, Mexico is among the countries that have the highest COVID- 19 fatality rates. Efforts were made to expand hospital capacity. However, there were significant disruptions in the health services. 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Doubova SV, Leslie HH, Kruk ME, Perez-Cuevas R, Arsenault C. Disruption in essential health services in Mexico during COVID-19: an interrupted time series analysis of health information system data. British Medical Journal Global Health. 2021 Sep; 6(9). 45. Sullivan DH (1995) The role of nutrition in increased morbidity and mortality. Clin Geriat Med 11(4):661–674 46. Downer S, Berkowitz SA, Harlan TS, Olstad DL, Mozaffarian D (2020Jun) Food is medicine: actions to integrate food and nutrition into healthcare. BMJ 29:369. https://doi.org/10.1136/ bmj.m2482.PMID:32601089;PMCID:PMC7322667 47. References Sandra Celada Martínez, MSc Collaborative Researcher, University Health Program, Univer- sidad Autonoma de Queretaro, Queretaro, Mexico Sandra Celada Martínez, a collaborator to health programs at the Universidad Autonoma de Queretaro, focuses on the implementation of new areas for healthcare systems oriented to adequate spaces and necessary sanitary measures. Sandra Celada Martínez, MSc Collaborative Researcher, University Health Program, Univer- sidad Autonoma de Queretaro, Queretaro, Mexico Sandra Celada Martínez, a collaborator to health programs at the Universidad Autonoma de Queretaro, focuses on the implementation of new areas for healthcare systems oriented to adequate spaces and necessary sanitary measures. Dr. Oscar San Roman Orozco Project Manager, The BORN Project, Universidad Autonoma de Queretaro, Queretaro, Mexico Oscar San Roman Orozco is a medical doctor from the Univer- sidad Autonoma de Queretaro (UAQ), Mexico. He has a Masters in Global Public Health and Advanced Certificate in Public Health Disaster Science, Policy and Practice from the New York University (NYU) School of Global Public Health. He has a University Expert Degree in Hospital Management from the Universidad of Cadiz, Spain. He is the Project Manager of the BORN Project Mexico at the Newborn Foundation. He has been on Expert Advisory Boards for devel- oping interventions like the Masimo Safety Net OPEN and the COVID-19 Clinic at UAQ where he has been actively publishing research on COVID-19 in the Mexican population including mobility measures, relationship with the reproductive number, models for re-opening the university and mental health impact among others. He was awarded the Lewis Blackman Leadership Award by the Patient Safety Movement. He is a member of the Delta Omega Honorary Public Health Society. He is a former Co-coordinator and to date, an Advisor to the Applied Global Public Health Initiative at the NYU School of Global Public Health and its satellite laboratory at the Universidad Autonoma de Queretaro Dr. Isidro Amadeo Gutiérrez Álvarez, Professor and Medical Coordinator, Applied Global Public Health Initiatives, Universidad Autonoma de Queretaro, Queretaro, Mexico is Professor of basic medical genetics at the school of medicine of the Autonomous University of Querétaro and is the academic coordinator of the laboratory of Applied Global Public Health Initiatives UAQ/NYU, a student’s organization in Querétaro, México that is dedicated to impacting health equity in underserved communities. References He has worked on health communications and policy for more than 30 years when he launched campaigns that focused on knowledge of congenital diseases, immunization, food insecurity, infant mortality, nutrition, and mental health. He is a peaker and author who blogs on congenital disease, environmental impacts on health and the connection between public health policies and clinical practice. Izarelly Rosillo Pantoja, PhD, Researcher, Universidad Autonoma de Queretaro, Mexico Doctor of Law, researcher, and trial lawyer in the field of human rights and the environ- ment. She elaborated 20 local legislations for the Mexican state and three international legislations that impacted on various plans and programs on environmental matters. She drafted the first Circular Economy law for Mexico in the state of Quintana Roo and also for the Dominican Republic. She worked for various international and national organizations including the World Bank, Inter-American Development Bank, ECLAC, UN Environment, German Cooperation Agency (GIZ), Chamber of Deputies in Mexico, and local governments, among others. Izarelly Rosillo Pantoja, PhD, Researcher, Universidad Autonoma de Queretaro, Mexico Doctor of Law, researcher, and trial lawyer in the field of human rights and the environ- ment. She elaborated 20 local legislations for the Mexican state and three international legislations that impacted on various plans and programs on environmental matters. She drafted the first Circular Economy law for Mexico in the state of Quintana Roo and also for the Dominican Republic. She worked for various international and national organizations including the World Bank, Inter-American Development Bank, ECLAC, UN Environment, German Cooperation Agency (GIZ), Chamber of Deputies in Mexico, and local governments, among others. She received several distinctions, including an honorary appointment as a member of the World Committee on Sustainable Development of the Organization Founded by Nobel Peace Prize winner Rajenda Kumar Pachauri. She is a member of the National System of Researchers of the National Council of Science and Technology in Mexico. She is the author of several books and articles published in national and international journals. She received several distinctions, including an honorary appointment as a member of the World Committee on Sustainable Development of the Organization Founded by Nobel Peace Prize winner Rajenda Kumar Pachauri. She is a member of the National System of Researchers of the National Council of Science and Technology in Mexico. She is the author of several books and articles published in national and international journals. Dr. Nuri G. References Brugliera L, Spina A, Castellazzi P, Cimino P, Arcuri P, Deriu MG, Zanetta C, Angelone SM, Capitanio JF, Alemanno F, Meloni C, D’Angelo G, Houdayer E, Abutalebi J, Mortini P, Iannac- cone S (2020) Rehabilitative of COVID-19 patients with acute lower extremity Ischemia and amputation. J Rehab Med 52(9). https://doi.org/10.2340/16501977-2714. PMID: 32720698. 48. Zhou F, Yu T, Du R, Fan G, Liu Y, Liu Z, Xiang J, Wang Y, Song B, Gu X, Guan L, Wei Y, Li H, Wu X, Xu J, Tu S, Zhang Y, Chen H, Cao B (2020) Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet. 395(10229): 1054–1062. https://pubmed.ncbi.nlm.nih.gov/32171076/ p p g 49. Galván-Tejada CE, Herrera-García CF, Godina-González S, Villagrana-Bañuelos KE, Amaro JDL, Herrera-García K, Rodríguez-Quiñones C, Zanella-Calzada LA, Ramírez-Barranco J, Avila JLR, Reyes-Escobedo F, Celaya-Padilla JM, Galván-Tejada JI, Gamboa-Rosales H, Martínez-Acuña M, Cervantes-Villagrana A, Rivas-Santiago B, Gonzalez-Curiel IE (2020) Persistence of COVID-19 symptoms after recovery in Mexican population. Int J Environ Resilience Public Health. 17(24):9367. https://doi.org/10.3390/ijerph17249367.PMID:333 27641;PMCID:PMC7765113 50. Fishbein AB, Knutson KL, Zee PC (2021Oct 1) Circadian disruption and human health. J Clin Investig 131(19):148286. https://doi.org/10.1172/JCI148286.PMID:34596053;PMCID:PMC 8483747 51. Leone MJ, Sigman M, Golombek DA (2020) Effects of lockdown on human sleep and chrono- type during the COVID-19 pandemic. Current Biol 30(16): 930–931. https://doi.org/10.1016/ j.cub.2020.07.015. Epub 2020 Jul 8. PMID: 32810450; PMCID: PMC734078 52. Ray S, Reddy AB (2020Sep) COVID-19 management in light of the circadian clock. Nat Rev Mol Cell Biol 21(9):494–495. https://doi.org/10.1038/s41580-020-0275-3.PMID:32699357; PMCID:PMC7374068 53. Cheikh Ismail L, Osaili TM, Mohamad MN, Al Marzouqi A, Jarrar AH, Abu Jamous DO, Magriplis E, Ali HI, Al Sabbah H, Hasan H, AlMarzooqi LMR, Stojanovska L, Hashim M, Shaker Obaid RR, Saleh ST, Al Dhaheri AS (2020) Eating Habits and Lifestyle during COVID- 19 Lockdown in the United Arab Emirates: A Cross-Sectional Study. Nutrients 12(11):3314. https://doi.org/10.3390/nu12113314.PMID:33137947;PMCID:PMC7693610 Adriana Aguilar Galarza, PhD Collaborative Researcher, University Health Program, Univer- sidad Autonoma de Queretaro, Queretaro, Mexico Adriana Aguilar Galarza, a Collaborative Researcher from the University Health Program, Universidad Autonoma de Queretaro, focuses on the impact on health of the university community through integral diagnostics and first level medical care, promoting healthy spaces and forming health promoters. She has worked on epidemiology studies related to metabolic risk in young adults and the impact of circadian rhythms in metabolic health. 60 A. Aguilar Galarza et al. Teaching, Bachelor of Mexican Folk Dance, Bachelor of Acting, Faculty of Fine Arts, and Faculty of Medicine, UAQ. She has published extensively on COVID-19 and other important issues in international and national journals. Comprehensive Care Response and Systematic Management … References Villaseñor Cuspinera, MSc, School of Medicine, Universidad Autónoma de Queretaro, Mexico is a full-time teacher-research. She has a Degree in Medicine from the Faculty of Medicine, UNAM, and a Master Degree in Bioethics. She is a Faculty of Bioethics, Universidad Anáhuac. Specialty in Nuclear Medicine. National Medical Center 20 de Noviembre. ISSSTE, PhD student in sciences, Faculty of Medicine, UAQ. She held the position of Tutoring Coordinator of the Faculty of Medicine, UAQ. She is professor of the Bachelor of Medicine, Bachelor of Art Comprehensive Care Response and Systematic Management … 61 Teaching, Bachelor of Mexican Folk Dance, Bachelor of Acting, Faculty of Fine Arts, and Faculty of Medicine, UAQ. She has published extensively on COVID-19 and other important issues in international and national journals. 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Portuguese
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Silagem de cereais de inverno submetidos ao manejo de duplo propósito
Revista Brasileira de Zootecnia
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Palavras-chave: capacidade tampão, forragem conservada, integração lavoura-pecuária, proteína bruta Palavras-chave: capacidade tampão, forragem conservada, integração lavoura-pecuária, proteína bruta Recebido em 14/6/2010 e aprovado em 15/12/2010. Correspondências devem ser enviadas para: gilmarmeinerz@yahoo.com.br Silage of winter cereals submitted to double purpose management ABSTRACT - The objective of this research was to evaluate the silage of 12 genotypes of six species of double purpose winter cereals (forage and grains) submitted to harvest at Depressão Central region of Rio Grande do Sul state. Species and genotypes tested were: BRS 277, BRS Guatambu, BRS Tarumã and BRS Umbu wheat; Agro Zebu, UPFA 21 - Moreninha and Commom black-oat; UPF 18 white oat; BR 1 and BRS Serrano rye; BRS Marciana barley; and BRS 148 triticale. Genotypes were submitted to tree harvests with the objective to simulate grazing, according to double purpose management. After the third harvest, the development of cultures was permitted for silage production. The genotypes were distributed in 36 experimental plots in a completely randomized experimental design with 12 treatments (genotypes) and three replications (plots). Silages were made in PVC experimental silos when forages achieved phenological stage of soft dough. Fermentative parameters, dry matter production, structural and botanical composition of ensilage material and nutritive value of silages were determined. BR 1 rye and UPF 18 white oat presented the highest dry matter yield. BRS Umbu wheat produced silage with higher grain participation and better nutritive value. All genotypes presented satisfactory conditions for ensilage. Key Words: buffering capacity, conserved forage, crude protein, livestock-crop production system Silagem de cereais de inverno submetidos ao manejo de duplo propósito1 Gilmar Roberto Meinerz2, Clair Jorge Olivo3, Julio Viégas3, José Laerte Nörnberg4, Carlos Alberto Agnolin2, Rudolf Brand Scheibler5, Tiago Horst5, Renato Serena Fontaneli6 Correspondências devem ser enviadas para: gilmarmeinerz@yahoo.com.br Gilmar Roberto Meinerz2, Clair Jorge Olivo3, Julio Viégas3, José Laerte Nörnberg4, Carlos Alberto Agnolin2, Rudolf Brand Scheibler5, Tiago Horst5, Renato Serena Fontaneli6 1 Pesquisa financiada com recursos do Conselho Nacional de Desenvolvimento Científico e Tecnológico. 2 Programa de Pós-graduação em Zootecnia, Universidade Federal de Santa Maria (UFSM), RS, Brasil, 97105-900. 3 Departamento de Zootecnia, UFSM, RS, Brasil. 4 Departamento de Tecnologia e Ciência dos Alimentos, UFSM, RS, Brasil. 5 Acadêmicos do curso de Zootecnia - UFSM. 1 Pesquisa financiada com recursos do Conselho Nacional de Desenvolvimento Científico e Tecnológico. 2 Programa de Pós-graduação em Zootecnia, Universidade Federal de Santa Maria (UFSM), RS, Brasil, 97105-900. 3 Departamento de Zootecnia, UFSM, RS, Brasil. epa ta e to de ootec a, U S , S, as 4 Departamento de Tecnologia e Ciência dos Alimentos, UFSM, RS, Brasil. 5 Acadêmicos do curso de Zootecnia - UFSM. 6 Centro Nacional de Pesquisa do Trigo (CNPT - EMBRAPA). RESUMO - Esta pesquisa foi conduzida com o objetivo de avaliar a silagem de 12 genótipos de seis espécies de cereais de inverno de duplo propósito (forragem e grãos), submetidos ao corte na região da Depressão Central do Rio Grande do Sul. As espécies e os genótipos testados foram: trigo BRS 277, BRS Guatambu, BRS Tarumã, BRS Umbu; aveia-preta Agro zebu, UPFA 21 - Moreninha e Comum; aveia-branca UPF 18; centeio BR 1 e BRS Serrano; Cevada BRS Marciana; e triticale BRS 148. Os genótipos foram submetidos a três cortes com o objetivo de simular o pastejo, conforme manejo indicado para duplo propósito. Após o terceiro corte, permitiu-se o desenvolvimento das culturas para a ensilagem. As silagens foram feitas em silos experimentais de PVC quando as forrageiras atingiram o estádio fenológico de grão pastoso. O delineamento experimental foi o inteiramente casualizado, com 12 tratamentos (genótipos) e três repetições (parcelas). Foram determinadas a produção de matéria seca e a composição estrutural do material ensilado, o valor nutritivo e os parâmetros fermentativos das silagens. O centeio BR 1 e a aveia- branca UPF 18 apresentaram as maiores produções de matéria seca. O trigo BRS Umbu produziu silagem com maior participação de grãos e valor nutritivo mais elevado. Todos os genótipos apresentaram condições satisfatórias para a ensilagem. Recebido em 14/6/2010 e aprovado em 15/12/2010. Revista Brasileira de Zootecnia © 2011 Sociedade Brasileira de Zootecnia ISSN 1806-9290 www.sbz.org.br Revista Brasileira de Zootecnia © 2011 Sociedade Brasileira de Zootecnia ISSN 1806-9290 www.sbz.org.br R. Bras. Zootec., v.40, n.10, p.2097-2104, 2011 Introdução A adubação nitrogenada, foi de 120 kg/ha de N para todas as espécies, sendo aplicado 10 kg/ha na semeadura e 110 kg/ha em cobertura, divididos em 4 aplicações. As principais culturas usadas para produção de silagem são o milho e o sorgo, culturas típicas de verão. Entretanto, há necessidade de se estudar a utilização de novas culturas na confecção da silagem, no sentido de reduzir os custos de produção (Pinto et al., 2007). Com a expansão dos sistemas de produção baseados na integração lavoura-pecuária, a utilização de cereais de inverno de duplo propósito (forragem e grãos) surge como uma alternativa para produção de silagem de qualidade, com baixo custo, considerando-se que no final do ciclo dessas culturas, normalmente há um excedente de massa de forragem. A silagem de cereais de inverno apresenta, geralmente, maiores teores de proteína bruta do que a silagem de milho, mas com valor energético inferior (Scheffer-Basso et al., 2003). As principais espécies utilizadas para ensilagem são aveia-preta e azevém, colhidos no estádio de elongamento e submetidos ao pré-murchamento antes da ensilagem, tendo em vista que o excesso de umidade é prejudicial ao processo de fermetação e conservação do material ensilado. O estádio fenológico indicado para ensilagem de cereais de inverno sem pre-murchamento é o de grão pastoso (Fontaneli et al., 2009). Há, no entanto, carência de informações sobre diversos fatores envolvidos na ensilagem destes materiais. Assim, o objetivo neste trabalho foi avaliar silagens de cereais de inverno submetidos ao manejo de duplo propósito na Depressão Central de Rio Grande do Sul. O estádio fenológico indicado para ensilagem de cereais de inverno sem pre-murchamento é o de grão pastoso (Fontaneli et al., 2009). Há, no entanto, carência de informações sobre diversos fatores envolvidos na ensilagem destes materiais. Assim, o objetivo neste trabalho foi avaliar silagens de cereais de inverno submetidos ao manejo de duplo propósito na Depressão Central de Rio Grande do Sul. As forrageiras foram submetidas ao manejo de cortes indicado para duplo propósito. Foram realizados três cortes com o objetivo de simular o pastejo, em intervalos variando entre 16 e 38 dias. Após o terceiro corte, foi realizado o diferimento, permitindo-se o desenvolvimento final das culturas. No estádio fenológico de grão pastoso, metade da área de cada parcela foi destinada à confecção das silagens e a outra metade foi utilizada para avaliação do rendimento de grãos. Introdução inverno ainda não estão prontas para a utilização (Scheffer- Basso et al., 2004). O Sul do Brasil possui condições edafoclimáticas favoráveis ao cultivo de muitas espécies de plantas forrageiras. No entanto, a estacionalidade na produção de forragem da maioria das espécies utilizadas ocasiona um vazio forrageiro no outono e início do inverno, quando as espécies de verão já completaram seu ciclo e as de Neste sentido, a utilização de forragens conservadas na alimentação de vacas leiteiras é uma prática bastante usual nos períodos de carência de forragem e visa fornecer alimento volumoso de boa qualidade e em quantidades suficientes para a manutenção dos níveis de produtividade. Dentre as formas de conservação da forragem destaca-se a Meinerz et al. 2098 ensilagem, que consiste no corte da planta forrageira em momento ideal e posterior armazenamento da massa verde picada em silos (Novaes et al., 2004). Este processo tem como principal objetivo conservar a forragem e, desde que seja realizado em condições ideais, manter a qualidade do material original (Van Soest, 1994). Foram testados 12 genótipos de seis espécies de cereais de inverno de duplo propósito de utilização: trigo (BRS 277, BRS Guatambu, BRS Tarumã, BRS Umbu); aveia-preta (Agro Zebu, UPFA 21 - Moreninha e Comum); aveia-branca (UPF 18); centeio (BR 1 e BRS Serrano); cevada (BRS Marciana); e triticale (BRS 148). A área experimental foi dividida em 36 parcelas experimentais, com dimensões de 5 m de comprimento e 3 m de largura. Os dados da análise do solo foram os seguintes: índice SMP 5,2; P 2,2 mg/dm; K 0,12 cmolc/dm³; Al3+ 2,7 cmolc/dm³; Ca2+ 3,1 cmolc/dm³; Mg 2+ 1,5 cmolc/dm³; MO 2,3%; saturação de bases 30,0% e saturação por alumínio 36%. A semeadura foi feita em 10 de abril, em linhas com espaçamento de 17cm e com sementes provenientes do Centro Nacional de Pesquisa do Trigo (CNPT-EMBRAPA), em Passo Fundo, Rio Grande do Sul. A densidade de semeadura foi de 400 sementes viáveis/m2. Trinta dias antes da semeadura, foi realizada a correção da acidez, conforme a análise do solo, mediante a aplicação de calcário dolomítico do tipo Filler, incorporado mediante escarificação do solo. A adubação potássica e fosfórica foi realizada conforme as recomendações da Comissão de Química e Fertilidade do Solo – RS/SC (2004), individualmente para cada espécie. Introdução Para fins de amostragem e coleta de material para ensilagem foram desconsiderados 15 cm de cada um dos lados das parcelas (bordaduras). R. Bras. Zootec., v.40, n.10, p.2097-2104, 2011 Material e Métodos A pesquisa foi conduzida no Laboratório de Bovinocultura de Leite do Departamento de Zootecnia da UFSM, localizado na região fisiográfica denominada Depressão Central do Rio Grande do Sul, com altitude de 95 m, latitude 29° 43' Sul e longitude 53° 42' Oeste, no período entre março e outubro de 2008. As médias de temperatura e a precipitação pluviométrica do período foram de 14,86 °C e 985,2 mm (168,89 mm mensais). Esses valores são similares às médias climáticas normais da região. O solo da área experimental é classificado como Argissolo Vermelho distrófico arênico, pertencente à unidade de mapeamento São Pedro (Embrapa, 1999) e o clima da região é o Cfa (subtropical úmido), conforme classificação de Köppen (Moreno, 1961). O rendimento de forragem foi estimado através de cinco amostras por parcela, cortadas rente ao solo, sendo utilizado um quadro com dimensões de 50 × 30 cm. As amostras foram homogeneizadas, sendo retirada uma subamostra para estimativa das composições botânica e estrutural da forragem, fazendo-se manualmente a separação da lâmina foliar, colmo+bainha, material senescente, espigueta/panículas, grãos e outras espécies. Estes componentes foram secos em estufa de ar forçado a 55 ºC até peso constante para determinação dos teores de matéria parcialmente seca. Estes componentes foram secos em estufa de ar forçado a 55 ºC até peso constante para determinação dos teores de matéria parcialmente seca. R. Bras. Zootec., v.40, n.10, p.2097-2104, 2011 Silagem de cereais de inverno submetidos ao manejo de duplo propósito 2099 com o uso de α-amilase termoestável, descontando-se a proteína insolúvel em detergente neutro e as cinzas residuais. Também foi determinada a fibra em detergente ácido corrigida para cinzas (FDAc) e para proteína (FDAcp). O delineamento experimental utilizado foi o inteiramente casualizado, com 12 tratamentos (genótipos), três repetições (parcelas). Os resultados foram submetidos à análise de variância e as médias comparadas entre si pelo teste Tukey a 5% de probabilidade do erro. Os dados foram submetidos à análise de correlação, pelo coeficiente de Pearson. com o uso de α-amilase termoestável, descontando-se a proteína insolúvel em detergente neutro e as cinzas residuais. Também foi determinada a fibra em detergente ácido corrigida para cinzas (FDAc) e para proteína (FDAcp). A confecção das silagens foi realizada entre 20 de setembro e 24 de outubro. A forragem foi cortada rente ao solo, sendo triturada em moinho forrageiro regulado para fragmentar o material em partículas de 1,5 cm. Resultados e Discussão Os genótipos testados apresentaram ciclos distintos, sendo o triticale BRS 148 e o centeio BR 1 os mais precoces para produção de silagem, com 150 dias entre a emergência e o estádio de grão pastoso (Tabela 1). Os genótipos mais tardios foram os trigos BRS 277, BRS Guatambu, BRS Tarumã, o centeio BRS Serrano e as aveias UPF 18 e UPFA 21 - Moreninha, que apresentaram ciclo superior a 180 dias. A estatura das plantas no momento da ensilagem foi distinta entre os genótipos, sendo que o genótipo que apresentou maior altura foi o centeio BRS Serrano e o trigo BRS Tarumã a menor. Não foi observado acamamento nos materiais estudados. As determinações de matéria seca (MS), matéria orgânica (MO), fibra em detergente ácido corrigida para cinzas (FDAc), lignina em detergente ácido (ácido sulfúrico), proteína bruta (PB), nitrogênio insolúvel em detergente neutro (NIDN) e nitrogênio insolúvel em detergente ácido (NIDA) foram realizadas segundo procedimentos descritos por Silva & Queiroz (2002). Para determinação da fibra em detergente neutro corrigida para cinzas (FDNc) não foi utilizado sulfito de sódio na solução em detergente neutro, sendo empregada α-amilase termoestável. Por isso, a proteína remanescente na FDNc foi subtraída após a multiplicação do fator 6,25 pelo teor do NIDN. A abreviação FDNcp expressa o teor de fibra em detergente neutro determinada Para os teores de matéria seca das silagens (Tabela 1), foram verificados resultados distintos entre os genótipos, que variaram de 24 a 42% de MS. Os valores mais elevados foram observados para os trigos BRS 277 e BRS Guatambu, que apresentaram teores de matéria seca superiores à 40%. No entanto, não foi constatada presença de mofo ou desenvolvimento de fungos. O teor de MS afeta a qualidade fermentativa da silagem, que está relacionada tanto ao potencial de ingestão quanto à eficiência de utilização de nutrientes para produção animal (McDonald, 1981). Material e Métodos O material foi compactado e hermeticamente fechado em silos experimentais de PVC com 100 mm de diâmetro e 50 cm de altura, com tampas com válvulas de Bunsen para permitir o escape dos gases, e acondicionados em sala protegida da radiação solar. O delineamento experimental utilizado foi o inteiramente casualizado, com 12 tratamentos (genótipos), três repetições (parcelas). Os resultados foram submetidos à análise de variância e as médias comparadas entre si pelo teste Tukey a 5% de probabilidade do erro. Os dados foram submetidos à análise de correlação, pelo coeficiente de Pearson. A abertura dos silos foi realizada após 40 dias de fermentação, desprezando-se a porção superior de cada um. O restante do material foi homogeneizado, retirando-se uma subamostra para a determinação do pH em potenciômetro digital (Silva & Queiroz, 2002) e da capacidade tampão (Playne & McDonald, 1966). Com auxílio de uma prensa, foi retirado suco para a determinação do nitrogênio amoniacal (N-NH3) por destilação com óxido de magnésio (Chaney & Marbach, 1962). O restante da amostra foi parcialmente seca em estufa de ventilação forçada, a 55 ºC até peso constante, sendo posteriormente moída em moinho do tipo Willey em peneira com malha de 1mm e acondicionada para a realização das análises laboratoriais. R. Bras. Zootec., v.40, n.10, p.2097-2104, 2011 Meinerz et al. 2100 Meinerz et al. e material morto. A participação de lâminas foliares foi maior para o trigo BRS Umbu e para a cevada BRS Marciana. exceção da aveia-preta Comum e da cevada BRS Marciana, os teores de MS ficaram próximos aos preconizados por Noller et al. (1954) para que ocorram as menores perdas durante o processo fermentativo. No momento da abertura dos silos, não foi observado odor de amônia ou mesmo de forragem apodrecida, indicando que o processo de fermentação ocorreu de maneira satisfatória. Os valores de pH das silagens (Tabela 3) foram diferentes entre os genótipos e mantiveram-se dentro do preconizado para que ocorra fermentação adequada, com valores de pH entre 3,7 e 4,2 (Kung Júnior & Stokes, 2003), exceto o centeio BR 1, que apresentou pH de 4,39. Apesar da grande variabilidade entre os teores de matéria seca das silagens, o teor de matéria seca dos grãos foi similar entre os genótipos testados: em média de 54%. Este resultado indica que não houve diferença no estádio fenológico de desenvolvimento entre os genótipos no momento da ensilagem. A estabilização do pH na silagem deve-se às interações entre a concentração da matéria seca, da capacidade tamponante (Fisher & Burns, 1987), das concentrações de carboidratos solúveis, do teor de lactato e das condições de anaerobiose do meio (Moisio & Heikonen, 1994). A correlação negativa encontrada entre o pH e o teor de matéria seca (r = –0,35; P = 0,03), embora baixa, indica a associação existente entre estas duas variáveis. Com relação à produção de matéria seca (Tabela 1), os maiores rendimentos foram obtidos com o centeio BR 1 e a aveia-branca UPF 18, com valores próximos a 12 t/ha de MS. Estes resultados são superiores aos relatados por Primavesi et al. (2001) em estudo no qual avaliaram diferentes cultivares de aveia-branca. Floss et al. (2003), também com aveia- branca, obtiveram 11,4 t/ha de MS, no estádio de grão em massa dura. Os menores rendimentos, inferiores a 7 t/ha de MS foram obtidos com o trigo BRS 277, a aveia-preta Comum, UPFA 21-Moreninha e a cevada BRS Marciana, que, por sua vez, não diferiram entre si. A capacidade tamponante das silagens (Tabela 3), que consiste na capacidade do material em resistir às alterações de pH, foi diferente entre os genótipos e teve estreita correlação com os teores de matéria seca (r = 0,91; P<0,0001). R. Bras. Zootec., v.40, n.10, p.2097-2104, 2011 Resultados e Discussão À Espécie Genótipo Ciclo (dias) Estatura (cm) MS (%) MS grãos (%) Produção de MS (kg/ha) Triticale BRS 148 150 94ef 28,77de 52,47 9267bc Cevada BRS Marciana 156 72hi 24,60e 53,79 6648d Centeio BR 1 150 128b 29,12de 51,97 12136a BRS Serrano 186 142a 38,98abc 53,08 9058bc Aveia-branca UPF 18 182 114cd 35,79abcd 53,18 11913a Aveia-preta UPFA 21 – Moreninha 182 124bc 33,20cd 54,35 6808d Agro-zebu 169 116bc 30,22de 58,53 9075bc Comum 163 103de 24,45e 53,05 6247d Trigo BRS 277 185 70hi 42,50a 58,19 6095d BRS Guatambu 186 87fg 42,04ab 53,28 7101cd BRS Tarumã 184 67i 35,12bcd 51,39 9278bc BRS Umbu 156 80gh 28,63de 54,61 10577b CV (%) - - 4,29 7,56 5,12 8,52 Médias seguidas por letras distintas na coluna diferem entre si pelo teste de Tukey a 5% de probabilidade. Tabela 1 - Rendimento de matéria seca (MS) e características da forragem pré-ensilada de cereais de inverno submetidos ao manejo de duplo propósito Meinerz et al. Os maiores valores de capacidade tampão foram observados para a aveia-preta Comum e para a cevada BRS Marciana, indicando que estes materiais apresentam maior resistência à elevação do pH. Este parâmetro é de fundamental importância para a conservação da qualidade da silagem após a abertura do silo, uma vez que com a rápida elevação do pH e a exposição ao ar, propiciam-se condições para que certos microrganismos indesejáveis tornem-se metabolicamente ativos, produzindo calor e consumindo nutrientes da silagem (Ranjit & Kung Júnior, 2000). Para a composição estrutural do material pré-ensilado (Tabela 2) foram observadas diferenças significativas entre os genótipos. As menores participações de grãos foram obtidas nos genótipos de aveia-preta e no centeio BR1. Os trigos BRS Umbu, BRS Tarumã, BRS 277 e a aveia UPF 18 apresentaram maiores participações de grãos na massa ensilada. Segundo Mayombo et al. (1997), a maior proporção de grãos na forragem confere uma melhor qualidade à silagem. No entanto, a qualidade da fração fibrosa do caule, folhas, espiga e palhas, combinada com o percentual de cada uma dessas partes na planta, também determina o valor nutritivo do material ensilado (Barrière et al., 1997). As silagens foram compostas principalmente por colmos e grãos, com pequenas contribuições de lâminas foliares O teor de nitrogênio amoniacal foi diferente entre os genótipos, mas manteve-se abaixo do limite máximo de 10% do nitrogênio total, preconizado por Ferreira (2001) para Médias seguidas por letras distintas na coluna diferem entre si pelo teste Tukey a 5% de probabilidade. Meinerz et al. Os maiores valores foram observados para o triticale BRS 148 e os menores para a aveia-preta UPFA 21- Moreninha, sendo que os demais genótipos apresentaram teores intermediários. Os valores observados foram inferiores aos relatados por Coan et al. (2001), que, trabalhando com cultivares de aveia-preta e triticale ensilados no estádio de grão pastoso a farináceo, obtiveram teores médios de 10,85 e 10,45% de PB, respectivamente. Os resultados deste trabalho são semelhantes aos relatados por Dumont et al. (1989), que observaram valores de 7,3% de PB para a aveia-branca, colhida no estádio de grão pastoso. Excetuando-se o trigo BRS 277, a aveia-branca UPF Os teores de FDNc e FDNcp (Tabela 5) apresentaram as mesmas diferenças entre os genótipos estudados, sendo Tabela 3 - Parâmetros fermentativos da silagem de cereais de inverno submetidos ao manejo de duplo propósito Espécie Genótipo pH Capacidade tampão (eq.mg NaOH/100g MS) N-NH3* Triticale BRS 148 4,13ab 18,80abc 5,80bcd Cevada BRS Marciana 3,92bcd 22,92a 4,78abcd Centeio BR 1 4,39a 17,56abc 5,92abc BRS Serrano 3,78cd 14,87bc 5,72abc Aveia-branca UPF 18 3,78cd 14,72bc 3,21d Aveia-preta UPFA 21 - Moreninha 3,98cd 16,81bc 4,67bcd Agro-zebu 4,11ab 18,00abc 3,97cd Comum 3,76cd 23,24a 3,49d Trigo BRS 277 3,73d 13,80c 5,22abcd BRS Guatambu 3,87bcd 13,57c 6,76a BRS Tarumã 3,96bcd 16,03bc 6,33ab BRS Umbu 4,06bc 19,75ab 5,65abc CV (%) - 2,66 11,51 13,68 Médias seguidas por letras distintas na coluna diferem entre si pelo teste Tukey a 5% de probabilidade. * N-NH3 – Nitrogênio amoniacal em % do nitrogênio total. a silagem de cereais de inverno submetidos ao manejo de duplo propósito Médias seguidas por letras distintas na coluna diferem entre si pelo teste Tukey a 5% de probabilidade. * N-NH3 – Nitrogênio amoniacal em % do nitrogênio total. bruta, nitrogênio insolúvel em detergente ácido (NIDA) e nitrogênio insolúvel em detergente neutro Espécie Genótipo Proteína bruta NIDA* NIDN* Triticale BRS 148 8,45a 14,25 9,74 Cevada BRS Marciana 7,41ab 15,01 9,13 Centeio BR 1 7,21ab 18,05 14,36 BRS Serrano 6,31ab 17,19 12,87 Aveia-branca UPF 18 6,85ab 17,30 13,88 Aveia-preta UPFA 21 - Moreninha 5,97b 16,26 13,70 Agro-zebu 6,20ab 18,17 13,10 Comum 7,25ab 15,30 12,02 Trigo BRS 277 6,87ab 16,81 12,94 BRS Guatambu 7,96ab 15,51 10,14 BRS Tarumã 7,32ab 15,60 10,82 BRS Umbu 7,44ab 14,25 10,12 CV (%) - 12,25 19,92 15,68 * Relativo ao nitrogênio total. Meinerz et al. Espécie Genótipo Lâminas foliares Colmo + bainha Material morto Espiga/panícula Grãos Outras espécies Triticale BRS 148 3,80bc 41,91ab 4,69c 15,68ab 24,24bcd 9,66ab Cevada BRS Marciana 6,14ab 40,36ab 12,83abc 4,06d 17,10ef 19,49a Centeio BR 1 3,99abc 49,58a 9,78abc 8,50bcd 15,67fg 12,45ab BRS Serrano 2,74c 41,44ab 13,8ab 11,84abcd 20,06def 10,09ab Aveia-branca UPF 18 3,16bc 42,05ab 15,58a 5,65cd 25,68abcd 7,86ab Aveia-preta UPFA 21 - Moreninha 3,09bc 44,43a 13,88ab 4,74d 16,17fg 17,67ab Agro-zebu 3,63bc 45,17a 9,31abc 9,96abcd 14,10fg 17,82ab Comum 2,84c 48,09a 8,79abc 12,09abcd 9,88g 18,29a Trigo BRS 277 3,26bc 27,00b 9,76abc 12,18abcd 29,46abc 18,32a BRS Guatambu 2,55c 35,66ab 9,80abc 17,63a 23,01cde 11,32ab BRS Tarumã 3,6bc 27,31b 6,46bc 13,84abc 30,89ab 17,85ab BRS Umbu 7,06a 42,76ab 5,14bc 9,00abcd 31,17a 4,86b CV (%) - 27,2 13,62 30,81 29,25 10,63 32,77 Tabela 2 - Composição botânica e estrutural (% da massa seca total) da forragem pré-ensilada de cereais de inverno submetidos ao manejo de duplo propósito trutural (% da massa seca total) da forragem pré-ensilada de cereais de inverno submetidos ao Silagem de cereais de inverno submetidos ao manejo de duplo propósito 2101 18, as aveias-pretas UPFA 21-Moreninha, Agro-zebu e o centeio BRS Serrano, os teores de PB observados situaram-se acima do limite mínimo de 7%, considerado por Van Soest (1994), para que não ocorram limitações ao crescimento microbiano, permitindo adequada fermentação ruminal. silagens de boa qualidade. Os teores observados no presente trabalho indicam que houve pequena degradação da proteína no processo de ensilagem. Isto se deve, provavelmente, ao fato de que a atividade proteolítica diminui com o aumento do teor de MS do material ensilado e com o rápido abaixamento do pH (Pereira & Reis, 2001). Considerando os valores de NIDA e NIDN, não foram observadas diferenças entre os genótipos, sendo os teores de NIDA das silagens bastante elevados. Van Soest (1994) sugere que variações de 3 a 15% desta fração na MS estariam dentro da normalidade. Geralmente, os teores mais elevados de NIDA estão associados à formação de compostos de Mailard, em decorrência da elevação da temperatura nos silos (Evangelista et al., 2004). Os teores de NIDN observados são inferiores aos relatados por Oliveira (2008), que verificou valor médio de 20,3% para silagens de milho, sorgo sudão, sorgo forrageiro e girassol. O NIDN corresponde à fração do nitrogênio que se disponibiliza lentamente em ambiente ruminal. Para a porcentagem de PB (Tabela 4) foram observadas diferenças entre os genótipos. R. Bras. Zootec., v.40, n.10, p.2097-2104, 2011 * Relativo ao nitrogênio total. Médias seguidas por letras distintas na coluna diferem entre si pelo teste Tukey a 5% de probabilidade. Meinerz et al. Médias seguidas por letras distintas na coluna diferem entre si pelo teste Tukey a 5% de probabilidade Tabela 4 - Percentuais de proteína bruta, nitrogênio insolúvel em detergente ácido (NIDA) e nitrogênio insolúvel em detergente neutro (NIDN) Tabela 4 - Percentuais de proteína bruta, nitrogênio insolúvel em detergente ácido (NIDA) e nitrogênio i (NIDN) R. Bras. Zootec., v.40, n.10, p.2097-2104, 2011 2102 Meinerz et al. que a FDNcp foi, em média, 1,13 unidade percentual inferior à FDNc. Esta diferença numérica é oriunda da subtração da proteína insolúvel em detergente neutro (NIDN × 6,25) da FDNc, e a sua amplitude indica a importância desta correção para classificação das silagens quanto à qualidade. Os valores de FDAc e FDAcp também apresentaram comportamento similar, com diferença de 1,03 unidade percentual. Lopes et al. (2008), avaliando silagens de triticale em diferentes idades de corte, observaram valores de 48,20 e 29,40% de FDN e FDA, bem inferiores aos observados neste trabalho, enquanto Coan et al. (2001), avaliando silagens de aveia amarela do genótipo São Carlos e aveia- preta Comum, obtiveram valores médios de 60,5 e 37,25% para estas variáveis, respectivamente, mais próximos aos deste trabalho. Os menores valores de FDNcp e de FDAcp foram obtidos para os trigo BRS Umbu, BRS Guatambu, BRS Tarumã, para a cevada BRS Marciana para o triticale BRS 148. Foi encontrada correlação negativa entre o percentual de grãos e os teores de FDNcp (r–0,35; P = 0,03) e de FDAcp (r = –0,42; P = 0,008). Esta associação confirma que a maior participação deste componente confere maior qualidade nutricional às silagens (Barrière et al., 1997). Ressalta-se que os teores de FDAcp observados, excetuando-se os genótipos de centeio e a aveia-preta UPFA21-Moreninha, são inferiores a 40%, valor apontado por Nussio et al. (1998) como limitante ao consumo voluntário dos bovinos. Quanto ao teor de celulose (Tabela 6), o centeio BR 1 apresentou a maior concentração deste componente. O elevado valor de celulose observado para este genótipo está diretamente relacionado com a maior participação da fração FDAcp, uma vez que a celulose é importante componente desta fração. Esta afirmação é confirmada pela correlação encontrada entre estes dois componentes (r = 0,95; P<0,0001). Verificou-se também correlação negativa da celulose com a participação de grãos (r = 0,42; P = 0,01), indicando que quanto maior a participação desse componente, menor o percentual de carboidratos estruturais na silagem. R. Bras. Zootec., v.40, n.10, p.2097-2104, 2011 Meinerz et al. Com relação à hemicelulose, o maior valor foi observado para a aveia Agro Zebu. Os valores de tro e fibra em detergente ácido, corrigidas para cinzas (FDNc, FDAc) e para proteína (FDNcp, FDAcp) e inverno submetidos ao manejo de duplo propósito Espécie Genótipo FDNc FDNcp FDAc FDAcp Triticale BRS 148 62,65abcd 61,45abcd 37,55bcd 33,73bcd Cevada BRS Marciana 58,15cd 57,04cd 32,50d 31,83d Centeio BR 1 72,07ab 70,77a 45,93a 44,90a BRS Serrano 69,84ab 68,76ab 41,31abc 40,50abc Aveia-branca UPF 18 68,38abc 67,26abc 39,86abcd 38,95abcd Aveia-preta UPFA 21 – Moreninha 72,63a 71,67a 44,13ab 43,30ab Agro-zebu 70,80ab 69,67a 40,35abcd 39,54abcd Comum 65,36abcd 64,25abcd 39,31abcd 38,46abcd Trigo BRS 277 68,40abc 67,29abc 38,82abcd 37,96abcd BRS Guatambu 59,65bcd 58,41bcd 34,18cd 33,73cd BRS Tarumã 63,08abcd 61,96abcd 35,39cd 34,81cd BRS Umbu 55,59d 54,53d 32,81d 32,06d CV - 5,82 5,91 6,93 7,06 Médias seguidas por letras distintas na coluna diferem entre si pelo teste Tukey a 5% de probabilidade. Tabela 5 - Fibra em detergente neutro e fibra em detergente ácido, corrigidas para cinzas (FDNc, FDAc) e para proteína (FDNcp, FDAcp) da silagem de cereais de inverno submetidos ao manejo de duplo propósito Espécie Genótipo Celulose Hemicelulose Lignina Sílica Triticale BRS 148 33,00abc 25,10bc 4,26bc 1,50 Cevada BRS Marciana 30,10bc 25,65abc 3,21c 1,36 Centeio BR 1 40,39a 26,13abc 5,52ab 1,14 BRS Serrano 35,97abc 28,53ab 5,78a 1,31 Aveia-branca UPF 18 34,42abc 28,52ab 5,45ab 1,55 Aveia-preta UPFA 21 – Moreninha 37,78ab 28,50ab 5,36ab 1,23 Agro-zebu 35,38abc 30,45a 5,04ab 1,44 Comum 34,18abc 26,04abc 4,42abc 1,65 Trigo BRS 277 33,55abc 29,57ab 5,19ab 1,44 BRS Guatambu 29,76bc 25,47abc 4,90ab 1,69 BRS Tarumã 31,59bc 27,49abc 4,93ab 1,60 BRS Umbu 29,00c 22,78c 4,17bc 1,05 CV (%) - 8,13 6,62 9,65 24,41 Médias seguidas por letras distintas na coluna diferem entre si pelo teste Tukey a 5% de probabilidade. Tabela 6 - Percentuais de celulose, hemicelulose, lignina e sílica em silagens de cereais de inverno submetidas ao manejo de duplo propósito Silagem de cereais de inverno submetidos ao manejo de duplo propósito 2103 FERREIRA, J.J. Estágio de maturação ideal para ensilagem do milho e do sorgo. In: CRUZ, J.C.; PEREIRA FILHO, I.A.; RODRIGUES, J.A.S. et al. (Eds). Produção e utilização de silagem de milho e sorgo. Sete Lagoas: Embrapa Milho e Sorgo, 2001. p.405-428. celulose e hemicelulose encontrados no presente trabalho foram semelhantes aos observados por Coan et al. (2001), trabalhando com silagem de aveia pré-emurchecida. Conclusões LOPES, F.C.F.; SILVA E OLIVEIRA, J.; LANES, E.C.M. et al. Valor nutricional do triticale (X Triticosecale Wittmack) para uso como silagem na Zona da Mata de Minas Gerais. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v.60, n.6, p.1484-1492, 2008. Todos os genótipos testados apresentam condições para ser ensilados e produzem silagens com características fermentativas desejáveis. O triticale BRS 148 e o centeio BR 1 são os genótipos mais precoces para produção de silagem. O centeio BR 1 e a aveia UPF 18 apresentam o maior rendimento de massa seca de pré-ensilagem. O trigo BRS Umbu tem maior participação de grãos e de lâminas foliares na massa total, o que proporciona silagem com maior qualidade nutricional. MAYOMBO, A.P.; DUFRASNE, I.; HORNICK, J.L et al. Influencie du stade de maturité de la plante de may recolteé pour ensilage sur la composition, la digestibilité aparente, les caractéristiques de fermentation dans le rume et les performances zootechniques chez le taurillon à I’engraissement. Animal Zootech, v.46, n.1, p.43-55, 1997. McDONALD, P. The biochemistry of silage. New York: John Wiley & Sons, 1981. p.226 MOISIO, T.; HEIKONEN, M. Lactic acid fermentation in silage preserved with formic acid. Animal Feed Science and Technology, v.47, n.1-2, p.107-124, 1994. Meinerz et al. Os teores mais elevados de lignina foram observados para o centeio BRS Serrano, sendo semelhantes aos relatados por Hernández et al. (2002), de 5,35 e 5,29% da MS, respectivamente para silagens de milho sem e com inoculante. A lignina está associada à FDAcp, condição essa confirmada no presente trabalho (R = 0,74; P<0,0001) e diretamente relacionada com a digestibilidade da forragem, uma vez que é a fração da fibra totalmente indigestível. O menor teor de lignina foi observado para a cevada BRS Marciana. Coan et al. (2001), trabalhando com silagem pré-emurchecida, observaram teores de lignina de 5,2% em genótipos de triticale e aveia, semelhantes aos observados no presente trabalho. Para os teores de sílica, os resultados foram similares entre os genótipos testados. FISHER, D.S.; BURNS, J.C. Quality analysis of summer-annual forages. II. Effects of forage carbohydrate constituents on silage FISHER, D.S.; BURNS, J.C. Quality analysis of summer-annual forages. II. Effects of forage carbohydrate constituents on silage fermentation. Agronomy Journal, v.79, n.2, p.242-248, 1987. forages. II. Effects of forage carbohydrate constituents on silage fermentation. Agronomy Journal, v.79, n.2, p.242-248, 1987. g g y g fermentation. Agronomy Journal, v.79, n.2, p.242-248, 1987. FONTANELI, Ren.S.; FONTANELI, Rob.S.; SANTOS, H.P. et al. Rendimento e valor nutritivo de cereais de inverno de duplo propósito: forragem verde e silagem ou grãos. Revista Brasileira de Zootecnia, v.38, n.11, p.2116-2120, 2009. FLOSS, L.F.; BOIN, C.; PALHANO, A.L. et al. Efeito do estádio de maturação sobre o rendimento e valor nutritivo da aveia- branca no momento da ensilagem. Boletim da Industria Animal, v.60, n.2, p.117-126, 2003. branca no momento da ensilagem. Boletim da Industria Animal, v.60, n.2, p.117-126, 2003. HERNÁNDEZ, F.I.L.; VALADARES FILHO, S.C.; PAULINO, M.F. et al. Avaliação da composição de vários alimentos e determinação da cinética ruminal da proteína, utilizando o método de produção de gás e amônia in vitro. Revista Brasileira de Zootecnia, v.31, n.1, p.243-255, 2002. KUNG JUNIOR, L.; STOKES, M.R.; LIN, C.J. Silage additives. In: BUXTON, D.R.; MUCK, R.E.; HARRISON, J.H. (Eds.) Silage science and technology. Madison: American Society of Agronomy; Crop Science Society of America; Soil Science Society of America, 2003. p.251-304. R. Bras. Zootec., v.40, n.10, p.2097-2104, 2011 Referências MORENO, J.A. Clima do Rio Grande do Sul. Porto Alegre: Secretaria da Agricultura, 1961. 41p. BARRIÈRE, Y.; ARGILLIER, O.; MICHALET-DOREAU, B. et al. Relevant traits, genetic variation and breeding strategies in early silage maize. Agronomie, v.17, n.5, p.395-411, 1997. NOLLER, C.H.; STILLONS, M.C.; MARTZ, F.A. et al. Digestion studies with oat silages using a new fecal collection technique. Journal of Animal Science, v.18, n.2, p.671-675, 1954. NOVAES, L.P.; LOPES, F.C.F.; CARNEIRO, J.C. Silagens: pontos críticos e oportunidades. Brasília: Embrapa Cerrados; Juiz de Fora: Embrapa Gado de Leite, 2004. 10p. CHANEY, A.L.; MARBACH, E.P. Modified reagents for determination of urea and ammonia. Clinical Chemistry, v.8, n.2, p.130-162, 1962. COAN, R.M.; FREITAS, D.; REIS, R.A. et al. Composição bromatológica das silagens de forrageiras de inverno submetidas ou não ao emurchecimento e ao uso de aditivos. ARS Veterinária v 17 n 1 p 58-63 2001 NUSSIO, L.G.; MANZANO, R.P.; PEDREIRA, C.G.S. Valor alimentício em plantas do gênero Cynodon. In: PEIXOTO, A.M.; MOURA, J. C.; FARIA, V.P. (Eds.) Manejo de pastagens de tifton, coastcross e estrela. Piracicaba: FEALQ, 1998. p.203-242. p COMISSÃO DE QUÍMICA E FERTILIDADE DO SOLO. Manual de adubação e calagem para os estados do Rio Grande do Sul e de Santa Catarina. 10.ed. Porto Alegre: SBCS-NRS, 2004. 400p. OLIVEIRA, L.B. Produção e valor nutritivo de diferentes forrageiras e as suas respectivas silagens. 2008. 46f. Dissertação (Mestrado em Agronomia) – Universidade Estadual do Sudoeste da Bahia, Vitória da Conquista. DUMONT, L.J.C.; LANUZA, A.F.; ELIZALDE, V.H.F. Use of ensiled oats harvest at two growth stages and effects of protein supplementation in dairy cows. Agricultura Técnica, v.49, n.31, p.5-13, 1989. PEREIRA, J.R.A.; REIS. R.A. Produção de silagem pré-secada com forrageiras temperadas e tropicais. In: SIMPÓSIO SOBRE PRODUÇÃO E UTILIZAÇÃO DE FORRAGENS CONSERVADAS, 2011, Maringá. Anais... Maringá: Universidade Estadual de Maringá, 2001. p.64-86. EMPRESA BRASILEIRA DE PESQUISA E AGROPECUÁRIA - EMBRAPA. Sistema brasileiro de classificação de solos. Brasília: Centro Nacional de Pesquisa de Solos; Rio de Janeiro: Embrapa, 1999. 412p. PLAYNE, M.J.; McDONALD, P. The buffering constituints of herbage and of silage. Journal Science Food and Agriculture, v.17, n.2, p.264-268, 1966. p p EVANGELISTA, A.R.; ABREU, J.G.; AMARAL, P.N.C. et al. Produção de silagem de capim-marandu (Brachiaria brizantha Stapf cv. Marandu) com e sem emurchecimento. Ciência e Agrotecnologia, v.28, n.2, p. 443-44, 2004. PINTO, A.P.; MIZUBUTI, I.Y.; RIBEIRO, E.L.A. Avaliação da silagem de bagaço de laranja e silagem de milho em diferentes R. Bras. R. Bras. Zootec., v.40, n.10, p.2097-2104, 2011 Passo Fundo: Universidade de Passo Fundo – Centro de Pesquisa em Alimentação, 2003. 31p. Passo Fundo: Universidade de Passo Fundo – Centro de Pesquisa em Alimentação, 2003. 31p. SCHEFFER-BASSO, S.M.; AGRANIONIK, H.; FONTANELI, R.S. Acúmulo de biomassa e composição bromatológica de milhetos das cultivares comum e africano. Revista Brasileira de Agrociência, v.10, n.4, p.483-486, 2004. SILVA, D.J.; QUEIROZ, A.C. Análise de alimentos: métodos químicos e biológicos. 3.ed. Viçosa, MG: UFV, 2002. 235p. VAN SOEST, P.J. Nutritional ecology of the ruminant. Ithaca: Cornell University, 1994. 476p. Referências Zootec., v.40, n.10, p.2097-2104, 2011 Meinerz et al. 2104 períodos de armazenamento. Acta Scientiarum Animal Sciences, v.29, n.4, p.371-377, 2007. períodos de armazenamento. Acta Scientiarum Animal Sciences, v.29, n.4, p.371-377, 2007. Passo Fundo: Universidade de Passo Fundo – Centro de Pesquisa em Alimentação, 2003. 31p. PRIMAVESI, A. C.; PRIMAVESI, O.; CHINELLATO, A. et al. Indicadores de determinação de cortes de cultivares de aveia forrageira. Scientia Agricola, v.58, n.1, p.79-89, 2001. SCHEFFER-BASSO, S.M.; AGRANIONIK, H.; FONTANELI, R.S. Acúmulo de biomassa e composição bromatológica de milhetos das cultivares comum e africano. Revista Brasileira de Agrociência, v.10, n.4, p.483-486, 2004. RANJIT, N.K.; KUNG JR., L. The effect of Lactobacillus buchneri, Lactobacillus plantarum, or a chemical preservative on the fermentation and aerobic stability of corn silage. Journal of Dairy Science, v.83, n.3, p.526-535, 2000. SILVA, D.J.; QUEIROZ, A.C. Análise de alimentos: métodos químicos e biológicos. 3.ed. Viçosa, MG: UFV, 2002. 235p. VAN SOEST, P.J. Nutritional ecology of the ruminant. Ithaca: Cornell University, 1994. 476p. SCHEFFER-BASSO, S.M.; DÜRR, J.W.; FONTANELI, R.S. Valor nutritivo de forragens: concentrados, pastagens e silagens.
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Polymorphisms in Chemokine Receptor 5 and Toll-Like Receptor 3 Genes Are Risk Factors for Clinical Tick-Borne Encephalitis in the Lithuanian Population
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Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright:  2014 Mickiene et al. This is an open-access article distributed under the terms of the Creative Commons Attr unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The authors confirm that all data underlying the findings are fully available without restriction. All data are included within the pape Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All data are inc Funding: AM and JP received the research grant from Research Council of Lithuania (grant number MIP-11174) URL: http://www.lmt.lt/en/about.html. LS received grant from Swedish Research Council (grant No: 3485). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * Email: aukse.mickiene@lsmuni.lt * Email: aukse.mickiene@lsmuni.lt * Email: aukse.mickiene@lsmuni.lt * Email: aukse.mickiene@lsmuni.lt . These authors contributed equally to this work. . These authors contributed equally to this work. After an infected tick bites a person, TBE viruses first multiply locally in the skin dendritic cells and subsequently are transported to regional nymph nodes. After replication in the lymph nodes, TBE viruses pass into the blood stream. Various organs are invaded after a hematogenic spread of the viruses, and the release of TBEV from various tissues enables the viremia to continue for several days. Although in the majority of cases the TBEV infection is terminated at the extraneural stage, in some cases high virus replication in the primarily affected organs leads to a progressive Aukse? Mickiene?1,2*., Jolita Pakalniene?1., Johan Nordgren3, Beatrice Carlsson3, Marie Hagbom3, Lennart Svensson3,4, Lars Lindquist2 Aukse? Mickiene?1,2*., Jolita Pakalniene?1., Johan Nordgren3, Beatrice Carlsson3, Marie Hagbom3, Lennart Svensson3,4, Lars Lindquist2 1 Department of Infectious Diseases, Lithuanian University of Health Sciences, Kaunas, Lithuania, 2 Unit for Infectious Diseases, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden, 3 Division of Molecular Virology, Medical Faculty, Linko¨ping University, Linko¨ping, Sweden 4 Department of Clinical Microbiology, Linko¨ping University Hospital, Linko¨ping, Sweden Linköping University Post Print Linköping University Post Print N.B.: When citing this work, cite the original article. N.B.: When citing this work, cite the original article. Original Publication: Aukse Mickiene, Jolita Pakalniene, Johan Nordgren, Beatrice Carlsson, Marie Hagbom, Lennart Svensson and Lars Lindquist, Polymorphisms in Chemokine Receptor 5 and Toll-Like Receptor 3 Genes Are Risk Factors for Clinical Tick-Borne Encephalitis in the Lithuanian Population, 2014, PLoS ONE, (9), 9, e106798. http://dx.doi.org/10.1371/journal.pone.0106798 Copyright: Public Library of Science http://www.plos.org/ Postprint available at: Linköping University Electronic Press http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-112653 Aukse Mickiene, Jolita Pakalniene, Johan Nordgren, Beatrice Carlsson, Marie Hagbom, Lennart Svensson and Lars Lindquist, Polymorphisms in Chemokine Receptor 5 and Toll-Like Receptor 3 Genes Are Risk Factors for Clinical Tick-Borne Encephalitis in the Lithuanian Population, 2014, PLoS ONE, (9), 9, e106798. http://dx.doi.org/10.1371/journal.pone.0106798 Copyright: Public Library of Science http://www.plos.org/ Aukse Mickiene, Jolita Pakalniene, Johan Nordgren, Beatrice Carlsson, Marie Hagbom, Lennart Svensson and Lars Lindquist, Polymorphisms in Chemokine Receptor 5 and Toll-Like Receptor 3 Genes Are Risk Factors for Clinical Tick-Borne Encephalitis in the Lithuanian Population, 2014, PLoS ONE, (9), 9, e106798. h //d d i /10 1371/j l 0106798 p http://dx.doi.org/10.1371/journal.pone.0106798 Copyright: Public Library of Science http://www.plos.org/ Abstract Background: Tick-borne encephalitis virus (TBEV) infections can be asymptomatic or cause moderate to severe injuries of the nervous system. We previously reported that a nonfunctional chemokine receptor 5 (CCR5) and a functional Toll-like receptor 3 (TLR3) predispose adults to clinical tick-borne encephalitis (TBE). This study expands our previous findings and further examines polymorphisms in CCR5 and TLR3 genes in different age and disease severity groups. Methods: 117 children and 129 adults, stratified into mild, moderate and severe forms of TBE, and 103 adults with severe TBE were analyzed. 135 healthy individuals and 79 patients with aseptic meningoencephalitis served as controls. CCR5 delta 32 and rs3775291 TLR3 genotypes were established by pyrosequencing, and their frequencies were analyzed using recessive genetic, genotype and allelic models. Findings: The prevalence of CCR5D32 homozygotes was higher in children (2.5%), in adults with severe TBE (1.9%), and in the combined cohort of TBE patients (2.3%) than in controls (0%) (p,0.05). The nonfunctional homozygous TLR3 genotype was less prevalent among the combined TBE cohort (11.5%) than among controls (19.9%) (p = 0.025), but did not differ between children TBE and controls. The genotype and allele prevalence of CCR5 and TLR3 did not differ in children nor adult TBE cohorts stratified by disease severity. However, in the severe adult TBE cohort, homozygous functional TLR3 genotype and wt allele were less prevalent compared to the adult cohort with the whole disease severity spectrum (44.4% vs 59.8% p = 0.022 and 65.2% vs 76.4% p = 0.009; respectively). Conclusions: Independently of age, nonfunctional CCR5D32 mutation is a significant risk factor for development of clinical TBE, but not for disease severity. The polymorphism of TLR3 gene predisposes to clinical TBE in adults only and may be associated with disease severity. Further studies are needed to clarify the role of these polymorphisms in susceptibility to TBEV infection. Citation: Mickiene? A, Pakalniene? J, Nordgren J, Carlsson B, Hagbom M, et al. (2014) Polymorphisms in Chemokine Receptor 5 and Toll-Like Receptor 3 Genes Are Risk Factors for Clinical Tick-Borne Encephalitis in the Lithuanian Population. PLoS ONE 9(9): e106798. doi:10.1371/journal.pone.0106798 Editor: Ulrike Gertrud Munderloh, University of Minnesota, United States of America Editor: Ulrike Gertrud Munderloh, University of Minnesota, United States of America Received April 15, 2014; Accepted August 1, 2014; Published September 16, 2014 Received April 15, 2014; Accepted August 1, 2014; Published September 16, 2014 Copyright:  2014 Mickiene et al. September 2014 | Volume 9 | Issue 9 | e106798 Study population TBE cases. All patients included in the study had clinical signs of neuroinfection and pleocytosis in cerebrospinal fluid (CSF) $86106/l. TBE was diagnosed by the demonstration of specific IgM activity by the two-step ELISA method in serum samples [13]. The clinical presentation of TBE was classified as mild, moderate or severe, as previously described [14]. Briefly, mild form showed predominantly meningeal symptoms; moderate form showed monofocal encephalitic symptoms (ataxia, dysphasia, tremor, single cranial nerve paralysis) and/or moderate diffuse brain dysfunction (Glasgow Coma Scale (GCS) score .9); severe form showed multifocal encephalitic symptoms and/or GCS #9. For all study participants, CSF specimens were obtained by lumbar puncture in a standardized manner on a median of 1 day (range, 0–11 days) after admission to the hospital. Immediately after collection, all CSF samples were examined for cell count, differential count, total protein and glucose concentration. For PCR, we used 5–7.5 mL DNA, 2.5 mL 10xPCR buffer II (Applied Biosystems), 2.5 mL 50 mM MgCl (Applied Biosystems), 1 uL 10 mM GeneAmp dNTP mix with dTTP (Applied Biosystems), 1(CCR5) or 2 (TLR3) mL 10 mM Fw, 1 (CCR5) or 2 (TLR3) mL 10 mM Rev primer (Table 2), and 0.2 mL AmpliTaq Gold DNA Polymerase per reaction. The final volume was adjusted with ultraclean water to a final volume of 25 mL. The PCR reaction was performed at 95uC for 5 min followed by 50 cycles of 15 s at 95uC, 30 s at 55 or 65uC depending on the amplified sequence (Table 2), and 30 s at 72uC, and finally 1 cycle of 5 min at 72uC. Pyrosequencing to distinguish between homozygous wild type (wt/wt), heterozygous (wt/mut), and homozygous mutant (mut/ mut) of CCR5D32 and TLR3 rs3775291 genotypes was performed in a PSQ 96 MA Instrument (Biotage) as described previously [6]. Sequencing primers and dispension orders are shown in Table 2. The CCR5D32 genotype of a subset of DNA samples (n = 30) were verified by gel electrophoresis, by separation of the 100- and 132-bp PCR amplicons in a 2% agarose gel. The TLR3 genotype for 8 DNA samples could not be determined by pyrosequencing and were determined by sanger sequencing using the same amplicon with forward and reverse primers (Table 2) as sequencing primers. Three TBE cohorts were used in this study. Three TBE cohorts were used in this study. CCR5 and TLR3 Polymorphisms in Tick-Borne Encephalitis infection when TBEV crosses the blood-brain barrier and invades the brain [4]. Three combined cohorts of TBE cases were made for statistical analysis. The first combined cohort was created in order to cover the entire age and disease severity spectrum in usual proportions of mild, moderate, and severe forms, and consisted of the 117 children recruited in the present study and 129 adults reported previously [6,9,14]. The clinical spectrum of TBEV infection varies considerably from asymptomatic to mild meningitis, severe encephalitis or encephalomyelitis [5]. Why certain individuals respond with severe clinical symptoms while the majority either remains asymptomatic or only develops mild disease is largely unknown. The second combined cohort was a total sample of all three TBE cohorts described above. y p y p g y Recently, we have shown an association between development of TBE and the polymorphisms of two genes modulating the innate immune response. The first genetic risk factor identified for TBE was a 32-bp deletion in the coding region of the chemokine receptor 5 (CCR5) gene [6]. The CCR5 gene encodes a protein expressed on the surfaces of leukocytes that is involved in regulation of leukocyte migration [7,8]. The second risk factor was a rs3775291 single nucleotide polymorphism (SNP) Leu412Phe in exon 4 of the Toll-like receptor 3 (TLR3) gene [9]. TLR3 protein is expressed in dendritic cells, a variety of epithelial cells, and in the brain, and is localized primarily in endosomal membranes [10]. The TLR3 protein binds to virus- derived dsRNA and activates the transcription factors, NF-kB and IRF3. This in turn induces the production of type 1 interferons and pro-inflammatory cytokines, in particular TNF-a, which decreases the integrity of the blood-brain barrier and allows the passage of the virus into the brain [10–12]. The third combined cohort was used to investigate the association of gene polymorphisms and severity of TBE, and consisted of an overall cohort of adults with TBE (129 reported previously and 103 severe TBE cases). Controls. Two control cohorts from our previous studies on TBE were included for comparison. Controls. Two control cohorts from our previous studies on TBE were included for comparison. The first control cohort was 135 healthy Lithuanians with no documented TBEV infection matched geographically and by age and gender. The second control cohort was 79 geographically matched adult patients with aseptic meningoencephalitis (AME), negative for IgM antibody for TBE virus. CCR5D32 and TLR3 rs3775291 genotyping All serum samples were analyzed for a 32-bp deletion in the coding region of the chemokine receptor CCR5 gene and for missense mutation rs3775291 (G/A, Leu412Phe) in exon 4 of the Toll-like receptor TLR3 gene by polymerase chain reaction (PCR) and pyrosequencing, essentially as described in a previous articles [6,9]. DNA was extracted from serum by QiaAmp 96 DNA Blood Kit (Qiagen), in accordance with the manufacturer’s instructions and the DNA samples were eluted into 100 mL of AE buffer (Qiagen) and stored at 220uC, until further analyzed. Study population The first cohort consisted of all consecutive patients under 18 years of age admitted from 1999 through 2009 (n = 117), 73 of which were classified as mild, 40 as moderate, and 4 as severe TBE; and is called ‘‘Children TBE cohort’’ in the manuscript. The second cohort consisted of all adults meeting the criteria of severe form of disease from the database of 831 consecutive adult TBE patients admitted from 2004 through 2010 (n = 103); and is called ‘‘Adult severe TBE cohort’’ in the manuscript. The third TBE patient cohort was composed of 129 adult TBE patients, stratified by the same clinical classification into mild (n = 56), moderate (n = 57), and severe (n = 16) form of TBE, and reported earlier in our previous studies [6,9,14]. CCR5 and TLR3 Polymorphisms in Tick-Borne Encephalitis As no difference in CCR5D32 and TLR3 rs3775291 genotype and allele distribution was found between these two control cohorts [6,9], they were considered as one combined control cohort when statistical calculations were performed. The aim of this study was to extend our previous findings by enlarging our data set, to understand the role of these polymor- phisms in different age groups, and to investigate if they determine the severity of TBE. Introduction Tick-borne encephalitis (TBE) is a zoonotic disease caused by a RNA virus of the genus Flavivirus within the family Flaviviridae. Like Japanese encephalitis virus and West Nile virus (WNV), TBE virus (TBEV) is one of the major neurotropic flaviviruses [1]. The endemic area of TBEV ranges from Western Europe to China and Japan, and the virus is taxonomically classified into three subtypes: European, Siberian, and Far Eastern. Members of the European subtype are transmitted primarily by Ixodes ricinus ticks and members of the latter two subtypes by I. persulcatus [2,3]. September 2014 | Volume 9 | Issue 9 | e106798 1 PLOS ONE | www.plosone.org September 2014 | Volume 9 | Issue 9 | e106798 Statistical methods Statistical analysis included descriptive statistics with frequency analysis (percentages) for categorical variables and means with standard deviations (SD) for continuous variables. The two-sample Demographic data of all three TBE cohorts are presented in Table 1. PLOS ONE | www.plosone.org September 2014 | Volume 9 | Issue 9 | e106798 2 CCR5 and TLR3 Polymorphisms in Tick-Borne Encephalitis t, Mann-Whitney or Kruskal-Wallis tests were used for continuous variables as appropriate. Proportions were compared using x2 or Fisher’s exact tests. One way analysis of variance (ANOVA) was used to compare more than two independent groups. Multivariate logistic regression model was used to investigate associations of risk factors with disease severity. Statistical analyses were performed using SPSS (Statistical Package for the Social Science for Windows; Table 1. Demographic data of children TBE, adult severe TBE, and adult TBE cohorts. Characteristic Value p Children TBE (n = 117) Gender, n (%): Boys 70 (59.2) p = 0.033 Girls 47 (40.2) Age, mean (years)6SD: Boys 12.0763.857 p = 0.376 Girls 11.4363.855 Mild form 11.3663.987 p = 0.179 Moderate form 12.7363.289 Severe form 11.0065.888 Adult severe TBE (n = 103) Gender, n (%): Male 55 (53.4) p = 0.490 Female 48 (46.6) Age, mean (years)6SD: Male 51.93615.419 p = 0.080 Female 57.27615.108 Adult TBE (n = 129) Gender, n (%): Male 70 (54.3) p = 0.333 Female 59 (45.7) Age, mean (years)6SD: Male 43.56615.284 p = 0.176 Female 46.97612.756 Mild form 41.34614.895 p = 0.021 Moderate form 47.30613.450 Severe form 50.56611.696 doi:10.1371/journal.pone.0106798.t001 Table 2. Primers, annealing temperatures and dispension orders used for amplification and sequencing of the different mutations. doi:10.1371/journal.pone.0106798.t001 logistic regression model was used to investigate associations of risk factors with disease severity. Statistical analyses were performed using SPSS (Statistical Package for the Social Science for Windows; logistic regression model was used to investigate associations of risk factors with disease severity. Statistical analyses were performed using SPSS (Statistical Package for the Social Science for Windows; t, Mann-Whitney or Kruskal-Wallis tests were used for continuous variables as appropriate. Proportions were compared using x2 or Fisher’s exact tests. One way analysis of variance (ANOVA) was used to compare more than two independent groups. Multivariate Table 2. Primers, annealing temperatures and dispension orders used for amplification and sequencing of the different mutations. CCR5 and TLR3 polymorphisms and severity of TBE An association between CCR5 and TLR3 polymorphisms and the severity of TBE was analysed in the children cohort and the overall cohort of adults, stratified by severity of disease. We did not detect any significant difference in CCR5 genotype distribution and in CCR5D32 allele prevalence among the cohort of children (n = 117) and adults (n = 232), stratified by severity of disease (Table 5). Combined TBE cohorts. Altogether, 8 CCR5D32 homozy- gotes were found among the combined cohort of all patients with TBE (2.3% (8/349)). The prevalence of CCR5D32 homozygotes was higher in this cohort than among the AME and the Lithuanian TBE-naive control cohorts (Table 3). The difference was statistically significant when both the cohort of all TBE patients (n = 349) and the combined cohort of children and adult TBE cases (n = 246) were compared with the combined control cohort (n = 210; p = 0.027 and p = 0.023, respectively) (Table 3). For TLR3 rs3775291, neither the genotype distribution nor the wild type allele prevalence differed in children (n = 105) and adult (n = 226) TBE cohorts (Table 6). Interestingly, the cohort of adults with severe TBE (n = 99) had a significantly lesser prevalence of both homozygous wild genotype and wt allele compared with the cohort of adult TBE cases (n = 127), (44.4% vs 59.8% p = 0.022 and 65.2% vs 76.4% p = 0.009; respectively) (Table 4). Further- more, the prevalence of homozygous mutation was higher in adults with severe or moderate TBE compared to adults with mild TBE (p = 0.071, Table 6). The CCR5D32 allele prevalence was also higher in the cohort of all patients with TBE (n = 349) (12.3% (86/698)) than in the AME cohort, and the Lithuanian TBE-naive control cohort (Table 3). The difference was observed when the combined children and adult TBE cohort (n = 246) was compared with the cohort of Lithuanian TBE-naive individuals (p = 0.046), which suggests CCR5D32 allele being a risk factor for clinical TBEV infection (Odds ratio (OR) 1.672; 95% confidence interval (CI) 1.005–2.782; p = 0.048) (Table 3). The same trend was observed when the cohort of all TBE cases (n = 349) was compared with the CCR5D32 polymorphism in clinical TBE Adult severe TBE cohort. TLR3 rs3775291 genotypes were successfully obtained for 99 out of the cohort of 103 (96.1%) adults with severe TBE. Children TBE cohort. All child patients with TBE (n = 117) were successfully genotyped. Children TBE cohort. All child patients with TBE (n = 117) were successfully genotyped. The prevalence of CCR5D32 homozygotes was higher in the children TBE cohort (2.5% (3/117) than in the Lithuanian TBEV- naive control (0% (0/134) and in the AME cohort (0% (0/76). The difference was also observed when the children TBE cohort was compared with the combined control cohort (p = 0.045) (Table 3). In this TBE cohort, the genotype distribution of TLR3 rs3775291 was 44.4% for homozygous wild type, 41.4% for heterozygous, 14.1% for mutant homozygous genotype, and was in concordance with the distribution among the cohort of Lithuanian TBE-naive controls and among the AME cohort, and did not differ when compared with the combined control cohort (p = 0.135). The wt allele distribution between the TBE and the control cohorts did not differ either (Table 4). The CCR5D32 allele prevalence was also higher in the children TBE cohort (12.4% (29/234) than in the Lithuanian TBEV-naive control (8.2% (22/268) and in the AME cohort (10.5% (16/152) (Table 3) but the difference was not statistically significant when the children TBE cohort was compared with the control cohorts. The CCR5D32 allele prevalence was also higher in the children TBE cohort (12.4% (29/234) than in the Lithuanian TBEV-naive control (8.2% (22/268) and in the AME cohort (10.5% (16/152) (Table 3) but the difference was not statistically significant when the children TBE cohort was compared with the control cohorts. Combined TBE cohorts. The mutant homozygous genotype for TLR3 rs3775291 was found significantly less frequently among TBE patients in both the combined cohort of children and adults (n = 232) and the overall combined cohort of TBE cases (n = 331), compared to the combined control cohort (p = 0.02 and p = 0.025, respectively) (Table 4). The wild allele was found to be a risk factor for clinical TBEV infection when comparing the children and adult TBE cohort (n = 232) with the combined control cohort (OR 1.449, 95% CI 1.085–1.936, p = 0.012). Combined TBE cohorts. TLR3 polymorphism in clinical TBE Children TBE cohort. 105 out of 117 (89.7%) children with TBE were successfully genotyped. y g yp For TLR3 rs3775291, the genotype distribution in children cohort was 51.4% for homozygous wild type, 34.3% for heterozygous, 14.3% for mutant homozygous genotype, and was in concordance with the distribution in the cohort of the Lithuanian TBE-naive controls (51.6% wt/wt, 29.4% heterozy- gous, 19.0% mut/mut, respectively), as well as in the AME cohort (46.7% wt/wt, 32.0% heterozygous, 21.3% mut/mut, respective- ly). When the children TBE cohort was compared with the combined control cohort, we did not find any significant differences (p = 0.453). The wt allele prevalence between the children TBE and the control cohorts did not differ either (Table 4). Ethical approval The study was approved by Kaunas Regional Research Ethics Committee (No. BE-2-15) and a written informed consent was obtained from each study participant or from a parent/legally authorised representative for all children. CCR5 and TLR3 Polymorphisms in Tick-Borne Encephalitis Chicago, USA) 13.1 software. p,0.05 was considered statistically significant. Lithuanian TBE-naive cohort (p = 0.069) as well as in comparison of the combined children and adult TBE cohort (n = 246) with the combined control cohort (p = 0.059) (Table 3). For the CCR5 gene, a recessive genetic model (i.e., CCR5D32 homozygotes vs wild-type CCR5 plus CCR5D32 heterozygotes) and an allelic model (mut vs wt) were applied. For TLR3 rs3775291, genotype (362 contingency analysis) was used as well as allelic model (mut vs wt). For the cohort of all adult patients stratified by severity of TBE, a recessive genetic model was also applied. Statistical calculations included only samples that were successfully genotyped. CCR5D32 polymorphism in clinical TBE The mutant homozygous genotype for TLR3 rs3775291 was found significantly less frequently among TBE patients in both the combined cohort of children and adults (n = 232) and the overall combined cohort of TBE cases (n = 331), compared to the combined control cohort (p = 0.02 and p = 0.025, respectively) (Table 4). The wild allele was found to be a risk factor for clinical TBEV infection when comparing the children and adult TBE cohort (n = 232) with the combined control cohort (OR 1.449, 95% CI 1.085–1.936, p = 0.012). p Adult severe TBE cohort. All adult patients with severe TBE (n = 103) were successfully genotyped. In this cohort, 2 CCR5D32 homozygotes were found, making the prevalence of CCR5D32 homozygotes higher in the TBE cohort (1.9% (2/103) than in the Lithuanian TBEV-naive control and in the AME cohorts (Table 3). Also, the prevalence of CCR5D32 homozygotes differed when the TBE cohort was compared with the combined control cohort (p = 0.043) (Table 3). The CCR5D32 allele prevalence was also higher in the cohort of adult patients with severe TBE (11% (22/206) than in the Lithuanian TBEV-naive control and in the AME cohorts (Table 3), but no statistical differences were found between the TBE cohort compared to the control cohorts. In this cohort, 2 CCR5D32 homozygotes were found, making the prevalence of CCR5D32 homozygotes higher in the TBE cohort (1.9% (2/103) than in the Lithuanian TBEV-naive control and in the AME cohorts (Table 3). Also, the prevalence of CCR5D32 homozygotes differed when the TBE cohort was compared with the combined control cohort (p = 0.043) (Table 3). The CCR5D32 allele prevalence was also higher in the cohort of adult patients with severe TBE (11% (22/206) than in the Lithuanian TBEV-naive control and in the AME cohorts (Table 3), but no statistical differences were found between the TBE cohort compared to the control cohorts. Statistical methods Mutation (gene) PCR and sequencing primers PCR annealing temperature Dispension order D32 CCR5 65uC GACAGTCAGA Fw: 59-CACCTGCAGCTCTCATTTTCC-39 Rev: 59-BIOTIN- TTTTTAGGATTCCCGAGTAGCA-39 Seq: 59-CAGCTCTCATTTTCCAT-39 rs3775291 TLR3 55uC GAGTATGT Fw: 59-TCATTAAGGCCCAGGTCAAG-39 Rev:59-BIOTIN-TGGCTAAAATGTTTGGAGCA-39 Seq: 59- TTATTCTTGGTTAGGTTGA-39 PCR – polymerase chain reaction, Fw – forward, Rev – reverse, Seq – sequencing. doi:10.1371/journal.pone.0106798.t002 PLOS ONE | www.plosone.org 3 September 2014 | Volume 9 | Issue 9 | e106798 Table 2. Primers, annealing temperatures and dispension orders used for amplification and sequencing of the different mutations. Mutation (gene) PCR and sequencing primers PCR annealing temperature Dispension order D32 CCR5 65uC GACAGTCAGA Fw: 59-CACCTGCAGCTCTCATTTTCC-39 Rev: 59-BIOTIN- TTTTTAGGATTCCCGAGTAGCA-39 Seq: 59-CAGCTCTCATTTTCCAT-39 rs3775291 TLR3 55uC GAGTATGT Fw: 59-TCATTAAGGCCCAGGTCAAG-39 Rev:59-BIOTIN-TGGCTAAAATGTTTGGAGCA-39 Seq: 59- TTATTCTTGGTTAGGTTGA-39 PCR – polymerase chain reaction, Fw – forward, Rev – reverse, Seq – sequencing. doi:10.1371/journal.pone.0106798.t002 PLOS ONE | www.plosone.org 3 September 2014 | Volume 9 | Issue 9 | e106798 September 2014 | Volume 9 | Issue 9 | e106798 3 CCR5 and TLR3 Polymorphisms in Tick-Borne Encephalitis CCR5 and TLR3 Polymorphisms in Tick-Borne Encephalitis September 2014 | Volume 9 | Issue 9 | e106798 Correlation between demographic, laboratory, and genetic parameters and severity of TBE CSF leukocyte and mononuclear cell count and total protein level in TBE patient cohorts stratified by severity of disease September 2014 | Volume 9 | Issue 9 | e106798 PLOS ONE | www.plosone.org 4 CCR5 and TLR3 Polymorphisms in Tick-Borne Encephalitis Table 3. Genotype distribution of CCR5 and D32 allele prevalence among TBE patients, Lithuanian TBE virus-naive control subjects, and patients with aseptic meningoencephalitis (AME) of non-TBEV etiology. Cohort Population CCR5 genotype, n (%) Failed, n (%) Allele prevalence (wt/D32 allele) Reference wt/wt wt/D32 D32/D32 TBE1 Children TBE (n = 117) 91 (77.8) 23 (19.7) 3 (2.5)a 0 (0) 0.876/0.124 TBE2 Adult severe TBE (n = 103) 83 (80.6) 18 (17.5) 2 (1.9)b 0 (0) 0.890/0.110 TBE3 Adult TBE (n = 129) 97 (75.2) 29 (22.5) 3 (2.3) 0 (0) 0.864/0.136 [6] TBE1+TBE3 Children and adult TBE (n = 246) 188 (78.4) 52 (21.1) 6 (2.4)c 0 (0) 0.870/0.130f,g TBE1+TBE2+TBE3 All TBE cases (n = 349) 271 (77.7) 70 (20.1) 8 (2.3)d 0 (0) 0.877/0.123h,i C1 Lithuanian controls (n = 134) 112 (83.6) 22 (16.4) 0 (0) 0 (0) 0.918/0.082f,h [6] C2 Adult AME (n = 79) 60 (78.9) 16 (21.1) 0 (0) 3 (3.8) 0.895/0.105 [6] C1+C2 Lithuanian controls and adult AME (n = 213) 172 (81.9) 38 (18.1) 0 (0)a,b,c,d 3 (1.4) 0.910/0.090g,i [6] wt – wild type. a TBE1 vs C1+C2: p = 0.045 (Pearson’s x2 test). b TBE2 vs C1+C2: p = 0.043 (Pearson’s x2 test). c TBE1+TBE3 vs C1+C2: p = 0.023 (Pearson’s x2 test). d TBE1+TBE2+TBE3 vs C1+C2: p = 0.027 (Pearson’s x2 test). f TBE1+TBE3 vs C1: p = 0.046 (Pearson’s x2 test) and OR = 1.672 (95% CI 1.005–2.782; p = 0.048). g TBE1+TBE3 vs C1+C2: p = 0.059 (Pearson’s x2 test). h TBE1+TBE2+TBE3 vs C1: p = 0 069 (Pearson’s x2 test) PLOS ONE | www.plosone.org 5 Genotype distribution of CCR5 and D32 allele prevalence among TBE patients, Lithuanian TBE virus-naive control subjects, and patients with aseptic meningoencephalitis non-TBEV etiology. September 2014 | Volume 9 | Issue 9 | e106798 September 2014 | Volume 9 | Issue 9 | e106798 September 2014 | Volume 9 | Issue 9 | e106798 Correlation between demographic, laboratory, and genetic parameters and severity of TBE Population CCR5 genotype, n (%) Failed, n (%) Allele prevalence (wt/D32 allele) Reference wt/wt wt/D32 D32/D32 Children TBE (n = 117) 91 (77.8) 23 (19.7) 3 (2.5)a 0 (0) 0.876/0.124 Adult severe TBE (n = 103) 83 (80.6) 18 (17.5) 2 (1.9)b 0 (0) 0.890/0.110 Adult TBE (n = 129) 97 (75.2) 29 (22.5) 3 (2.3) 0 (0) 0.864/0.136 [6] Children and adult TBE (n = 246) 188 (78.4) 52 (21.1) 6 (2.4)c 0 (0) 0.870/0.130f,g TBE3 All TBE cases (n = 349) 271 (77.7) 70 (20.1) 8 (2.3)d 0 (0) 0.877/0.123h,i Lithuanian controls (n = 134) 112 (83.6) 22 (16.4) 0 (0) 0 (0) 0.918/0.082f,h [6] Adult AME (n = 79) 60 (78.9) 16 (21.1) 0 (0) 3 (3.8) 0.895/0.105 [6] Lithuanian controls and adult AME (n = 213) 172 (81.9) 38 (18.1) 0 (0)a,b,c,d 3 (1.4) 0.910/0.090g,i [6] September 2014 | Volume 9 | Issue 9 | e106798 5 CCR5 and TLR3 Polymorphisms in Tick-Borne Encephalitis Table 4. Genotype distribution of TLR3 rs3775291 and allele prevalence among TBE patients, Lithuanian TBEV-naive control subjects, and patients with aseptic meningoencephalitis (AME) of non-TBEV etiology. Cohort Population TLR3 rs3775291 genotype, n (%) Failed, n (%) Allele prevalence (wt/mut allele) Reference wt/wt wt/mut mut/mut TBE1 Children TBE (n = 117) 54 (51.4) 36 (34.3) 15 (14.3)a 12 (10.3) 0.686/0.314 TBE2 Adult severe TBE (n = 103) 44 (44.4)f 41 (41.4) 14 (14.1)b 4 (3.9) 0.652/0.348g TBE3 Adult TBE (n = 128) 76 (59.8)f 42 (33.1) 9 (7.1) 1 (1.0) 0.764/0.236g [9] TBE1+TBE3 Children and adult TBE (n = 245) 130 (56.0) 78 (33.6) 24 (10.4)c 13 (5.3) 0.728/0.272e TBE1+TBE2+TBE3 All TBE cases (n = 348) 174 (52.5) 119 (36.0) 38 (11.5)d 17 (4.8) 0.705/0.295 C1 Lithuanian controls (n = 135) 65 (51.6) 37 (29.4) 24 (19.0) 9 (7.0) 0.663/0.337 [9] C2 Adult AME (n = 77) 35 (46.7) 24 (32.0) 16 (21.3) 2 (3.0) 0.627/0.373 [9] C1+C2 Lithuanian controls and adult AME (n = 212) 100 (49.8) 61 (30.3) 40 (19.9)a,b,c,d 11 (5.2) 0.649/0.351e [9] wt – wild type. September 2014 | Volume 9 | Issue 9 | e106798 6 CCR5 and TLR3 Polymorphisms in Tick-Borne Encephalitis Table 5. Genotype distribution of CCR5 and D32 allele prevalence among patients with TBE, stratified by severity of disease. Table 5. Genotype distribution of CCR5 and D32 allele prevalence among patients with TBE, stratified by severity of disease. Correlation between demographic, laboratory, and genetic parameters and severity of TBE Population Clinical form of TBE CCR5 genotype, n (%) Allele prevalence (wt/D32 allele) wt/wt wt/D32 D32/D32 Children TBE* Mild (n = 73) 58 (79.5) 14 (19.2) 1 (1.3) 0.890/0.110 Moderate (n = 40) 30 (75.0) 8 (20.0) 2 (5.0) 0.850/0.150 Severe (n = 4) 3 (75.0) 1 (25.0) 0 (0) 0.875/0.125 Total (n = 117) 91 (77.8) 23 (19.7) 3 (2.5) 0.876/0.124 Adult TBE** Mild (n = 56) 43 (76.8) 13 (23.2) 0 (0) 0.884/0.116 Moderate (n = 57) 44 (77.2) 10 (17.5) 3 (5.3) 0.860/0.140 Severe (n = 119) 93 (78.1) 24 (20.2) 2 (1.7) 0.882/0.118 Total (n = 232) 180 (77.6) 47 (20.2) 5 (2.2) 0.877/0.123 wt – wild type; M – Mild, Mo – Moderate, S – Severe form. * (wt/wt + wt/D32) vs D32/D32, M vs Mo vs S: p = 0.479; M vs (Mo+S): p = 0.292; wt vs D32, M vs Mo vs S: p = 0.571; M vs (Mo+S): p = 0.391. ** (wt/wt + wt/D32) vs D32/D32, M vs Mo vs S: p = 0.137; M vs (Mo+S): p = 0.202; wt vs D32, M vs Mo vs S: p = 0.806; M vs (Mo+S): p = 0.802. doi:10.1371/journal.pone.0106798.t005 wt – wild type; M – Mild, Mo – Moderate, S – Severe form. * (wt/wt + wt/D32) vs D32/D32, M vs Mo vs S: p = 0.479; M vs (Mo+S): p = 0.292; wt vs D32, M vs Mo vs S: p = 0.571; M vs (Mo+S): p = 0.391. ** (wt/wt + wt/D32) vs D32/D32, M vs Mo vs S: p = 0.137; M vs (Mo+S): p = 0.202; wt vs D32, M vs Mo vs S: p = 0.806; M vs (Mo+S): p = 0.802. doi:10.1371/journal.pone.0106798.t005 wt wild type; M Mild, Mo Moderate, S Severe form. * (wt/wt + wt/D32) vs D32/D32, M vs Mo vs S: p = 0.479; M vs (Mo+S): p = 0.292; wt vs D32, M vs Mo vs S: p = 0.571; M vs (Mo+S): p = 0.391. ** (wt/wt + wt/D32) vs D32/D32, M vs Mo vs S: p = 0.137; M vs (Mo+S): p = 0.202; wt vs D32, M vs Mo vs S: p = 0.806; M vs (Mo+S): p = 0.802. doi:10 1371/journal pone 0106798 t005 (excluding CSF samples with red blood cells due to traumatic lumbar puncture) are presented in Table 7. Correlation between demographic, laboratory, and genetic parameters and severity of TBE independently of age but does not determine the severity of TBE. In contrast to CCR5, the polymorphism of TLR3 gene plays a role in the development of clinical TBE in adults only and could also be associated with disease severity. In the overall combined cohort of TBE cases (n = 349), CSF cell count, homozygous CCR5D32 genotype, D32 allele, homozygous wild type, homozygous mutant TLR3 rs3775291 genotype and wt allele did not correlate with the severity of TBE. The rationale to extend our previous studies was based on the hypothesis that genetic factors predispose to the important features of TBE: the disease being much more common in adults than in children [5,15,16], and having a very broad spectrum of disease severity. The study was designed to avoid selection bias combining two large prospective and consecutive cohorts of children and adults with severe TBE using the same clinical classification by the same investigators. Three independent predictors of the encephalitic (moderate and severe) form of illness in this cohort – age, gender and total protein in CSF – were assessed using the multivariate logistic regression model. Increased age (with each year added, OR = 1.045; 95% CI 1.031–1.059, p,0.001), increased total protein in CSF (with each g/l added, OR = 2.353; 95% CI 1.039–5.328, p = 0.04) and being female (OR = 1.714; 95% CI 1.019–2.880, p = 0.042) were associated with the risk for encephalitic (moderate and severe) form of TBE. In this report, the frequency of CCR5D32 homozygotes was higher in TBE patients than in controls (2.5% in children, 1.9% in adults with severe TBE, and 0% in controls), as in agreement with the results of our first study on the association between CCR5D32 and TBE [6]. Furthermore, demonstrating this association in the large combined children and adult TBE cohort, covering the whole age and disease severity spectrum, strongly suggests that functional CCR5 protein plays a role in the host defense against TBE infection. The association between functional/wild type TLR3 polymorphism and TBE in the combined cohorts was – Moderate, S – Severe form. t, M vs Mo vs S: p = 0.757; M vs (Mo+S): p = 0.485; wt vs mut, M vs Mo vs S: p = 0.476; M vs (Mo+S): p = 0.280. * wt/wt vs wt/mut vs mut/mut, M vs Mo vs S: p = 0.757; M vs (Mo+S): p = 0.485; wt vs mut, M vs Mo vs S: p = 0.476; M vs (Mo+S): p = 0.280. ** wt/wt vs wt/mut vs mut/mut, M vs Mo vs S: p = 0.264; M vs (Mo+S): p = 0.180; (wt/wt + wt/mut) vs mut/mut, M vs Mo vs S: p = 0.197; M vs (Mo+S): p = 0.071; wt vs mut, M vs Mo vs S: p = 0.157; M vs (Mo+S): p = 0.096. doi:10.1371/journal.pone.0106798.t006 , p ; ( ) p ; , p ; ( ) p t, M vs Mo vs S: p = 0.264; M vs (Mo+S): p = 0.180; (wt/wt + wt/mut) vs mut/mut, M vs Mo vs S: p = 0.197; M vs (Mo+S): p = 0.071; wt vs mu (Mo+S): p = 0.096. 798 t006 pe; M – Mild, Mo – Moderate, S – Severe form. t/mut vs mut/mut, M vs Mo vs S: p = 0.757; M vs (Mo+S): p = 0.485; wt vs mut, M vs Mo vs S: p = 0.476; M vs (Mo+S): p = 0.280. wt/mut vs mut/mut M vs Mo vs S: p = 0 264; M vs (Mo+S): p = 0 180; (wt/wt + wt/mut) vs mut/mut M vs Mo vs S: p = 0 197; M vs (Mo+S): Discussion TLR3 polymorphism in TBE in children has never been investigated before, and our results indicate that, in contrast to adults, this particular gene polymorphism is not a risk factor predisposing to clinical TBE in children. A special focus of this study was gene polymorphisms predisposing to disease severity. No evidence of such association for CCR5D32 in neither of our cohorts was found. In contrast, a recent study on WNV established CCR5D32 homozygosity as a predictor of severity of clinical presentation [27]. Although being large, our study may still have been inadequate in terms of the number of CCR5D32 homozygotes to demonstrate this associa- tion. An unexpected finding of our study was that both homozygous wild TLR3 genotype and wt allele were significantly less prevalent in the cohort of adults with severe form of TBE compared to the cohort of adults with the entire clinical spectrum of TBE. We further found a trend of lower prevalence of homozygous wild genotype (p = 0.071) and wt allele (p = 0.096) in the adults with moderate and severe TBE. Therefore, it is tempting to speculate that TLR3 may play both beneficial and detrimental roles in pathogenesis of TBE: the carriers of wild type allele are more prone to develop clinical TBE; however, when the virus is already in the brain, TLR3 seems to play a protective role. This could also provide an explanation as to why no association between TLR3 polymorphisms and clinical TBE was found in the cohort of adults with severe TBE when compared to the controls. It is striking that similar findings were observed in animal models with WNV [28,29]. TLR3-deficient mice were more resistant to WNV after intraperitoneal inoculation but not after direct injection of WNV into the brain [28]. Our findings further support a benefit from the therapies directed at restoring the functional defect of TLR3. In addition to genotype-disease association, the importance of the mutated allele has also been shown in our study. As the prevalence of both CCR5D32 and wild type TLR3 alleles were higher among TBE patients than the controls, we believe that a gene-dosage effect of those proteins exists in TBEV infection, and that heterozygote carriers are predisposed to clinical TBE as well. Discussion This study both confirms and extends our previous findings that a nonfunctional CCR5 protein and a functional TLR3 receptor are associated with the clinical expression of TBE. The nonfunctional CCR5 protein predisposes to the clinical TBE Table 6. Genotype distribution of TLR3 rs3775291 and allele prevalence among patients with TBE, stratified by severity of disease. Population Clinical form of TBE TLR3 rs3775291 genotype, n (%) Allele prevalence (wt/mut allele) wt/wt wt/mut mut/mut Children TBE* Mild (n = 66/73) 31 (46.9) 25 (37.8) 10 (15.2) 0.659/0.341 Moderate (n = 36/40) 21 (58.3) 10 (27.8) 5 (13.9) 0.722/0.278 Severe (n = 3/4) 2 (66.7) 1 (33.3) 0 (0) 0.833/0.167 Total (n = 105/117) 54 (51.4) 36 (34.3) 15 (14.3) 0.686/0.314 Adult TBE** Mild (n = 54/56) 32 (59.3) 20 (37.0) 2 (3.7) 0.778/0.222 Moderate (n = 57/57) 33 (57.9) 17 (29.8) 7 (12.3) 0.728/0.272 Severe (n = 115/119) 55 (47.8) 46 (40.0) 14 (12.2) 0.678/0.322 Total (n = 226/232) 120 (53.1) 83 (36.7) 23 (10.2) 0.715/0.285 wt – wild type; M – Mild, Mo – Moderate, S – Severe form. * wt/wt vs wt/mut vs mut/mut, M vs Mo vs S: p = 0.757; M vs (Mo+S): p = 0.485; wt vs mut, M vs Mo vs S: p = 0.476; M vs (Mo+S): p = 0.280. ** wt/wt vs wt/mut vs mut/mut, M vs Mo vs S: p = 0.264; M vs (Mo+S): p = 0.180; (wt/wt + wt/mut) vs mut/mut, M vs Mo vs S: p = 0.197; M vs (Mo+S): p = 0.071; wt vs mut, M vs Mo vs S: p = 0.157; M vs (Mo+S): p = 0.096. doi:10.1371/journal.pone.0106798.t006 n of TLR3 rs3775291 and allele prevalence among patients with TBE, stratified by severity of disease. Table 6. Genotype distribution of TLR3 rs3775291 and allele prevalence among patients with TBE, stratif September 2014 | Volume 9 | Issue 9 | e106798 PLOS ONE | www.plosone.org 7 CCR5 and TLR3 Polymorphisms in Tick-Borne Encephalitis Table 7. CSF leukocyte and mononuclear cell count and total protein level in TBE patient cohorts, stratified by severity of disease. Table 7. CSF leukocyte and mononuclear cell count and total protein level in TBE patient cohorts, stratif Table 7. CSF leukocyte and mononuclear cell count and total protein level in TBE patient cohorts, stratified by severity of disease. Discussion Population Clinical form, (n) Leukocyte count (106/l), mean±SD (min–max) Mononuclear cells (106/l), mean±SD (min–max) Total protein (g/l), mean±SD (min–max) Children TBE (n = 117)* 182.066149.14 (8–853) 111.15687.85 (1–428) 0.58560.309 (0.137–1.630) Mild (n = 73) 167.716132.53 (8–612) 104.816 86.96 (5–428) 0.56960.307 (0.137–1.630) Moderate (n = 40) 217.206176.53 (10–853) 128.51690.69 (1–367) 0.63060.324 (0.230–1.620) Severe (n = 4) 92.50633.79 (64–141) 53.37624.05 (19–71) 0.43060.178 (0.190–0.580) Adult severe TBE (n = 89/103) 180.356218.14 (8–1600) 106.09688.74 (7–418) 0.89260.409 (0.214–2.540) Adult TBE (n = 117/129)** 154.496163.77 (9–995) 113.79694.59 (9–512) 0.68860.319 (0.237–1.908) Mild (n = 52) 151.926170.27 (9–995) 118.306105.13 (9–428) 0.68860.282 (0.241–1.543) Moderate (n = 52) 143.446145.17 (10–682) 106.05690.45 (10–512) 0.65260.324 (0.237–1.526) Severe (n = 13) 208.926206.71 (30–832) 123.98669.87 (19–245) 0.83360.424 (0.388–1.908) All TBE cases (n = 323/349)*** 171.606175.63 (8–1600) 110.57690.16 (1–512) 0.70760.364 (0.137–2.540) Mild (n = 125) 161.146148.94 (8–995) 110.02694.19 (5–428) 0.61860.301 (0.137–1.630) Moderate (n = 92) 175.516162.85 (10–853) 116.75690.72 (1–512) 0.64360.322 (0.230–1.620) Severe (n = 106) 180.546212.58 (8–1600) 106.29685.55 (7–418) 0.86860.412 (0.190–2.540) Note. Normal CSF cell count range: 0–56106/l leukocytes (all mononuclear cells), no red blood cells; normal total protein range: 0.15–0.45 g/l. M – Mild, Mo – Moderate, S – Severe form. * M vs Mo vs S, Leukocyte count: p = 0.114; Mononuclear cells: p = 0.160; Total protein: p = 0.362. ** M vs Mo vs S, Leukocyte count: p = 0.434; Mononuclear cells: p = 0.763; Total protein: p = 0.191. *** M vs Mo vs S, Leukocyte count: p = 0.684; Mononuclear cells: p = 0.729; Total protein: p,0.001. doi:10.1371/journal.pone.0106798.t007 Note. Normal CSF cell count range: 0–5610 /l leukocytes (all mononuclear cells), no red blood cells; M – Mild, Mo – Moderate, S – Severe form. * M vs Mo vs S, Leukocyte count: p = 0.114; Mononuclear cells: p = 0.160; Total protein: p = 0.362. ** M vs Mo vs S, Leukocyte count: p = 0.434; Mononuclear cells: p = 0.763; Total protein: p = 0.191. *** M vs Mo vs S, Leukocyte count: p = 0.684; Mononuclear cells: p = 0.729; Total protein: p,0.001. doi:10.1371/journal.pone.0106798.t007 report were in agreement with our data, major differences in design make comparison of the results difficult. consistent with the previous observations. However, in children and in adults with severe TBE, the homozygous wild TLR3 genotype was not more prevalent than in the controls. September 2014 | Volume 9 | Issue 9 | e106798 References 1. Lindquist L, Vapalahti O (2008) Tick-borne encephalitis. Lancet 371: 1861– 1871. 18. Lim JK, Louie CY, Glaser C, Jean C, Johnson B, et al. (2008) Genetic deficiency of chemokine receptor CCR5 is a strong risk factor for symptomatic West Nile virus infection: a meta-analysis of 4 cohorts in the US epidemic. J Infect Dis 197: 262–265. 2. Mansfield KL, Johnson N, Phipps LP, Stephenson JR, Fooks AR, et al. (2009) Tick-borne encephalitis virus - a review of an emerging zoonosis. J Gen Virol 90: 1781–1794. 19. Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, et al. (1996) Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86: 367–377. 3. Suss J (2011) Tick-borne encephalitis 2010: epidemiology, risk areas, and virus strains in Europe and Asia-an overview. Ticks Tick Borne Dis 2: 2–15. 20. Gorbea C, Makar KA, Pauschinger M, Pratt G, Bersola JL, et al. (2010) A role for Toll-like receptor 3 variants in host susceptibility to enteroviral myocarditis and dilated cardiomyopathy. J Biol Chem 285: 23208–23223. 4. Robertson SJ, Mitzel DN, Taylor RT, Best SM, Bloom ME (2009) Tick-borne flaviviruses: dissecting host immune responses and virus countermeasures. Immunol Res 43: 172–186. 5. Mickiene A, Lindquist L, Laiskonis A (2005) Tick-borne encephalitis – clinical course and outcome. Review of the literature. In: Ebert RA, editor. Progress in encephalitis research. Hauppauge N.Y.: Nova Science Publishers. pp. 1–30. 21. Lee SO, Brown RA, Razonable RR (2013) Association between a functional polymorphism in Toll-like receptor 3 and chronic hepatitis C in liver transplant recipients. Transpl Infect Dis 15: 111–119. 6. Kindberg E, Mickiene A, Ax C, Akerlind B, Vene S, et al. (2008) A deletion in the chemokine receptor 5 (CCR5) gene is associated with tickborne encephalitis. J Infect Dis 197: 266–269. 22. Dhiman N, Ovsyannikova IG, Vierkant RA, Ryan JE, Pankratz VS, et al. (2008) Associations between SNPs in toll-like receptors and related intracellular signaling molecules and immune responses to measles vaccine: preliminary results. Vaccine 26: 1731–1736. 7. Wong MM, Fish EN (2003) Chemokines: attractive mediators of the immune response. Semin Immunol 15: 5–14. 23. O9Dwyer DN, Armstrong ME, Trujillo G, Cooke G, Keane MP, et al. (2013) The Toll-like receptor 3 L412F polymorphism and disease progression in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 188: 1442–1450. 8. Glass WG, Lim JK, Cholera R, Pletnev AG, Gao JL, et al. Acknowledgments We kindly acknowledge Ylva Ro¨nnelid for greatly helping us with pyrosequencing. References (2005) Chemokine receptor CCR5 promotes leukocyte trafficking to the brain and survival in West Nile virus infection. J Exp Med 202: 1087–1098. 24. Barkhash AV, Perelygin AA, Babenko VN, Myasnikova NG, Pilipenko PI, et al. (2010) Variability in the 29-59-oligoadenylate synthetase gene cluster is associated with human predisposition to tick-borne encephalitis virus-induced disease. J Infect Dis 202: 1813–1818. J p 9. Kindberg E, Vene S, Mickiene A, Lundkvist A, Lindquist L, et al. (2011) A functional Toll-like receptor 3 gene (TLR3) may be a risk factor for tick-borne encephalitis virus (TBEV) infection. J Infect Dis 203: 523–528. 10. Kumar H, Kawai T, Akira S (2009) Toll-like receptors and innate immunity. Biochem Biophys Res Commun 388: 621–625. 25. Barkhash AV, Perelygin AA, Babenko VN, Brinton MA, Voevoda MI (2012) Single nucleotide polymorphism in the promoter region of the CD209 gene is associated with human predisposition to severe forms of tick-borne encephalitis. Antiviral Res 93: 64–68. 11. Kumar H, Kawai T, Akira S (2009) Pathogen recognition in the innate immune response. The Biochemical journal 420: 1. p j 12. Lafon M, Megret F, Lafage M, Prehaud C (2006) The innate immune facet of brain: human neurons express TLR-3 and sense viral dsRNA. J Mol Neurosci 29: 185–194. 26. Barkhash AV, Voevoda MI, Romaschenko AG (2013) Association of single nucleotide polymorphism rs3775291 in the coding region of the TLR3 gene with predisposition to tick-borne encephalitis in a Russian population. Antiviral Res 99: 136–138. 13. Hofmann H, Heinz FX, Dippe H (1983) ELISA for IgM and IgG antibodies against tick-borne encephalitis virus: quantification and standardization of results. Zentralbl Bakteriol Mikrobiol Hyg A 255: 448–455. 27. Lim JK, McDermott DH, Lisco A, Foster GA, Krysztof D, et al. (2010) CCR5 deficiency is a risk factor for early clinical manifestations of West Nile virus infection but not for viral transmission. J Infect Dis 201: 178–185. infection but not for viral transmission. J Infect Dis 201: 178–185. 14. Mickiene A, Laiskonis A, Gunther G, Vene S, Lundkvist A, et al. (2002) Tickborne encephalitis in an area of high endemicity in lithuania: disease severity and long-term prognosis. Clin Infect Dis 35: 650–658. 28. Wang T, Town T, Alexopoulou L, Anderson JF, Fikrig E, et al. (2004) Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis. Nat Med 10: 1366–1373. y g p g 15. CCR5 and TLR3 Polymorphisms in Tick-Borne Encephalitis demographic, and genetic findings with disease severity. Age, gender, and total protein in CSF were established as associated with severity of TBE, which is in line with other clinical studies [1,5,14,31]. A major limitation of our study is the lack of a TBEV seropositive asymptomatic cohort. Therefore, it remains unknown if CCR5 and TLR3 polymorphisms predispose to an increased susceptibility to TBEV infection or to the development of clinical illness. However, because TBEV seropositivity and mutations under consideration, especially CCR5D32, are both uncommon, a study including a sufficient number of asymptomatic TBEV seropositive individuals will need a large-scale multicentral approach. [ ] In animal models, other factors contributing to disease severity have been shown besides the host-dependent risk factors, such as the virulence of the particular strain, which could be inconsistent with the TBEV subtype [32], the inoculation dose [33], and the immunomodulatory compounds of tick saliva inoculated with virus into the skin [4]. Animal models are also commonly used to elucidate the mechanism of disease development following TBEV infection in vivo and suggest that CNS pathology during TBE is primarily driven by immune response and inflammatory reactions. A key role of CD8+ T cells in the immunopathology of TBE, as demonstrated by the prolonged survival of severe combined immunodeficiency (SCID) or CD82/2 mice following infection compared with immunocompetent mice or mice with transferred CD8+ T cells has been shown by Ruzek et al. [34]. Another study found that TNF-a levels were significantly increased in the brains and in serum of mice that died following TBEV infection [35]. The most recent animal study showed that neutralizing antibody response is crucial for preventing fatality, while high expression of various cytokines/chemokines during TBE mediates immunopa- thology and is associated with a more severe course of the infection [36]. Furthermore, this study demonstrated that mice of the same age, gender, nutritious status, and TBEV inoculum, showed different TBE severity depending on genetic background, strongly supporting our findings. In conclusion, this study confirms that polymorphisms in the CCR5 and TLR3 genes are risk factors for the development of clinical TBE. It also shows that the role of TLR3 polymorphism differs in different age groups, and may be linked to disease severity. Further studies are needed to clarify if CCR5 and TLR3 polymorphisms play a role in susceptibility to TBEV infection and to further elucidate its influence on the severity of TBE. Author Contributions Conceived and designed the experiments: LL LS. Performed the experiments: JN BC AM JP MH. Analyzed the data: AM JP JN. Contributed reagents/materials/analysis tools: LS. Contributed to the writing of the manuscript: AM JP JN LL. Discussion Our findings on CCR5D32 contradict those of clinical studies on WNV infection which claim that both CCR5D32 alleles are needed to confer a deficit [17,18], but are in agreement with the data on HIV that show slower rates of disease progression to AIDS in heterozygous CCR5D32 individuals [19]. Data on TLR3, and on functional SNP, characterized by amino acid substitution from phenylalanine to leucine at position 412 in particular, are also inconsistent and depend on the virus under consideration. Gorbea et al. could only prove the wild type homozygosity to be significantly associated with enteroviral myocarditis [20]. Howev- er, a recent study on chronic hepatitis C in liver transplant patients demonstrated that heterozygous carriers of the mutated allele are predisposed to higher mortality after liver transplantation [21]. Also, heterozygous variant of the same SNP in TLR3 gene was associated with low antibody and lymphoproliferative responses to measles vaccination [22] and with early mortality and an accelerated decline in lung function in idiopathic pulmonary fibrosis patients [23]. TBE is associated with the infiltration of T cells and the macrophages (cell types known to express CCR5) into the brains of patients infected with TBEV [30]. The amount of the total protein in CSF is usually increased during TBE, and its elevation indirectly reflects the degree of the permeability of blood brain barrier [31]. Multivariate logistic regression model was employed to investigate the association of these laboratory parameters, To the best of our knowledge, only one other research group has studied the role of human genetics in TBE [24–26]. In the recently published study, no association between CCR5 polymor- phism and predisposition to TBE was found in the Russian population [26]. Although the findings on TLR3 in the Russian September 2014 | Volume 9 | Issue 9 | e106798 PLOS ONE | www.plosone.org 8 31. Gunther G, Haglund M, Lindquist L, Forsgren M, Skoldenberg B (1997) Tick- bone encephalitis in Sweden in relation to aseptic meningo-encephalitis of other etiology: a prospective study of clinical course and outcome. J Neurol 244: 230– 238. 32. Luat le X, Tun MM, Buerano CC, Aoki K, Morita K, et al. (2014) Pathologic potential of variant clones of the oshima strain of far-eastern subtype tick-borne encephalitis virus. Trop Med Health 42: 15–23. p p 33. Hayasaka D (2011) The Development of Encephalitis Following Tick-Borne Encephalitis Virus Infection in a Mouse Model. In: Ruzek D, editor. Flavivirus Encephalitis. Rijeka: InTech. pp. 157–166. 36. Palus M, Vojtiskova J, Salat J, Kopecky J, Grubhoffer L, et al. (2013) Mice with different susceptibility to tick-borne encephalitis virus infection show selective neutralizing antibody response and inflammatory reaction in the central nervous system. J Neuroinflammation 10: 77. 35. Hayasaka D, Nagata N, Fujii Y, Hasegawa H, Sata T, et al. (2009) Mortality following peripheral infection with Tick-borne encephalitis virus results from a combination of central nervous system pathology, systemic inflammatory and stress responses. Virology 390: 139–150. 34. Ruzek D, Salat J, Palus M, Gritsun TS, Gould EA, et al. (2009) CD8+ T-cells mediate immunopathology in tick-borne encephalitis. Virology 384: 1–6. CCR5 and TLR3 Polymorphisms in Tick-Borne Encephalitis References Arnez M, Avsic-Zupanc T (2009) Tick-borne encephalitis in children: an update on epidemiology and diagnosis. Expert Rev Anti Infect Ther 7: 1251–1260. 15. Arnez M, Avsic-Zupanc T (2009) Tick-borne encephalitis in child p 29. Daffis S, Samuel MA, Suthar MS, Gale M, Jr., Diamond MS (2008) Toll-like receptor 3 has a protective role against West Nile virus infection. J Virol 82: 10349–10358. 16. Lindquist L (2008) Tick-borne encephalitis (TBE) in childhood. Acta Paediatr 97: 532–534. 17. Glass WG, McDermott DH, Lim JK, Lekhong S, Yu SF, et al. (2006) CCR5 deficiency increases risk of symptomatic West Nile virus infection. J Exp Med 203: 35–40. 30. Gelpi E, Preusser M, Garzuly F, Holzmann H, Heinz FX, et al. (2005) Visualization of Central European tick-borne encephalitis infection in fatal human cases. J Neuropathol Exp Neurol 64: 506–512. PLOS ONE | www.plosone.org September 2014 | Volume 9 | Issue 9 | e106798 9 September 2014 | Volume 9 | Issue 9 | e106798 CCR5 and TLR3 Polymorphisms in Tick-Borne Encephalitis PLOS ONE | www.plosone.org September 2014 | Volume 9 | Issue 9 | e106798 PLOS ONE | www.plosone.org 10
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High Aerobic Intensity Training and Psychological States in Patients with Depression or Schizophrenia
Frontiers in psychiatry
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Jørn Heggelund 1,2*, Kim Daniel Kleppe3, Gunnar Morken2,4 and Einar Vedul-Kjelsås 2,4 1 Division of Psychiatry, Department of Østmarka, St. Olavs University Hospital, Trondheim, Norway 2 Department of Neuroscience, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway 3 Hamar District Psychiatric Centre, Innlandet Hospital Trust, Ottestad, Norway 4 Division of Psychiatry, Department of Research and Development (AFFU), St. Olavs University Hospital, Trondheim, Norway Jørn Heggelund 1,2*, Kim Daniel Kleppe3, Gunnar Morken2,4 and Einar Vedul-Kjelsås 2,4 1 Division of Psychiatry, Department of Østmarka, St. Olavs University Hospital, Trondheim, Norway 2 Department of Neuroscience, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway 3 Hamar District Psychiatric Centre, Innlandet Hospital Trust, Ottestad, Norway 4 Division of Psychiatry, Department of Research and Development (AFFU), St. Olavs University Hospital, Trondheim, Norway Aim: To explore changes in psychological states in response to a bout of high aerobic intensity training (HIT) in patients with depression or schizophrenia compared to healthy individuals. ClinicalTrials.gov identifier: NCT01310998. Keywords: exercise, intensity, affect, anxiety, transitory emotions *Correspondence: p Jørn Heggelund, Department of Østmarka, Division of Psychiatry, St. Olavs University Hospital, Box 3008 Lade, Trondheim N-7441, Norway e-mail: Jorn.Heggelund@ntnu.no Results: All three groups improved in positive affect and well-being 15 min after HIT (p < 0.01), but only patients with depression had maintained the effect after 3 h (p = 0.007, p = 0.012).The duration of the improved positive affect was longer in depression (p = 0.002) and schizophrenia (p = 0.025) than in healthy individuals (F 2.50 = 5.83, p < 0.01). Patients with depression or schizophrenia had reduced distress and state anxiety 15 min after HIT and 3 h after HIT (p < 0.05). The improvement in distress 15 min after HIT was larger in patients with depression (p = 0.028) compared to healthy individuals (F 2.50 = 5.05, p < 0.01). No changes were found during the no-training day (p > 0.05). Conclusion: High aerobic intensity training used as an acute intervention improved positive affect and well-being and reduced distress and state anxiety in patients with depression and schizophrenia. www.frontiersin.org Edited by: Jan Hoff, Norwegian University of Science and Technology, Norway Jan Hoff, Norwegian University of Science and Technology, Norway Reviewed by: Eivind Wang, Norwegian University of Science and Technology, Norway Øyvind Støren, Telemark University College, Norway *Correspondence: Jørn Heggelund, Department of Østmarka, Division of Psychiatry, St. Olavs University Hospital, Box 3008 Lade, Trondheim N-7441, Norway e-mail: Jorn.Heggelund@ntnu.no Reviewed by: Eivind Wang, Norwegian University of Science and Technology, Norway Øyvind Støren, Telemark University College, Norway Methods: After familiarization training of HIT, 20 patients with schizophrenia, 13 patients with depression, and 20 healthy individuals performed a no-training day followed by a train- ing day. HIT was 4 × 4 min intervals at 85–95% of peak heart rate, intermitted by 3 min active rest periods at 70% of peak heart rate. Self-evaluation questionnaires of positive affect, negative affect, state anxiety, well-being, distress, and fatigue were completed before training, 15 min after, and 3 h after training. The two latter measures were also completed the no-training day. PSYCHIATRY PSYCHIATRY ORIGINAL RESEARCH ARTICLE O INTRODUCTION for increasing aerobic fitness in terms of peak oxygen uptake (VO2peak). High aerobic intensity training (HIT) performed as 4 × 4 min intervals at 85–95% of the maximal heart rate improves VO2peak more than long slow distance training at 70% of the max- imal heart rate (8–11). HIT also improves VO2peak in patients with schizophrenia and could be used as an intervention to reduce the risk of cardiovascular disease in a high risk group, such as patients with severe mental illnesses (12–14). Despite a broad understanding of physiological mediators of improved VO2peak after HIT, it is unknown whether HIT yields supplementary short- term psychological benefits. Long-term reductions in depressive scoreareplausibleafterregularHIT(14).Merely,afewstudieshave explored interval based exercise as an intervention to improve psy- chological states (7). Thus, it is reasonable to explore the influence of HIT on post-exercise psychological states. Patients with severe mental illnesses suffer from long-term mental health problems that often persist in spite of extensive psychiatric treatments. Thus, acute affective improvements and relief of trou- bling symptoms may be beneficial, even though they would persist for a limited period of time. Improved self-reported affectiv- ity such as mood enhancements, increased subjective well-being, decreased state anxiety, decreased negative affect, and decreased psychological stress may be brought about by aerobic endurance training in both healthy individuals and patients with severe mental illness (1–3). These affective improvements normally per- sist for 2–4 h following the cessation of exercise (4), but mood enhancements up to 12 h post-exercise have been reported (5). To maximize the affective post-exercise improvements, it has been recommended that the exercise intensity is self-selected rather than prescribed (2, 6). Furthermore, increasing intensity is associated with reduced positivity of affect (7). These rec- ommendations are somewhat in contrast to recommendations The acute psychological benefits of aerobic exercise are widely explored in healthy individuals but to a lesser degree in patients with schizophrenia and depression. Patients with depressive October 2014 | Volume 5 | Article 148 | 1 Acute psychological responses to aerobic exercise Heggelund et al. Table 1 | Characteristics of the subjects. INTRODUCTION Schizophrenia (n = 20) Depression (n = 13) Healthy (n = 20) Men/women, n 13/7 8/5 10/10 Age (years), mean (SD) 37.7 (11.8) 41.0 (10.9) 40.8 (11.8) Body weight (kg), mean (SD) 88.9 (17.3) 87.87 (26.6) 78.9 (14.2) BMI (kg m−2), mean (SD) 24.79 (3.99) 24.95 (7.03) 22.42 (3.41) Inpatient/outpatient, n 10/10 2/11 ICD-10 diagnoses, n Schizophrenia 11 Schizotypal disorder 1 Delusional disorder 2 Schizoaffective disorder 5 Unspecified non-organic psychosis 1 Bipolar depression 1 Depressive disorder 11 Dysthymia 1 Difference in inpatient/outpatient distribution between patient groups (p < 0.05). Table 1 | Characteristics of the subjects. disorders may score relatively high on negative affect, distress, fatigue, and state anxiety whereas low on positive affect and well-being, contrary to healthy individuals (15). Patients with depression may therefore have a larger window of improvement compared to healthy individuals. Decreased state-anxiety and neg- ative affect along with improved positive well-being have been reported for patients with depression and anxiety (2, 16). Con- versely, patients with schizophrenia may not be perceptive of psychological states and are known to have a lack of insight into their illness (17). Eight weeks of HIT did not improve chronic symptoms of schizophrenia (12). Acute improvements in state anxiety, psychological stress, and well-being have though been reported (3), and observations suggest that patients are less irrita- ble, depressive, and psychotic as well as more socially interested and competent on the days they have exercised (18). Patients have also reported use of exercise as a coping strategy to deal with positive symptoms (19). HIT may therefore have important therapeutic benefits for patients with depression or schizophrenia. Thus, it is paramount to compare these groups to decide whether patients respond differently from healthy individuals and whether differences in responses exist between patient groups. To explore the acute psychological benefits of HIT, we included patients with depression, schizophrenia, and healthy individuals to a controlled clinical trial. We hypothesized that a bout of HIT would improve psychological states 15 min and 3 h post-exercise, and patients would have greater acute benefits of HIT than healthy individuals. particular moment in time. The scale ranges from 1 to 4. When evaluating the scores, six of the items are reversed so that higher scores indicate higher levels of anxiety. The test–retest correlations range from 0.16 to 0.54 in college students (21). INSTRUMENTS AND PROCEDURES Borg rating of perceived exertion (RPE) was applied to deter- mine subjects’ perception of physical exertion during HIT and VO2peak testing (26). The scale ranges from 6 to 20. Six indicate “no exertion at all” whereas 20 is “maximal exertion.” During a period of two consecutive days, each participant com- pleted self-report questionnaires’ about their psychological states. A bout of HIT was performed on the second day. Question- naires’ were completed 10 min before (pre-) HIT, 15 min after (post-) HIT, and 3 h after HIT. The two latter measures were also completed at the same hour on the first day (no-training day). Par- ticipants were instructed to refrain from exercising 2 days before entering the data acquisition. SUBJECTS Fifty-three participants volunteered to take part in one of three study groups: (1) patients with schizophrenia or psychotic dis- orders, (2) patients with depression, or (3) healthy individuals. Patients were in- and out-patients at the University hospital psy- chiatric department. The schizophrenia/psychotic group was 20 patients with ICD-10 schizophrenia, schizotypal, or delusional disorders (ICD-10, F20–29) (20). The group of patients with depression comprised 1 with bipolar depression (F31.3), 11 with depressive episode (F32) or recurrent depression (F33),and 1 with dysthymia (F34.1). Twenty healthy controls with no history of psychiatric illness and with a training frequency less than three times per week were enrolled. Characteristics of the subjects are presented in Table 1. Positive well-being, psychological distress, and fatigue were measured by the Subjective Exercise Experiences Scale (SEES). SEES is a 12-item questionnaire that is validated to measure effects of exercise on psychological states (25). Each subscale contains the scores from four items and the scale ranges from 1 to 7. Higher scores indicate a higher perception of the particular factor. INTRODUCTION A low level of reli- ability is expected since the scale reflects the influence of transient situational factors. MATERIALS AND METHODS SUBJECTS Positive affect and negative affect were measured by the Positive and Negative Affect Schedule (PANAS). The PANAS incorporates 10-itemsof positiveaffectand10-itemsof negativeaffect.Thescale ranges from 1 to 5 and higher score indicate higher perception of the factor. The subjects were asked to evaluate whether they expe- rienced a particular emotion in the present moment. The positive affect and negative affect scores correlate negatively and positively, respectively, with the Beck Depression Inventory (BDI) (15, 23). Test–retest intraclass correlation is 0.79 and 0.93 for the positive and negative scales, respectively (24). HIGH AEROBIC INTENSITY TRAINING High aerobic intensity training was performed on a treadmill according to the procedures described elsewhere (8, 12). Four minutes intervals at a fixed workload were repeated 4 times, inter- mitted with 3 min active break periods between each interval. The intensity was 85–95% of peak heart rate (HRpeak) during inter- vals and similar to warming-up work load (i.e., taxing about 70% of HRpeak) during break periods. Subjects either walked or ran a State anxiety was measured by the State-Trait Anxiety Inven- tory (STAI) form X-1 (21). A 12-item short form of the original 20-items was used (22). The STAI is a self-evaluation question- naire and the instruction is to indicate how the subject feels at a October 2014 | Volume 5 | Article 148 | 2 Frontiers in Psychiatry | Schizophrenia Acute psychological responses to aerobic exercise Heggelund et al. Table 2 | Mean (SD) work load of intervals during the high aerobic intensity training. Schizophrenia (n = 20) Depression (n = 13) Healthy (n = 20) Speed (km h−1) 7.4 (1.7) 7.2 (1.9) 8.5 (2.3) Incline (%) 5.5 (1.3) 6.7 (3.4) 6.3 (1.0) Watt 95 (29) 106 (45) 121 (29) No differences between groups. Table 2 | Mean (SD) work load of intervals during the high aerobic intensity training. minimum of 5% incline. Warm-up was 5 min. Heart rate was con- tinuously measured using a Polar S610i heart rate monitor (Polar Electro, Finland). Heart rate and RPE was collected 3 min within each interval. All subjects performed a familiarization period of 3 HIT bouts within 2 weeks prior to the study. During familiar- ization training, the speed and incline were adjusted to attain the exact heart rate intensity (Table 2). All subjects performed a test of HRpeak in order to calcu- late the HIT intensity (Table 3). The test was carried out as HIT interval training. The first interval was performed without an experience of leg stiffness. After a 3 min active rest, the sec- ond interval was carried out with >1 km h−1 incensements each minute until exhaustion (preferentially within 3–5 min). HRpeak was the highest recorded HR. A measure of blood lactate was taken 1 min after discontinuation using a Lactate Pro blood lac- tate test meter (Arkray, Inc, Japan). A peak test was accepted at a blood lactate level above 8 mmol L−1. Borgs RPE was collected at discontinuation of the test. STATISTICAL ANALYSES Within-group changes were analyzed using paired t-tests. One- way between-groups ANOVA was used to analyze baseline dif- ferences and differences in change between the three groups. Separate analysis were performed on the delta changes (pre- to 15 min post-HIT) and (pre- to 3 h post-HIT). LSD or Tamhane’s post hoc analyses were performed during significant main analysis. Cohens’s d effect sizes were calculated using the formula described in Nakagawa and Cuthill (27). The effect was considered small, medium, and large at 0.2, 0.5, and 0.8, respectively. Chi-square test was used to evaluate categorical data. respectively. Positive well-being was different between the healthy individuals and patients with depression (p < 0.001) but not between healthy individuals and patients with schizophrenia (p > 0.05). The patients with schizophrenia and patients with depression scored differently in positive affect (p = 0.013) and positive well-being (p < 0.001) but not in negative affect, psycho- logical distress, fatigue, and state anxiety (p > 0.05) at baseline. Statistical significance was reached at p < 0.05. Data are described as mean and SD, unless otherwise noted. IBM SPSS Statistics, version 20 (SPSS Inc.), was applied to analyze results. The study was approved by the regional committees for med- ical and health research ethics, middle Norway and conducted according to the Helsinki declaration. Written informed consent was obtained from all the included patients after the procedures were fully explained. RESULTS Characteristics of the patients are presented in Table 1. There were no differences in age, bodyweight, or BMI between the groups (p > 0.05). There were no differences between the three groups in HRpeak, speed, or incline during the peak treadmill test (p > 0.05; Table 3). All three groups performed the HIT at the prescribed intensity and duration. Work load of the intervals are presented in Table 2. There were between-group differences at baseline in posi- tive affect (F2.50 = 9.67, p < 0.01), negative affect (F2.50 = 17.21, p < 0.01), positive well-being (F2.50 = 19.84, p < 0.01), psycho- logical distress (F2.50 = 12.64, p < 0.01), fatigue (F2.50 = 8.36, p < 0.01), and state anxiety (F2.50 = 21.74, p < 0.01). Post hoc analyses revealed that the positive affect (p = 0.046 and p < 0.001), negative affect (p = 0.005 and p < 0.001), psychological distress (p = 0.006 and p < 0.001), fatigue (p = 0.012 and p < 0.001), and state anxiety (p = 0.001 and p < 0.001) were different between health individuals and patients with schizophrenia and depression, HIGH AEROBIC INTENSITY TRAINING Table 3 | Mean (SD) values of the peak heart rate test. Schizophrenia (n = 20) Depression (n = 13) Healthy (n = 20) HRpeak (beats min−1) 177 (20) 186 (9) 186 (12) Peak speed (km h−1) 9.4 (2.8) 9.5 (2.5) 11.4 (3.5) Peak incline (%) 9.2 (3.8) 8.9 (3.8) 9.6 (3.8) Watt 192 (75) 190 (86) 218 (79) La−(mmol l−1) 11.02 (3.60) 11.2 (2.2) 11.78 (3.16) BORG RPE 19 (2) 19 (1) 19 (1) No differences between groups. HRpeak, peak heart rate; min, minute; RPE, rating of perceived exertion. Table 3 | Mean (SD) values of the peak heart rate test. POSITIVE AFFECT AND POSITIVE WELL-BEING All three groups increased their experience of positive affect and well-being from pre- to 15 min post-HIT (Figure 1). The Cohen’s d effect size indicated a medium (0.66), large (0.91), and small (0.45) effect on the positive affect in the patients with schizophre- nia,patients with depression,and healthy individuals,respectively. The corresponding Cohen’s d effect sizes for positive well-being was medium (0.74), large (1.05), and medium (0.58), respectively. After 3 h, the Cohen’s d effect sizes for positive affect and posi- tive well-being were still large (0.96 and 0.90) in the patients with depression (Figure 2). The change in positive affect from pre-HIT to 3 h post-HIT was different between the groups (F2.50 = 5.83, p < 0.01), and the post hoc analyses revealed that the change in healthy individuals was different from patients with schizophrenia (p = 0.025) and patients with depression (p = 0.008). Frontiers in Psychiatry | Schizophrenia PSYCHOLOGICAL DISTRESS The patients with schizophrenia and depression reduced their experience of psychological distress with a small (0.39) and medium (0.71) Cohen’s d effect size, respectively, from pre- to 15 min post-HIT (Figure 1). The Cohen’s d effect size for distress indicated a medium effect 3 h post-HIT (Figure 2) in the patients with schizophrenia (0.48) and depression (0.56). Cohen’s d effect October 2014 | Volume 5 | Article 148 | 3 www.frontiersin.org www.frontiersin.org Acute psychological responses to aerobic exercise Heggelund et al. FIGURE 1 | Perception of psychological states before and 15 min after HIT. PANAS, Positive and Negative Affect Schedule; SEES, Subjective Exercise Experiences Scale; STAI, State-Trait Anxiety Inventory Within groups; *p < 0.05; **p < 0.01; ***p < 0.001 different from before HIT. Between groups; different change from healthy #p < 0.05. FIGURE 1 | Perception of psychological states before and 15 min after HIT. PANAS, Positive and Negative Affect Schedule; SEES, Subjective Exercise Experiences Scale; STAI, State-Trait Anxiety FIGURE 1 | Perception of psychological states before and 15 min after HIT. PANAS, Positive and Negative Affect Schedule; SEES, Subjective Exercise Experiences Scale; STAI, State-Trait Anxiety Inventory Within groups; *p < 0.05; **p < 0.01; ***p < 0.001 different from before HIT. Between groups; different change from healthy #p < 0.05. FIGURE 1 | Perception of psychological states before and 15 min after HIT. PANAS, Positive and Negative Affect Schedule; SEES, Subjective Exercise Experiences Scale; STAI, State-Trait Anxiety Frontiers in Psychiatry | Schizophrenia October 2014 | Volume 5 | Article 148 | 4 Acute psychological responses to aerobic exercise Heggelund et al. FIGURE 2 | Perception of psychological states before and after 3 h after HIT. PANAS, Positive and Negative Affect Schedule; SEES, Subjective Exercise Experiences Scale; STAI, State-Trait Anxiety Inventory. Within groups; *p < 0.05; **p < 0.01; ***p < 0.001 different from before HIT. Between groups; different change from healthy #p < 0.05; ##p < 0.01. Inventory. Within groups; *p < 0.05; **p < 0.01; ***p < 0.001 different from before HIT. Between groups; different change from healthy #p < 0.05; ##p < 0.01. FIGURE 2 | Perception of psychological states before and after 3 h after HIT. PANAS, Positive and Negative Affect Schedule; SEES, Subjective Exercise Experiences Scale; STAI, State-Trait Anxiety October 2014 | Volume 5 | Article 148 | 5 www.frontiersin.org Acute psychological responses to aerobic exercise Heggelund et al. NEGATIVE AFFECT The negative affect was unchanged following HIT in all groups (Figures 1 and 2). The negative affect was already reduced in the patients with depression before the HIT bout compared to the no-training day (p = 0.024; Cohen’s d = 0.59). STATE ANXIETY The Cohen’s d effect sizes for state anxiety for patients with schiz- ophrenia and patients with depression was medium (0.47 and 0.48) 15 min following the HIT (Figure 1). Cohen’s d indicated a medium (0.66) effect in patients with schizophrenia as well as in patients with depression (0.68). The healthy individuals had a higher level of state anxiety immediately before the HIT com- pared with the no-training day (p = 0.025; Cohen’s d = 0.59). This increase in state anxiety was unchanged from pre- to 15 min post- HIT (p = 0.408) but had returned to no-training day levels 3 h post-HIT as indicated by a reduction in state anxiety from pre-HIT values (p = 0.038; Cohen’s d = 0.50; Figure 2). NO-TRAINING DAY During the no-training day, the two measures were not different in either group (p > 0.05), which indicate a stable experience of all six psychological state variables. The between groups analyses confirm some beneficial responses in patients compared to healthy individuals. The dura- tion of the improved positive affect was longer for those with depression or schizophrenia than in the healthy individuals. Fur- ther, the 15 min post-HIT effect on distress was larger in patients with depression compared to healthy individuals. These findings support the hypothesis that the benefits are largest in those with the most unbalanced affect and underscore the particular role of exercise in the treatment of patients with depression (28). It has been suggested that HIT does not improve chronic symptoms in patients with schizophrenia (12). Nevertheless, it now appears that patients are perceptive of certain affective improvements that exceed those of the healthy individuals. This might have important implications for daily functioning and quality of life, particularly at the days of exercise. Patients with schizophrenia (p = 0.038; Cohen’s d = 0.45), depression (p = 0.009; Cohen’s d = 0.81), and healthy individ- uals (p = 0.007; Cohen’s d = 0.48) experienced a lower level of fatigue before performing the HIT session compared to the no-training day. PSYCHOLOGICAL DISTRESS Patients with depression or schizophrenia sustained the reduc- tion in distress and state anxiety more than 3 h after HIT but the improved positive affect and well-being did only persist in the patients with depression. The negative affect were unaltered in all groups at both of the post-HIT assessments. Together, these find- ings replicate previous results suggesting that the psychological benefits are sustained for some hours after the exercise is ter- minated (4, 5). The findings are tentative evidence that patients with depression might have particularly beneficial and sustainable responses in positive affect and well-being after HIT compared to the patients with schizophrenia and the healthy individuals. Patients with depression also have the lowest baseline scores in positive affect and well-being, which provides a large window of improvement. Positive affect and BDI score are in fact nega- tively correlated, which indicate that patients with enhancements in positive affect also would experience a reduction in BDI scores (23). Further, positive affect is more strongly related to depression than to anxiety (15). Thus, patients with high levels of depres- sion might have important therapeutic benefits from HIT (16). Chamove (18) found a reduction in depression 2 h after exercise in patients with schizophrenia. Chamove (18) found that patients were“less tense”and“less irritable,”which probably reflect similar changes as the reduced distress and state anxiety in the present study. Chamove (18) applied a wider range of physical activities, illustrating that affective improvements likely appear through a multifactorial mechanism. size indicated a small effect (0.26) on distress in the healthy individuals, 15 min post-HIT. The three groups changed differ- ently in distress from pre-HIT to 15 min post-HIT (F2.50 = 5.05, p < 0.01) and the post hoc analyses reviled a difference between the healthy individuals and the patients with depression (p = 0.028). size indicated a small effect (0.26) on distress in the healthy individuals, 15 min post-HIT. The three groups changed differ- ently in distress from pre-HIT to 15 min post-HIT (F2.50 = 5.05, p < 0.01) and the post hoc analyses reviled a difference between the healthy individuals and the patients with depression (p = 0.028). STATE ANXIETY REFERENCES a range of psychosocial stimuli such as diversion from stress- ful stimuli, attention form the coach, improved self-image, feel- ings of control, social interaction, and social support (29). It is also suggested that exercise improves depression through a neurobiological mechanism. Regular exercise may exert a regu- latory influence on the neurotransmitter system or/and affect the hypothalamic–pituitary–adrenal cortical (HPA) axis (29). 1. Hale BS, Koch KR, Raglin JS. State anxiety responses to 60 minutes of cross training. Br J Sports Med (2002) 36(2):105–7. doi:10.1136/bjsm.36.2.105 2. Knapen J, Sommerijns E, Vancampfort D, Sienaert P, Pieters G, Haake P, et al. State anxiety and subjective well-being responses to acute bouts of aerobic exer- cise in patients with depressive and anxiety disorders. Br J Sports Med (2009) 43(10):756–9. doi:10.1136/bjsm.2008.052654 3. Vancampfort D, De Hert M, Knapen J, Wampers M, Demunter H, Deckx S, et al. State anxiety,psychological stress and positive well-being responses to yoga and aerobic exercise in people with schizophrenia: a pilot study. Disabil Rehabil (2011) 33(8):684–9. doi:10.3109/09638288.2010.509458 y y This study did not aim to compare HIT with other types of exercise interventions. Thus, it is not possible to advocate the use of HIT merely based on its effect on psychological states. Although the finding that a single HIT session is beneficial, it is appropriate to implement HIT on a regular basis in order to improve fitness and overall health. Better VO2peak is associated with decreased depressive symptoms,quality of life,better capacity to cope with stress as well as reduced mortality from cardiovascular disease (30–33). It is evidential that HIT performed as 4 × 4 min intervals induces larger improvements in VO2peak compared to other aerobic endurance training interventions (8–11). This large improvement caused by HIT contribute risk reduction of cardio- vascular disease (12, 34) and likely influence depressive score (14). Thus, HIT should be implemented in clinical practice to improve VO2peak in patients with severe mental illnesses. 4. Raglin JS,Wilson GS. Exercise and its effects on mental health. 2 ed. In:Bouchard C, Blair S, Haskell WL, editors. Physical Activity and Health. (Vol. 331–342), Champaign, IL: Human Kinetics (2012). p. 247–57. 5. Sibold JS, Berg KM. Mood enhancement persists for up to 12 hours follow- ing aerobic exercise: a pilot study. Percept Mot Skills (2010) 111(2):333–42. doi:10.2466/02.06.13.15.PMS.111.5.333-342 6. Szabo A. Acute psychological benefits of exercise performed at self selected workloads: implications for theory and practice. J Sports Sci Med (2003) 2(3): 77–87. 7. AUTHOR CONTRIBUTIONS Jørn Heggelund, Kim Daniel Kleppe, Gunnar Morken, and Einar Vedul-Kjelsås took part in the concept and design of the study. Jørn Heggelund conducted data analyses and prepared the manuscript. Kim Daniel Kleppe recruited participants and collected data, per- formed preliminary analyses and preparation of the manuscript. Gunnar Morken assisted data analyses, interpretation, and prepa- ration of the manuscript. Einar Vedul-Kjelsås assisted with data collection, data analyses, and interpretation, and was involved in the preparation of the manuscript. All authors approved the final manuscript. 16. Bartholomew JB, Morrison D, Ciccolo JT. Effects of acute exercise on mood and well-being in patients with major depressive disorder. Med Sci Sports Exerc (2005) 37(12):2032–7. doi:10.1249/01.mss.0000178101.78322.dd 17. Linaker OM, Moe A. The COOP/WONCA charts in an acute psychiatric ward. Validity and reliability of patients’ self-report of functioning. Nord J Psychiatry (2005) 59(2):121–6. doi:10.1080/08039480510022918 18. Chamove AS. Positive short-term effects of activity on behaviour in chronic schizophrenic patients. Br J Clin Psychol (1986) 25(Pt 2):125–33. doi:10.1111/j. 2044-8260.1986.tb00681.x 19. Faulkner G, Sparkes A. Exercise as therapy for schizophrenia: an ethnographic study. J Sport Exerc Psychol (1999) 21(1):52–70. 20. World Health Organization.The ICD-10 Classification of Mental and Behavioural Disorders: Diagnostic Criteria for Research. (Vol. XIII). Geneva: World Health Organization (1993). 248 p. CONCLUSION Supervised HIT performed at the Hospitals Exercise Training Clinic increased positive affect and well-being and reduced state anxiety and distress. It is therefore possible that patients could have twofold benefits from HIT. Training effectively to improve VO2peak gave acute psychological benefits. Reductions in distress and state anxiety were sustained for more than 3 h after HIT and patients with depression also sustained the improved posi- tive affect and well-being. The duration of the improved positive affect was longer for those with depressive and schizophrenia disorders than in the healthy individuals. The reduction in dis- tress was also larger in patients with depression compared to healthy individuals with a low distress at baseline. It is there- fore reasonable to conclude that patients with reduced affectiv- ity had benefits from HIT that exceeded those in the healthy individuals. 10. Wisloff U, Stoylen A, Loennechen JP, Bruvold M, Rognmo O, Haram PM, et al. Superior cardiovascular effect of aerobic interval training versus moderate con- tinuous training in heart failure patients: a randomized study. Circulation (2007) 115(24):3086–94. doi:10.1161/CIRCULATIONAHA.106.675041 11. Tjonna AE, Lee SJ, Rognmo O, Stolen TO, Bye A, Haram PM, et al. Aer- obic interval training versus continuous moderate exercise as a treatment for the metabolic syndrome: a pilot study. Circulation (2008) 118(4):346–54. doi:10.1161/CIRCULATIONAHA.108.772822 12. Heggelund J, Nilsberg GE, Hoff J, Morken G, Helgerud J. Effects of high aer- obic intensity training in patients with schizophrenia-A controlled trial. Nord J Psychiatry (2011) 65(4):269–75. doi:10.3109/08039488.2011.560278 13. Hennekens CH. Increasing global burden of cardiovascular disease in general populations and patients with schizophrenia. J Clin Psychiatry (2007) 68(Suppl 4):4–7. doi:10.4088/JCP.0507e12 14. Flemmen G, Unhjem R, Wang E. High-intensity interval training in patients with substance use disorder. Biomed Res Int (2014) 2014:616935. doi:10.1155/ 2014/616935 15. Crawford JR, Henry JD. The positive and negative affect schedule (PANAS): construct validity, measurement properties and normative data in a large non-clinical sample. Br J Clin Psychol (2004) 43(Pt 3):245–65. doi:10.1348/ 0144665031752934 REFERENCES Ekkekakis P, Petruzzello SJ. Acute aerobic exercise and affect: current sta- tus, problems and prospects regarding dose-response. Sports Med (1999) 28(5):337–74. doi:10.2165/00007256-199928050-00005 8. Helgerud J, Hoydal K, Wang E, Karlsen T, Berg P, Bjerkaas M, et al. Aerobic high-intensity intervals improve VO2max more than moderate training. Med Sci Sports Exerc (2007) 39(4):665–71. doi:10.1249/mss.0b013e3180304570 9. Rognmo O, Hetland E, Helgerud J, Hoff J, Slordahl SA. High intensity aerobic interval exercise is superior to moderate intensity exercise for increasing aerobic capacity in patients with coronary artery disease. Eur J Cardiovasc Prev Rehabil (2004) 11(3):216–22. doi:10.1097/01.hjr.0000131677.96762.0c DISCUSSION The results from the present study support the hypothesis that supervised HIT improves the psychological state by increasing positive affect and well-being and reduce state anxiety and dis- tress 15 min post-exercise in patients with schizophrenia and in patients with depression. The exact mechanism that led to the improved psychological state is not possible to elucidate from this study. Social, cognitive, and biological mechanisms of perform- ing HIT in a supervised fashion in an Exercise Training Clinic contribute to the psychological response. However, when HIT is presented in this clinical wrapping, the high aerobic intensity falls within the range of intensities that improves psychological states beyond baseline levels already 15 min after cessation. The present study also finds that a prescribed intensity enhances the affective responses. Thus, it is possible to achieve these beneficial affective responses from prescribing an aerobic endurance training pro- gram that is developed for optimizing VO2peak. Exercising at a high aerobic intensity (i.e., 85–95% HRpeak) is fatigable, as should be expected. All subjects rated the intervals in the range from somewhat hard – very hard (heavy), which is reasonably and at a fair distance from maximal exertion. The perception of fatigue, from the SEES questionnaire, is evident 15 min after cessation but is attenuated sometime within 3 h. The psychological states were stable during the no-exercise day, whichsupportstheinfluenceof HIT.Further,thefindingthatsome variables sustained elevated/reduced 3 h post-HIT also supports the hypothesis that they were influenced by HIT rather than being spontaneous changes. Studies also suggest that fitness level might influence the psychological response to exercise (7). VO2peak mea- surements were not collected to explore exact differences between the three groups. However, the speed, incline, and watt load dur- ing the peak exercise test and the HIT intervals did not reveal any significant changes between the groups. We also assume that the familiarization training limited some of the affective responses of attending to an exercise regimen for the first time. This study investigated supervised HIT at the hospitals Exer- cise Training Clinic. Thus, a combination of social, cognitive, and biologicalmechanismslikelyhasbeenresponsiblefortheimprove- ments (29). Performing supervised HIT exposes the patient to Frontiers in Psychiatry | Schizophrenia October 2014 | Volume 5 | Article 148 | 6 Acute psychological responses to aerobic exercise Heggelund et al. REFERENCES Acute psychological responses to aerobic exercise Acute psychological responses to aerobic exercise Heggelund et al. 22. Bodin T, Martinsen EW. Mood and self-efficacy during acute exercise in clin- ical depression. A randomized, controlled study. J Sport Exerc Psychol (2004) 26:623–33. 32. Sloan RA, Sawada SS, Martin CK, Church T, Blair SN. Associations between cardiorespiratory fitness and health-related quality of life. Health Qual Life Out- comes (2009) 7:47. doi:10.1186/1477-7525-7-47 23. Watson D, Clark LA, Tellegen A. Development and validation of brief mea- sures of positive and negative affect: the PANAS scales. J Pers Soc Psychol (1988) 54(6):1063–70. doi:10.1037/0022-3514.54.6.1063 33. Sui X, Laditka JN, Church TS, Hardin JW, Chase N, Davis K, et al. Prospective study of cardiorespiratory fitness and depressive symptoms in women and men. J Psychiatr Res (2009) 43(5):546–52. doi:10.1016/j.jpsychires. 2008.08.002 24. Ostir GV, Smith PM, Smith D, Ottenbacher KJ. Reliability of the positive and negative affect schedule (PANAS) in medical rehabilitation. Clin Rehabil (2005) 19(7):767–9. doi:10.1191/0269215505cr894oa 34. Heggelund J, Hoff J, Helgerud J, Nilsberg GE, Morken G. Reduced peak oxygen uptake and implications for cardiovascular health and quality of life in patients uptake and implications for cardiovascular health and quality of life in patients with schizophrenia. BMC Psychiatry (2011) 11(1):188. doi:10.1186/1471-244X- 11-188 25. McAuley E, Courneya KS. Sport psychology the subjective exercise experiences scale (SEES): development and preliminary validation. J Sport Exerc Psychol (1994) 16(2):163–77. doi:10.1016/j.jbmt.2008.11.005 with schizophrenia. BMC Psychiatry (2011) 11(1):188. doi:10.1186/1471-244X- 11-188 26. Borg G. Perceived exertion as an indicator of somatic stress. Scand J Rehabil Med (1970) 2(2):92–8. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 27. Nakagawa S, Cuthill IC. Effect size, confidence interval and statistical sig- nificance: a practical guide for biologists. Biol Rev Camb Philos Soc (2007) 82(4):591–605. doi:10.1111/j.1469-185X.2007.00027.x Received: 16 May 2014; accepted: 11 October 2014; published online: 30 October 2014. Citation: Heggelund J, Kleppe KD, Morken G andVedul-Kjelsås E (2014) High aerobic intensity training and psychological states in patients with depression or schizophrenia. Front. Psychiatry 5:148. doi: 10.3389/fpsyt.2014.00148 28. Rimer J, Dwan K, Lawlor DA, Greig CA, McMurdo M, Morley W, et al. Exercise for depression. Cochrane Database Syst Rev (2012) 7:CD004366. October 2014 | Volume 5 | Article 148 | 8 ACKNOWLEDGMENTS We acknowledge the participants who took part in the study. We thank physiotherapist Aksel Skjelbred and psychiatric nurse Kari Kristiansen Herre for the contribution in data acquisition. 21. Spielberger CD, Gorsuch RL, Lushene R, Vagg PR, Jacobs GA. Manual for the State-Trait Anxiety Inventory. Palo Alto, CA: Consulting Psychologists Press (1983). October 2014 | Volume 5 | Article 148 | 7 www.frontiersin.org www.frontiersin.org Acute psychological responses to aerobic exercise doi:10.1002/14651858.CD004366 29. Buckworth J, Dishman RK, O’Connor PJ, Tomporowski PD. Depression. In: Schrag M, Zavala M, Cox K, Feeney J, Fortney P, editors. Exercise Psychology. Champaign, IL: Human Kinetics (2013). p. 185–219. This article was submitted to Schizophrenia, a section of the journal Frontiers in Psychiatry. This article was submitted to Schizophrenia, a section of the journal Frontiers in Psychiatry. 30. Gerber M, Lindwall M, Lindegard A, Borjesson M, Jonsdottir IH. Cardiorespira- tory fitness protects against stress-related symptoms of burnout and depression. Patient Educ Couns (2013) 93(1):146–52. doi:10.1016/j.pec.2013.03.021 Copyright © 2014 Heggelund, Kleppe, Morken and Vedul-Kjelsås. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 31. Rieck T, Jackson A, Martin SB, Petrie T, Greenleaf C. Health-related fitness, body mass index, and risk of depression among adolescents. Med Sci Sports Exerc (2013) 45(6):1083–8. doi:10.1249/MSS.0b013e3182831db1 October 2014 | Volume 5 | Article 148 | 8 Frontiers in Psychiatry | Schizophrenia Frontiers in Psychiatry | Schizophrenia
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¿Obligatoriedad de los estudios de precios de transferencia?
Contabilidad y negocios
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cc-by
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Revista del Departamento Académico de Ciencias Administrativas año 2, número 3 julio 2007 TRIBUTACIÓN ¿Obligatoriedad de los estudios de precios de transferencia? Milton Álvarez Eguiluz Pontificia Universidad Católica del Perú Departamento Académico de Ciencias Administrativas Los precios de transferencia aparecieron como consecuencia del proceso de liberalización económica y comercial aplicado en la mayoría de los países desarrollados o en vías de desarrollo, lo que originó un incremento en las transacciones internacionales; además, las empresas multinacionales empezaron a jugar un rol cada vez más importante dentro del comercio mundial. Al existir distintos regímenes impositivos en diversas jurisdicciones, se hacía necesario contar con directrices generales que sean aplicadas por los países de manera uniforme. Con esta inquietud, la Organización para la Cooperación y el Desarrollo Económico (OCDE), organismo no gubernamental que agrupa a más de treinta países del mundo dentro de los que se encuentran las economías más grandes, emitió en 1979 unas directrices sobre precios de transferencia que tuvieron gran acogida y cuya versión aprobada en 1995 y actualizada hasta 1999, constituye hoy por hoy un referente mundial. Es así que con el objeto de evitar que las empresas multinacionales trasladen indebidamente las utilidades de una empresa rentable de un país a otras empresas filiales, afiliadas, sucursales, etcétera, domiciliadas en países donde hayan menores tasas impositivas y que de esta manera eludan el Impuesto a la Renta en el país en donde tales utilidades se generan, la legislación tributaria internacional recogió el Arms Lenght Principle, o principio Julio 2007 de libre o plena concurrencia o del precio justo sin ventaja. Este principio establece que los precios acordados en transacciones efectuadas entre empresas vinculadas económicamente deben realizarse de acuerdo a su valor de mercado, que es el valor que normalmente se obtiene en las operaciones que la empresa realiza con terceros no vinculados en condiciones iguales o similares, o en su defecto se considera el valor que se obtenga en una operación entre sujetos no vinculados en condiciones iguales y similares. Las administraciones tributarias de más de treinta países en el mundo aplican este principio desde el año 1996 para fiscalizar el valor de los precios de los bienes y servicios de las transacciones realizadas con empresas relacionadas. Así verifican que se hayan efectuado a su valor de mercado, es decir, que no se hayan subvaluado o sobrevaluado. En el presente trabajo vamos a desarrollar en forma conceptual y objetiva los aspectos siguientes: - ¿Qué son los precios de transferencia? ¿Cuáles son las transacciones de compraventa de bienes y prestación de servicios más frecuentes con empresas vinculadas del exterior? La obligatoriedad legal de las empresas en el Perú de realizar estos estudios a partir del ejercicio 2001. El desarrollo de los estudios de precios de transferencia. Contabilidad y negocios 15 - - Los métodos de valoración internacionalmente aceptados. Los ajustes a los resultados tributarios. Obligaciones tributarias formales. Infracciones tributarias y sanciones. La importancia de los estudios de precios de transferencia para las empresas peruanas. ¿Quiénes están obligados a presentar la declaración jurada y los informes de estudios de precios de transferencia a partir del 2006? ¿Quiénes están exceptuados de presentar la declaración jurada y los informes de estudios de precios de transferencia en los años 2006 y 2007? Las consecuencias de no realizar los estudios de precios de transferencia. 1. ¿Qué son los precios de transferencia? En términos de la Organización para la Cooperación y el Desarrollo Económico (OCDE), «los precios de transferencia son los precios a los que una empresa transfiere bienes físicos, propiedad intelectual o presta servicios a una empresa relacionada». 2. ¿Cuáles son las transacciones de compraventa de bienes y prestación de servicios más frecuentes con empresas vinculadas del exterior? a) Compraventa de materia prima o de productos terminados. b) Prestación de servicios administrativos o de servicios de asistencia técnica. c) Transferencia de tecnología. d) Arrendamiento de bienes muebles e inmuebles. e) Transferencias o cesión en uso de intangibles —patentes, marcas, gastos de investigación y desarrollo, know how—. f) Operaciones financieras y bursátiles —préstamos, compraventa de acciones-subcapitalización, operaciones de financiamiento, futuros, derivados, warrants, entre otras—. g) Compraventa de divisas —sport, forward, entre otras—. 16 Contabilidad y negocios h) Distribución de la asunción de costos generales de dirección y administración —management fee, gerenciamiento—. 3. La obligatoriedad legal de las empresas en el Perú de realizar estudios de precios de transferencia a partir del ejercicio 2001 En el Perú, a partir del 1 de enero de 2001, para los efectos de la determinación de las bases imponibles del Impuesto a la Renta y del Impuesto General a las Ventas, los precios de transferencia de bienes que los contribuyentes acuerden en las transacciones con empresas vinculadas económicamente y con empresas con residencia en territorios con baja o nula imposición, deberán estar sustentados con la documentación e información sobre los métodos de valoración utilizados para la determinación de sus precios de transferencia, señalando los criterios y los elementos objetivos considerados. Similar obligación se ha establecido a partir del 1 de enero de 2003 para las prestaciones de servicios a terceros independientes o compañías no vinculadas. La legislación peruana de precios de transferencia vigente, hasta el 31 de diciembre de 2003, establece un orden de prelación para su aplicación, debiéndose contar con un comparable interno producto de las operaciones independientes con terceros no vinculados. Excepcionalmente, se debe buscar el precio comparable de terceros no vinculados entre sí, vale decir, un referente externo en condiciones iguales o similares. Los métodos del costo incrementado, precio de reventa y, en su caso, los métodos basados en las utilidades, fueron considerados de aplicación supletoria como una obligación exclusiva de la SUNAT. Hasta el 31 de diciembre de 2003 el ajuste de valor establecido por la administración tributaria era un ajuste primario unilateral que no compensaba a la otra parte implicada en la transacción, aun tratándose de operaciones nacionales. En este supuesto, se afecta el principio de igualdad en tanto se genera una doble tributación. El tratamiento dispuesto por la Ley del Impuesto a la Renta (LIR) hasta el 31 de diciembre de 2003 se aplica a las transacciones locales e internacionales sin ningún prerrequisito. En este período no se consideraron transacciones comparables a aquellas no producidas en el curso ordinario de los negocios. entre partes vinculadas celebradas a título oneroso o gratuito. Debido a ello, las empresas que realicen transferencias gratuitas de bienes a sus partes vinculadas deberán estimar una ganancia presunta basada en uno de los métodos de precios de transferencia. Bajo la legislación sobre precios de transferencia vigente hasta el 31 de diciembre de 2003, resultaba conveniente contar con un estudio técnico basado en las políticas de precios y descuentos de la empresa, como una aplicación del método del precio comparable no controlado. Desde el 1 de enero de 2004 el ajuste primario será bilateral para las transacciones entre partes vinculadas con efectos para el transferente y para el adquiriente cuando ambos se encuentran domiciliados. Esta circunstancia no es aplicable para las transacciones internacionales con distinta jurisdicción para efectos tributarios, con excepción de las transacciones efectuadas con empresas de países con los que el Perú ha suscrito un convenio para evitar la doble imposición. La diferencia entre el tratamiento de precios de transferencia para partes vinculadas y el valor de mercado de aplicación general a todas las empresas se encuentra determinada por la aplicación de métodos de valuación y la incorporación de ajustes a las operaciones. Los métodos no solo se refieren a precios, sino a la comparación con información financiera de empresas independientes, en condiciones iguales o similares. El artículo 24 del Reglamento excede los alcances de la LIR cuando establece un criterio de vinculación comercial no considerado por esta, lo que atenta contra los principios de legalidad y jerarquía de las normas. La vinculación comercial puede significar una dependencia económica del proveedor, pero no implica necesariamente administración, capital o control de sus decisiones. Por resolución de la SUNAT, se puede exceptuar del tratamiento de precios de transferencia a determinadas operaciones entre partes vinculadas a criterio de esta entidad. Esta facultad razonablemente utilizada, debe contribuir a garantizar una mejor administración del impuesto. A partir del 1 de enero de 2004 las normas sobre precios de transferencia se aplican a las transacciones Julio 2007 4. El desarrollo de los estudios de precios de transferencia Para realizar los estudios de precios de transferencia se necesita evaluar la documentación e información financiera y descriptiva de las operaciones que las empresas realicen con partes relacionadas residentes en el extranjero. Este estudio se divide en dos partes: (a) El análisis funcional. Que brinda un panorama preciso de la industria, del negocio y del mercado dentro del cual se desarrollan las transacciones sujetas a análisis, identificando los riesgos asumidos por las partes involucradas y los recursos empleados en las actividades desarrolladas. (b) El análisis económico. Que constituye el informe central de la documentación y respaldo probatorio para demostrar a las autoridades tributarias el cumplimiento del valor de mercado por cada transacción involucrada, mediante la aplicación de los métodos previstos en la legislación tributaria vigente. Contabilidad y negocios 17 5. Métodos de valoración internacionalmente aceptados Para determinar el valor de mercado de las transacciones con empresas vinculadas, el literal e del artículo 32-A del decreto legislativo 945 de la LIR, vigente a partir del 1 de enero de 2004, ha establecido los siguientes métodos de valoración internacionalmente aceptados, que a partir de 1979 fueron aportados por la Organización para la Cooperación y el Desarrollo Económico (OCDE): a) El método del precio comparable no controlado. Es el más apropiado para el principio de libre concurrencia, en tanto permite comparar los precios de bienes o servicios que se transfieren en una operación entre partes vinculadas con el precio obtenido en una operación independiente. b) El método del precio de reventa. Resulta más exacto cuando existe similitud en los bienes transferidos y en las funciones de comercialización. Método plenamente aplicable cuando el revendedor no incorpora valor al producto, ejecutando una simple distribución. c) El método del costo incrementado. Es el más adecuado para operaciones entre partes vinculadas de bienes semiterminados, suministros de larga duración y servicios. d) El método del margen neto transaccional. Es utilizado en operaciones complejas en las que existen prestaciones o funciones estrechamente relacionadas, los márgenes brutos no son identificados con claridad o resulta difícil obtener información confiable de alguna de las partes involucradas en la transacción. e) El método de partición de utilidades. Para su aplicación será necesario efectuar un análisis riguroso de las operaciones, considerando hechos y circunstancias concretas, las funciones desempeñadas por cada parte, los riesgos asumidos y los activos implicados. Se deberá aplicar el método de valoración que resulte más apropiado para reflejar la realidad económica de la operación bajo análisis. 18 Contabilidad y negocios 6. Los ajustes a los resultados tributarios Si la administración tributaria tuviera información de otras operaciones similares entre empresas no vinculadas, por las que se hayan pactado precios distintos a los acordados por las empresas relacionadas, entonces podría ajustar los valores pactados por ellas, determinando un ajuste unilateral de las bases imponibles de los IR e IGV, notificando a la empresa fiscalizada las resoluciones de determinación y de multa correspondientes. Dichas resoluciones contendrían una deuda tributaria conformada por el importe de tales tributos, más multas, que pueden alcanzar hasta el 50% de los mencionados tributos, e intereses moratorios, a razón de 1,5% mensual, sobre tales tributos y sanciones administrativas fiscales. 7. Obligaciones tributarias formales a) Los contribuyentes sujetos a las reglas de precios de transferencia deben presentar una declaración jurada anual de sus transacciones con partes vinculadas o con sujetos residentes en países o territorios de baja o nula imposición. b) Toda la documentación de sustento de las transacciones a precios de transferencia, incluyendo papeles de trabajo que demuestren que las operaciones son efectuadas a valores de mercado, debe ser mantenida por los contribuyentes durante el plazo de prescripción del tributo, traducida al castellano, de ser el caso. c) Los contribuyentes que se encuentren dentro del ámbito de aplicación de precios de transferencia deberán contar con un estudio técnico que respalde las operaciones efectuadas y los métodos utilizados. 8. Infracciones tributarias y sanciones El incumplimiento en la presentación de la declaración jurada especial anual y de contar con un estudio de precios de transferencia, así como la ausencia de la documentación requerida por la LIR, constituyen infracciones formales que pueden ser sancionadas con multas por la administración tributaria. En efecto, de acuerdo con el decreto legislativo 953 del 5 de febrero de 2004, que modificó el Código Tributario, se han establecido sanciones administrativas por cometer las siguientes infracciones: a) No contar con la documentación e información que respalde el cálculo de precios de transferencia —multa de 2 UIT, equivalentes a S/. 6.900—. b) No contar con el estudio de precios de transferencia —multa de 30 UIT, equivalentes a S/. 103.500—. Esta sanción solo es aplicable a partir del 2006. c) No conservar la documentación e información que respalde el cálculo de precios de transferencia —multa de 30 UIT, equivalentes a S/. 103.500—. d) No presentar el estudio técnico de precios de transferencia —multa de 15 UIT, equivalentes a S/. 51.750—. 9. La importancia de los estudios de precios de transferencia para las empresas peruanas Es evidente que este tema se ha tornado sumamente importante: (a) por la posibilidad de que las empresas puedan adoptar un enfoque estratégico preventivo de precios de transferencia que abarque tanto el planeamiento operacional como el impositivo; y (b) por el riesgo de las elevadas multas que se derivan de su incumplimiento. Por todo ello, las empresas deben sustentar con estudios de precios de transferencia el valor de mercado de las transacciones de compra y venta de bienes y servicios con empresas vinculadas y de servicios con terceros no vinculados o entidades independientes, a fin de minimizar la carga impositiva global, mejorar la rentabilidad, y a la vez, evitar que en una fiscalización de los impuestos a la renta y general a las ventas correspondientes a los ejercicios abiertos a revisión fiscal, la administración tributaria ajuste tales valores por subvaluación o sobrevaluación. Julio 2007 10. ¿Quiénes están obligados a presentar la declaración jurada y los informes de estudios de precios de transferencia a partir del 2006? El sábado 14 de octubre de 2006 se publicó en el diario oficial El Peruano la resolución de superintendencia 167-2006-SUNAT, que establece qué contribuyentes deben presentar declaración jurada informativa de Precios de Transferencia (PT) y cuáles deberán contar con un estudio técnico de PT. 10.1. ¿Quiénes deberán presentar una declaración jurada de PT? Deberán presentar esta declaración jurada anual los contribuyentes que en el ejercicio 2006 hayan realizado operaciones con empresas vinculadas por un total de más de doscientos mil nuevos soles o hubieran realizado al menos una transacción desde, hacia o a través de países de baja o nula imposición fiscal. Como se puede apreciar, la obligación de informar las operaciones con compañías establecidas en países de baja o nula imposición, corre por separado de la obligación de informar operaciones con compañías vinculadas. En efecto, de realizar ambos tipos de operaciones, el contribuyente necesariamente deberá informar las primeras, pero solo deberá informar las segundas si estas superan los doscientos mil nuevos soles. Asimismo, cabe destacar que para el cálculo del monto de las operaciones con compañías vinculadas, se deben sumar tanto las que dan lugar a ingresos como las que dan lugar a gastos, en vez de calcular un monto neto. Por otra parte, no se ha establecido un nivel de materialidad individual por transacción, sino que una vez que la sumatoria supera el límite establecido, deberán declararse todas las transacciones, incluso aquellas de muy bajo monto. Contabilidad y negocios 19 Hemos sido informados que el nuevo Programa de Declaración Telemática (PDT) para precios de transferencia, que está preparando la SUNAT, incluiría tres grandes campos: información sobre el declarante, sobre los informados —empresas vinculadas— y sobre cada transacción. El PDT de precios de transferencia comprendería puntos como: nombres completos de las partes vinculadas o sujetos domiciliados en paraísos fiscales; razón social o número de identificación personal o tributaria del país de origen; el registro del monto de las transacciones, tanto en la moneda en la cual se llevó a cabo la transacción como en aquella en la cual se registró contablemente. De la misma manera, los préstamos tendrían un formato de declaración exclusivo, en el cual se identificaría el capital, la tasa de interés pactada, los intereses pagados y el saldo al cierre del ejercicio. Adicionalmente, por cada transacción declarada se deberá especificar la metodología escogida para evaluar cada operación, según lo establecido en el nuevo reglamento de precios de transferencia, expedido mediante el decreto supremo 190-2005-EF. excepción, pero solo deberán analizarse las primeras cuando se superen los límites establecidos. 10.2. ¿Quiénes están obligados a contar con un estudio técnico de PT? a) El monto de operaciones al que se refiere el inciso a del indicado artículo 4, se determinará sin tomar en cuenta las transacciones que los contribuyentes domiciliados en el país realicen con sus partes vinculadas domiciliadas. b) No será de aplicación respecto a las transacciones que los contribuyentes domiciliados en el país realicen con sus partes vinculadas domiciliadas. Deben contar con dicho estudio los contribuyentes cuyos ingresos devengados superen los seis millones de nuevos soles —en el ejercicio 2006— y cuyo monto de operaciones con compañías vinculadas —se entiende— supere el millón de nuevos soles. Asimismo, deben contar con el estudio de PT las compañías que realicen una o más operaciones hacia, desde o a través de compañías radicadas en países de baja o nula imposición fiscal, sin importar el monto de las mismas. Nuevamente, la obligación de contar con el estudio de PT es distinta para transacciones celebradas con compañías vinculadas —locales o del exterior—, de la existente para transacciones celebradas con compañías radicadas en países de baja o nula imposición —vinculadas o no vinculadas—. En este último caso se debe contar con el estudio técnico sin 20 Contabilidad y negocios 11. ¿Quiénes están exceptuados de presentar la declaración jurada y los informes de estudios de precios de transferencia en los años 2006 y 2007? El domingo 7 de enero de 2007 se publicó en el diario oficial El Peruano la resolución de superintendencia 008-2007-SUNAT, que amplía las excepciones a la obligación de contar con estudio técnico de precios de transferencia (PT). Mediante dicha resolución, la SUNAT —haciendo uso de sus facultades para efectuar excepciones relacionadas con la obligación de contar con el estudio técnico de PT— ha establecido que la obligación a la que hacía referencia el artículo 4 de la resolución de superintendencia 167-2006-SUNAT, respecto de las operaciones realizadas por los contribuyentes durante los ejercicios gravables 2006 y 2007, estará sujeta a: Como se sabe, el inciso a del artículo 4 establecía la obligación de contar con dicho estudio a los contribuyentes cuyos ingresos devengados superen los seis millones de nuevos soles —por ejercicio gravable— y cuyo monto de operaciones con compañías vinculadas supere el millón de nuevos soles. Por tanto, según lo establecido por la mencionada resolución recientemente publicada, la obligación de contar con dicho estudio técnico no será aplicable para las operaciones realizadas entre contribuyentes locales. Asimismo, tampoco se tomarán en cuenta las transacciones que se realicen entre contribuyentes locales para el cálculo del monto de operaciones con vinculadas —un millón de nuevos soles— que determina la obligación de contar con estudio técnico. Es decir, si una empresa tiene transacciones con compañías vinculadas locales por valor de dos millones de nuevos soles y transacciones con compañías vinculadas del exterior por medio millón de nuevos soles, solo se contabilizará, para la determinación de la obligación de contar con el estudio, las transacciones con vinculadas del exterior —medio millón de nuevos soles—, de modo que dicha empresa estaría exenta de la obligación. si sus operaciones se celebraron como lo hubiesen hecho partes independientes. 12. Algunas reflexiones sobre las consecuencias de no realizar los estudios de precios de transferencia Finalmente, cabe tomar en consideración que la SUNAT está especializándose desde hace varios años en esta normatividad, y que se espera que los procesos de fiscalización en esta materia empiecen este año. Por tanto, al documentar los precios de transferencia, aun si no se estuviera obligado a contar con el estudio técnico de PT, se estaría trasladando la carga de la prueba al fisco, forzado a demostrar que el análisis efectuado por el contribuyente no es el correcto, mediante la elaboración de otro estudio de precios de transferencia. No obstante la indicada excepción de contar con el estudio técnico, es importante considerar que todos los contribuyentes que mantienen operaciones con vinculadas deben actuar bajo el principio de valor de mercado, es decir, los contribuyentes deberán determinar si compraron o vendieron a valor de mercado de acuerdo con los métodos de valoración indicados en el artículo 32-A inciso e de la Ley del Impuesto a la Renta. Por tanto, el hecho de que existan contribuyentes exceptuados de contar con el estudio técnico no significa que estos estén excluidos de la normatividad de PT. Dado ello, el contribuyente no está exento de ser acotado por problemas de PT si la administración tributaria determinara que no operó con valor de mercado. Por ello, aun en el supuesto de no estar obligados a contar con un estudio técnico de PT, es recomendable que los contribuyentes tengan la seguridad de que sus operaciones se pactaron a valores de mercado, para lo cual es sumamente importante que evalúen bajo los métodos de PT Julio 2007 Un punto importante por mencionar es el hecho de que si bien la resolución de superintendencia 167-2006-SUNAT exceptúa de contar con el estudio técnico, ella no realiza excepciones respecto de la presentación de la declaración jurada de PT, con lo cual los precios y montos de las contraprestaciones acordados por el contribuyente con sus vinculadas serán informados al fisco, quien, de presumir que pudiera existir algún problema de PT, podría realizar una fiscalización que derive en la emisión y notificación de resoluciones de determinación y de multa. A partir de lo expuesto, consideramos que las empresas deben contar con un estudio de precios de transferencia que demuestre a las autoridades tributarias que las transacciones de bienes y servicios efectuadas entre partes relacionadas, como las de servicios efectuadas con terceros no vinculados, se han realizado con valores de mercado. Bibliografía consultada Organización para la Cooperación y el Desarrollo Económico 1995 Directrices aplicables en materia de precios de transferencia para empresas multinacionales y administraciones tributarias. México D.F.: OECD. Contabilidad y negocios 21 Legislación peruana vigente sobre precios de transferencia «Decreto Legislativo Nº 945» El Peruano, 23 de diciembre de 2003 (vigente a partir de 1 de enero de 2004). «Decreto Legislativo Nº 953. Modifica el Código Tributario para establecer sanciones administrativas por cometer infracciones tributarias». El Peruano, 5 de febrero de 2004 (vigente a partir de 1 de enero de 2006). 22 Contabilidad y negocios «Decreto Supremo Nº 179-2004-EF. Texto Único Ordenado de la Ley de Impuesto a la Renta». El Peruano, 8 de diciembre de 2004. «Decreto Supremo Nº 190-2005-EF. Reglamento de la Ley del Impuesto a la Renta». El Peruano, 31 de diciembre de 2005 (vigente a partir de 1 de enero de 2006). «Resolución de Superintendencia Nº 167-2006SUNAT». El Peruano, 14 de octubre de 2006. «Resolución de Superintendencia Nº 008-2007SUNAT». El Peruano, 7 de enero de 2007
https://openalex.org/W3138492038
https://www.nature.com/articles/s41598-021-85670-z.pdf
English
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Administration of β-lactam antibiotics and delivery method correlate with intestinal abundances of Bifidobacteria and Bacteroides in early infancy, in Japan
Scientific reports
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www.nature.com/scientificreports www.nature.com/scientificreports Administration of β‑lactam antibiotics and delivery method correlate with intestinal abundances of Bifidobacteria and Bacteroides in early infancy, in Japan Naruaki Imoto1*, Chie Kano2, Yumi Aoyagi2, Hiroto Morita2, Fumitaka Amanuma3, Hidekazu Maruyama3, Shuko Nojiri4, Naoyuki Hashiguchi5 & Shin Watanabe1 The intestinal microbiome changes dynamically in early infancy. Colonisation by Bifidobacterium and Bacteroides and development of intestinal immunity is interconnected. We performed a prospective observational cohort study to determine the influence of antibiotics taken by the mother immediately before delivery on the intestinal microbiome of 130 healthy Japanese infants. Faecal samples (383) were collected at 1, 3, and 6 months and analysed using next-generation sequencing. Cefazolin was administered before caesarean sections, whereas ampicillin was administered in cases with premature rupture of the membranes and in Group B Streptococcus-positive cases. Bifidobacterium and Bacteroides were dominant (60–70% mean combined occupancy) at all ages. A low abundance of Bifidobacterium was observed in infants exposed to antibiotics at delivery and at 1 and 3 months, with no difference between delivery methods. A lower abundance of Bacteroides was observed after caesarean section than vaginal delivery, irrespective of antibiotic exposure. Additionally, occupancy by Bifidobacterium at 1 and 3 months and by Bacteroides at 3 months differed between infants with and without siblings. All these differences disappeared at 6 months. Infants exposed to intrapartum antibiotics displayed altered Bifidobacterium abundance, whereas abundance of Bacteroides was largely associated with the delivery method. Existence of siblings also significantly influenced the microbiota composition of infants. The human body is inhabited by 100–1000 trillion bacteria in the oral cavity, skin, and intestine, influencing the biological health of the ­host1,2. There are large differences in the composition of the human intestinal microbiome depending on race, country, and ­lifestyle3–5. Although the predominance of Bifidobacterium in the intestinal microbiome has been identified in Japanese children, there are very few studies on the intestinal microbiome specifically in Japanese ­infants5–10. i Many studies have indicated an association between the intestinal microbiome and ­disease11–13. Colonisation by intestinal bacteria in early infancy is known to have a major effect on intestinal ­immunity14–16. In particular, colonisation by the dominant bacteria such as Bifidobacteria and Bacteroides17–23, resulting from the start of food ingestion at 6 months after birth has been implied to be linked with the onset of diseases such as ­allergies24–26. Scientific Reports | (2021) 11:6231 www.nature.com/scientificreports/ and antibiotic administration in the late perinatal ­period30. These studies, however, have reported differences in the administration and screening period, and offer inconclusive results. The influence of the delivery method on the Bifidobacteria and Bacteroides populations in infants has also been extensively ­studied16,31–33. It is a widely accepted hypothesis that as infants delivered by caesarean section do not pass through the birth canal, they do not come in contact with the maternal bacterial microbiome, thus influencing the microbiome of the ­infant34,35. However, the actual influence of caesarean sections on infant intestinal microbiome differs among studies and is, therefore, uncertain. Moreover, the prophylactic administration of antibiotics immediately before surgery in caesarean sections, while the foetus and umbilical cord are connected, has not been considered in most ­studies36,37. A recent ­study38 investigated the effect of exposure to cefloxime prior to caesarean section on the intestinal microbiome of infants at 10 days and 9 months of age. No distinct difference in gut microbiota composition was observed at 10 days postnatal. The nutrition ­method35,39, presence or absence of ­siblings19,40,41, and gestational ­age33,42,43 may also influence the intestinal microbiome.l g g yl We previously examined the influence of antibiotics administered immediately before delivery on intestinal colonisation of Bifidobacteria in a pilot study of 1-month-old healthy Japanese ­infants44. This study showed that antibiotic administration to the mother at the time of delivery has a strong influence on the Bifidobacteria population and that this influence may even be more substantial than that of caesarean section. l Unlike previous studies of IAP, this pilot study was unique in a sense that the antibiotics were used for GBS and IAP in cases of premature rupture of the membranes (PROM) and routinely before caesarean section. i.e., antibiotics were used in the mother immediately before delivery, while the umbilical cord was still intact. Inter- estingly, the results revealed a significantly higher bifidobacterial occupancy in infants with siblings. In contrast, Bacteroides occupancy was substantially influenced by delivery mode compared to antibiotic exposure at delivery. As the pilot ­study43 had a cross-sectional design and was limited to 33 1-month-old infants, a continuous study comprising a larger group of infants was needed to confirm the results.i i Based on the above background, the present study defined antibiotic exposure of infants through antibiotics administered to their mothers immediately before delivery as antimicrobial exposure at delivery (AED). Results S bj t Subject data. A total of 142 mother and infant pairs were registered in the study, and 424 samples were obtained. Among these, 130, 127, and 126 samples collected at 1, 3, and 6 months, respectively, adhered to the inclusion criteria of the study. Subjects were excluded due to the following reasons: three premature babies dropped out by 1 month of age, one infant received antibiotic administration for fever in the neonatal period, and there were eight cases at 1 month with samples that could not be analysed. While dropouts from 1 to 3 months were due to antibiotic administration in eight cases, no samples were received in one case, and a non- analysable sample was received at 3 months in one case, dropouts from 3 to 6 months were due to antibiotic administration in one case and a non-analysable sample at 6 months in one case.hl The background information of the mothers and infants at 1 month, which were influencing factors, are shown in Table 1. As the dropout cases resulted in no major change in the mean value of the background factors, the data at 3 and 6 months are shown in Supplementary Table S1 online. Antibiotics were used immediately before deliv- ery in about 55% of cases at each age. caesarean section, GBS-positive, and PROM cases accounted for about 20%, 15%, and 15% of all cases at each age, respectively. The PROM cases included one emergency and 6 GBS-positive caesarean sections. These seven cases were included in the caesarean section group because cefazolin (CEZ) was administered as an antibiotic. In 5 PROM cases, delivery progressed rapidly, and no antibiotic was used. Of the infants born by caesarean section, the Apgar score at birth was low in two cases, oxygen was administered after birth for mild neonatal respiratory disorder in one case, phototherapy was performed for neonatal jaundice in one case, the mothers had diabetes in two cases, the mothers had hyperthyroidism in two cases, and one infant was admitted for respiratory syncytial virus (RSV) infection during the observation period and discharged after symptomatic treatment. Regarding the nutritional method, the infants enrolled in this study were those that were solely breastfed and those that were mix-fed, and none were fed milk only. All infants were confirmed to be healthy via a health examination at each age by physicians of the paediatrics and neonatology department. www.nature.com/scientificreports/ Based on the hypothesis that AED may have a strong influence on the intestinal microbiome of healthy Japanese children in early infancy, especially on the dominant bacterial genera Bifidobacteria and Bacteroides, samples were collected from infants until 6 months after birth, and the influence of AED and other factors in the infant population were investigated. Administration of β‑lactam antibiotics and delivery method correlate with intestinal abundances of Bifidobacteria and Bacteroides in early infancy, in Japan Various factors are known to influence bacterial colonisation, including maternal exposure to antibiotics admin- istered as intrapartum antimicrobial prophylaxis (IAP) in Group B Streptococcus (GBS)-positive ­mothers27–29 1Department of Microbiome Research, School of Medical Science, Juntendo University, Hongou 2‑1‑1, Bunkyo Ward, Tokyo  113‑8421, Japan. 2Core Technology Laboratories, Asahi Group Holdings, Ltd., Sagamihara, Kanagawa, Japan. 3Department of Paediatrics, Department of Neonatology, Iwate Prefectural Iwai Hospital, Ichinoseki, Iwate, Japan. 4Juntendo Clinical Research Support Centre, Juntendo University, Bunkyo Ward, Tokyo, Japan. 5Department of Emergency and Disaster Medicine, School of Medical Science, Juntendo University, Bunkyo Ward, Tokyo, Japan. *email: nimoto@juntendo.ac.jp Scientific Reports | (2021) 11:6231 | https://doi.org/10.1038/s41598-021-85670-z Scientific Reports | (2021) 11:6231 www.nature.com/scientificreports/ Results S bj t Background factors Data Number of infants 130 Number of females 70 (54.0%) Gestational age at ­birtha 275.1 ± 9.3 Birth ­weighta 3038.9 ± 339.7 Maternal antimicrobial use at delivery 74 (56.9%) Caesarean section 31 (23.8%) Premature rupture of membrane 22 (16.9%) Group B Streptococcus-positive status 33 (25.4%) Infants with older siblings 67 (51.5%) Exclusive Breast feeding 80 (61.5%) Age of ­Mothersa 31.6 ± 5.1 Maternal history of allergy 55 (42.3%) Neonatal respiratory disorder 2 (1.5%) Neonatal jaundice 4 (3.1%) RSV infection 3 (2.3%) Maternal history of smoking 9 (6.9%) Maternal history of Hyperthyroidism 2 (1.5%) Maternal history of Diabetes Mellitus 2 (1.5%) Background factors Data Number of infants 130 Number of females 70 (54.0%) Gestational age at ­birtha 275.1 ± 9.3 Birth ­weighta 3038.9 ± 339.7 Maternal antimicrobial use at delivery 74 (56.9%) Caesarean section 31 (23.8%) Premature rupture of membrane 22 (16.9%) Group B Streptococcus-positive status 33 (25.4%) Infants with older siblings 67 (51.5%) Exclusive Breast feeding 80 (61.5%) Age of ­Mothersa 31.6 ± 5.1 Maternal history of allergy 55 (42.3%) Neonatal respiratory disorder 2 (1.5%) Neonatal jaundice 4 (3.1%) RSV infection 3 (2.3%) Maternal history of smoking 9 (6.9%) Maternal history of Hyperthyroidism 2 (1.5%) Maternal history of Diabetes Mellitus 2 (1.5%) Background factors Data Number of infants 130 Number of females 70 (54.0%) Gestational age at ­birtha 275.1 ± 9.3 Birth ­weighta 3038.9 ± 339.7 Maternal antimicrobial use at delivery 74 (56.9%) Caesarean section 31 (23.8%) Premature rupture of membrane 22 (16.9%) Group B Streptococcus-positive status 33 (25.4%) Infants with older siblings 67 (51.5%) Exclusive Breast feeding 80 (61.5%) Age of ­Mothersa 31.6 ± 5.1 Maternal history of allergy 55 (42.3%) Neonatal respiratory disorder 2 (1.5%) Neonatal jaundice 4 (3.1%) RSV infection 3 (2.3%) Maternal history of smoking 9 (6.9%) Maternal history of Hyperthyroidism 2 (1.5%) Maternal history of Diabetes Mellitus 2 (1.5%) Table 1. Background factors of 1-month-old infants and their mothers. RSV respiratory syncytial virus. a Gestational age at birth, birth weight, and maternal age are shown as the mean ± standard deviation. Other factors are given as the number of subjects and a percentage. Table 1. Background factors of 1-month-old infants and their mothers. RSV respiratory syncytial virus. a Gestational age at birth, birth weight, and maternal age are shown as the mean ± standard deviation. Other factors are given as the number of subjects and a percentage. Figure 1. Results S bj t y g y y gy As for the sequencing data, input numeric totaled 15,957,768 reads, an average of 41,665 reads, a maximum of 261,246 reads, and a minimum of 17,884 reads. In addition, non-chimeric numeric reads totaled 11,055,110, an average of 28,864, a maximum of 180,762, and a minimum of 12,828.h g The top 20 bacterial genera constituting the intestinal microbiome at each age are shown in Fig. 1. At 1 month, based on the occupancy, the Bifidobacteria population was found to be overwhelmingly dominant (49.7% ± 34.1%), whereas the Bacteroides population was found to be the third most dominant (7.7% ± 12.6%), but its occupancy was almost equivalent to the second most dominant bacteria, Streptococcus (7.8% ± 12.6%). The Bifidobacteria population was also the most dominant at 3 months (61.7% ± 28.0%), whereas the Bacteroides population was the second most dominant (6.5% ± 10.9%). The Bifidobacteria population continued to be the most dominant at 6 months (66.2% ± 21.6%), followed by the Bacteroides population (5.7% ± 9.2%). Analysis at 1 month. The effects of background factors on the five most dominant bacterial genera of the intestinal microbiome of 1-month-old infants are shown in Table 2. Bifidobacteria occupancy was significantly dependent on the exposure to AED (non-AED: Odds Ratio (OR), 0.11; 95% Confidence Interval (CI), 0.03– 0.39) and the existence of siblings (no sibling: OR 3.03; 95% CI 1.09–8.4), whereas that of Bacteroides signifi- Scientific Reports | (2021) 11:6231 | https://doi.org/10.1038/s41598-021-85670-z www.nature.com/scientificreports/ Table 1. Background factors of 1-month-old infants and their mothers. RSV respiratory syncytial virus. a Gestational age at birth, birth weight, and maternal age are shown as the mean ± standard deviation. Other factors are given as the number of subjects and a percentage. Results S bj t Variances Bifidobacterium Streptococcus Bacteroides Clostridium Escherichia Odds Ratio 95% CI Odds Ratio 95% CI Odds Ratio 95% CI Odds Ratio 95% CI Odds Ratio 95% CI Gestational ­daysa  1 0.96 0.91 1.02 1.05 0.99 1.11 0.93 0.87 0.98 * 1.09 1.03 1.14 ** 0.92 0.87 0.98 **  2 1.03 0.97 1.09 1.03 0.98 1.09 0.89 0.83 0.94 ** 1.06 1.002 1.13 * 0.93 0.88 0.99 *  3 0.95 0.90 1.00 1.01 0.96 1.07 1.00 0.95 1.07 1.03 0.97 1.10 0.96 0.91 1.02 Birth ­weighta  1 0.999 0.998 1.00 1.001 0.99 1.002 0.999 0.998 1.00 1.002 1.00 1.003 0.998 0.996 0.999 **  2 0.999 0.998 1.00 1.001 0.99 1.002 0.998 0.99 1.00 1.001 1.00 1.003 0.998 0.99 1.00  3 0.999 0.998 1.00 0.999 0.99 1.001 0.999 0.99 1.00 1.001 1.00 1.003 0.999 0.998 1.001 Age of ­mothersa  1 1.00 0.91 1.11 1.02 0.93 1.13 1.09 0.94 1.14 0.91 0.83 0.99 * 1.07 0.97 1.18  2 0.96 0.87 1.06 1.00 0.91 1.11 1.03 0.94 1.14 0.94 0.85 1.04 1.13 1.02 1.25  3 0.98 0.89 1.08 1.03 0.94 1.14 1.10 1.00 1.21 0.96 0.86 1.07 1.05 0.96 1.16 Male 1.47 0.57 3.75 1.20 0.57 2.53 1.35 0.56 3.23 0.85 0.33 2.18 1.45 0.67 3.14 Vaginal delivery 1.84 0.49 6.92 1.06 0.32 3.54 0.026 0.003 0.23 ** 4.94 1.10 22.23 * 0.48 0.14 1.68 Infants without siblings 3.03 1.09 8.43 * 1.19 0.56 2.57 0.96 0.40 2.33 0.22 0.072 0.66 ** 0.84 0.38 1.87 Non-AED 0.11 0.03 0.39 *** 1.22 0.53 2.82 0.65 0.27 1.56 4.98 1.69 14.69 ** 0.75 0.32 1.77 Mothers without allergy 1.33 0.51 3.50 1.27 0.60 2.72 1.14 0.46 2.78 0.85 0.33 2.23 1.62 0.73 3.58 Exclusively breast-fed 1.06 0.41 2.73 2.06 0.96 4.41 1.61 0.66 3.94 5.88 2.24 15.43 *** 0.97 0.44 2.13 Table 2. Influence of background factors of the mother and child at 1 month on occupancies of the five most dominant bacterial genera in the intestinal microbiome of the infant. The bacterial genera are shown from left to right in the order of higher occupancy (mean). For the continuous variables (a gestational age at birth, birth weight, and maternal age), occupancy was classified from 1 to 4 in ascending order from low occupancy with the occupancy of each bacterial species rounded to four decimal places. Results S bj t The bacterial genera are shown from left to right in the order of higher occupancy (mean). For the continuous variables (a gestational age at birth, birth weight, and maternal age), occupancy was classified from 1 to 4 in ascending order from low occupancy with the occupancy of each bacterial species rounded to four decimal places. For the high occupancy group (group 4), a multinomial logistic regression analysis was performed as the category standard and the occupancy was classified into two groups based on the median for each genus for the logistic regression analysis for categorical variables. The odds ratio and 95% confidence interval were calculated using the logistic regression analysis and the multinomial regression analysis. The significance level was set at 5%. *p < 0.05, **p < 0.01, ***p < 0.001. Table 2. Influence of background factors of the mother and child at 1 month on occupancies of the five most dominant bacterial genera in the intestinal microbiome of the infant. The bacterial genera are shown from left to right in the order of higher occupancy (mean). For the continuous variables (a gestational age at birth, birth weight, and maternal age), occupancy was classified from 1 to 4 in ascending order from low occupancy with the occupancy of each bacterial species rounded to four decimal places. For the high occupancy group (group 4), a multinomial logistic regression analysis was performed as the category standard and the occupancy was classified into two groups based on the median for each genus for the logistic regression analysis for categorical variables. The odds ratio and 95% confidence interval were calculated using the logistic regression analysis and the multinomial regression analysis. The significance level was set at 5%. *p < 0.05, **p < 0.01, ***p < 0.001. cantly depended on the delivery method (vaginal delivery: OR 0.03; 95% CI 0.003–0.23), but not on the exposure to AED or the existence of siblings. The fourth dominant genera, Clostridium (6.2% ± 15.5%), showed significant effects of the exposure to AED (non-AED: OR 4.98; 95% CI 1.69–14.7), delivery method (vaginal delivery: OR 4.94; 95% CI 1.1–22.2), the existence of siblings (no sibling: OR 0.22; 95% CI 0.07–0.66), and feeding methods (exclusive breastfeeding: OR 5.88; 95% CI 2.24–15.4). cantly depended on the delivery method (vaginal delivery: OR 0.03; 95% CI 0.003–0.23), but not on the exposure to AED or the existence of siblings. Results S bj t For the high occupancy group (group 4), a multinomial logistic regression analysis was performed as the category standard and the occupancy was classified into two groups based on the median for each genus for the logistic regression analysis for categorical variables. The odds ratio and 95% confidence interval were calculated using the logistic regression analysis and the multinomial regression analysis. The significance level was set at 5%. *p < 0.05, **p < 0.01, ***p < 0.001. Variances Bifidobacterium Streptococcus Bacteroides Clostridium Escherichia Odds Ratio 95% CI Odds Ratio 95% CI Odds Ratio 95% CI Odds Ratio 95% CI Odds Ratio 95% CI Gestational ­daysa  1 0.96 0.91 1.02 1.05 0.99 1.11 0.93 0.87 0.98 * 1.09 1.03 1.14 ** 0.92 0.87 0.98 **  2 1.03 0.97 1.09 1.03 0.98 1.09 0.89 0.83 0.94 ** 1.06 1.002 1.13 * 0.93 0.88 0.99 *  3 0.95 0.90 1.00 1.01 0.96 1.07 1.00 0.95 1.07 1.03 0.97 1.10 0.96 0.91 1.02 Birth ­weighta  1 0.999 0.998 1.00 1.001 0.99 1.002 0.999 0.998 1.00 1.002 1.00 1.003 0.998 0.996 0.999 **  2 0.999 0.998 1.00 1.001 0.99 1.002 0.998 0.99 1.00 1.001 1.00 1.003 0.998 0.99 1.00  3 0.999 0.998 1.00 0.999 0.99 1.001 0.999 0.99 1.00 1.001 1.00 1.003 0.999 0.998 1.001 Age of ­mothersa  1 1.00 0.91 1.11 1.02 0.93 1.13 1.09 0.94 1.14 0.91 0.83 0.99 * 1.07 0.97 1.18  2 0.96 0.87 1.06 1.00 0.91 1.11 1.03 0.94 1.14 0.94 0.85 1.04 1.13 1.02 1.25  3 0.98 0.89 1.08 1.03 0.94 1.14 1.10 1.00 1.21 0.96 0.86 1.07 1.05 0.96 1.16 Male 1.47 0.57 3.75 1.20 0.57 2.53 1.35 0.56 3.23 0.85 0.33 2.18 1.45 0.67 3.14 Vaginal delivery 1.84 0.49 6.92 1.06 0.32 3.54 0.026 0.003 0.23 ** 4.94 1.10 22.23 * 0.48 0.14 1.68 Infants without siblings 3.03 1.09 8.43 * 1.19 0.56 2.57 0.96 0.40 2.33 0.22 0.072 0.66 ** 0.84 0.38 1.87 Non-AED 0.11 0.03 0.39 *** 1.22 0.53 2.82 0.65 0.27 1.56 4.98 1.69 14.69 ** 0.75 0.32 1.77 Mothers without allergy 1.33 0.51 3.50 1.27 0.60 2.72 1.14 0.46 2.78 0.85 0.33 2.23 1.62 0.73 3.58 Exclusively breast-fed 1.06 0.41 2.73 2.06 0.96 4.41 1.61 0.66 3.94 5.88 2.24 15.43 *** 0.97 0.44 2.13 Table 2. Influence of background factors of the mother and child at 1 month on occupancies of the five most dominant bacterial genera in the intestinal microbiome of the infant. Results S bj t Mean occupancies of the top 20 bacterial genera constituting the intestinal microbiome at 1, 3, and 6 months after birth. At each age, the occupancies of the top 20 genera (others are indicated as ‘Others’) are shown as 100% stacked columns. The genera are shown in the order of higher occupancy from the bottom to the top. This figure was generated using Microsoft Excel for Mac 2016 (https​://www.micro​soft.com). Figure 1. Mean occupancies of the top 20 bacterial genera constituting the intestinal microbiome at 1, 3, and 6 months after birth. At each age, the occupancies of the top 20 genera (others are indicated as ‘Others’) are shown as 100% stacked columns. The genera are shown in the order of higher occupancy from the bottom to the top. This figure was generated using Microsoft Excel for Mac 2016 (https​://www.micro​soft.com). https://doi.org/10.1038/s41598-021-85670-z Scientific Reports | (2021) 11:6231 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ cantly depended on the delivery method (vaginal delivery: OR 0.03; 95% CI 0.003–0.23), but not on the exposure to AED or the existence of siblings. The fourth dominant genera, Clostridium (6.2% ± 15.5%), showed significant effects of the exposure to AED (non-AED: OR 4.98; 95% CI 1.69–14.7), delivery method (vaginal delivery: OR 4.94; 95% CI 1.1–22.2), the existence of siblings (no sibling: OR 0.22; 95% CI 0.07–0.66), and feeding methods (exclusive breastfeeding: OR 5.88; 95% CI 2.24–15.4). Analysis at 3 months. The effects of background factors on the five most dominant bacterial genera and other high-ranking genera of the intestinal microbiome of 3-month-old infants are shown in Supplementary Table S2 online. Bifidobacteria occupancy was significantly dependent on the exposure to AED (non-AED: OR 0.3; 95% CI 0.09–0.9) and the existence of siblings (no sibling: OR 3.73; 95% CI 1.1–12.6), similar to that seen at 1 month (p < 0.05), whereas that of Bacteroides was significantly dependent on the delivery method (vaginal delivery: OR 0.14; 95% CI 0.03–0.63), also similar to that at 1 month (p < 0.05). The fifth dominant genus, Rumi- nococcus (2.9% ± 8.5%), showed a significant dependence on feeding methods (exclusive breastfeeding: OR 2.5; 95% CI 1.13–5.62). Analysis at 6 months. The effects of background factors on the five most dominant bacterial genera and on other high ranking genera of the intestinal microbiome in 6 month old infants are shown in Supplementary Table 2. Results S bj t Influence of background factors of the mother and child at 1 month on occupancies of the five most dominant bacterial genera in the intestinal microbiome of the infant. The bacterial genera are shown from left to right in the order of higher occupancy (mean). For the continuous variables (a gestational age at birth, birth weight, and maternal age), occupancy was classified from 1 to 4 in ascending order from low occupancy with the occupancy of each bacterial species rounded to four decimal places. For the high occupancy group (group 4), a multinomial logistic regression analysis was performed as the category standard and the occupancy was classified into two groups based on the median for each genus for the logistic regression analysis for categorical variables. The odds ratio and 95% confidence interval were calculated using the logistic regression analysis and the multinomial regression analysis. The significance level was set at 5%. *p < 0.05, **p < 0.01, ***p < 0.001. Results S bj t The fourth dominant genera, Clostridium (6.2% ± 15.5%), showed significant effects of the exposure to AED (non-AED: OR 4.98; 95% CI 1.69–14.7), delivery method (vaginal delivery: OR 4.94; 95% CI 1.1–22.2), the existence of siblings (no sibling: OR 0.22; 95% CI 0.07–0.66), and feeding methods (exclusive breastfeeding: OR 5.88; 95% CI 2.24–15.4). Analysis at 3 months. Analysis at 3 months. The effects of background factors on the five most dominant bacterial genera and other high-ranking genera of the intestinal microbiome of 3-month-old infants are shown in Supplementary Table S2 online. Bifidobacteria occupancy was significantly dependent on the exposure to AED (non-AED: OR 0.3; 95% CI 0.09–0.9) and the existence of siblings (no sibling: OR 3.73; 95% CI 1.1–12.6), similar to that seen at 1 month (p < 0.05), whereas that of Bacteroides was significantly dependent on the delivery method (vaginal delivery: OR 0.14; 95% CI 0.03–0.63), also similar to that at 1 month (p < 0.05). The fifth dominant genus, Rumi- nococcus (2.9% ± 8.5%), showed a significant dependence on feeding methods (exclusive breastfeeding: OR 2.5; 95% CI 1.13–5.62). Analysis at 6 months. The effects of background factors on the five most dominant bacterial genera and on other high-ranking genera of the intestinal microbiome in 6-month-old infants are shown in Supplementary Table S2 online. Bifidobacteria occupancy showed no significant dependence on any factor, and Bacteroides only showed a significant dependence on exclusive breastfeeding (p < 0.05). There were significant effects of the deliv- ery method (vaginal delivery: OR 0.26; 95% CI 0.07–0.91) and the existence of siblings (no sibling: OR 0.26; 95% CI 0.07–0.91) on the third most dominant genus, Streptococcus (4.8% ± 6.9%). The mothers’ allergy history (with history of allergy: OR 2.67; 95% CI 1.20–5.94) significantly affected the fourth most dominant genus, Enterobac- teriaceae; and feeding methods (exclusive breastfeeding: OR 0.29; 95% CI 0.12–0.66) significantly affected the fifth most dominant genus, Ruminococcus. https://doi.org/10.1038/s41598-021-85670-z Scientific Reports | (2021) 11:6231 | www.nature.com/scientificreports/ Effects of AED. Infants at each age were divided based on the AED status and delivery method, and back- ground factors of the mother and child were compared. The results are shown in Supplementary Table S3 online. In infants born via caesarean section, the existence of siblings and age of the mother were significantly higher due to the influence of the previous caesarean section, and the gestational age and birth weight were significantly lower because the date of caesarean delivery was decided beforehand unless performed as an emergency. This tendency also existed between the two types of delivery methods in the AED group, but a sub-group analysis of the vaginal delivery group, excluding the influence of caesarean section, showed no difference in background factors between the AED and non-AED groups. Analysis at 3 months. The effect of background factors that influenced the dominant bacterial genera at each age (AED, delivery method, siblings) and the occupancies by Bifidobacteria and Bacte- roides were then analysed.if AED had a significant effect on the overall diversity of the intestinal microbiome at 1 and 3 months (Fig. 2a). Bifidobacteria occupancy was significantly lower in AED cases than in non-AED cases in 1-month-old infants, regardless of the use of ampicillin (ABPC) or CEZ (p < 0.001). In contrast, in 3-month-old infants, occupancy was not affected by AED. The Bacteroides population was markedly lower in the CEZ group at both 1 and 3 months than in the non-AED group (p < 0.001) and this tendency was also noted in the ABPC group. A significant dif- ference was also found between AED and the non-AED groups (p < 0.05) (Fig. 2b). g p p g In a sub-group analysis of AED in vaginal delivery cases without CEZ administration (i.e., excluding infants born by caesarean section), there was a significant difference in diversity (p = 0.03) (Fig. 3a). Bifidobacteria occupancy in 1-month-old infants was significantly lower in the AED group (all were included in the ABPC group) (p < 0.001), and a significant difference was also noted at 3 months (p < 0.05). In contrast, occupancy of Bacteroides did not differ between these two groups (Fig. 3b). In the AED group, the exposure to antibiotics did not affect the Bifidobacteria and Bacteroides populations in the PROM and GBS-positive groups (Supplementary Fig. S1 online). Effects of the delivery method. The delivery method significantly influenced the diversity of the intes- tinal microbiome at 1 month (Fig. 4a). The occupancy of Bifidobacteria did not differ with age, whereas that of Bacteroides was significantly lower in 1- and 3-month-old infants born via caesarean section (p < 0.001) (Fig. 4b). A comparison of delivery methods within the AED group gave similar findings (Fig. 5a,b). Effects of siblings. The presence of siblings significantly changed the diversity of the intestinal microbiome at 1 and 3 months (Fig. 6a). Bifidobacteria occupancy was significantly higher in 1-month-old (p = 0.001) and 3-month-old (p < 0.001) infants with siblings. Occupancy of Bacteroides did not differ at 1 month but was sig- nificantly lower in infants with siblings at 3 months (p < 0.05). At 6 months, there was no significant difference in occupancy for either genus (Fig. 6b). Analysis at 3 months. Sub-group analysis within the AED group also showed a significant change in the diversity of the intestinal microbiome at 1 and 3 months (Fig. 5a), and bifidobacterial occupancy was significantly higher in 1- and 3-month-old infants with siblings (p < 0.01 and p < 0.001, respectively) (Fig. 5b). Time‑course changes in Bifidobacteria and Bacteroides (linear mixed‑effect model). Com- parison of bifidobacterial occupancy in AED and non-AED infants using a linear mixed-effects model (Fig. 7a) showed a significant difference over 6 months after birth, with lower occupancy in the AED group, but the dif- ference in occupancy decreased with time. There was, however, no difference seen due to the delivery method. Occupancy appeared to be higher in infants with siblings, but there was no significant difference by the sixth month. Occupancy of Bacteroides did not differ significantly between the AED and non-AED groups (Fig. 7b), but the occupancy was lower in the caesarean section group and in infants with siblings. Discussionh The results of this study indicated that the diversity of the intestinal microbiome was influenced by AED, delivery method, and siblings, with significant effects on Bifidobacteria and Bacteroides populations, which remained dominant at a combined occupancy of 60–70% in infants aged up to 6 months. These findings confirm the results of an earlier pilot ­study44 in 1-month-old infants.li p y AED to β-lactamase antibiotics has a major influence on Bifidobacteria population in early infants regardless of the use of ABPC or CEZ, with the influence being especially marked in 1-month-old infants. The influence of ABPC persisted until 3 months, but then gradually weakened and mostly disappeared by the sixth month. These results also conform with those of the pilot ­study44. Several previous studies have suggested a minor effect of IAP on the Bifidobacteria occupancy. However, these studies were limited to use of antibiotics for specific reasons, such as for GBS-positive ­mothers45,46 or in the late stage of ­delivery30, and the study design and screening time- line of the intestinal microbiome were inconsistent. Although there has been a previous ­study38 on the effect of cefuroxime administration to mothers on the gut composition of infants immediately after delivery (10 days) and after weaning (9 months), the present study is the first, to the best of our knowledge, to evaluate the effects of antibiotic administration to mothers immediately before delivery on 1-, 3-, and 6-month-old infants, includ- ing the cases of caesarean section. The study comprised a statistically appropriate number of samples, thereby allowing sub-group analyses. The effects were evaluated until 6 months after birth using a 16S rRNA-targeting next-generation sequencer.hfi g q The delivery method (vaginal vs caesarean section) did not affect the Bifidobacteria occupancy in 1-month old infants, but there was a significant difference in the CEZ and non-AED groups. The delivery method did affect the Bifidobacteria population between the ABPC and CEZ groups. Thus, Bifidobacteria occupancy was sig- nificantly influenced in the caesarean section and CEZ groups compared to non-AED infants, but this effect did not change due to AED in the vaginal delivery group (i.e., the ABPC group). These results suggest that β-lactam Scientific Reports | (2021) 11:6231 | https://doi.org/10.1038/s41598-021-85670-z www.nature.com/scientificreports/ Figure 2. Comparison of the β diversity and occupancy of the top two bacteria species in the AED and non- AED groups. Discussionh (a) Comparison of β diversity of the intestinal microbiome between infants at each age with (AED) and without (non-AED) antibiotic exposure at delivery. (b) Bifidobacteria and Bacteroides occupancies in infants in the AED and non-AED groups, shown as box-whisker plots at each age. The AED group was also divided into ampicillin-treated (ABPC) and cefazolin-treated (CEZ) groups. Comparison of occupancy among these groups and the non-AED group was performed using Bonferroni multiple comparison test. Comparison between the AED and non-AED groups was performed using Mann–Whitney U-test. The ABPC, CEZ, and non-AED groups included 43, 31, and 56 infants at 1 month (130 in total); 42, 27, and 58 infants at 3 months (127 in total); and 40, 28, and 58 infants at 6 months (126 in total). The significance level was set at 5%. *p < 0.05, ***p < 0.001 Thi fi t d i SAS i 9 4 (htt // ) Figure 2. Comparison of the β diversity and occupancy of the top two bacteria species in the AED and non- AED groups. (a) Comparison of β diversity of the intestinal microbiome between infants at each age with (AED) and without (non-AED) antibiotic exposure at delivery. (b) Bifidobacteria and Bacteroides occupancies in infants in the AED and non-AED groups, shown as box-whisker plots at each age. The AED group was also divided into ampicillin-treated (ABPC) and cefazolin-treated (CEZ) groups. Comparison of occupancy among these groups and the non-AED group was performed using Bonferroni multiple comparison test. Comparison between the AED and non-AED groups was performed using Mann–Whitney U-test. The ABPC, CEZ, and non-AED groups included 43, 31, and 56 infants at 1 month (130 in total); 42, 27, and 58 infants at 3 months (127 in total); and 40, 28, and 58 infants at 6 months (126 in total). The significance level was set at 5%. *p < 0.05, ***p < 0.001 This figure was generated using SAS version 9.4 (https​://www.sas.com). Figure 2. Comparison of the β diversity and occupancy of the top two bacteria species in the AED and non- AED groups. (a) Comparison of β diversity of the intestinal microbiome between infants at each age with (AED) and without (non-AED) antibiotic exposure at delivery. (b) Bifidobacteria and Bacteroides occupancies in infants in the AED and non-AED groups, shown as box-whisker plots at each age. The AED group was also divided into ampicillin-treated (ABPC) and cefazolin-treated (CEZ) groups. Discussionh The AED and non-AED groups included 43 and 56 infants at 1 month (99 in total), 42 and 58 infants at 3 months (100 in total), and 41 and 58 infants at 6 months (99 in total). The significance level was set at 5%. *p < 0.05, ***p < 0.001 using Mann–Whitney U-test. This figure was generated using SAS version 9.4 (https​://www.sas.com). Figure 3. Comparison of the β diversity and occupancy of the top two bacteria species in the AED and non- AED groups of the vaginal delivery group. (a) Comparison of β diversity of the intestinal microbiome between infants at each age with (AED) and without (non-AED) antibiotic exposure at vaginal delivery. (b) Comparison of Bifidobacteria and Bacteroides occupancies in infants in the AED and non-AED groups with vaginal delivery, shown as box-whisker plots at each age. The AED and non-AED groups included 43 and 56 infants at 1 month (99 in total), 42 and 58 infants at 3 months (100 in total), and 41 and 58 infants at 6 months (99 in total). The significance level was set at 5%. *p < 0.05, ***p < 0.001 using Mann–Whitney U-test. This figure was generated using SAS version 9.4 (https​://www.sas.com). Figure 3. Comparison of the β diversity and occupancy of the top two bacteria species in the AED and non- AED groups of the vaginal delivery group. (a) Comparison of β diversity of the intestinal microbiome between infants at each age with (AED) and without (non-AED) antibiotic exposure at vaginal delivery. (b) Comparison of Bifidobacteria and Bacteroides occupancies in infants in the AED and non-AED groups with vaginal delivery, shown as box-whisker plots at each age. The AED and non-AED groups included 43 and 56 infants at 1 month (99 in total), 42 and 58 infants at 3 months (100 in total), and 41 and 58 infants at 6 months (99 in total). The significance level was set at 5%. *p < 0.05, ***p < 0.001 using Mann–Whitney U-test. This figure was generated using SAS version 9.4 (https​://www.sas.com). Bifidobacteria colonisation was also influenced by the presence or absence of siblings. Bifidobacteria occu- pancy in AED infants was significantly higher in those with siblings. Discussionh Comparison of occupancy among these groups and the non-AED group was performed using Bonferroni multiple comparison test. Comparison between the AED and non-AED groups was performed using Mann–Whitney U-test. The ABPC, CEZ, and non-AED groups included 43, 31, and 56 infants at 1 month (130 in total); 42, 27, and 58 infants at 3 months (127 in total); and 40, 28, and 58 infants at 6 months (126 in total). The significance level was set at 5%. *p < 0.05, ***p < 0.001 This figure was generated using SAS version 9.4 (https​://www.sas.com). antibiotics may directly influence bifidobacterial occupancy. Influence of the delivery method on Bifidobacteria population from immediately after birth to early infancy has been shown ­previously40,47–50, but the influence of antibiotics administered before caesarean ­section36,37,51 was not considered in these studies, and this effect may have been simultaneously observed. The present study is not capable of judging whether the observed effect is due to the infant not passing through the birth canal in caesarean section or CEZ. Further studies with the use of the same antibiotics for vaginal delivery and caesarean section are therefore warranted. antibiotics may directly influence bifidobacterial occupancy. Influence of the delivery method on Bifidobacteria population from immediately after birth to early infancy has been shown ­previously40,47–50, but the influence of antibiotics administered before caesarean ­section36,37,51 was not considered in these studies, and this effect may have been simultaneously observed. The present study is not capable of judging whether the observed effect is due to the infant not passing through the birth canal in caesarean section or CEZ. Further studies with the use of the same antibiotics for vaginal delivery and caesarean section are therefore warranted. https://doi.org/10.1038/s41598-021-85670-z Scientific Reports | (2021) 11:6231 | www.nature.com/scientificreports/ Figure 3. Comparison of the β diversity and occupancy of the top two bacteria species in the AED and non- AED groups of the vaginal delivery group. (a) Comparison of β diversity of the intestinal microbiome between infants at each age with (AED) and without (non-AED) antibiotic exposure at vaginal delivery. (b) Comparison of Bifidobacteria and Bacteroides occupancies in infants in the AED and non-AED groups with vaginal delivery, shown as box-whisker plots at each age. Discussionh The results suggested that at least until 3 months after birth, the presence of an elder sibling promoted the colonisation of Bifidobacteria, even in infants exposed to antibiotics at delivery, i.e., there may be mutual interference of the microbiome between siblings. This may explain the maintenance of high Bifidobacteria occupancy in infants, even in the AED group. In a previous pilot ­study44, the presence of siblings was suggested to influence IAP, and the present study confirmed this effect. Previous studies on the effects of siblings have reported that Bifidobacteria colonisation occurs more easily in Scientific Reports | (2021) 11:6231 | https://doi.org/10.1038/s41598-021-85670-z www.nature.com/scientificreports/ Figure 4. Comparison of the β diversity and occupancy of the top two bacteria species in the vaginal delivery (VD) and caesarean section (CS) groups. (a) Comparison of β diversity of the intestinal microbiome at each age between vaginal delivery (VD) and caesarean section (CS) (b) Bifidobacteria and Bacteroides occupancies between delivery methods, shown as box-whisker plots at each age. The VD and CS groups included 99 and 31 infants at 1 month (130 in total), 100 and 27 infants at 3 months (127 in total), and 99 and 27 infants at 6 months (126 in total). The significance level was set at 5%. *p < 0.05, ***p < 0.001 using Mann–Whitney U-test. This figure was generated using SAS version 9.4 (https​://www.sas.com). Figure 4. Comparison of the β diversity and occupancy of the top two bacteria species in the vaginal delivery (VD) and caesarean section (CS) groups. (a) Comparison of β diversity of the intestinal microbiome at each age between vaginal delivery (VD) and caesarean section (CS) (b) Bifidobacteria and Bacteroides occupancies between delivery methods, shown as box-whisker plots at each age. The VD and CS groups included 99 and 31 infants at 1 month (130 in total), 100 and 27 infants at 3 months (127 in total), and 99 and 27 infants at 6 months (126 in total). The significance level was set at 5%. *p < 0.05, ***p < 0.001 using Mann–Whitney U-test. This figure was generated using SAS version 9.4 (https​://www.sas.com). infants with ­siblings19,40,41. However, this effect has been scarcely studied as compared to studies on factors such as AED and the delivery method, particularly in Japanese infants. Thus, the present study is significant in showing the effect of siblings in a large cohort study. Discussionh This effect was confirmed using a sub-group analysis within the AED group to allow interpretation in the context of AED. The association of this effect with the intestinal microbiome of siblings requires a continuous study, including siblings living together.hlhf infants with ­siblings19,40,41. However, this effect has been scarcely studied as compared to studies on factors such as AED and the delivery method, particularly in Japanese infants. Thus, the present study is significant in showing the effect of siblings in a large cohort study. This effect was confirmed using a sub-group analysis within the AED group to allow interpretation in the context of AED. The association of this effect with the intestinal microbiome of siblings requires a continuous study, including siblings living together.hlhf g q y g g g g The influence of the caesarean section was more substantial than that of AED in Bacteroides. This effect was firmly maintained at 3 months and persisted at least until 6 months after birth. The same tendency was observed in the sub-analysis of the AED group. This confirmed that birth via caesarean section is an important factor in the occupancy of Bacteroides, compared to AED. A similar persistence of the influence of the caesarean section on Bacteroides until the weaning period and thereafter has been previously ­reported34,40,50,52,53. However, as described above, all caesarean section cases received preoperative CEZ. Thus, although the Bacteroides population was Scientific Reports | (2021) 11:6231 | https://doi.org/10.1038/s41598-021-85670-z www.nature.com/scientificreports/ Figure 5. Comparison of the β diversity and occupancy of the top two bacteria species in the vaginal delivery (VD) and caesarean section (CS) groups of the AED group. (a) Comparison of β diversity of the intestinal microbiome at each age between delivery methods (1) and with and without siblings (2) in the AED group. VD: vaginal delivery, CS: caesarean section, Non-Siblings: infants without a sibling, Siblings: infants with older siblings. (b) Bifidobacteria and Bacteroides occupancies in the AED group compared between delivery methods (1) and presence of absence of siblings (2), shown as box-whisker plots at each age. (1) The vaginal delivery (VD) and caesarean section (CS) groups included 43 and 31 infants at 1 month (74 in total); 42 and 27 infants at 3 months (69 in total), and 42 and 27 infants at 6 months (68 in total). Discussionh Comparison of the β diversity and occupancy of the top two bacteria species between the infants with siblings and those without siblings (a) Comparison of β diversity of the intestinal microbiome between infants with and without siblings. (b) Bifidobacteria and Bacteroides occupancies between infants with siblings (Sib) and without siblings (Non-Sib), shown as box-whisker plots at each age. The Sib and non-Sib groups included 67 and 63 infants at 1 month (130 in total), 61 and 66 infants at 3 months (127 in total), and 66 and 60 infants at 6 months (126 in total). The significance level was set at 5%. *p < 0.05, ***p < 0.001 using Mann–Whitney U-test. This figure was generated using SAS version 9.4 (https​://www.sas.com). Figure 6. Comparison of the β diversity and occupancy of the top two bacteria species between the infan Figure 6. Comparison of the β diversity and occupancy of the top two bacteria species between the infants with siblings and those without siblings (a) Comparison of β diversity of the intestinal microbiome between infants with and without siblings. (b) Bifidobacteria and Bacteroides occupancies between infants with siblings (Sib) and without siblings (Non-Sib), shown as box-whisker plots at each age. The Sib and non-Sib groups included 67 and 63 infants at 1 month (130 in total), 61 and 66 infants at 3 months (127 in total), and 66 and 60 infants at 6 months (126 in total). The significance level was set at 5%. *p < 0.05, ***p < 0.001 using Mann–Whitney U-test. This figure was generated using SAS version 9.4 (https​://www.sas.com). of this study until 6 months of ­age54. Similar to the effect seen on Bifidobacteria, the effect on Bacteroides until 3 months may reflect mutual interference among siblings.ii l Many studies have examined the clinical significance of colonisation of the intestinal microbiome with Bifi- dobacteria and Bacteroides in early ­infancy17–22. The present study evaluated the influence of AED on early infants in terms of the effects on their subsequent health. The samples from this study were also used to assess the relation of allergies with changes in the intestinal microbiome. This study was performed as a part of a cohort study evaluating the clinical significance of changes in the intestinal microbiome during early infancy in healthy Japanese infants and their effect on the intestinal microbiome later in life. Discussionh (2) The Sib and non-Sib groups included 44 and 30 infants at 1 month (74 in total), 42 and 27 infants at 3 months (69 in total), and 41 and 27 infants at 6 months (68 in total). The significance level was set at 5%. **p < 0.01, ***p < 0.001 using Mann–Whitney U-test. This figure was generated using SAS version 9.4 (https​://www.sas.com). Figure 5. Comparison of the β diversity and occupancy of the top two bacteria species in the vaginal delivery (VD) and caesarean section (CS) groups of the AED group. (a) Comparison of β diversity of the intestinal microbiome at each age between delivery methods (1) and with and without siblings (2) in the AED group. VD: vaginal delivery, CS: caesarean section, Non-Siblings: infants without a sibling, Siblings: infants with older siblings. (b) Bifidobacteria and Bacteroides occupancies in the AED group compared between delivery methods (1) and presence of absence of siblings (2), shown as box-whisker plots at each age. (1) The vaginal delivery (VD) and caesarean section (CS) groups included 43 and 31 infants at 1 month (74 in total); 42 and 27 infants at 3 months (69 in total), and 42 and 27 infants at 6 months (68 in total). (2) The Sib and non-Sib groups included 44 and 30 infants at 1 month (74 in total), 42 and 27 infants at 3 months (69 in total), and 41 and 27 infants at 6 months (68 in total). The significance level was set at 5%. **p < 0.01, ***p < 0.001 using Mann–Whitney U-test. This figure was generated using SAS version 9.4 (https​://www.sas.com). not influenced by ABPC, it may have been markedly influenced by CEZ. This possibility should be examined in future studies.hfif The Bacteroides population was not affected by siblings at 1 month, but a significant effect was seen at 3 months, with no influence at 6 months. In contrast to Bifidobacteria, siblings negatively influenced the occu- pancy of Bacteroides. The influence of siblings on the Bacteroides population has been shown ­before40, with the occupancy by Bacteroides at 18 months after birth being higher in infants with siblings, in contrast to the findings https://doi.org/10.1038/s41598-021-85670-z Scientific Reports | (2021) 11:6231 | www.nature.com/scientificreports/ Figure 6. Discussionh Data at 1, 3, and 6 months after birth were used in this study, but data related to longer-term time-course changes in the same individuals are needed https://doi.org/10.1038/s41598-021-85670-z Scientific Reports | (2021) 11:6231 | www.nature.com/scientificreports/ Figure 7. Time-course changes in bifidobacterial (a) and Bacteroides (b) occupancies (from 1 to 6 months after birth) based on AED or non-AED, delivery method, and the presence or absence of siblings, using a linear mixed-effect model. The analysis set at 1 month included 130 infants. Dropouts at 3 and 6 months were handled as missing values. The standard deviation by age in months is shown in bar form in each figure. Significant differences in occupancies between the two groups at each time point are also indicated in each comparison. The significance level was set at 5%. *p < 0.05, ***p < 0.001 using the Mann–Whitney U-test. This figure was generated using SAS version 9.4 (https​://www.sas.com). Figure 7. Time-course changes in bifidobacterial (a) and Bacteroides (b) occupancies (from 1 to 6 months after birth) based on AED or non-AED, delivery method, and the presence or absence of siblings, using a linear mixed-effect model. The analysis set at 1 month included 130 infants. Dropouts at 3 and 6 months were handled as missing values. The standard deviation by age in months is shown in bar form in each figure. Significant differences in occupancies between the two groups at each time point are also indicated in each comparison. The significance level was set at 5%. *p < 0.05, ***p < 0.001 using the Mann–Whitney U-test. This figure was generated using SAS version 9.4 (https​://www.sas.com). for a complete and comprehensive investigation. The composition of the intestinal microbiome shows substan- tial similarities at different time-points in the same ­individual55, but external factors influencing the intestinal microbiome increase with growth, such as the increase in baby food intake, interaction with siblings and other infants in group nursing, and further use of antibiotics for various diseases. However, this study is significant as a cohort study with a statistically significant large number of samples of the intestinal microbiome of infants in the first 6 months of life, which is a crucial period for the development of the immune system as this is a time at which external factors have the least influence in the entire ­lifetime24–26. l Despite its importance, the present study is not without limitations. Discussionh With regard to the possibility of the AED and differences in delivery modes in this study affecting Bifidobacterium and Bacteroides, a strong “relationship” was observed. However, this study is a prospective cohort follow-up observation study, and comparative experi- mental studies are needed to confirm more robust causal relationship, together with follow-up observations. This study was conducted in a single hospital, which does not allow for extrapolation of the obtained results to the population from other regions within Japan and beyond, with differences in ethnicity or geography. Although the protocol for the use of antimicrobial agents before the delivery was similar to that in previous studies on ­IAP27,29, multi-centre studies are needed for validation of such extrapolations. Additionally, while this study focused on several factors that could have a significant impact on the gut microbiota of infants, it did not account for some factors such as feeding methods as infants fed solely on milk were not included; thus, comparison of the feeding methods is not adequate. These points will likely cause a selection bias, which could affect colonisation characteristics of certain bacteria in the intestinal microbiota of infants. Inclusion of another group of infants that were milk-fed for the most part along with top feed is imperative to understand the factors influencing gut microbiota in infants. Moreover, occupancy was used in the comparison of bacteria in this study. A qualitative assessment based on qPCR is required to accurately assess the effect of factors such as AED and delivery method, rather than a relative assessment based on occupancy as was done in this ­study56,57. Methods hi Ethics approval and consent to participate. All experimental procedures used in the study complied with the ethical standards of national guidelines of the Japanese government on human experimentation and the Declaration of Helsinki 1975, as revised in 2008. The study was approved by the institutional review boards of Iwate Prefectural Iwai Hospital (No. 1234) and Juntendo University (No. 2017127). Written informed consent for participation and publication was obtained from all mothers. Subjects. This prospective cohort study was performed as a part of a study investigating the association of allergic diseases with time-course changes observed in the microbiome in infants. The subjects included 142 infants and their mothers, who gave consent to registration before delivery or 2 weeks after delivery at Iwate Prefectural Iwai Hospital between February 2018 and March 2019. This hospital is in the Tohoku (Northeast) region of Japan and handles about 800 deliveries each year as a core perinatal medical care centre in the southern Iwate prefecture. p Inclusion criteria were as follows: infants born at full-term with natural delivery or caesarean section, whose mothers had not been exposed to antibiotics for 1 month before delivery, except for the antimicrobial used just before the delivery. Faecal samples were collected at 1, 3, and 6 months after birth by the parents. t Exclusion criteria were as follows: premature babies born before 37 weeks of gestation; if the babies received any antibiotics during first 6 months from their birth, the samples collected were handled as dropouts. If the sample collection was missed, the infant was considered as a dropout at that age and after that. If a sample was inappropriate for MiSeq analysis and could not be analysed, data for this infant were excluded from the analysis only at this time-point, whereas the analytical results for the infant at all other ages were used. y y g During IAP at delivery, a single dose of CEZ (1 g) was given systemically to mothers for all caesarean sections just before the surgery. ABPC (2 g) was given at least 4 h before delivery, followed by intermittent administration every 6 h until delivery in GBS-positive and PROM cases. Methods hi Antibiotics were administered to all subjects at the dose and time defined in the clinical protocol determined by the hospital board.t i Containers for collection of faecal samples at 1 month after birth were handed to mothers during hospitali- sation for delivery or at the infant health examination 2 weeks after delivery. The container for the collection of faecal samples at 3 months was sent by mail to the address of each registered infant from Core Technology Laboratories, Asahi Group Holdings, and the container for 6 months was sent by mail from the Department of Microbiome Research, Juntendo University. Each faecal sample collected by the infants’ parents was transferred to a test tube (Techno Suruga Laboratory, Shizuoka, Japan) containing 0.001% bromothymol and 100 mM Tris–HCl (pH 9), 40 mM EDTA, 4 M guanidine thiocyanate, and was mixed well as described in a previous ­study58. Mixed faecal samples were delivered to a laboratory of Asahi Group Holdings (Sagamihara, Kanagawa, Japan) within one or two days from Ichinoseki city, Iwate prefecture, where most of the enrolled infants resided (the distance between the two locations is approximately 480 km), and were stored at − 80 °C until processing for DNA extrac- tion. The test tubes containing the samples were dissolved in a solution (provided in the kit mentioned above), which ensured that the composition of the intestinal microbiome present within the body was stable even at room ­temperature58. DNA extraction. The processed samples were subjected to DNA extraction, as described ­previously44. Briefly, the samples (2 mL) were transferred to plastic tubes, centrifuged at 14,000× g for 3 min, washed in 1.0 mL of phosphate-buffered saline, and centrifuged at 14,000× g. Pellets were re-suspended in 500 μL of extraction buffer (166 mM Tris/HCl, 66 mM EDTA, 8.3% sodium dodecyl sulphate, pH 9.0) and 500 μL of TE buffer-satu- rated phenol. Next, 300 mg of zirconium beads (0.1 mm diameter) was added to the suspension, and the mixture was vortexed vigorously for 60 s × 3 times using a Multi-Beads Shocker (Yasui Kikai Corp., Osaka, Japan). After centrifugation at 14,000 × g for 5 min, 400 μL of the supernatant was purified using a Maxwell Instrument (Pro- mega KK, Tokyo, Japan). Sequencing and data processing. 16S rRNA gene sequencing was performed using a MiSeq V3 kit as per the manufacturer’s protocol (Illumina, San Diego, CA, USA). Conclusionsh This prospective cohort study confirmed the findings from a previous pilot study indicating that β-lactam anti- biotics administered to mothers immediately before delivery significantly influence the intestinal microbiome of healthy Japanese infants at 1 and 3 months after birth. This effect is more substantial than the effect of the deliv- ery method on the dominant bacterial genus Bifidobacterium. The evaluation of the influence of AED requires the inclusion of all antibiotics used immediately before delivery, including before the caesarean section. The https://doi.org/10.1038/s41598-021-85670-z Scientific Reports | (2021) 11:6231 | www.nature.com/scientificreports/ presence of siblings also affects Bifidobacteria colonisation, and this effect persists until 3 months and increases with time. In contrast, for Bacteroides, the influence of the delivery method is greater than that of AED. However, it is unclear whether this is an influence of delivery via caesarean section or CEZ administration. These results provide a new perspective on essential factors influencing the intestinal microbiome in early infancy, which is vital for the development of intestinal immunity. The clinical significance of the results in the later life of the infants requires further long-term studies. www.nature.com/scientificreports/ ing chimeric sequences and feature table construction were performed with the DADA2 ­plugin60. The primers sequence were trimmed and the remaining forward and reverse sequences were truncated to a final length of 280 bp. Phylogenetic diversity analyses were carried out with q2-phylogeny and q2-diversity, and beta diversity was visualised using principal coordinate analysis (PCoA). The feature classifiers were performed with q2-fea- ture-classifier plugin using “gg-13-8-99-nb-classifier.qza.” from the greengenes database as reference sequences. ing chimeric sequences and feature table construction were performed with the DADA2 ­plugin60. The primers sequence were trimmed and the remaining forward and reverse sequences were truncated to a final length of 280 bp. Phylogenetic diversity analyses were carried out with q2-phylogeny and q2-diversity, and beta diversity was visualised using principal coordinate analysis (PCoA). The feature classifiers were performed with q2-fea- ture-classifier plugin using “gg-13-8-99-nb-classifier.qza.” from the greengenes database as reference sequences. Data collection. The following data were collected from medical records at Iwate Prefectural Iwai Hospi- tal: delivery method, gender, body weight at birth, perinatal history, records of hospital visits, and treatments received by the infant up to 6 months after birth including the use of antibiotics after birth. Additional informa- tion related to age (days) at sample collection, feeding method (breastfeed exclusively or added top feed), and siblings were obtained from a questionnaire completed by the mothers. The following data about the mothers were also collected from medical records and a questionnaire: age, delivery method, history of allergies (food allergy, bronchial asthma, atopic dermatitis, allergic rhinitis), abnormal findings at delivery (including PROM and GBS-positive status), antimicrobial use during late pregnancy, and systemic antibiotics (including types) given at delivery. Statistical analysis. The significance of the difference between the two groups was analysed using a non- parametric ANOSIM (analysis of similarities) test based on unweighted UniFrac distances within QIIME2 (https​ ://qiime​2.org/). Acquired 16S rRNA gene data for bacteria were analysed using SAS 9.4 (SAS Institute Inc., Cary, NC, USA). Background factors of the mother and child were compared using the Mann–Whitney U-test for continuous variables and the Pearson’s chi-square test for categorical variables. The influence of each back- ground factor on occupancies by higher-rank dominant bacterial genera in the intestinal microbiome at each age was examined using logistic regression analysis and multinomial logistic regression for categorical variables and continuous variables, respectively. www.nature.com/scientificreports/ The dependence of occupancy of each bacterial genus on factors, for which a significant association was found in diversity analysis and logistic regression analysis, was examined using a Mann–Whitney U-test for between-group comparison and using a Kruskal–Wallis test with a Bonferroni cor- rection for multiple group comparison. Continuous comparative changes of dominant bacterial genera due to different factors were analysed using a linear mixed-effect model (random intercept and first-order autoregres- sion model). The significance level was set at p < 0.05 in all analyses. Data availabilityh y The data generated during the current study are available in the Figshare repository (https​://doi.org/10.6084/ m9.figsh​are.12000​255), and the sequencing data is deposited in the DNA Data Bank of Japan (DDBJ; accession number: DRA010467). Received: 22 July 2020; Accepted: 4 March 2021 Received: 22 July 2020; Accepted: 4 March 2021 References 1. Kundu, P., Blacher, E., Elinav, E. & Pettersson, S. Our gut microbiome: the evolving inner self. Cell 171, 1481–1493 (2017). g g 2. Sender, R., Fuchs, S. & Milo, R. Revised estimates for the number of human and bacteria cells in the body. PLoS Biol. 14, e1002533. https​://doi.org/10.1371/journ​al.pbio.10025​33 (2016).i https​://doi.org/10.1371/journ​al.pbio.10025​33 (2016). p g j p 3. Turroni, F. et al. Diversity of bifidobacteria within the infant gut microbiota. PLoS ONE 7, e36957. https​://doi.org/10.1371/journ​ al.pone.00369​57 (2012). p 4. Kumbhare, S. V. et al. 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Methods hi Briefly, the V3-V4 region of the bacterial 16S rDNA was amplified using PCR with forward and reverse primers (5′-TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGA GGC WGC AG-3′ and 5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3′) with the TaKaRa Ex Taq HS Kit (TaKaRa Bio, Shiga, Japan) from 5 ng of DNA from faecal samples. After the PCR products were purified with Agencourt AMPure XP (Beckman Coulter, Indianapolis, IN, USA) and amplified using a Nextera XT Index Kit v2 (Illumina, San Diego, CA, USA). After the second round of PCR, the products were again purified using Agencourt AMPure XP. The library was quantified, normalised, and pooled in equimolar amounts. Sequencing was conducted using a paired-end 2 × 300-bp cycle run on an Illumina MiSeq system with a MiSeq Reagent Kit v.3 (600 cycles). 16S rRNA‑based taxonomic and diversity analysis. QIIME2 (Quantitative Insights into Microbial Ecology, http://qiime​2.org/) v.2019.4.0. was used for sequence ­analysis59. The sequence quality control, remov- Scientific Reports | (2021) 11:6231 | https://doi.org/10.1038/s41598-021-85670-z www.nature.com/scientificreports/ www.nature.com/scientificreports/ 48 Bi i G t l M d f d li ff t th b t i l it i th b t E l H D 86(S l 1) 13 15 (2010 47. Huurre, A. et al. Mode of delivery effects on gut microbiota and humoral immunity. Neonatology 93, 236 240 (2008). 48 Biasucci G et al Mode of delivery affects the bacterial community in the newborn gut Early Hum Dev 86(Suppl 1) 13 15 (2010) , yf g y gy 9 , ( ) 48 Biasucci, G et al Mode of delivery affects the bacterial community in the newborn gut Early Hum Dev 86(Suppl 1), 13–15 (2010) f 48. Biasucci, G. et al. Mode of delivery affects the bacterial community in the newborn gut. Early Hum. Dev. 86(Suppl 1), 13–15 (2 f 9. Lee, E. et al. 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Impact of intrapartum and postnatal antibiotics on the gut microbiome and emergence of antimicrobial resist ance in infants. Sci. Rep. 9, 10635 (2019).f p 47. Huurre, A. et al. Mode of delivery - effects on gut microbiota and humoral immunity. Neonatology 93, 236–240 (2008).f 47. Huurre, A. et al. Mode of delivery - effects on gut microbiota and humoral immunity. Neonatology 93, 236–240 (2008). Author contributions Author contributions N.I., Y.A., H.M., and F.A. designed the study. S.W., N.H., and H.M. evaluated, corrected, and reviewed the study. N.I., Y.A., C.K., F.A., and H.M. implemented the study (explanation to parents, distributing sample containers, and collecting questionnaires). Y.A., C.K., and H.M. performed storage, processing, and analysis using MiSeq of samples. N.I., Y.A., and C.K. performed data management and statistical analysis. N.I. drafted the manuscript. S.W., N.H., F.A., and H.M. corrected and reviewed the manuscript. S.N. supervised the statistical methods and examined the validity of the analysis. All authors read and approved the content of the manuscript, including the accuracy of the data, ethical legitimacy, and validity of the results. p g The authors declare no competing interests. The authors declare no competing interests. www.nature.com/scientificreports/ https ://doi.org/10.1038/s4158​6-019-1560-1 (2019). 57. Shao, Y. et al. Stunted microbiota and opportun ://doi.org/10.1038/s4158​6-019-1560-1 (2019). 58. Nishimoto, Y. et al. High stability of faecal microbiome composition in guanidine thiocyanate solution at room temperature and robustness during colonoscopy. Gut 65, 1574–1575 (2016). 59. Bolyen, E. et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 37, 852–857 (2019). ( ) 0. Callahan, B. J. et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 13, 581–583 (2016). Scientific Reports | (2021) 11:6231 | https://doi.org/10.1038/s41598-021-85670-z www.nature.com/scientificreports/ Additional informationh Supplementary Information The online version contains supplementary material available at https​://doi. org/10.1038/s4159​8-021-85670​-z. Correspondence and requests for materials should be addressed to N.I. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Postpartum acute fatty liver of pregnancy: a case report
Journal of medical case reports
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© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. CASE REPORT Open Access * Correspondence: Husban48@yahoo.com Obstetrics & Gynecology Department, Faculty of Medicine, University of Jordan and Jordan University Hospital, P.O. Box 2194, Amman 11941, Jordan Background be needed in severe cases. Admission to the intensive care unit (ICU) is recommended [3]. In our literature research, we found no cases of AFLP reported to present initially as skin rash. In 1940, Sheehan first recognized acute fatty liver of pregnancy (AFLP) as a distinct clinical syndrome and reported a series of six cases from the Glasgow Royal Maternity Hospital [1]. AFLP can be a very dramatic clinical event with sudden and catastrophic conse- quences to healthy women. It remains a disease of unknown etiology and pathogenesis [2]. This serious condition usually occurs in the third trimester or in the immediate postpartum period [3]. There exists some medical evidence to suggest that AFLP may be due to disordered metabolism of fatty acids in the maternal mitochondria [4]. It is best treated in a center with expert- ise in high-risk obstetrics, maternal-fetal medicine, neonat- ology, and hepatology. Experts in liver transplantation may Naser Al-Husban*, Oqba Al-Kuran and Amal Al Helou Naser Al-Husban*, Oqba Al-Kuran and Amal Al Helou Al-Husban et al. Journal of Medical Case Reports (2018) 12:67 https://doi.org/10.1186/s13256-018-1593-3 Al-Husban et al. Journal of Medical Case Reports (2018) 12:67 https://doi.org/10.1186/s13256-018-1593-3 Abstract Background: Acute fatty liver of pregnancy can be a very dramatic clinical event with significant risk of mortality to healthy women. The pathogenesis is still unknown. It usually occurs in the third trimester or in the immediate postpartum period. The clinical presentation is very variable. Medical staff have to be very cautious even regarding a minor complaint of feeling unwell. Skin rash has not been reported as one of the initial presentations of acute fatty liver of pregnancy. It is best treated in a center with a multidisciplinary approach. Admission to the intensive care unit is recommended. Case presentation: We report a case of a 20-year-old Middle Eastern Arabic woman who developed an acute fatty liver of pregnancy. She was not known to have any medical disease. She had had two previous uncomplicated deliveries. She developed acute fatty liver of pregnancy on the first day after an uncomplicated normal vaginal delivery of a healthy male newborn. She started to have nonitchy skin rash over her abdomen and upper limbs. Then she started to feel unwell. Twelve hours later, she developed epigastric and right upper quadrant abdominal pain, followed by jaundice, nausea, and vomiting. She developed recurrent hypoglycemic attacks, hemolytic anemia, coagulopathy, and hepatorenal syndrome. Conclusions: The clinical presentation of acute fatty liver of pregnancy is very variable and nonspecific. Skin rash can be a new presenting symptom of acute fatty liver of pregnancy. Immediate suspicion of the diagnosis, appropriate investigations, and urgent initiation of therapy in an intensive care unit and by a multidisciplinary team resulted in a good outcome with no adverse health consequences for our patient. Keywords: Fatty liver, Pregnancy, Liver dysfunction, Postpartum, Skin rash Case presentation Our patient was a 20-year-old Arabic Middle Eastern woman. She was not known to have any medical illness. She had had two previous uneventful pregnancies with uncomplicated vaginal deliveries. Her only antenatal visit to our hospital was at 38 weeks of gestation, when she presented in early labor. Her general physical examin- ation was unremarkable. An ultrasound (US) scan showed a cephalic, normally grown fetus with decreased amniotic fluid. The patient’s whole blood platelet count was 182 × 109/L, white blood cell count (WBC) was 11 × 109/L, and whole blood hemoglobin (Hb) was 116 g/L. Her blood group was AB positive. Page 2 of 6 Al-Husban et al. Journal of Medical Case Reports (2018) 12:67 On vaginal examination, she was found to have a 3-cm dilated, 80% effaced cervix and intact membranes. She was augmented with artificial rupture of her membranes and syntocinon intravenous infusion. Six hours later, she had an uneventful vaginal delivery of a healthy male newborn weighing 3.06 kg. The baby’s Apgar scores at 1 and 5 minutes were 8 and 9, respectively. Fig. 2 Skin rash over the abdomen p y On the morning of her first postpartum day, the patient complained of a nonpruritic maculopapular skin rash over her upper limbs (Fig. 1), abdomen (Fig. 2), and back. It appeared suddenly as patchy lesions. It was not associated with pustules or vesicles. Her neck, face, and the palmar aspects of her hands and lower limbs were spared. There were no noticeable striae over her abdo- men. She was not known to have any allergic reactions, and she did not receive any medications that could explain the findings. Twelve hours later, she was feeling very unwell and tired. She then developed generalized abdominal pain that increased in severity and was asso- ciated with nausea and occasional vomiting. Her vital signs were normal (blood pressure [BP] 120/70 mmHg, pulse rate 83 beats/minute, and oral temperature 37.1 ° C). Her urine was yellow and turbid with 3+ proteinuria, and she had numerous WBC/high-power field (HPF) but no glycosuria. The same result was confirmed by testing a second urine sample that was obtained via a Foley catheter. A dermatologist’s review indicated non- specific maculopapular skin rash, and the dermatologist advised only observation with no specific therapy but to investigate further. Case presentation This advice alerted the medical staff to do further testing, which showed that her liver func- tion, kidney function, whole blood count, serum glucose, serum lactate dehydrogenase (LDH), and coagulation profile were within normal limits. Fig. 2 Skin rash over the abdomen tenderness. Her vital signs were stable. Investigations were repeated and showed thrombocytopenia (platelet count 54 × 109/L), hypoglycemia (serum glucose 2.11 mmol/L), renal impairment (serum creatinine 228.75 μmol/L), impaired liver function (serum alanine amino- transferase [ALT] 0.735 μkat/L, serum aspartate amino- transferase [AST] 1.15 μkat/L, serum LDH 19.8 μkat/L, serum total bilirubin 68.4 μmol/L, serum direct bilirubin 58.15 μmol/L), and coagulopathy (plasma prothrombin time [PT] 22 seconds, control 14 seconds, blood partial thromboplastin time [PTT] 36 seconds, control 26 sec- onds, international normalized ratio [INR] 1.85) with normal urinalysis and normal plasma D-dimer and fibrin degradation products. Acute fatty liver was suspected, and the patient was admitted to the ICU in the evening. In the ICU, her blood Hb was 88 g/L (dropped from 105 g/L), and her blood platelet count was 51 × 109/L. Internist, hematologist, and anesthetist consultants were involved in her care. Septic workup was done, including urine and blood cultures, as well as high vaginal and endocervical swabs for culture and sensitivity. Because she was critically ill in the ICU with too many intravenous catheters and an indwelling urinary catheter, and because patients with AFLP are at risk of infection, a decision was taken by the multidiscip- linary team to start her on a renal dose of imipenem/cilas- tatin. She was kept on intravenous fluid, normal saline (N/ S) 100 ml/hour, and dextrose infusion. Five units of fresh frozen plasma (FFP), 5 U of cryoprecipitate, and 2 U of packed red blood cells (PRBCs) were given. Day 2 postpartum was marked by persistence of nau- sea and vomiting and a decrease in the intensity of skin rash. On day 3 postpartum, she had nausea, vomiting, and abdominal pain. Her skin rash showed a further de- crease in intensity. She was very sick, pale, and jaundiced with epigastric and right upper quadrant abdominal Fig. 1 Skin rash over the right hand and forearm On the fourth day postpartum, the patient had persist- ent nausea, vomiting, and epigastric and right upper quadrant abdominal pain. Her vital signs were stable. She was jaundiced. Her skin rash had significantly decreased in distribution and intensity. Case presentation She had a strict fluid input-output observation. Her urine output remained around 45–60 ml/hour. Her investigations showed anemia and thrombocytopenia (blood Hb 79 g/L and blood platelet count 44 × 109/L), acute renal impair- ment (serum creatinine 316.4 μmol/L), very high serum Fig. 1 Skin rash over the right hand and forearm Al-Husban et al. Journal of Medical Case Reports (2018) 12:67 Page 3 of 6 Page 3 of 6 Page 3 of 6 LDH (19.7 μkat/L), elevated serum ALT (0.77 μkat/L), and elevated serum AST (1.52 μkat/L) with elevated serum direct and total bilirubin. Her serum glucose was 3.38 mmol/L (on dextrose infusion), and her total serum bile acids level was normal (6 μmol/L). Blood film showed hypochromic microcytic anemia, few schisto- cytes and acanthocytes, neutrophilia with toxic granula- tion of neutrophils, a majority of neutrophils that were hypersegmented, and thrombocytopenia. She received 2 U of PRBCs, 2 U of FFP, and 4 U of cryoprecipitate and was started on dexamethasone 4 mg intravenously every 8 hours. (very mild nausea, occasional vomiting, and mild abdom- inal pain) with stable V/S. Blood tests showed Hb 98 g/L, blood platelet count 60 × 109/L, blood WBC 16 × 109/L (76% neutrophils and 16% lymphocytes), serum glucose 6.1 mmol/L, serum creatinine 251.6 μmol/L, serum urea nitrogen 52.1 mmol/L, and serum LDH 11.6 μkat/L with normal electrolytes and liver enzymes. y y A CXR showed reticular shadowing bilaterally, a blunt left costophrenic angle, and a clear right costophrenic angle, which further supported the continuation of the antibiotic. She was given 4 U of FFP. On the eighth day postpartum (the sixth day in the ICU), the patient was very well with no nausea, vomiting, or ab- dominal pain. Her dextrose infusion was disconnected. She was started on oral intake of fluids. She remained normo- glycemic. She was prophylactically given 5 U of cryoprecipi- tate, 5 U of FFP, and 2 U of PRBCs for of her mild thrombocytopenia and anemia. In the evening, repeat blood test results were normal apart from mild elevation of serum creatinine. A decision was taken to discharge her from the ICU. In the afternoon, after transfusion of blood and blood products, her blood platelet count was 38 × 109/L, blood Hb 97 g/L, and blood WBC 14.9 × 109/L. Case presentation Other tests revealed plasma PT 17.5 seconds, blood PTT 29.7 sec- onds, and INR 1.4 (corrected by the infusion of the blood and blood products). An abdominopelvic computed tomographic (CT) scan without contrast enhancement revealed only hyperdense free fluid (ascites). A chest x-ray (CXR) showed congestive pulmonary changes and blunted bilateral costophrenic angles. She was started on furosemide 20 mg intraven- ously every 4 hours, intravenous fluid dextrose 25% 50 ml/hour, and N/S 0.9% 50 ml/hour. On the ninth day postpartum (the first day in the obstet- ric ward), the patient was very well with no complaints. She resumed breastfeeding in addition to artificial supple- ment. Her laboratory test results were normal. Her full sep- tic workup result was negative. Imipenem/cilastatin was discontinued. On the fifth day postpartum (the third day in the ICU), the patient still felt unwell with epigastric and right upper quadrant abdominal pain and recurrent attacks of hypoglycemia. She had no skin rash at all. She had normal BP readings with mild epigastric and right upper quadrant tenderness. Her laboratory tests showed anemia, thrombocytopenia, hypoglycemia, leukocytosis, renal im- pairment, hyperbilirubinemia, and elevated serum LDH. Urinalysis showed 1+ proteinuria and hematuria. The result of a viral hepatitis screen was negative. On the tenth day postpartum, the patient was very well and had no complaint. The results of her blood tests were normal apart from very mildly elevated serum creatinine. The patient’s 11th postpartum day was unremarkable; she had no complaints and normal laboratory test results. On the 12th day postpartum (4th day in the obstetric ward), the patient was very well with stable vital signs and no complaints. She had normal serum glucose, normal serum electrolytes, and normal liver enzymes and serum bilirubin (total and direct). Her serum LDH was 10.1 μkat/L, blood Hb 105 g/L, blood platelet count 584 × 109/L, blood WBC 11.6 × 106/L, and serum cre- atinine 1.43. In the afternoon, she was discharged to home receiving no medications. An abdominal U/S scan showed a marked amount of free fluid in the abdomen, liver span 17 cm, spleen span 14 cm, and a normal hepatobiliary tree with no stones or dilatation. A CXR was normal. She was given 5 U of FFP and kept on the antibiotic because of the ascitic fluid. On the sixth day postpartum (fourth day in the ICU), the patient showed significant clinical improvement with stable vital signs (V/S). Case presentation Her blood tests showed persist- ent anemia, thrombocytopenia, leukocytosis, elevated serum creatinine, elevated serum LDH, mild elevation of serum bilirubin, normal serum glucose, and normal liver enzymes and coagulation. A repeat blood film showed hypochromic microcytic anemia with mild anisocytosis, neutrophilic leukocytosis, few hypersegmented neutro- phils and thrombocytopenia with large forms. She was prophylactically given 5 U of FFP as suggested by the multidisciplinary team. The patient was seen in the clinic 1 week later. She was doing well with no complaints and was seeking contraception. One month later, she and her baby were doing well with no complaints. In the clinic, she had an intrauterine contraceptive device inserted. The chronological order of her symptomatology and laboratory results are shown in Tables 1 and 2, respectively. Discussion In our literature research, we did not come across skin rash preceding or being part of an AFLP presentation. Our pa- tient’s skin rash was different from pruritic urticarial papules On the seventh day postpartum (fifth day in the ICU), the patient started to show much clinical improvement Al-Husban et al. Journal of Medical Case Reports (2018) 12:67 Page 4 of 6 Table 1 Patient’s symptoms in chronological order postpartum Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 12 (discharged to home) Skin rash +++ ++ + + − − − − − − Nausea + +++ +++ +++ + Very mild Very mild − − − Vomiting + +++ +++ ++ + Occasional Very occasional − − − General feeling Very unwell Very unwell Extremely unwell Unwell Unwell Well Very well Very well Very well Very well Abdominal pain + +++ +++ ++ + Very mild − − − − + mild, ++ moderate, +++ severe, - no or nil + mild, ++ moderate, +++ severe, - no or nil of severe hypoglycemia. The usual presentation of AFLP is nonspecific [6]. The diagnosis of the condition is sug- gested by jaundice, mild liver enzyme elevation, elevated WBC, disseminated intravascular coagulation (DIC), and a clinically unwell patient [6]. All these features were very apparent and evident in our patient (raised serum and plaques of pregnancy because it was neither pruritic nor associated with striae, and it involved both upper limbs and the abdomen [5]. Abbreviations: ALT Alanine aminotransferase, AST Aspartate aminotransferase, FDP Fibrin degradation products, Hb Hemoglobin, HPF High-power field, INR International normalized ratio, LDH Lactate dehydrogenase, mg/L milligram/Litre, mmol/L millimole/Litre, N normal, nmol/L nanomole/ Litre, PT Prothrombin time, PTT Partial thromboplastin time, RBC Red blood cell, μmol/L micromole/Litre, μkat/L microkatal/Litre, WBC White blood cell, g/L gram/Litre, + 1 proteinuria Values are given in standard international units Discussion Initially, the diagnosis of AFLP was suspected because of the abrupt onset of feeling very unwell; abrupt onset of abdominal pain, nausea, and vomiting; and the attacks Table 2 Laboratory test results in chronological order postpartum Table 2 Laboratory test results in chronological order postpartum Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Hb, g/L 112 88 79, then 97 after transfusion 94 98 98 101 102 105 Platelets, ×109/L 161 54 then 51 44 then 38 30 34 60 80 482 584 WBC, ×109/L 10.0 7.0 9.0 then 14.9 14.9 15.0 16.0 12.0 14.0 11.6 Serum creatinine, μmol/L 79.5 265.2 366.8 485.3 371.2 291.7 236.0 203.3 190.0 147.6 126.8 Serum urea nitrogen, mmol/L 10.7 56.8 55.7 52.1 50.7 40.7 33.5 Serum ALT, μkat/L 0.27 0.735 0.77 0.42 N N Serum AST, μkat/L 0.22 1.15 1.52 0.57 N N Serum bilirubin direct, μmol/L 1.71 58.15 71.8 34.2 22.2 10.3 Serum bilirubin total, μmol/L 5.13 68.4 82.1 42.8 30.8 15.4 PT, seconds 12 22 17.5 14 14.5 PTT, seconds 26 36 29.7 26 28 INR 1.0 1.85 1.4 1.06 1.11 1.1 Plasma D-dimer, nmol/L 1.9 N 9.8 FDP, mg/L 7.0 N Plasma fibrinogen, μmol/L 9.0 N 3.36 LDH, μkat/L 5.37 19.8 19.7 19.39 14.5 11.6 14.16 10.1 Urinalysis 3+ proteinuria, numerous WBC/HPF, no glycosuria N N + Proteinuria, 8–10 RBC/HPF Serum glucose, mmol/L 3.4 2.11 3.38 3.0 4.1 6.1 3.9 5.1 4.2 5.2 4.7 Abbreviations: ALT Alanine aminotransferase, AST Aspartate aminotransferase, FDP Fibrin degradation products, Hb Hemoglobin, HPF High-power field, INR International normalized ratio, LDH Lactate dehydrogenase, mg/L milligram/Litre, mmol/L millimole/Litre, N normal, nmol/L nanomole/ Litre, PT Prothrombin time, PTT Partial thromboplastin time, RBC Red blood cell, μmol/L micromole/Litre, μkat/L microkatal/Litre, WBC White blood cell, g/L gram/Litre, + 1 proteinuria Values are given in standard international units Abbreviations: ALT Alanine aminotransferase, AST Aspartate aminotransferase, FDP Fibrin degradation products, Hb Hemoglobin, HPF High-power field, INR International normalized ratio, LDH Lactate dehydrogenase, mg/L milligram/Litre, mmol/L millimole/Litre, N normal, nmol/L nanomole/ Litre, PT Prothrombin time, PTT Partial thromboplastin time, RBC Red blood cell, μmol/L micromole/Litre, μkat/L microkatal/Litre, WBC White blood cell, g/L gram/Litre, + 1 proteinuria Values are given in standard international units Al-Husban et al. Funding Funding All authors declare that they have not received any external source of funding for the present study. Acknowledgements None. Acknowledgements None. Conclusions The clinical presentation of AFLP is very variable and nonspecific. Skin rash can be a new presenting symp- tom of AFLP. In our patient, immediate suspicion of the diagnosis, appropriate investigations, and urgent initiation of therapy in an ICU and by a multidisciplin- ary team resulted in a good outcome with no adverse health consequences. Our patient’s blood laboratory test results showed marked elevation of serum bilirubin and jaundice with only mild liver enzyme elevation. She also had leukocytosis and ascites. These results, in addition to the patient’s clinical symptoms and hypoglycemia, were consistent with the diagnosis of AFLP [16]. Availability of data and materials The data presented in this case report are the original patient’s data. Therefore, the authors will not share it in an additional file. Discussion Journal of Medical Case Reports (2018) 12:67 Page 5 of 6 Page 5 of 6 Page 5 of 6 bilirubin, raised blood WBC and DIC). The differential diagnosis includes preeclampsia, HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets), viral hepatitis, and obstetric cholestasis [3, 6–8]. Our pa- tient’s BP remained normal prior to delivery and all through her hospital stay until discharge. She had no itching to suggest obstetric cholestasis, and her serum bile acid level was normal (6 μmol/L). Her viral hepatitis screen result was negative. patient on an antibiotic and watched her carefully for the development of any sign of adult respiratory distress syndrome (ARDS). On the basis of her CXR, she needed an intravenous diuretic. ARDS might occur as a compli- cation of acute liver failure, septicemia, or transfusion of multiple blood products [23]. After the fifth day of the clinical onset of our patient’s presentation, she started to show clinical and hemato- biochemical improvement. In patients who develop AFLP antenatally, clinical recovery typically is seen within 3–4 days; however, laboratory abnormalities can persist much longer [24]. g At an early stage, these patients may have an upper gastrointestinal hemorrhage due to coagulation abnor- malities, acute renal failure, infection, pancreatitis, or hypoglycemia [9, 10]. Our patient had acute renal failure and persistent hypoglycemia with the need for strict input-output observation and intravenous dextrose infu- sion. She maintained a normal urine output. The associ- ation of transient diabetes insipidus and AFLP appears more common than previously recognized. Both may be part of the spectrum of preeclampsia [11]. Hypoglycemia and prolongation of PT helped us to differentiate our patient’s presentation from HELLP syndrome. DIC is relatively common in these cases [12, 13]. Our patient received appropriate infusions of FFP and cryoprecipi- tate. She also developed thrombocytopenia, which is a known complication of AFLP [14]. We obtained both a CT scan and a US scan. Both imaging modalities are noninvasive but have limited usefulness in the AFLP diagnosis [15]. Our patient made a quick, uncomplicated recovery. We found a case report of massive intrahepatic calci- fication [25]. AFLP can progress to fulminant hepatic failure with the need for liver transplant, encephalop- athy, coma, and death [26, 27]. The clinical manifes- tations of patients with mutations in enzymes of fatty acid metabolism may include AFLP that may mimic severe preeclampsia [28]. Authors’ contributions NAH was the patient’s consultant and the main writer of the manuscript. OAK contributed to the literature search and writing of the manuscript. AAH collected the patient’s clinical notes. All authors read and approved the final manuscript. In the ICU, our patient was conscious, alert, and did not need ventilator support. Patients who have received ventilator support or encephalopathy and failed to re- spond to conventional supportive therapy have benefited from plasma exchange alone or in combination with continuous hemodiafiltration [20–22]. We started our Abbreviations AFLP: Acute fatty liver of pregnancy; ALT: Alanine aminotransferase; ARDS: Adult respiratory distress syndrome; AST: Aspartate aminotransferase; BP: Blood pressure; CT: Computed tomographic; CXR: Chest x-ray; DIC: Disseminated intravascular coagulation; FDP: Fibrin degradation product; FFP: Fresh frozen plasma; Hb: Hemoglobin; HELLP syndrome: Hemolysis, elevated liver enzymes, and low platelets; HPF: High-power field; ICU: Intensive care unit; INR: International normalized ratio; LDH: Lactate dehydrogenase; N/S: Normal saline; PRBC: Packed red blood cell; PT: Prothrombin time; PTT: Partial thromboplastin time; RBC: Red blood cell; US: Ultrasound; WBC: White blood cell count The patient went through hemostatic dysfunction in the form of hemolytic anemia and DIC, as indicated by the hematological test results. Hemostatic dysfunction started very early in her condition and persisted for a few days thereafter. In those patients who develop AFLP prior to delivery, this dysfunction persists 4–5 days post- partum [17]. Our patient received infusions of PRBCs, FFP, and cryoprecipitate. Severe cases of AFLP can lead to coagulopathy, liver failure, and hypoglycemia. The pathological hepatic condition is usually self-limiting, with liver function returning to normal 7–9 days after delivery [18, 19]. Fluid therapy in our patient was very strict to avoid pulmonary edema caused by low plasma oncotic pressure. References 1. Sheehan HL. The pathology of acute yellow atrophy and delayed 1. Sheehan HL. The pathology of acute yellow atrophy and delayed chloroform poisoning. J Obstet Gynaecol. 1940;47:49–61. chloroform poisoning. J Obstet Gynaecol. 1940;47:49–61. 2. Riely CA. Acute fatty liver of pregnancy. Semin Liver Dis. 1987;7(1):47–54. 3. Ko H, Yoshida EM. Acute fatty liver of pregnancy. Can J Gastroenterol. 2006; 20(1):25–30. 4. Bellig LL. Maternal acute fatty liver of pregnancy and the associated risk for long-chain 3-hydroxyacyl-coenzyme a dehydrogenase (LCHAD) deficiency in infants. Adv Neonatal Care. 2004;4(1):26–32. in infants. Adv Neonatal Care. 2004;4(1):26–32. 5. Dehdashti AL, Wikas SM. Pruritic urticarial papules and plaques of pregnancy occurring postpartum. Cutis. 2015;95(6):344–7. 5. Dehdashti AL, Wikas SM. Pruritic urticarial papules and plaque pregnancy occurring postpartum. Cutis. 2015;95(6):344–7. 6. Wei Q, Zhang L, Liu X. Clinical diagnosis and treatment of acute fatty liver of pregnancy: a literature review and 11 new cases. J Obstet Gynaecol Res. 2010;36(4):751–6. 7. Riely CA. Liver disease in the pregnant patient. Am J Gastroenterol. 1999; 94(7):1728–32. 8. Pang WW, Lei CH, Chang DP, Yang TF, Chung YT, Huang MH. Acute jaundice in pregnancy: acute fatty liver or acute viral hepatitis? Acta Anaesthesiol Sin. 1999;37(3):167–70. 8. Pang WW, Lei CH, Chang DP, Yang TF, Chung YT, Huang MH. Acute jaundice in pregnancy: acute fatty liver or acute viral hepatitis? Acta Anaesthesiol Sin. 1999;37(3):167–70. 9. Kaplan MM. Acute fatty liver of pregnancy. N Engl J Med. 1985;313:367–70. 9. Kaplan MM. Acute fatty liver of pregnancy. N Engl J Med. 1985;313:367–70. 10. Vigil-De Gracia P, Lavergne JA. Acute fatty liver of pregnancy. Int J Gynaecol Obstet. 2001;72:193–5. 10. Vigil-De Gracia P, Lavergne JA. Acute fatty liver of pregnancy. Int J Gynaecol Obstet. 2001;72:193–5. 11. Kennedy S, Hall PM, Seymour AE, et al. Transient diabetes insipidus and acute fatty liver of pregnancy. Br J Obstet Gynaecol. 1994;101:387–91. 11. Kennedy S, Hall PM, Seymour AE, et al. Transient diabetes insipidus and acute fatty liver of pregnancy. Br J Obstet Gynaecol. 1994;101:387–91. 12. Holzbach RT. Acute fatty liver of pregnancy with disseminated intravascula coagulation. Obstet Gynecol. 1974;43:740–4. 12. Holzbach RT. Acute fatty liver of pregnancy wit coagulation. Obstet Gynecol. 1974;43:740–4. 13. Cano RI, Delman MR, Pitchumoni CS, Lev R, Rosenthal WS. Acute fatty liver of pregnancy. Complication by disseminated intravascular coagulation. JAMA. 1975;231:159–61. 14. Burroughs AK, Seong NH, Dojcinov DM, Scheuer PJ, Sherlock SV. Idiopathic acute fatty liver of pregnancy in 12 patients. Competing interests The authors declare that they have no competing interests. Publisher’s Note 28. Iruretagoyena JI, Shah D. A case of severe preeclampsia leading to the diagnosis of de novo abnormal fatty acid metabolism and ACE gene deletion. J Obstet Gynaecol Can. 2010;32(7):695–7. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Received: 1 September 2017 Accepted: 27 January 2018 Ethics approval and consent to participate pp p p Approval from the Department of Obstetrics and Gynecology at Jordan University Hospital was obtained for this case publication. Page 6 of 6 Page 6 of 6 Al-Husban et al. Journal of Medical Case Reports (2018) 12:67 24. Nelson DB, Yost NP, Cunningham FG. Acute fatty liver of pregnancy: clinical outcomes and expected duration of recovery. Am J Obstet Gynecol. 2013; 209(5):456–7. 24. Nelson DB, Yost NP, Cunningham FG. Acute fatty liver of pregnancy: clinical outcomes and expected duration of recovery. Am J Obstet Gynecol. 2013; 209(5):456–7. 25. Bhat KJ, Shovkat R, Samoon HJ. Postpartum Acute liver dysfunction: a case of acute fatty liver of pregnancy developing massive intrahepatic calcification. Gastroenterol Res. 2015;8(6):313–5. 26. Bacq Y. Liver diseases unique to pregnancy: a 2010 update. Clin Res Hepatol Gastroenterol. 2011;35:182–93. 27. Heneghan MA, Selzner M, Yoshida EM, Mullhaupt B. Pregnancy and sexual function in liver transplantation. J Hepatol. 2008;49(4):507–19. 28. Iruretagoyena JI, Shah D. A case of severe preeclampsia leading to the diagnosis of de novo abnormal fatty acid metabolism and ACE gene deletion. J Obstet Gynaecol Can. 2010;32(7):695–7. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. 25. Bhat KJ, Shovkat R, Samoon HJ. Postpartum Acute liver dysfunction: a case of acute fatty liver of pregnancy developing massive intrahepatic calcification. Gastroenterol Res. 2015;8(6):313–5. References Q J Med. 1982;51:481–97. 15. Castro MA, Ouzounian JG, Colletti PM, Shaw KJ, Stein SM, Goodwin TM. Radiologic studies in acute fatty liver of pregnancy: a review of the literature and 19 new cases. J Reprod Med. 1996;41:839–43. 16. Knight M, Nelson-Piercy C, Kurinczuk JJ, Spark P, Brocklehurst P. UK obstetric surveillance system: a prospective national study of acute fatty liver of pregnancy in the UK. Gut. 2008;57:951–6. 17. Nelson DB, Yost NP, Cunningham FG. Hemostatic dysfunction with acute fatty liver of pregnancy. Obstet Gynecol. 2014;124(1):40–6. 18. Bacq Y. Acute fatty liver of pregnancy. Semin Perinatol. 1998;22:134–40. 19. Castro MA, Fassett MJ, Reynolds TB, et al. Reversible peripartum liver failure: a new prospective on the diagnosis, treatment and cause of acute fatty liver of pregnancy, based on 28 consecutive cases. Am J Obstet Gynecol. 1999;181:389–95. 25. Bhat KJ, Shovkat R, Samoon HJ. Postpartum Acute liver dysfunction: a case of acute fatty liver of pregnancy developing massive intrahepatic calcification. Gastroenterol Res. 2015;8(6):313–5. 28. Iruretagoyena JI, Shah D. A case of severe preeclampsia leading to the diagnosis of de novo abnormal fatty acid metabolism and ACE gene deletion. J Obstet Gynaecol Can. 2010;32(7):695–7. Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: 20. Martin JN Jr, Briery CM, Rose CH, et al. Postpartum plasma exchange as adjunctive therapy for severe acute fatty liver of pregnancy. J Clin Apher. 2008;23(4):138–43. 21. Jin F, Cao M, Bai Y, et al. Therapeutic effects of plasma exchange for the treatment of 39 patients with acute fatty liver of pregnancy. Discov Med. 2012;13(72):369–73. 22. Chu YF, Mei M, Juan Z, et al. Effectiveness of combining plasma exchange with continuous hemodiafiltration on acute fatty liver of pregnancy complicated by multiple organ dysfunction. Artif Organs. 2012;36(6):530–4. 23. Kalpana S, Veena R, Geeta P, et al. Acute fatty liver of pregnancy: a case report of an uncommon disease. Indian J Crit Care Med. 2009;13(1):34–6.
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The Influence of Content Meaningfulness on Eye Movements across Tasks: Evidence from Scene Viewing and Reading
Frontiers in psychology
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ORIGINAL RESEARCH published: 01 March 2016 doi: 10.3389/fpsyg.2016.00257 Edited by: Guillaume A. Rousselet, University of Glasgow, UK Reviewed by: Sébastien M. Crouzet, Centre de Recherche Cerveau et Cognition, France Françoise Vitu, Aix-Marseille Université, France Keywords: eye movements, cognitive control, meaning, reading, scene perception, eye tracking, eye movement control *Correspondence: Steven G. Luke steven_luke@byu.edu *Correspondence: Steven G. Luke steven_luke@byu.edu The Influence of Content Meaningfulness on Eye Movements across Tasks: Evidence from Scene Viewing and Reading Steven G. Luke1* and John M. Henderson2,3 1 Department of Psychology and Neuroscience Center, Brigham Young University, Provo, UT, USA, 2 Department of Psychology, University of California Davis, Davis, CA, USA, 3 Center for Mind and Brain, University of California, Davis, Davis, CA, USA The present study investigated the influence of content meaningfulness on eye- movement control in reading and scene viewing. Texts and scenes were manipulated to make them uninterpretable, and then eye-movements in reading and scene-viewing were compared to those in pseudo-reading and pseudo-scene viewing. Fixation durations and saccade amplitudes were greater for pseudo-stimuli. The effect of the removal of meaning was seen exclusively in the tail of the fixation duration distribution in both tasks, and the size of this effect was the same across tasks. These findings suggest that eye movements are controlled by a common mechanism in reading and scene viewing. They also indicate that not all eye movements are responsive to the meaningfulness of stimulus content. Implications for models of eye movement control are discussed. INTRODUCTION When we are performing a visual task, such as reading, searching for an object, or just looking around at the world, our eyes make rapid movements, called saccades, several times a second. In between these saccades, the eyes make pauses (called fixations) to take in visual information. Researchers who study eye movements are interested in why we look where we do and why we move our eyes when we do. It is thought by many that eye movements are under cognitive control, meaning that the where and when of eye movements are influenced by cognitive processes related to perception, memory, and language (Rayner, 2009; Rayner and Reingold, 2015; but see Yang and McConkie, 2001; Vitu, 2003 for an alternate viewpoint). In short, cognitive control implies that our eye movements respond quickly to the nature of what we are looking at in any given moment. h d f d h b f l d f h fl f Specialty section: This article was submitted to Perception Science, a section of the journal Frontiers in Psychology Received: 19 October 2015 Accepted: 09 February 2016 Published: 01 March 2016 Specialty section: This article was submitted to Perception Science, a section of the journal Frontiers in Psychology Received: 19 October 2015 Accepted: 09 February 2016 Published: 01 March 2016 Citation: Luke SG and Henderson JM (2016) The Influence of Content Meaningfulness on Eye Movements Specialty section: This article was submitted to Perception Science, a section of the journal Frontiers in Psychology Received: 19 October 2015 Accepted: 09 February 2016 Published: 01 March 2016 Citation: Luke SG and Henderson JM (2016) The Influence of Content Meaningfulness on Eye Movements across Tasks: Evidence from Scene Viewing and Reading. Front. Psychol. 7:257. doi: 10.3389/fpsyg.2016.00257 The study of reading has proven to be fertile ground for investigating the influence of cognitive factors on eye movement control. This is primarily because words have multiple quantifiable cognitive properties such as frequency and predictability, properties that influence the processing of those words in multiple ways. Studies exploring the influence of these cognitive properties have shown that cognitive processing can influence eye movements on a moment-by-moment basis (Just and Carpenter, 1980; Rayner, 1998, 2009; Kliegl et al., 2006). March 2016 | Volume 7 | Article 257 1 Frontiers in Psychology | www.frontiersin.org Meaningfulness and Eye Movement Control Luke and Henderson Studying the influence of cognitive processing in other visual tasks, such as scene viewing, has proven more difficult, because the units of processing (i.e., objects) are not so clearly differentiated in visual scenes (they overlap) and their cognitive properties are not so easily defined or quantified. As a result, the influence of cognitive factors in scene viewing is less clearly understood (Henderson et al., 1999; Henderson, 2003; Võ and Henderson, 2009; Wang et al., 2010). Researchers have therefore been forced to rely more on manipulations of global image properties in order to investigate cognitive control of eye movements in these scene viewing tasks. These global manipulations, which are described below, reduce the possibility of cognitive control by removing or denying access to information needed to interpret and understand the image. paradigm thus simulates processing difficulty in a manner that is precisely controlled and easily applied to a variety of different visual tasks. This paradigm has been employed to study eye movement control in both reading (Rayner and Pollatsek, 1981; Morrison, 1984; Dambacher et al., 2013) and scene processing (Henderson and Pierce, 2008; Henderson and Smith, 2009). The SOD paradigm has also been used to compare reading and scene viewing directly (Nuthmann and Henderson, 2012; Luke et al., 2013), and equivalent effects of delay duration on fixation durations were observed in both tasks, strongly suggesting that reading and scene viewing share a common mechanism for the control of fixation duration. The SOD paradigm typically reveals two populations of fixations: some that outlast the delay and others that do not (Henderson and Pierce, 2008; Henderson and Smith, 2009; Luke et al., 2013). Citation: The first population of fixations appear to be under cognitive control, increasing linearly with the duration of the delay, while the second population of fixations does not lengthen in response to the presence of the mask, suggesting that these fixations are not under cognitive control. Thus, direct comparisons of eye movement control in different tasks are important not only because they tell us that these tasks share a common mechanism, but also because they reveal something about the nature of that mechanism. In some models, such as E–Z reader (Reichle et al., 1998), most or all reading saccades are initiated by successful completion of some stage of lexical access, while other models such as SWIFT (Engbert et al., 2002, 2005), CRISP (Nuthmann et al., 2010; Nuthmann and Henderson, 2012), and the competition-interaction model (Yang and McConkie, 2001) delay some saccades when cognitive processing difficulty is encountered. The existence of two different populations of fixations is more consistent with the latter class of models. Exploring eye movement control across multiple tasks can help to further adjudicate between these different proposals. These global image manipulations are also useful because they can also be applied to reading, permitting direct cross-task comparisons. This is important because reading is measurably different from other visual tasks, such as scene viewing. For example, fixation durations are significantly shorter in reading (Henderson and Hollingworth, 1998; Rayner, 2009; Luke et al., 2013). Saccade amplitudes tend to be shorter in reading as well (Rayner, 2009; Henderson and Luke, 2014). On the other hand, in reading the eyes are presumably guided by the same neural systems as in other visual tasks, and eye movements are made for the purpose of gathering information regardless of task. In support of this is the observation that aggregate measures of eye movement behavior correlate across tasks (Henderson and Luke, 2014), although not all researchers have found this relationship with regard to reading (Rayner et al., 2007). At the same time, while many core visual and eye movement control areas appear to be common to both tasks (Choi and Henderson, 2015), cognitive control in reading and scene viewing could be exerted by different cortical centers. Citation: For example, the parahippocampal place area (PPA; Epstein and Kanwisher, 1998; Epstein et al., 1999) appears to be involved in scene viewing but not in reading (Choi and Henderson, 2015; Henderson and Choi, 2015), while the visual word form area (VWFA; Cohen et al., 2000; McCandliss et al., 2003) and language areas in the left hemisphere contribute to eye movement control in reading but not in scene viewing (Choi and Henderson, 2015; Henderson et al., 2015). So, the underlying processes that control eye movements, making them sensitive to the meaning of words and objects, might differ in significant ways in reading compared to other tasks. Identifying which processes are common to all tasks and which are task-specific is an important goal of eye-movement research. Another global method for exploring eye movement control that has been employed exclusively in reading is the pseudo- reading paradigm, in which all letters in a text are replaced with block shapes or a single letter such as Z. This manipulation removes all meaning from the text, but preserves the visual- spatial layout of the words, sentences, and paragraphs. A finding from this technique is that fixations are typically longer in pseudo-reading than in reading. This finding seems rather paradoxical at first, as one might expect longer fixations when cognitive processing is engaged, not when it is absent, as no processing difficulty should occur when there is nothing to process; nevertheless this finding has been consistently shown across studies (Vitu et al., 1995; Rayner and Fischer, 1996). If we define processing difficulty simply as an inability to identify a stimulus, such as a word, then it makes sense that fixation durations should be lengthened when meaning is removed and identification is not possible. Or it could be that having meaning facilitates processing, thereby shortening fixations relative to the meaningless text condition (Reichle et al., 2012). Usually, the global image manipulations used to study eye movement control involve obscuring or removing the stimulus for an extended period of time. Sometimes participants are given a brief view of part of the text or of the scene before it is removed from view (Rayner et al., 2003, 2009). The results of these studies suggest that saccades are under cognitive control in both tasks. Frontiers in Psychology | www.frontiersin.org Materials The present study uses the principle behind the pseudo- reading paradigm, the removal of meaningfulness, to directly compare the cognitive control of eye movements in reading and in scene viewing. We manipulated text and scenes to create a pseudo-reading and a pseudo-scene viewing condition, and we then compared participants’ eye movements in the two tasks and in their pseudo-variants in a within-subjects design. Based on previous research on reading, we expected increased fixation durations for meaningless stimuli (Vitu et al., 1995; Rayner and Fischer, 1996; Luke and Henderson, 2013). Filtering scenes to remove high-frequency visual information, which makes object identification difficult, has also been shown to increase fixation durations (Mannan et al., 1997; Henderson et al., 2014b). We predict, therefore, that fixation durations will be longer for our pseudo-stimuli, indicating that at least some fixations are under cognitive control. Fifty-six short paragraphs (40–60 words) were taken from online news articles. These texts were used in Luke and Henderson (2013) and included a total of 1415 unique words: three one- letter words, 30 two-letter words, 93 three-letter words, 197 four-letter words, 247 five-letter words, 227 six-letter words, 218 seven-letter words, 159 eight-letter words, 110 nine-letter words, and 131 words 10 letters or longer. Two different versions of each text were created, a Normal Reading version, in which the text appeared on the screen in Courier New 16pt font, and a Mindless Reading version, in which the text was displayed in a custom font (also 16pt). This font transformed letters into block shapes (see Figure 1) while preserving overall word shape. Both fonts were monospace, and all letters, words, and lines of text appeared in exactly the same location regardless of font. g This manipulation permits us to test two more specific hypotheses about eye-movement control as well. The first is that the same systems control eye movements in reading and in scene viewing: If eye movements are controlled by the same systems across tasks, then eye movements should be influenced similarly by the removal of meaningfulness in both tasks. We note here that we use the term ‘meaningfulness’ because of the global nature of the manipulation; the pseudo-stimuli differ from the original text and scenes on many levels, but what is important is that they are not interpretable, meaning that cognitive control has little opportunity to influence eye movements for these stimuli (see Figures 1 and 2 below). Materials The second hypothesis is that, consistent with findings from the SOD paradigm and from pseudo-reading that not all fixations are under cognitive control (Henderson and Pierce, 2008; Henderson and Smith, 2009; Luke and Henderson, 2013; Luke et al., 2013; Henderson and Luke, 2014), the removal of meaningfulness will only affect some, and, not all, fixations. For the scene stimuli, pseudo-scenes were created that were analogous to the pseudo-texts in that they had a complex visual structure similar to that of their real scene counterparts but were not meaningful. These pseudo-scenes did not contain any identifiable objects and were not easily assigned to a particular scene category. In order to create a set of pseudo-scenes, we began with a large set of 840 images. The images depicted scenes from seven different categories, five outdoor and two indoor. The outdoor scenes were images of beaches, forests, mountains, cityscapes, and highways (Walther et al., 2009), while the indoor scenes were images of bedrooms and kitchens. g Our goal was to find a manipulation for scenes that was similar to the manipulation that we and others had previously used for text (see Figure 1), which still looked like text but was not interpretable. Specifically, we wanted to (1) preserve the spatial layout of the scene as much as possible, while (2) removing meaning. This proved to be a difficult task for visual scenes, and we tried and rejected several different methods. The manipulation we ultimately chose removed the meaning from these images via an extensive filtering process that extracted the edges from the images, expanded and distorted these edges, and then filled the empty areas within these newly warped edges with color, a process which also erased the edge lines. Then both the scenes and pseudo-scenes were transformed to grayscale. Citation: Another technique, called the stimulus onset delay (SOD) paradigm, involves covering all or part of the visual stimulus with a mask during predefined saccades, so that when the next fixation begins the stimulus is not visible. The mask remains on screen for a predetermined and varied delay and is then removed so that the stimulus again becomes visible. The SOD Like the SOD paradigm, the pseudo-reading technique has also shown that some eye movement behaviors in reading are under the influence of cognitive control, while others appear not March 2016 | Volume 7 | Article 257 2 Meaningfulness and Eye Movement Control Luke and Henderson to be (Nuthmann et al., 2007; Henderson and Luke, 2012; Luke and Henderson, 2013). The pseudo-reading technique has also been used in combination with EEG or MRI to explore the neural bases of cognitive control in reading (Henderson et al., 2013, 2014a, 2015). minimized with a chin and head rest. Although viewing was binocular, eye movements were recorded from the right eye. The experiment was controlled with SR Research Experiment Builder software. Participants Forty participants recruited from Brigham Young University completed the experiment. All participants were native English speakers with 20/20 corrected or uncorrected vision. Prior to participant recruitment, the institutional review board that Brigham Young University approved the study. This manipulation disguised the identities of the objects in the image and made the scene categories difficult to identify. The pseudo-scenes were then normed to see which ones were the most difficult to identify. All 840 meaningless images were posted on Amazon’s Mechanical Turk (Buhrmester et al., 2011). Participants were told that each image was created by altering a photograph of a scene, and asked to provide a short label identifying the category of the scene that the image was created from (or “Don’t Know” if they were unable to identify it) and to rate their confidence in the label they had provided. Each image was labeled by ten different participants. Apparatus Eye movements were recorded via an SR Research Eyelink 1000 plus tower mount eye tracker (spatial resolution of 0.01◦) sampling at 1000 Hz. Subjects were seated 60 cm away from a 24′′ LCD monitor with display resolution set to 1600 × 900, so that approximately three characters subtended 1◦of visual angle. Scenes (800 × 600 pixel images) subtended 21 by 16◦of visual angle. Head movements were March 2016 | Volume 7 | Article 257 Frontiers in Psychology | www.frontiersin.org 3 Luke and Henderson Meaningfulness and Eye Movement Control FIGURE 1 | Examples of text and pseudo-text. Normal text is above, with the corresponding pseudo-text below. Meaningfulness and Eye Movement Control Luke and Henderson FIGURE 1 | Examples of text and pseudo-text. Normal text is above, with the corresponding pseudo-text below. Based on these norming data, 56 pseudo-scenes were selected, eight from each scene category. Overall, participants in the norming study gave the “Don’t Know” response for 73% of the images, and for the minority of images that they did attempt to provide a label for, they provided a correct label only 10% of the time. Confidence ratings were very low (M = 1.34 on a 5- point scale). Therefore, these images were extremely difficult to identify, even for participants who knew that the images had been derived from actual scenes. Examples of the images used can be seen in Figure 2. were further told that they should view each image in preparation for a memory test that would be administered at the end of the experiment. Each trial involved the following sequence. Each trial began with a gaze trigger, which consisted of a black circle presented in the center of the screen. Once a stable fixation had been detected on the gaze trigger, the image was presented for 10 s. At the end of 10 s, a new gaze trigger appeared and the next trial began. Stimulus condition (Meaningful vs. Pseudo-Stimulus) was counterbalanced across two stimulus lists, separately for each task (Reading vs. Scene Viewing), and each participant saw only one of the lists. Thus, each participant saw 28 normal texts, 28 pseudo- texts, 28 normal scenes, and 28 pseudo-scenes, and no participant saw the same text or scene twice. The order of stimulus presentation was counterbalanced across participants, with half of the participants completing the scene viewing task first, and half the reading task. Apparatus Within each task, stimuli were presented in a random order for each participant. For the memory test, participants were presented with a random selection of novel and previously viewed texts, scenes, and pseudo-scenes, and asked to indicate via button-press if they had seen the stimulus before. The memory test was administered solely to ensure that participants attended carefully to the experimental tasks, and so the data from the memory task were not analyzed. Frontiers in Psychology | www.frontiersin.org Procedure For the reading task, participants were told that they would be reading short texts on a computer screen while their eye movements were recorded. Participants were also told that some of the texts would appear with blocks in place of letters, and that in those cases they should move their eyes as if they were reading. These are the standard instructions given in pseudo- reading experiments (Vitu et al., 1995; Rayner and Fischer, 1996; Nuthmann et al., 2007; Luke and Henderson, 2013). Participants were informed that their memory for the texts and pseudo-texts would be tested at the end of the experimental session. Each trial involved the following sequence. The trial began with a gaze trigger, a black circle presented in the position of the first character in the text. Once a stable fixation was detected on the gaze trigger, the text was presented. The participant read the text and pressed a button when finished. Then a new gaze trigger appeared and the next trial began. Saccade Amplitude Thus, although the findings of the present study with regard to mean saccade amplitudes may appear to contradict previous findings that saccades are shorter is pseudo-reading, the pattern of changes in the distribution of saccade amplitudes is the same. A by-participant 2 (TASK: Reading vs. Scene Viewing) × 2 (STIMULUS TYPE: Meaningful vs. Pseudo-Stimulus) ANOVA was conducted on saccade amplitude. In this analysis, both main effects were significant, as was the interaction (all Fs > 9.12, all ps < 0.0061). Follow-up t-tests showed that the significant interaction indicated that the effect of STIMULUS TYPE was present for reading but was not significant for scene viewing (Reading t(39) = −2.82, p = 0.0082, difference = 0.44; Scene Viewing t(39) = −0.63, p = 0.53, difference = 0.1). Interestingly, the mean saccade amplitude was shorter in reading than in pseudo-reading (see Table 1). This contradicts findings from previous studies using similar pseudo-reading tasks, where longer mean saccades were observed in normal reading than in pseudo- reading (Vitu et al., 1995; Rayner and Fischer, 1996; Luke and Henderson, 2013). Luke and Henderson (2013) observed that these mean differences were due to a greater proportion of very short and long fixations in pseudo-reading. Figure 3 shows that the same pattern of results was obtained for reading in the current study. A similar increase in the proportion of longer saccades is observable for pseudo-scene viewing, although this shift was not large enough to significantly influence the means. RESULTS Fixations shorter than 50 ms or longer than 1200 ms were removed as outliers, and saccades larger than 22◦were removed to exclude return sweeps in reading. Summary statistics for all dependent variables can be found in Table 1. For the scene task, participants were told that they would be viewing both photographs and patterns of blobs and shapes on the screen as their eye movements were monitored. Participants March 2016 | Volume 7 | Article 257 Frontiers in Psychology | www.frontiersin.org Meaningfulness and Eye Movement Control Luke and Henderson FIGURE 2 | Examples of the scene and pseudo-scene stimuli. Normal scenes are on the left, with the corresponding pseudo-scenes on the right. of the scene and pseudo-scene stimuli. Normal scenes are on the left, with the corresponding pseudo-scenes on the right. FIGURE 2 | Examples of the scene and pseudo-scene stimuli. Normal scenes are on the left, with the corresponding pseu Frontiers in Psychology | www.frontiersin.org March 2016 | Volume 7 | Article 257 Fixation Duration Figure 4 shows the distribution of fixation durations for both scenes and text in the meaningful and pseudo-stimulus conditions. This figure illustrates that fixation duration distributions are not normal but are skewed to the right. Any difference in means between the meaningful and pseudo-stimuli might reflect a difference in the center of the two distributions, which occurs when most fixations in one condition are longer. However, since means are strongly influenced by outliers and TABLE 1 | Means (and standard deviations) for the dependent variables. Reading Scene viewing Meaningful Pseudo-text Meaningful Pseudo-scenes Fixation duration 206 (89) 255 (126) 284 (143) 307 (169) Saccade amplitude 3.22 (2.09) 3.66 (2.92) 4.26 (3.47) 4.36 (3.55) March 2016 | Volume 7 | Article 257 Frontiers in Psychology | www.frontiersin.org 5 Meaningfulness and Eye Movement Control Luke and Henderson FIGURE 3 | Global Distribution of Saccade Amplitudes. There was a greater proportion of very short and long saccades for the pseudo-stimuli (dashed lines) than for the meaningful stimuli, especially for the reading task. FIGURE 3 | Global Distribution of Saccade Amplitudes. There was a greater proportion of very short and long saccades for the pseudo-stimuli (dashed lines) than for the meaningful stimuli, especially for the reading task. Previous research comparing fixation duration distributions in scene viewing and reading has observed that the distribution in scene viewing is both shifted to the right and more skewed to the right compared to reading (Luke et al. (2013); see also Henderson and Hollingworth (1998)). The removal of meaningfulness from a text stimulus has been shown to result in a “fatter tail”, skewing the distribution to the right compared to the normal, meaningful stimulus condition, but does not appear to influence µ or σ (Luke and Henderson, 2013). Figure 4 and Table 2 suggests that the primary difference between the Meaningful and Pseudo-Stimulus conditions in both tasks is indeed an increase in skew in the Pseudo-Stimulus condition, with no large shifts in the center of the distribution apparent. To look for any interactions between task and stimulus type that might indicate task-based differences in fixation duration control, especially in the analysis of the skew of the distribution (τ), by-participant 2 (TASK: Reading vs. Scene Viewing) × 2 (STIMULUS TYPE: Meaningful vs. Pseudo-Stimulus) ANOVAs were conducted on all three of the ex-Gaussian parameters. Frontiers in Psychology | www.frontiersin.org Fixation Duration The solid vertical lines represent overall means, while the dotted vertical lines represent the values for µ derived from the response time distribution analysis. (Meaning: both ts > 5.61, both ps < 0.0001; No meaning: both ts > 2.28, both ps < 0.025). When the effect of STIMULUS TYPE was considered separately for each task, no significant differences were found in the analysis of µ (Reading: t(39) < 1.78, p = 0.072; Scene Viewing: t(39) < 0.57, p > 0.57). The effect was significant (although numerically tiny, only 4 ms) for reading only in the analysis of σ (t(39) < 2.03, p = 0.046; Scene Viewing: t(39) < 0.07, p > 0.94). These results indicate that if semantic content has an influence on the center or spread of the fixation duration distributions, such influences are quite small (<10 ms; see Table 2) and mostly non-significant. Accordingly, between- task differences in the influence of semantic content on µ or σ, if they exist at all, are on the order of a few milliseconds. Thus, these results are consistent with previous studies (Luke and Henderson, 2013). (Meaning: both ts > 5.61, both ps < 0.0001; No meaning: both ts > 2.28, both ps < 0.025). When the effect of STIMULUS TYPE was considered separately for each task, no significant differences were found in the analysis of µ (Reading: t(39) < 1.78, p = 0.072; Scene Viewing: t(39) < 0.57, p > 0.57). The effect was significant (although numerically tiny, only 4 ms) for reading only in the analysis of σ (t(39) < 2.03, p = 0.046; Scene Viewing: t(39) < 0.07, p > 0.94). These results indicate that if semantic content has an influence on the center or spread of the fixation duration distributions, such influences are quite small (<10 ms; see Table 2) and mostly non-significant. Accordingly, between- task differences in the influence of semantic content on µ or σ, if they exist at all, are on the order of a few milliseconds. Thus, these results are consistent with previous studies (Luke and Henderson, 2013). globally manipulated the meaningfulness of both texts and scenes in a within-subjects design, enabling us to explore potential differences in eye movement control across two visual tasks. Saccade amplitudes were found to increase significantly in reading for pseudo-text. Fixation Duration This difference in mean saccade amplitude across conditions appears to result from an increase in very short and long saccade amplitudes when meaning is removed from text. This shift in the distribution has been observed in other studies (Luke and Henderson, 2013), although in these studies means decreased because the proportion of short saccades increased more than was observed here. In scene viewing there was a numeric trend toward an increase (see Table 1) but it was small and far from significant. There was some TABLE 2 | Parameters from the response time distributional analysis of fixation durations. Reading Scene viewing µ σ T µ σ τ Meaningful 139 38 66 164 52 122 Pseudo-stimulus 148 42 101 161 52 152 Effect 9 4 35 −3 0 30 The three parameters µ, σ, and τ are from the response time distributional analysis of the fixation duration distributions shown in Figure 3. The first two parameters represent the normal component of the distribution (µ, the mean, and σ, the standard deviation), and τ is the exponential parameter representing the skew of the distribution. Mean log likelihoods for the four different conditions are: Reading, Meaningful: −2460.22; Reading, Pseudo-Stimulus: −2015.7; Scenes, Meaningful: −1345.4; Scenes, Pseudo-Stimulus: −1173.18. TABLE 2 | Parameters from the response time distributional analysis of fixation durations. In the analysis of τ, both main effects (TASK and STIMULUS TYPE) were significant (both Fs > 99.06, both ps < 0.0001). The interaction of the two was not significant (p > 0.32). These findings indicate that while all three parameters of the fixation duration distribution were larger in scene viewing than in reading, consistent with previous research (Luke et al., 2013), meaningfulness only had a significant effect on the skew of the distribution (Luke and Henderson, 2013). The size of this effect was statistically the same in the two tasks. The three parameters µ, σ, and τ are from the response time distributional analysis of the fixation duration distributions shown in Figure 3. The first two parameters represent the normal component of the distribution (µ, the mean, and σ, the standard deviation), and τ is the exponential parameter representing the skew of the distribution. Mean log likelihoods for the four different conditions are: Reading, Meaningful: −2460.22; Reading, Pseudo-Stimulus: −2015.7; Scenes, Meaningful: −1345.4; Scenes, Pseudo-Stimulus: −1173.18. Fixation Duration In the analyses of µ and σ, there were main effects of TASK (both Fs > 43.75, all ps < 0.0046), indicating that both parameters were larger for scenes than for text. The main effects of STIMULUS TYPE were not significant (both Fs < 3.9, both ps > 0.056). The interaction of the two factors was significant in both analyses (both Fs > 6.9, all ps < 0.012), indicating that the effect of TASK was somewhat smaller in the Pseudo-Stimulus condition extreme scores, a difference between means can also occur because one distribution is more skewed than another, which occurs when some subset (but not all) of the fixations are longer. Mean differences can reflect either of these differences or both in combination (Balota and Yap, 2011). Since the center and skew of fixation duration distributions vary independently of each other and often reflect different processes (Staub and Benatar, 2013), it is important to consider them separately. To test whether the removal of meaningfulness had similar effects in the different tasks, the fixation duration distributions for each participant in each condition were analyzed using a response time distributional analysis (Balota and Yap, 2011). This analysis fits participants’ response time data with an ex- Gaussian distribution (Ratcliff, 1979), which is the convolution of a normal (Gaussian) distribution and an exponential distribution, with two parameters representing the normal component (µ, the mean, and σ, the SD), and a single exponential parameter (τ). Any changes in µ and σ indicate changes in the distribution’s normal component (i.e., the center), whereas increases in τ indicate increased skew to the right. Ex-Gaussian distributions fit eye-movement data quite well (Staub et al., 2010; Staub, 2011; White and Staub, 2012; Luke and Henderson, 2013; Luke et al., 2013). The ex-Gaussian distribution was fitted to the data from each participant in each task in each meaning condition using QMPE software (Heathcote et al., 2004). The mean ex-Gaussian parameters are found in Table 2. March 2016 | Volume 7 | Article 257 Frontiers in Psychology | www.frontiersin.org Frontiers in Psychology | www.frontiersin.org 6 Meaningfulness and Eye Movement Control Luke and Henderson FIGURE 4 | Global Distribution of Fixation Durations. The solid vertical lines represent overall means, while the dotted vertical lines represent the values for µ derived from the response time distribution analysis. FIGURE 4 | Global Distribution of Fixation Durations. DISCUSSION The present study investigated how the meaningfulness of a visual stimulus influences how our eyes move. More specifically, we March 2016 | Volume 7 | Article 257 Frontiers in Psychology | www.frontiersin.org 7 Meaningfulness and Eye Movement Control Luke and Henderson fixated stimulus (e.g., the competition-inhibition theory; Yang and McConkie (2001). suggestion of an increase in the proportion of longer fixations for pseudo-scenes as well. The absence of a significant effect of semantic content on saccades amplitudes in scene viewing may simply reflect a ceiling effect; saccades are larger by default in scene viewing, and it is probably not possible to increase them much more and still keep the eyes within the bounds of the stimulus. Regardless, these observed changes in saccade amplitude likely reflect a reduced need for foveal processing when the stimulus is not being processed for meaning. While the global manipulation employed here is useful for cross-task comparisons, it of course has certain limitations. Since our manipulation altered the stimuli in multiple ways, removing or changing some low-level visual features as well as obscuring the identity of words and objects, it is not possible to determine which cognitive processes (or which stage in processing) has the most influence on eye movements. This technique cannot therefore adjudicate cleanly between different proposals about the nature and source of cognitive control. The present study does, however, provide additional evidence, first, that reading, and scene viewing share a common control mechanism, and, second, that only some fixations are under the direct influence of the visual stimulus. Most models of eye-movement control apply to reading only (Reichle et al., 1998; Engbert et al., 2002, 2005), and so may not generalize to other tasks (but see Reichle et al. (2012) for an example of how E–Z reader can generalize to non-reading tasks). One model of eye-movement control that has been shown to successfully predict eye movements in both reading and scene viewing is the CRISP model (Nuthmann et al., 2010; Nuthmann and Henderson, 2012). CRISP also predicts that some eye movements will not be under cognitive control; fixation duration is determined by a random walk timer, after which a new saccade program is initiated. Prior to saccade program initiation, cognitive intervention can occur via inhibition of the saccade program when processing difficulty is encountered, but after the timer expires no cognitive intervention is possible. DISCUSSION Thus, CRISP is consistent with the findings of this and other studies. The results of the current study suggest that current and future models of eye movement control should, first, be able to account for eye movements across multiple tasks, and second, incorporate a mechanism for cognitive control that exempts some subset of fixations. g p g One goal of the present study was to investigate whether the influence of stimulus meaningfulness differs across tasks. A close look at the distribution parameters from the fixation duration distribution analysis (Table 2) shows that the removal of meaningfulness affected the fixation duration distributions in the same way in both tasks, influencing the skew (τ) but not consistently influencing the center (µ, σ) of the distributions. That is, the distribution analysis showed no consistent evidence of any significant effects of meaning on either µ or σ. There was, however, a main effect of stimulus type in the analysis of τ. Further, the interaction of stimulus type and task was not significant in the analysis of τ, indicating that the removal of meaningfulness from the stimulus had a statistically identical influence in reading and in scene viewing. Thus, it appears that the influence of cognitive control on eye movements in reading and scene viewing is both qualitatively and quantitatively similar; not only did the removal of meaningfulness influence the same component of the distribution in both tasks, the magnitude of that influence was nearly identical. This observation is highly consistent with other research with the SOD paradigm showing that eye movements respond similarly to processing difficulty in reading and in scene viewing (Luke et al., 2013). Increases in τ like those observed here occur when some, but not all, of the fixations are longer, which elongates the tail of the distribution but does not significantly shift its center. Thus, the fact that for the pseudo-stimuli τ was increased but the other parameters of the ex-Gaussian distribution were not indicates that not all fixations were affected by the removal of meaningfulness. This finding fits nicely with research using the SOD paradigm that reveals two populations of eye movements, providing converging evidence that longer duration fixations are under cognitive control and shorter duration fixations are not (Henderson and Pierce, 2008; Henderson and Smith, 2009; Nuthmann and Henderson, 2012; Luke et al., 2013). AUTHOR CONTRIBUTIONS Both authors (SL, JH) made substantial contributions to the conception or design of the work. SL was primarly responsible for the acquisition, analysis, or interpretation of data for the work. SL Drafted the work and JH revised it critically for important intellectual content. Both SL and JH gave final approval of the version to be published, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. 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Rev. 19, 1101–1107. doi: 10.3758/s13423-012-0274-2 Rayner, K., Liversedge, S. P., White, S. J., and Vergilino-Perez, D. (2003). Reading disappearing text: cognitive control of eye movements. Psychol. Sci. 14, 385–388. doi: 10.1111/1467-9280.24483 Rayner, K., and Pollatsek, A. (1981). Eye movement control during reading: evidence for direct control. Q. J. Exp. Psychol. 33, 351–373. doi: 10.1080/14640748108400798 Henderson, J. M., and Luke, S. G. (2014). Stable individual differences in saccadic eye movements during reading, pseudoreading, scene viewing, and scene search. J. Exp. Psychol. Hum. Percept. Perform. 40, 1390–1400. doi: 10.1037/a0036330 Rayner, K., and Reingold, E. M. (2015). Evidence for direct cognitive control of fixation durations during reading. Curr. Opin. Behav. Sci. 1, 107–112. doi: 10.1016/j.cobeha.2014.10.008 Henderson, J. M., Luke, S. G., Schmidt, J., and Richards, J. E. (2013). REFERENCES Balota, D. A., and Yap, M. J. (2011). Moving beyond the mean in studies of mental chronometry the power of response time distributional analyses. Curr. Dir. Psychol. Sci. 20, 160–166. doi: 10.1177/0963721411 408885 Buhrmester, M., Kwang, T., and Gosling, S. D. (2011). Amazon’s mechanical turk a new source of inexpensive, yet high-quality, data? Perspect. Psychol. Sci. 6, 3–5. doi: 10.1177/1745691610393980 March 2016 | Volume 7 | Article 257 Frontiers in Psychology | www.frontiersin.org 8 Meaningfulness and Eye Movement Control Luke and Henderson Engbert, R., Longtin, A., and Kliegl, R. (2002). A dynamical model of saccade generation in reading based on spatially distributed lexical processing. Vis. Res. 42, 621–636. doi: 10.1016/S0042-6989(01)00301-7 McCandliss, B. D., Cohen, L., and Dehaene, S. (2003). The visual word form area: expertise for reading in the fusiform gyrus. Trends Cogn. Sci. 7, 293–299. doi: 10.1016/S1364-6613(03)00134-7 Morrison, R. E. (1984). Manipulation of stimulus onset delay in reading: evidence for parallel programming of saccades. J. Exp. Psychol. Hum. Percept. Perform. 10, 667–682. Engbert, R., Nuthmann, A., Richter, E. M., and Kliegl, R. (2005). SWIFT: a dynamical model of saccade generation during reading. Psychol. Rev. 112, 777–813. doi: 10.1037/0033-295X.112.4.777 Epstein, R., Harris, A., Stanley, D., and Kanwisher, N. (1999). The parahippocampal place area: recognition, navigation, or encoding? Neuron 23, 115–125. doi: 10.1016/S0896-6273(00)80758-8 Nuthmann, A., Engbert, R., and Kliegl, R. (2007). The IOVP effect in mindless reading: experiment and modeling. Vision Res. 47, 990–1002. doi: 10.1016/j.visres.2006.11.005 Epstein, R., and Kanwisher, N. (1998). A cortical representation of the local visual environment. Nature 392, 598–601. doi: 10.1038/33402 Epstein, R., and Kanwisher, N. (1998). A cortical representat Nuthmann, A., and Henderson, J. M. (2012). Using CRISP to model global characteristics of fixation durations in scene viewing and reading with a common mechanism. Vis. Cogn. 20, 457–494. doi: 10.1080/13506285.2012.670142 environment. Nature 392, 598–601. doi: 10.1038/33402 Heathcote, A., Brown, S., and Cousineau, D. (2004). QMPE: estimating lognormal, wald, and weibull RT distributions with a parameter-dependent lower bound. Behav. Res. Methods Instrum. Comput. 36, 277–290. doi: 10.3758/ BF03195574 Nuthmann, A., Smith, T. J., Engbert, R., and Henderson, J. M. (2010). CRISP: a computational model of fixation durations in scene viewing. Psychol. Rev. 117, 382–405. doi: 10.1037/a0018924 Henderson, J. (2003). Human gaze control during real-world scene perception. Trends Cogn. Sci. 7, 498–504. doi: 10.1016/j.tics.2003.09.006 Ratcliff, R. (1979). Group reaction time distributions and an analysis of distribution statistics. Psychol. Bull. 86, 446–461. doi: 10.1037/0033-2909.86. 3.446 Henderson, J. Frontiers in Psychology | www.frontiersin.org REFERENCES doi: 10.3758/s13414-013-0482-5 Vitu, F., O’Regan, J. K., Inhoff, A. W., and Topolski, R. (1995). Mindless reading: eye-movement characteristics are similar in scanning letter strings and reading texts. Percept. Psychophys. 57, 352–364. doi: 10.3758/ BF03213060 Luke, S. G., Nuthmann, A., and Henderson, J. M. (2013). Eye movement control in scene viewing and reading: evidence from the stimulus onset delay paradigm. J. Exp. Psychol. Hum. Percept. Perform. 39, 10–15. doi: 10.1037/ a0030392 Võ, M. L. H., and Henderson, J. M. (2009). Does gravity matter? Effects of semantic and syntactic inconsistencies on the allocation of attention during scene perception. J. Vis. 9, 24.1–24.15. doi: 10.1167/9.3.24 Mannan, S. K., Ruddock, K. H., and Wooding, D. S. (1997). Fixation patterns made during brief examination of two-dimensional images. Perception 26, 1059–1072. doi: 10.1068/p261059 Walther, D. B., Caddigan, E., Fei-Fei, L., and Beck, D. M. (2009). Natural scene categories revealed in distributed patterns of activity in the human March 2016 | Volume 7 | Article 257 Frontiers in Psychology | www.frontiersin.org 9 Meaningfulness and Eye Movement Control Luke and Henderson brain. J. Neurosci. 29, 10573–10581. doi: 10.1523/JNEUROSCI.0559-09. 2009 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Wang, H.-C., Hwang, A. D., and Pomplun, M. (2010). Object frequency and predictability effects on eye fixation durations in real-world scene viewing. J. Eye Move. Res. 3, 3.1–3.10. Copyright © 2016 Luke and Henderson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. White, S. J., and Staub, A. (2012). The distribution of fixation durations during reading: effects of stimulus quality. J. Exp. Psychol. Hum. Percept. Perform. 38, 603–617. doi: 10.1037/a0025338 Yang, S.-N., and McConkie, G. W. (2001). Eye movements during reading: a theory of saccade initiation times. Vision Res. 41, 3567–3585. doi: 10.1016/S0042- 6989(01)00025-6 March 2016 | Volume 7 | Article 257 Frontiers in Psychology | www.frontiersin.org 10
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Revistas de Buenos Aires durante la guerra de 1898. La Biblioteca, La Ilustración Sud-Americana y Revista de Derecho, Historia y Letras, entre la «cuestión palpitante» y las encrucijadas identitarias
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23 ISSN: 1510-5024 (papel) - 2301-1629 (en línea) Humanidades: revista de la Universidad de Montevideo, No 9, Junio 2021, pp. 23-58 Para citar este artículo / To reference this article / Para citar este artigo Bruno, Paula. “Revistas de Buenos Aires durante la guerra de 1898. La Biblioteca, La Ilustración Sud-Americana y Revista de Derecho, Historia y Letras, entre la «cuestión palpitante» y las encrucijadas identitarias”. Humanidades: revista de la Universidad de Montevideo, nº 9, (2021): 23-58. https://doi.org/10.25185/9.3 Palabras claves: vida intelectual, revistas, anti-imperialismo, Calibán, guerra de 1898, sociabilidades, Buenos Aires. ..................................................................................................................................................... PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS - doi: https://doi.org/10.25185/9.3 Revistas de Buenos Aires durante la guerra de 1898. La Biblioteca, La Ilustración Sud-Americana y Revista de Derecho, Historia y Letras, entre la «cuestión palpitante» y las encrucijadas identitarias Resumen: En el contexto de la guerra de 1898 entre España y Estados Unidos por las últimas posesiones coloniales españolas, Buenos Aires se convirtió en un centro en el que se realizaron diversos eventos, movilizaciones y colectas. La presencia de inmigrantes españoles en la ciudad propició estas dinámicas. A su vez, los ámbitos de sociabilidad cultural fueron escenario de conferencias sobre qué significaba la guerra para la vida política e intelectual argentina y latinoamericana. Las nuevas preguntas sobre el orden geopolítico que surgiría de la guerra habilitaron la aparición de nuevas formas de intervención intelectual en América Latina. Figuras de distintas latitudes propusieron interpretaciones y análisis que conformaron repertorios identitarios como el latinoamericanismo, el hispanoamericanismo y anti-imperialismo latinoamericano. A la luz de estos eventos y del surgimiento de nuevas formas de intervención intelectual, este artículo analiza cómo algunas revistas publicadas en Buenos Aires dieron cuenta en sus páginas de los eventos de la guerra de 1898 y sus efectos. Se analizan de manera central La Biblioteca (1896-1898), La Ilustración Sud-Americana (1892-1917), Revista de Derecho, Historia y Letras (1898-1924) con el objetivo de ver qué modalidades adoptaron para intervenir en ese contexto y cómo se tramitaron en sus páginas distintos repertorios identitarios. Palabras claves: vida intelectual, revistas, anti-imperialismo, Calibán, guerra de 1898, sociabilidades, Buenos Aires. PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS Palavras-chave: vida intelectual, revistas, anti-imperialismo, Calibã, guerra de 1898, sociabilidades, Buenos Aires. Buenos Aires journals during the 1898 war. La Biblioteca, La Ilustración Sud-Americana y Revista de Derecho, Historia y Letras, between the «throbbing issue» and identities crossroads Abstract: During the 1898 war between Spain and the United States caused by the last Spanish colonial possessions, Buenos Aires became the setting of different initiatives related to the war, such as conferences and mobilizations. The significant Spanish immigration in the city encouraged these events. In turn, the cultural circles and groups were the milieu of dissertations on the meaning of the war for the Argentine and Latin American public and intellectual life. The new questions about the geopolitical order that would appear after the war promoted new ways and forms of intellectual reflection in Latin America. Personalities from different latitudes proposed interpretations and analysis that shaped identity repertoires, such as Latino Americanism, Spanish- Americanism, and Latin American Anti-imperialism. Considering these events and ways of intellectual intervention, this article analyses how the episodes of 1898 war were covered by some journals published in Buenos Aires. It analyses La Biblioteca (1896-1898), La Ilustración Sud-Americana (1892-1917), Revista de Derecho, Historia y Letras. The main purpose is to show how these publications intervened in this context and how they managed the identity repertoires. Keywords: intellectual life, journals, Anti- Imperialism, Caliban, 1898 war, sociability, Buenos Aires. Keywords: intellectual life, journals, Anti- Imperialism, Caliban, 1898 war, sociability, Buenos Aires. Revistas de Buenos Aires durante a guerra de 1898. La Biblioteca, La Ilustración Sud-Americana e Revista de Derecho, Historia y Letras, entre a «questão palpitante» e as encruzilhadas de identidade Revistas de Buenos Aires durante a guerra de 1898. La Biblioteca, La Ilustración Sud-Americana e Revista de Derecho, Historia y Letras, entre a «questão palpitante» e as encruzilhadas de identidade Resumo: No contexto da guerra de 1898 entre Espanha e os Estados Unidos pelas últimas possessões coloniais espanholas, Buenos Aires tornou-se um centro no qual foram realizados vários eventos, mobilizações e coletas. A presença de imigrantes espanhóis na cidade favoreceu essas dinâmicas. Por sua vez, os âmbitos de sociabilidade cultural foram o cenário de conferências sobre o significado da guerra para a vida política e intelectual argentina e latino-americana. As novas questões sobre a ordem geopolítica após a guerra permitiram a aparição de novas formas de intervenção intelectual na América Latina. Figuras de diferentes latitudes propuseram interpretações e análises que conformaram repertórios identitários como o latino-americanismo, o hispano-americanismo e o anti-imperialismo latino-americano. À luz desses eventos e do surgimento de novas formas de intervenção intelectual, este artigo analisa como algumas revistas publicadas em Buenos Aires deram conta na suas páginas dos eventos da guerra de 1898 e seus efeitos. Analisam-se principalmente La Biblioteca (1896-1898), La Ilustración Sud- Americana (1892-1917), Revista de Derecho, Historia y Letras, (1898-1924) com o objetivo de ver que modalidades adotaram para intervir neste contexto e como foram configurados na suas páginas diferentes repertórios de identidade. Palavras-chave: vida intelectual, revistas, anti-imperialismo, Calibã, guerra de 1898, sociabilidades, Buenos Aires. PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS 1 Puede verse sobre esta cronología: Paula Bruno, “Un momento latinoamericano. Voces intelectuales entre la I Conferencia Panamericana y la Gran Guerra”, en Ideas comprometidas. Los intelectuales y la política, eds. Ferran Archilés y Maximiliano Fuentes (Madrid: Akal, 2018), 57-77. 3 Roberto Fernández Retamar, Todo Calibán (La Habana: Editorial Letras Cubanas, 2000), 28. 2 Sobre estos aspectos pueden consultarse: Ángel Rama, “La modernización literaria latinoamericana (1870- 1910)”, Hispamérica 12, nº 36 (1983): 3-19; Agustín Martínez, Figuras. La modernización intelectual de América Latina: 1850-1930 (Caracas: Fondo Editorial Topykos, 1995); Julio Ramos, “Hemispheric Domains: 1898 and the Origins of Latin Americanism”, Journal of Latin American Cultural Studies 10, nº 3 (2001): 237-251. 1 Puede verse sobre esta cronología: Paula Bruno, “Un momento latinoamericano. Voces intelectuales entre la I Conferencia Panamericana y la Gran Guerra”, en Ideas comprometidas. Los intelectuales y la política, eds. Ferran Archilés y Maximiliano Fuentes (Madrid: Akal, 2018), 57-77. 2 Sobre estos aspectos pueden consultarse: Ángel Rama, “La modernización literaria latinoamericana (1870- 1910)”, Hispamérica 12, nº 36 (1983): 3-19; Agustín Martínez, Figuras. La modernización intelectual de América Latina: 1850-1930 (Caracas: Fondo Editorial Topykos, 1995); Julio Ramos, “Hemispheric Domains: 1898 and the Origins of Latin Americanism”, Journal of Latin American Cultural Studies 10, nº 3 (2001): 237-251. 3 Roberto Fernández Retamar, Todo Calibán (La Habana: Editorial Letras Cubanas, 2000), 28. 1898 en Buenos Aires: voces, sociabilidades y empresas editoriales Las décadas finales del siglo XIX fueron intensas en América Latina en lo que se refiere a la conformación de identidades en disputa. Junto con los repertorios nacionales de cada país, se esbozaron identidades regionales — como el latinoamericanismo y el panamericanismo—, atlánticas —se cuentan aquí el americanismo surgido en España y el hispanoamericanismo propuesto en espacios de América Latina—, y constelaciones de ideas definidas en oposición a otras —se destacan el anti-imperialismo latinoamericano y el anti-yankismo—. Estos procesos estuvieron enmarcados en un ciclo que se extendió entre las apreciaciones de José Martí sobre la I Conferencia Panamericana realizada en Washington (1889-1890), que terminaron dando forma al ya clásico Nuestra América, y la publicación del Ariel de José Enrique Rodó (1900).1 En particular, el año 1898 fue un año destacado en este ciclo para la vida cultural latinoamericana y habilitó la aparición de nuevas formas de intervención intelectual.2 La guerra entre Estados Unidos y España por el control de las últimas colonias españolas en América generó un clima de opinión cargado de tensiones que se manifestó en distintos ámbitos públicos. Como indicó Fernández Retamar: «‘el 98’ no es sólo una fecha española, que da nombre a un complejo equipo de escritores y pensadores de aquel país, sino también, y acaso sobre todo, una fecha hispanoamericana, la cual debía servir para designar un conjunto no menos complejo de escritores y pensadores de este lado del Atlántico».3 En este marco, se gestaron y consolidaron una serie de repertorios de ideas e imágenes sobre España, Estados Unidos y América PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS Latina,4 y se destacaron voces como las de Paul Groussac, Rubén Darío y José Enrique Rodó. Aunque predominantes, sus intervenciones convivieron con —y articularon— otras formas de expresión en ámbitos y empresas culturales. En este clima, hubo una capital latinoamericana que ofició de centro privilegiado para poner en circulación y amplificar estas ideas. Como se estudió desde distintas perspectivas, Buenos Aires se convirtió en un escenario efervescente en la coyuntura de la guerra de 1898. Se realizaron diversos eventos, movilizaciones y colectas. 4 Sobre las relaciones entre España y Argentina véase: Beatriz Figallo Lascano, Argentina y España. Entre la pasión y el escepticismo (Buenos Aires: Teseo, 2014). Acerca de los vínculos culturales entre España y América Latina véase Carlos Rama, Historia de las relaciones culturales entre España y América Latina. Siglo XIX (México: Fondo de Cultura Económica, 1982). Para aproximaciones sobre las miradas acerca de Estados Unidos en América Latina pueden consultarse: Carlos Marichal y Alexandra Pita González, coords., Pensar el antiimperialismo. Ensayos de historia intelectual latinoamericana, 1900-1930 (México: COLMEX/Universidad de Colima, 2012); Andrés Kozel, Florencia Grossi, y Delfina Moroni, coords., El imaginario antiimperialista en América Latina (Buenos Aires: CLACSO/Centro Cultural de la Cooperación, 2015). 5 Entre otros trabajos, pueden consultarse: Ignacio García, “Apoyo a los españoles a la causa de la Cuba española. El caso argentino”, Estudios Sociales, nº 19 (2000): 85-104; Marcela García Sebastiani, “España fuera de España. El patriotismo español en la emigración argentina: una aproximación”, Hispania 73, nº 244 (2013): 469-500. 6 Pueden verse sobre estos asuntos las contribuciones reunidas en Paula Bruno, dir., Sociabilidades y vida cultural. Buenos Aires, 1860-1930 (Bernal: Editorial de la Universidad Nacional de Quilmes, 2014). Puede leerse este comentario en la sección “Cosas y Quiscosas”, La Ilustración Sudamericana, 1 de mayo, 1898, 1 1898 en Buenos Aires: voces, sociabilidades y empresas editoriales La presencia de inmigrantes españoles en la ciudad propició estas dinámicas.5 A su vez, los ámbitos de sociabilidad cultural fueron escenarios de conferencias y debates sobre qué significaba la guerra para la vida política e intelectual argentina y latinoamericana. Recintos como el Teatro Politeama y Teatro de la Victoria, y ámbitos culturales como el Ateneo y la Sociedad Científica Argentina, devinieron centros que acogieron a conferencistas de distintas latitudes para que disertaran sobre la guerra y sus efectos.6 Un cronista de la época daba cuenta de cómo el fervor bélico se había adueñado de la ciudad: Los diarios más renombrados y los de menor fuste en las veleidades de la opinión pública, todos son arrebatados por la curiosidad de las gentes: los telegramas se comentan y discuten: las fiestas y reuniones de la comunidad española se aplauden y dan pie de ‘conversación bélica’, y por todas partes, y en todos los lugares, suposiciones, hipótesis, soluciones guerreras, planes de campaña, asuntos de estrategia y militares motivos, ruedan por las conversaciones de todo el mundo que constituye la opinión pública, hoy favorable, favorabilísima, en esta dolorosa contienda de dos grandes pueblos, a la nación hispana, con todos los países ibero-americanos.7 PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS A la luz de estos eventos, del surgimiento de nuevas formas de intervención intelectual, de dinámicas de sociabilidad cultural atravesadas por la coyuntura, y del despliegue de novedosas estrategias de circulación de la información, este artículo analiza cómo algunas revistas publicadas en Buenos Aires dieron cuenta en sus páginas de los eventos de la guerra de 1898.8 Existe un considerable consenso historiográfico que señala que la guerra de 1898 fue un punto de inflexión en los modos periodísticos de cobertura de los conflictos bélicos. También se ha destacado que la expresión «prensa amarilla» surgió durante el conflicto, y tuvo que ver con el nivel de detalle con que se presentaban las atrocidades de la guerra. A su vez, se atribuye responsabilidad a los medios de prensa en la declaración misma del conflicto por el presidente William McKinley, sobre todo del New York World (comandado por Joseph Pulitzer) y del New York Journal (bajo responsabilidad de William Hearst). 11 Puede consultarse el siguiente volumen: Adriana Claudia Rodríguez, ed., Argentina y Cuba frente al 98 cubano: miradas cruzadas en torno al advenimiento del nuevo siglo nuestroamericano (Buenos Aires: Ediciones F.E.P.A.I., 2017). Se encuentran allí artículos acerca de cómo se cubrió en varios periódicos la guerra de 1898. Entre los diarios estudiados se encuentran La Nación (Claudio Gallegos), Buenos Aires Herald (Elena Torre), La Patria degli Italiani (Paolo Galassi) y La Protesta Humana (María Eugenia Chedrese). Referencias de cada artículo en la bibliografía. 8 Para realizar este análisis tuve en cuenta las consideraciones metodológicas para estudiar las relaciones entre revistas del siglo XIX y climas intelectuales de varios aportes. Destaco entre ellos los reunidos en los siguientes volúmenes colectivos: Aimer Granados, coord., Las revistas en la historia intelectual de América Latina: redes, intelectuales, política y sociedad (México: UAM-Cuajimalpa, 2012); Hanno Ehrlicher y Nanette Rißler-Pipka, eds., Almacenes de un tiempo en fuga. Revistas culturales en la modernidad hispánica (Berlín: Shaker Verlag, 2014). 9 Pueden consultarse como visiones generales sobre este fenómeno: Joseph Campbell, Yellow Journalism: Puncturing the Myths, Defining the Legacies (Westport: Praeger, 2001); Joseph Campbell, The Spanish-American War: American Wars and the Media in Primary Documents (Westport: Greenwood Press, 2005). 10 Manqing Qin, “La Guerra de Cuba. Un análisis desde diferentes perspectivas” (Tesis doctoral, Universidad Complutense de Madrid, 2018). La tesis analiza los siguientes periódicos españoles: El Liberal, El Imparcial y Heraldo de Madrid. Puede verse también Félix Santos, 1898: la prensa y la guerra de Cuba (Bilbao, Vizcaya: Asociación Julián Zugazagoitia, 1998). 1898 en Buenos Aires: voces, sociabilidades y empresas editoriales Ya con la guerra desatada, la participación de Estados Unidos estuvo apuntalada por una campaña de prensa sostenida, encabezada por Hearst, que fomentó en la ciudadanía norteamericana un sentimiento de rechazo a España.9 La prensa española, por su parte, desplegó distintas formas de expresión para generar sentimientos de adhesión en el contexto de la guerra.10 En América Latina los diarios cubrieron la guerra con distintas estrategias; y en Argentina, en particular, periódicos de comunidades étnicas y de tirada nacional se ocuparon de dar cuenta del conflicto.11 El impacto de la guerra en las publicaciones periódicas tuvo efectos a escala trasnacional. Este ensayo pretende ser una contribución para comprender esos efectos al analizar cómo en Buenos Aires se mostró y analizó el conflicto en algunas revistas. Los enfrentamientos se dieron en un contexto en el que las PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS empresas editoriales de la vida cultural y diplomática no contaban con plumas expertas para cubrir temas de geopolítica internacional, como el conflicto en cuestión.12 La hipótesis central de este artículo es que en torno a 1898 se instrumentaron varias opciones de intervención desde páginas de revistas que, a diferencia de los periódicos, no contaban con la presión cotidiana de tener que dar información sobre la guerra en sí, al menos en lo que se vinculaba con los acontecimientos diarios.13 Optaron, en cambio, por utilizar distintas modalidades de narrar el conflicto y, a la vez, propusieron alternativas para pensar las identidades en disputa en la misma. 28 ISSN: 1510-5024 (papel) - 2301-1629 (en línea) No 9, Junio 2021, pp. 23-58 Las revistas que se analizan de manera central son La Biblioteca (1896-1898), La Ilustración Sudamericana (1892-1917), y la Revista de Derecho, Historia y Letras (1898-1924).14 Algunas cuestiones previas para puntualizar: La Biblioteca y la Revista de Derecho, Historia y Letras compartieron un espíritu común. Intentaron ser revistas que, por medio de artículos eruditos, intervenían en la vida cultural y en ciertos debates políticos e intelectuales; ambas trataron de eludir las temáticas de coyuntura y los ritmos cotidianos. Sus respectivos directores, Paul Groussac y Estanislao Zeballos, tuvieron un rol central a la hora de definir líneas de interés y de despliegue de las publicaciones. 1898 significó, además, para estas revistas, un año significativo. 12 Aunque este artículo no versa sobre la profesionalización periodística o sobre el rol de los expertos en revistas y periódicos de fines del siglo XIX, pueden verse constataciones sobre la ausencia de plumas especializadas en Paula Bruno, Martín García Mérou. Vida intelectual y diplomática en las Américas (Bernal: Universidad Nacional de Quilmes, 2018); y en Paula Bruno y Emiliano Sánchez, “Argentina frente al espejo norteamericano. Definiciones sobre el escenario internacional en el fin-de-siglo. Intelectuales, revistas y prensa periódica”, Ponencia Presentada en la Jornada “Saberes que desbordan. Intersecciones entre conocimientos expertos y sentido común”, Instituto de Desarrollo Económico y Social, Buenos Aires, 19 y 20 de noviembre de 2017. 14 Revisé también las siguientes publicaciones: El sol del domingo, Almanaque Sud-Americano y Caras y Caretas, entre otras, con el objetivo de captar contrapuntos entre las publicaciones analizadas de manera sistemática y algunas que convivían con ellas. 13 Los periódicos recurrieron a varias estrategias para contar con información actualizada en distintos contextos. Puede verse al respecto: Lila Caimari, “El mundo al instante. Noticias y temporalidades en la era del cable submarino (1860-1900), Redes 21, nº 40 (2015): 125-146. 15 Se señalan en cada ocasión referencias bibliográficas útiles para ampliar la información sobre las publicaciones en alguna de estas direcciones, en caso de que estén disponibles. 16 Para un acercamiento descriptivo a la revista: Héctor Lafleur, Sergio Provenzano y Fernando Alonso, Las revistas literarias argentinas, 1893-1967 (Buenos Aires: CEDAL, 1967). Visiones más recientes pueden encontrarse en: Alejandro Eujanián, “Paul Groussac y una empresa cultural de fines del siglo XIX: la revista La Biblioteca, 1896- 1898”, Historia de revistas argentinas, tomo II (Buenos Aires: Asociación Argentina de Editores de Revistas, 1997), 9-44; Paula Bruno, “Paul Groussac y La Biblioteca (1896-1898)”, Hispamérica. Revista de literatura, nº 94 (2003): 87-94. 1898 en Buenos Aires: voces, sociabilidades y empresas editoriales Para La Biblioteca fue su año de cierre; para la Revista de Derecho, Historia y Letras fue su año fundacional. Por su parte, la revista La Ilustración Sudamericana, que se publicaba en Buenos Aires desde 1892 —contaba con una edición paralela en Montevideo— no respondía a las características de una revista cultural de corte erudito; era, en cambio, una revista ilustrada. Sin embargo, a diferencia de otras empresas de su tipo, mostró desde sus inicios un interés particular por dar cuenta de las novedades internacionales y, en particular, por los eventos de la vida diplomática latinoamericana. PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS En suma, las revistas seleccionadas tienen características diferentes entre sí, formatos variables y, probablemente, eran leídas por públicos diversos. En este artículo no realizo un análisis formal de las mismas como soportes, tampoco estudio la recepción de esas revistas como objetos culturales o de consumo.15 Apunto, en cambio, a dos objetivos: por un lado, analizar comparativamente los ecos que en sus páginas se pueden observar de las voces de figuras de la vida intelectual americana y europea que se pronunciaron en el contexto de la guerra. Por otro, cartografiar las modalidades que estas revistas desplegaron en sus páginas para narrar y analizar el conflicto bélico de 1898. Por una cuestión de claridad expositiva, en las siguientes secciones hago foco en cada una de las publicaciones y sus dinámicas en el contexto de la guerra y, posteriormente, en la sección final, propongo una interpretación general sobre las formas de intervención que estaban disponibles y por las que optaron. 29 ISSN: 1510-5024 (papel) - 2301-1629 (en línea) o 9, Junio 2021, pp. 23-58 La Biblioteca y Calibán: entre la pluma y la voz de Groussac En el mes de junio de 1896 apareció por primera vez en Buenos Aires la revista La Biblioteca, dirigida por Paul Groussac, quien ejercía el cargo de Director de la Biblioteca Nacional de la Argentina desde 1885. Se presentaba con intenciones de convertirse en portavoz de las novedades de carácter científico, histórico y literario. Se anunció en el prefacio como órgano mensual destinado a publicar artículos inéditos sobre estas áreas. En líneas generales, el formato de la revista permite inscribirla en una tradición que había empezado a difundirse en el ambiente intelectual porteño durante las décadas anteriores, con la Revista de Buenos Aires (1863-1871) y la Revista Argentina (1868-1872/1880-1882), y que continuaría luego de la experiencia de La Biblioteca, en publicaciones como otra de las aquí tratadas, la Revista de Derecho, Historia y Letras (que comenzó a publicarse en 1898).16 PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS Los artículos de la revista tratan cuestiones científicas y culturales en el sentido amplio y decimonónico de ambos términos; quedan fuera temas vinculados con los sucesos estrictamente coyunturales del mundo político (disputas entre facciones políticas, debates cotidianos en la Cámara de Diputados o en la Cámara de Senadores) y los de orden económico; tienen un corte erudito, lo cual diferencia a esta revista de otras en las que el tono estaba más ligado a la contribución periodística, sintética y de opinión. La publicación recibía un subsidio oficial por ser, al menos como sugería su nombre, un órgano de difusión de la Biblioteca Nacional. En la práctica, actuó como un medio de consagración y prestigio intelectual; en ella se publicaron escritos de destacados hombres de cultura de la época como Joaquín V. González, Miguel Cané, Rubén Darío, Juan Agustín García (h.), Lucio Vicente López, Leopoldo Lugones, Bartolomé Mitre, Lucio V. Mansilla, Ernesto Quesada, Luis M. Drago y Antonio Dellepiane, entre tantos otros. La revista fue considerada como una empresa modernizadora por figuras como Miguel Cané o Rubén Darío, quien se refería a ella como «nuestra Revue de Deux Mondes». La experiencia de esta publicación cesó en 1898. 17 Paul Groussac, “La desaparición de La Biblioteca”, La Biblioteca 8, abril-mayo de 1898, 247. La Biblioteca y Calibán: entre la pluma y la voz de Groussac Ese año Groussac fue instado por Luis Beláustegui, Ministro de Justicia, Culto e Instrucción, a dejar de criticar las labores intelectuales de un funcionario público clave para la coyuntura, Norberto Piñeiro, quien estaba comisionado entonces en funciones diplomáticas en las discusiones limítrofes con Chile. El director de la revista entendió este hecho como un acto de «censura ministerial» y decidió interrumpir la publicación, que consideraba «una empresa civilizadora» que bajo ningún punto de vista admitiría censuras.17 El último número de la revista es usualmente citado para dar cuenta de esta trifulca. Reviso aquí, en cambio, esa última aparición de la revista a la luz de los sucesos de la guerra de 1898. Como anticipé, Groussac fue una de las voces destacadas en ese contexto. Adquirió este estatus por su participación en un evento ocurrido en Buenos Aires y que tuvo amplia repercusión en América Latina. En el marco del clima bélico, el 2 de mayo de 1898, se realizó en el Teatro de La Victoria un acto organizado y patrocinado por el Club Español en el que participaron como oradores Roque Sáenz Peña, Paul Groussac y José Tarnassi. Mientras que el último presentó una oda al pueblo español y la guerra en forma de poema, Groussac y Sáenz Peña retomaron en sus PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS discursos distintos ángulos de observación para analizar la contienda entre Estados Unidos y España. Las intervenciones de los tres oradores fueron publicadas en un folleto con un prólogo de Severiano Lorente.18 En este acto, el discurso de Groussac presentó en términos contundentes la guerra de 1898 como conflicto cultural, además de geopolítico. Se trataba, desde su perspectiva, de un enfrentamiento de los valores de la latinidad versus los del yanquismo. El discurso está cargado de imágenes contundentes que ya había esbozado o desarrollado en su libro Del Plata al Niágara (1897).19 El volumen recogía ensayos y reflexiones surgidos al calor de un viaje que concretó en 1893 por Chile, Perú, México, algunos puntos de América Central y Estados Unidos, en su travesía para llegar a representar como comisionado a Argentina en el World’s Congress de Chicago de 1893. La publicación de Del Plata al Niágara suscitó interés en la comunidad intelectual iberoamericana. 18 España y Estados Unidos. Función dada en el Teatro de la Victoria el 2 de mayo de 1898 bajo el patrocinio del Club Español de Buenos Aires, a beneficio de la Suscripción Nacional Española. Conferencias de los Señores Dr. Roque Sáenz Peña, Paul Groussac y Dr. José Tarnassi. Prólogo del Dr. Severiano Llorente (Buenos Aires: Compañía General de Billetes de Banco, 1898). 19 Paul Groussac, Del Plata al Niágara (Buenos Aires: Administración de La Biblioteca, 1897). 20 Eduardo Gómez de Baquero, “Crónica literaria”, La España Moderna 10, nº. 118 (1898): 168 [Cita textual]. 0 Eduardo Gómez de Baquero, “Crónica literaria”, La España Moderna 10, nº. 118 (1898): 168 [Cita textual] 9 Paul Groussac, Del Plata al Niágara (Buenos Aires: Administración de La Biblioteca, 1897). La Biblioteca y Calibán: entre la pluma y la voz de Groussac Tres reseñas de plumas destacadas de la época así lo constatan. La primera fue publicada en las páginas de La España Moderna (Madrid) y firmada por Eduardo Gómez de Baquero, crítico literario español de amplio prestigio. En la misma se subraya que el libro de Groussac venía a suplir una ausencia de conocimiento: a pesar de ‘los estrechos lazos’ que unen a España con las Repúblicas hispanoamericanas, lazos de los que se habla mucho de algún tiempo a esta parte y que en realidad existen o deben existir, atendidas la filiación y lengua de aquellas naciones, la verdad es que la mayoría de los españoles sabemos muy poco de ellas […] Son, pues, de utilidad para el público español los libros capaces de llenar esta laguna. Entre ellos merece un puesto señalado el que con el título Del Plata al Niágara ha publicado recientemente Mr. Paul Groussac.20 a pesar de ‘los estrechos lazos’ que unen a España con las Repúblicas hispanoamericanas, lazos de los que se habla mucho de algún tiempo a esta parte y que en realidad existen o deben existir, atendidas la filiación y lengua de aquellas naciones, la verdad es que la mayoría de los españoles sabemos muy poco de ellas […] Son, pues, de utilidad para el público español los libros capaces de llenar esta laguna. Entre ellos merece un puesto señalado el que con el título Del Plata al Niágara ha publicado recientemente Mr. Paul Groussac.20 Otra recensión que ponderaba el libro de Groussac se publicó Revue Hispanique (París). La misma reviste interés por varios aspectos: en primer lugar, la redactó el director de la revista, Raymond Foulché-Delbosc, un reconocido filólogo hispanista. En segundo lugar, se encargaba de señalar que el libro era superador en relación a otros relatos de viaje de europeos PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. La Biblioteca y Calibán: entre la pluma y la voz de Groussac LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS en América; justamente porque se trataba de un libro escrito por un francés afincado hacía tiempo en un país americano, este hecho le otorgaba otro tipo de espesor y óptica a sus reflexiones a la hora de contar las realidades americanas.21 La tercera reseña que me interesa destacar se publicó en la Revista Brazileira (Río de Janeiro), y está firmada por Manuel de Oliveira Lima, figura central de la vida intelectual y diplomática de entonces; el autor señalaba algunos contrapuntos con las consideraciones sociológicas que Groussac esbozaba en el libro, pero encontraba en sus miradas sobre el continente interesantes consideraciones para comparar países como Chile, Perú y México. Por su parte, destaca que la voz del autor se diferenciaba de las de sus contemporáneos porque no estaba atravesada por las pasiones del patriotismo. En este sentido, reivindicaba a Groussac por estar alejado tanto del patriotismo francés como del argentino.22 Las sugerencias de estas reseñas permiten notar que la voz de Groussac, gracias a la publicación de Del Plata al Niágara, generó interés en una comunidad letrada atraída por asuntos hispano o iberoamericanos. La publicación lo había posicionado como un conocedor del despliegue político y cultural del continente. En la misma, sus reflexiones sobre Estados Unidos proyectaron una imagen peyorativa del país del Norte, descrito de manera recurrente como una nación que carecía de historia y tradiciones, que suplía su falta de espesor cultural con el gigantismo de sus edificios, y cuya carencia de ideales le vedaban la posibilidad de ser una nación que articulara las relaciones del continente americano. Pero si bien estas ideas estaban esbozadas en el libro, fue en el discurso que pronunció en el Teatro de la Victoria donde la intensidad dada por la oralidad en el contexto del conflicto generó imágenes contundentes y acentos efectistas. El evento asumió una espectacularidad cubierta en varias crónicas de periódicos. Por ejemplo, en la sección «Noticias Argentinas», de El Courrier Franco Oriental (Montevideo) se subrayaba: Anoche efectúese en el Teatro de la Victoria la conferencia política que los doctores Roque Saénz Peña y José Tarnassi dedicaron a la colectividad española. La concurrencia fue enorme. El teatro estaba profusamente adornado con flores y banderas. 22 Manuel de Oliveira Lima, “Do Prata ao Noagara”, Revista Brazileira 4, tomo 14 (1898): 90-96. 21 Raymond Foulché-Delbosc, “Comtes rendu. Paul Groussac. Del Plata al Niágara, Buenos Aires; Administración de La Biblioteca, 1897, XXIII-487 pp.”, Revue Hispanique, Cinquieme Année (1898): 270. y g de La Biblioteca, 1897, XXIII-487 pp.”, Revue Hispanique, Cinquieme Année (1898): 270. 21 Raymond Foulché-Delbosc, “Comtes rendu. Paul Groussac. Del Plata al Niágara, Buenos Aires; Administración de La Biblioteca, 1897, XXIII-487 pp.”, Revue Hispanique, Cinquieme Année (1898): 270. 22 Manuel de Oliveira Lima, “Do Prata ao Noagara”, Revista Brazileira 4, tomo 14 (1898): 90-96. 21 Raymond Foulché-Delbosc, “Comtes rendu. Paul Groussac. Del Plata al Niágara, Buenos Aires de La Biblioteca, 1897, XXIII-487 pp.”, Revue Hispanique, Cinquieme Année (1898): 270. 23 Courrier Franco Oriental, 3 de mayo de 1898. 25 Los personajes de La Tempestad fueron motivos recurrentes para pensar en el rol de Latinoamérica en el contexto internacional. Ariel, Calibán y Próspero forman parte de una galería de posibilidades para tematizar rasgos y problemas latinoamericanos. Pueden verse, entre otros, Jorge Dubatti, comp., Peregrinaciones de Shakespeare en la Argentina: testimonios y lecturas de teatro comparado (Buenos Aires: Centro Cultural Rector Ricardo Rojas, 1996); y Emir Rodríguez Monegal, “Las metamorfosis de Calibán”, Vuelta 3, nº 25 (1978): 23-26. 26 Nuevas contribuciones sobre Darío y sus percepciones acerca de Estados Unidos pueden verse en los trabajos reunidos en Gerardo Piña-Rosales, Carlos Paldao y Graciela Tomassini, Rubén Darío y los Estados Unidos (New York: Academia Norteamericana de la Lengua Española, 2017). 23 Courrier Franco Oriental, 3 de mayo de 1898. 24 España y Estados Unidos, 50. 25 Los personajes de La Tempestad fueron motivos recurrentes para pensar en el rol de Latinoamérica en el contexto internacional. Ariel, Calibán y Próspero forman parte de una galería de posibilidades para tematizar rasgos y problemas latinoamericanos. Pueden verse, entre otros, Jorge Dubatti, comp., Peregrinaciones de Shakespeare en la Argentina: testimonios y lecturas de teatro comparado (Buenos Aires: Centro Cultural Rector Ricardo Rojas, 1996); y Emir Rodríguez Monegal, “Las metamorfosis de Calibán”, Vuelta 3, nº 25 (1978): 23-26. 26 Nuevas contribuciones sobre Darío y sus percepciones acerca de Estados Unidos pueden verse en los trabajos reunidos en Gerardo Piña-Rosales, Carlos Paldao y Graciela Tomassini, Rubén Darío y los Estados Unidos (New York: Academia Norteamericana de la Lengua Española, 2017). 24 España y Estados Unidos, 50. La Biblioteca y Calibán: entre la pluma y la voz de Groussac Roque Sáenz Peña protestó enérgicamente contra la intervención de los Estados Unidos en los asuntos de las colonias PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS de España. Paul Groussac hizo un magnífico panegírico de España y recordó las efemérides que tiene en su historia evocadora.23 Mientras describía en su elocución una España hidalga, valiente y conquistadora, Groussac desprestigiaba la grandeza material y superficial de los norteamericanos, criticaba demoledoramente su concepción del gobierno libre —considerándola una distorsión caricaturizada de los principios políticos ingleses— y comparaba todo lo que sucedía en el país del norte con un organismo amorfo y bestial. Estas imágenes, que en Del Plata al Niágara fueron sintetizadas varias veces con la expresión «mammoth», se condensaron en la imagen del Calibán: «desde la guerra de Secesión y la brutal invasión del Oeste, se ha desprendido libremente el espíritu yankee del cuerpo informe y ‘calibanesco’; y el viejo mundo ha contemplado con inquietud y terror a la novísima civilización que pretende suplantar a la nuestra, declarada caduca».24 El uso de la figura del Calibán para definir a Estados Unidos, inspirada en el famoso personaje de La tempestad de Shakespeare,25 fue amplificado días después del evento del Teatro de la Victoria en un artículo firmado por quién ya la había utilizado con distintas modulaciones en los últimos años: Rubén Darío.26 Nacía de este modo el texto conocido como «El triunfo de Calibán». Se encuentran en sus líneas elocuentes trazos para describir a los Estados Unidos y sus habitantes «los aborrecedores de la sangre latina», «los Bárbaros». Entre otras consideraciones, se destacan las siguientes: Y los he visto a esos yankees, en sus abrumadoras ciudades de hierro y piedra y las horas que entre ellos he vivido las he pasado con una vaga angustia. Parecíame sentir la opresión de una montaña, sentía respirar en un país de cíclopes, comedores de carne cruda, herreros bestiales, habitadores de casas de mastodontes. Colorados, pesados, groseros, van por sus calles empujándose PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS y rozándose animalmente, a la caza del dollar. Paul Groussac, El viaje intelectual. Impresiones de naturaleza y arte. Primera Serie (Madrid: Librería General d toriano Suárez, 1904). 31 Almanaque Sud-Americano (1899): 202. 29 El propio Darío narra el éxito y las censuras que sufrió el artículo en Rubén Darío, España contemporánea (París: Garnier, 1907), 92. 27 “El triunfo de Calibán (Edición y notas de Carlos Jaúregui)”, Revista Iberoamericana, Número especial: Balance de un siglo (1898-1998), nº 184-185 (1998): 451-455. 28 En general se mencionan las apariciones en El Tiempo y El Cojo Ilustrado; aunque puede que haya habido más reproducciones de las que logré rastrear en esta investigación, intento aquí mostrar que la difusión del texto tuvo impacto trasnacional. 27 “El triunfo de Calibán (Edición y notas de Carlos Jaúregui)”, Revista Iberoamericana, Número especial: Balance de un siglo (1898-1998), nº 184-185 (1998): 451-455. 28 En general se mencionan las apariciones en El Tiempo y El Cojo Ilustrado; aunque puede que haya habido más 27 “El triunfo de Calibán (Edición y notas de Carlos Jaúregui)”, Revista Iberoamericana, Número especial: Balance de un siglo (1898-1998), nº 184-185 (1998): 451-455. 28 En general se mencionan las apariciones en El Tiempo y El Cojo Ilustrado; aunque puede que haya habido más reproducciones de las que logré rastrear en esta investigación, intento aquí mostrar que la difusión del texto tuvo impacto trasnacional. 29 El propio Darío narra el éxito y las censuras que sufrió el artículo en Rubén Darío, España contemporánea (París: Garnier, 1907), 92. 30 Paul Groussac, El viaje intelectual. Impresiones de naturaleza y arte. Primera Serie (Madrid: Librería General de Victoriano Suárez, 1904). 31 Almanaque Sud-Americano (1899): 202. En general se mencionan las apariciones en El Tiempo y El Cojo Ilustrado; aunque puede que haya habido má roducciones de las que logré rastrear en esta investigación, intento aquí mostrar que la difusión del texto tuv pacto trasnacional. La Biblioteca y Calibán: entre la pluma y la voz de Groussac El ideal de esos calibanes está circunscripto a la bolsa y a la fábrica.27 y rozándose animalmente, a la caza del dollar. El ideal de esos calibanes está circunscripto a la bolsa y a la fábrica.27 El texto de Darío, publicado el 30 de mayo en el periódico El Tiempo de Buenos Aires, fue profusamente republicado ese año. Algunas de las reproducciones se encuentran en La Época de Madrid (20 de agosto), La Vanguardia de Barcelona (22 de agosto), El Cojo Ilustrado de Caracas —con el título «Rubén Darío combatiente»— (1 de octubre), Don Quijote de Madrid —bajo el título «¡Los yanquis!»— (25 de noviembre).28 El efecto multiplicador y las controversias que generó el texto de Darío,29 se vieron reforzados por la reproducción de la conferencia de Groussac en periódicos y revistas. Además de transcribirse total o parcialmente en periódicos argentinos, tuvo inmediata recepción en el marco rioplatense. Se publicó en La Razón de Montevideo (5 y 6 de mayo) y se comentó en el ya mencionado Courrier Franco Oriental. Se publicó el mismo año en folleto y, años después, el mismo Groussac lo sumó a la edición de 1904 de su libro El viaje intelectual.30 En lo que se refiere a las reproducciones en revistas, además de dos de las aquí analizadas, fue publicado un extracto de la conferencia bajo el título «Llegada de Colón a Barcelona», en Almanaque Sud-Americano.31 De este modo, la publicación de Del Plata al Niágara y las declaraciones de Groussac en el Teatro de la Victoria, amplificadas por la crónica de Darío, tuvieron un impacto de dimensiones americanas y europeas. Como sugirió hace ya varias décadas Real de Azúa, Groussac fue una de las voces predominantes a la hora de condensar «núcleos temáticos» en los que Estados Unidos devino recurrentemente «el polo dialéctico de la negatividad» —piénsese, por ejemplo, en los ecos del par yanquismo-latinidad que replican en otros opuestos: bárbaros-civilizados, materialismo-espiritualismo, advenedizos de la historia-portadores de la tradición, cultura-naturaleza—. A la vez que su voz PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. 32 Carlos Real de Azúa, “Ariel libro porteño”, en Historia visible e historia esotérica. Personajes y claves del debate latinoamericano (Montevideo: Arca/Calicanto, 1975), 165. 36 Pueden verse: Oscar Terán, “El primer antiimperialismo latinoamericano”, en En busca de la ideología argentina (Buenos Aires: Catálogos, 1986): 85-97. 32 Carlos Real de Azúa, “Ariel libro porteño”, en Historia visible e historia esotérica. Personajes y claves del debate latinoamericano (Montevideo: Arca/Calicanto, 1975), 165. 33 Real de Azúa ha sugerido que la circulación de las ideas de Groussac en el marco de una comunidad intelectual rioplatense es indiscutible, a tal punto que señala que puede descontarse la lectura de Rodó de Del Plata al Niágara y que “no hay una sola clave del desarrollo ariélico (y su misma índole indirecta, literaria ayuda a explicarlo) que no se halle en el libro de Groussac”. Real de Azúa, “Ariel libro porteño”, 165. 34 Pueden verse, por ejemplo, los testimonios de Roberto Giusti acerca de cómo la lectura de Groussac y Rodó articuló las percepciones de sus contemporáneos sobre Estados Unidos: Roberto Giusti, Visto y vivido: anécdotas, semblanzas, confesiones y batallas (Buenos Aires: Losada, 1965), 90. 35 “Por España”, La Biblioteca 8, abril-mayo de 1898. El discurso de Roque Saénz Peña se reproduce en: 213 a 226; el de Groussac en: 227-240. 36 Pueden verse: Oscar Terán, “El primer antiimperialismo latinoamericano”, en En busca de la ideología argentina (Buenos Aires: Catálogos, 1986): 85-97. 5 “Por España”, La Biblioteca 8, abril-mayo de 1898. El discurso de Roque Saénz Peña se reproduce en: 21 26; el de Groussac en: 227-240. 32 Carlos Real de Azúa, Ariel libro porteño , en Historia visible e historia esotérica. Personajes y claves del debate latinoamericano (Montevideo: Arca/Calicanto, 1975), 165. 33 Real de Azúa ha sugerido que la circulación de las ideas de Groussac en el marco de una comunidad intelectual rioplatense es indiscutible, a tal punto que señala que puede descontarse la lectura de Rodó de Del Plata al Niágara y que “no hay una sola clave del desarrollo ariélico (y su misma índole indirecta, literaria ayuda a explicarlo) que no se halle en el libro de Groussac”. Real de Azúa, “Ariel libro porteño”, 165. 34 Pueden verse, por ejemplo, los testimonios de Roberto Giusti acerca de cómo la lectura de Groussac y Rodó articuló las percepciones de sus contemporáneos sobre Estados Unidos: Roberto Giusti, Visto y vivido: anécdotas, semblanzas, confesiones y batallas (Buenos Aires: Losada, 1965), 90. 34 Pueden verse, por ejemplo, los testimonios de Roberto Giusti acerca de cómo la lectura de Groussac y Rodó articuló las percepciones de sus contemporáneos sobre Estados Unidos: Roberto Giusti, Visto y vivido: anécdotas, semblanzas, confesiones y batallas (Buenos Aires: Losada, 1965), 90. 33 Real de Azúa ha sugerido que la circulación de las ideas de Groussac en el marco de una comunidad intelectual rioplatense es indiscutible, a tal punto que señala que puede descontarse la lectura de Rodó de Del Plata al Niágara y que “no hay una sola clave del desarrollo ariélico (y su misma índole indirecta, literaria ayuda a explicarlo) que no se halle en el libro de Groussac”. Real de Azúa, “Ariel libro porteño”, 165. 34 Pueden verse, por ejemplo, los testimonios de Roberto Giusti acerca de cómo la lectura de Groussac y Rodó articuló las percepciones de sus contemporáneos sobre Estados Unidos: Roberto Giusti, Visto y vivido: anécdotas, semblanzas, confesiones y batallas (Buenos Aires: Losada, 1965), 90. 35 “Por España”, La Biblioteca 8, abril-mayo de 1898. El discurso de Roque Saénz Peña se reproduce en: 213 a 226; el de Groussac en: 227-240. semblanzas, confesiones y batallas (Buenos Aires: Losada, 1965), 90. 35 “Por España”, La Biblioteca 8, abril-mayo de 1898. El discurso de Roque Saénz Peña se reproduce en: 213 a 226; el de Groussac en: 227-240. 36 Pueden verse: Oscar Terán, “El primer antiimperialismo latinoamericano”, en En busca de la ideología argentina 37 El título completo de la publicación es La Ilustración Sud-Americana. Periódico Ilustrado de las Repúblicas Sudamericanas. Para una caracterización de la revista puede verse Sandra Szir, “De la cultura impresa a la cultura de lo visible. Las publicaciones periódicas ilustradas en Buenos Aires en el Siglo XIX. Colección Biblioteca Nacional”, en Marcelo Garabedian, Sandra Szir y Miranda Lida, Prensa argentina siglo XIX. Imágenes, textos y contextos (Buenos Aires: Teseo- Biblioteca Nacional, 2009), 53-84. Para una mirada general sobre los semanarios ilustrados en el Río de la Plata puede consultarse: Eduardo Romano, Revolución en la lectura. El discurso periodístico-literario de las primeras revistas ilustradas rioplatenses (Buenos Aires: Catálogos, 2004). Aunque con foco en el análisis de Caras y Caretas, pueden encontrarse algunas referencias a La Ilustración Sud-Americana en Sandra Szir, “El semanario popular ilustrado Caras y Caretas y las transformaciones del paisaje cultural de la modernidad. Buenos Aires, 1898-1908” (Tesis Doctoral, Facultad de Filosofía y Letras, Universidad de Buenos Aires, 2011). 38 “A los lectores”, La Ilustración Sud-Americana, nº 1, 1 de diciembre de 1892, 2. 39 “A los lectores”, 2. La Biblioteca y Calibán: entre la pluma y la voz de Groussac LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS adquirió una proyección rioplatense,32 marcó un momento de cristalización de ideas e imágenes para pensar las disputas identitarias entre América Latina y Estados Unidos que se proyectó entre los contemporáneos,33 y tuvo ecos en las generaciones posteriores.34 Es decir, en el mismo año en el que Groussac estaba protagonizando problemas en la dirección de La Biblioteca por ser considerado un adversario intelectual severo de un representante diplomático en el marco de las discusiones con Chile por las cuestiones limítrofes, devino una voz que articulaba un discurso de proyección transnacional y de llamamiento de unidad hispanoamericana para hacer frente al avance de Estados Unidos. En esta coyuntura, el gesto último de Groussac en las páginas de la revista que había fundado y manejado de manera personal y donde había publicado numerosos trabajos de su autoría, fue intervenir replicando su propia voz. En el último tomo de su empresa editorial, se encuentra una sección entera dedicada a reproducir las conferencias pronunciadas por Roque Sáenz Peña y por él mismo el 2 de mayo en el Teatro de la Victoria —no se reproduce, en cambio, el poema de José Tarnassi.35 Mientras que sus opiniones parecían articular intenciones identitarias de proyección hispanoamericana, en su revista, estas conferencias se reprodujeron bajo un sobrio título: «Por España». Apenas termina la reproducción de los discursos, se encuentra el artículo de cierre de la revista. Desaparecía La Biblioteca, una empresa que había convertido a su director en un árbitro de la vida intelectual argentina; surgía la voz de Groussac como figura clave en la organización de entramados de un ideario antiimperialista latinoamericano e hispanoamericanista.36 36 Pueden verse: Oscar Terán, “El primer antiimperialismo latinoamericano”, en En busca de la ideología argentina (Buenos Aires: Catálogos, 1986): 85-97. PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS 38 “A los lectores”, La Ilustración Sud-Americana, nº 1, 1 de diciembre de 1892, 2. La Ilustración Sud-Americana: opinión americana, relatos premonitorios y conferencias La Ilustración Sud-Americana. Publicación quincenal de las Repúblicas Sud- Americanas se publicó por primera vez en diciembre de 1892 (el primer número indica que la ciudad de edición es Buenos Aires, años después se anunciaba una edición paralela en Montevideo) y estaba dirigida y fundada por Rafael Contell y Francisco María Conte; además, se mencionaba como director literario a Antonio Atienza y Medrano (a lo largo de los años hubo cambios en la dirección y en la redacción).37 Desde su primer número dejaba planteada una agenda que intentaba mantenerse al margen de los problemas de coyuntura. Como meta, señalaban sus editores: «registrará, pues, cuidadosamente esta publicación todos los acontecimientos concernientes a la vida política de los pueblos sudamericanos; pero no traspasará jamás los límites de la crónica, ni invadirá terrenos que atiende están vedados por su misma naturaleza».38 Luego de realizar una evaluación sobre la prensa diaria y las revistas abocadas a la ciencia y a la cultura, los redactores dejaban claro su programa: «tomando por punto de partida esos meritorios ensayos y por modelo los periódicos ilustrados más notables en la prensa europea y norteamericana, esta publicación se esforzará en corresponder a su título, no solo bajo su aspecto científico y literario, sino también bajo el artístico».39 A tono con lo que sus promotores señalaban, varios estudios han planteado que la revista compartía rasgos con otras publicaciones ilustradas contemporáneas americanas y europeas. En las páginas de la misma convivían notas de opinión sin firma, una notable cantidad de fotografías y láminas, noticias sobre los países de la América hispana y algunos textos firmados que, por lo general, eran reproducciones parciales o totales de textos PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS generados en otros formatos, o para otras publicaciones. Así, aunque se pueden rastrear firmas como las de Calixto Oyuela, Rafael Obligado, Lucio V. Mansilla o Estanislao Zeballos, los textos de autoría de estas y otras figuras intelectuales estaban, en general, pensados para destinos diferentes a los de sus páginas. Es notable, además, el esfuerzo por replicar textos de voces de la vida latinoamericana, como Ricardo Palma, Rubén Darío, Enrique Gómez Carrillo y otros. 40 “Cosas y quiscosas”, La Ilustración Sud-Americana, nº 129, 1 de mayo de 1898, 162. La Ilustración Sud-Americana: opinión americana, relatos premonitorios y conferencias Desde mi perspectiva, la revista es una fuente privilegiada para el estudio de la vida diplomática y cultural en las Américas, dado que en sus páginas se ve la intención de pensar más allá de las fronteras nacionales —ya desde su título— y es visible un esfuerzo por dar cuenta de conexiones y relaciones americanas e hispanoamericanas. Por su parte, se ve una clara intención de cubrir los movimientos de figuras de la vida diplomática entre países —por medio de semblanzas de diplomáticos y otros comisionados a labores en países diferentes al de origen— y es evidente la determinación de dar cuenta de eventos de convivencia entre naciones, como congresos americanos y panamericanos, exposiciones de proyección continental, y otros momentos de encuentro entre naciones. Por estos motivos, la revista tenía una tendencia a mostrar lazos y afinidades de dimensión regional de manera sistemática. Con estas intenciones de subrayar la confraternidad, la guerra de 1898 se presentó como un desafío. En sus páginas, el 1 de mayo, los redactores expresaban su plena conciencia acerca de la guerra como asunto de centralidad avasalladora. Se lee en las columnas de apertura, tituladas «Cosas y quiscosas»: Es el asunto del día, el de la quincena; y lo será tal vez por algunos meses, en todos nuestros círculos sociales. A la cuestión de límites con Chile; a los cabildeos de la política y las murmuraciones sobre la marcha financiera de los gobiernos que administran nuestros intereses; a los arreglos chileno-peruanos que el telégrafo nos transmite […] a los acuerdos y desacuerdos de las cosas uruguayas, y a las dificultades, revoluciones, y demás percances de los demás países sudamericanos; a todo cuanto constituía base o fundamento de parleras disquisiciones, ha venido a sustituir el tema de la guerra hispano-americana.40 Según señalaba la publicación, «gente pensadora en los países americanos» parecía estar, indefectiblemente, a favor de España. Los redactores señalaban —con un dejo de ironía— que la «cuestión de Cuba» había quedado atrás; PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS había llegado el momento de los «¡Viva España!». La pregunta quedaba claramente planteada: ¿cómo participar en ese clima de opinión desde las páginas de una revista ilustrada? Las respuestas a este interrogante fueron varias. 41 “La Guerra inicua. Mackinley [sic] ante el derecho. La constitución y las leyes violadas”, La Ilustración Sud- Americana, nº 129, 1 de mayo de 1898, 163-164. 42 “La guerra hispano-yankee. Opinión de autoridad”, La Ilustración Sud-Americana, nº 130, 16 de mayo de 1898, 183-185. La Ilustración Sud-Americana: opinión americana, relatos premonitorios y conferencias Pueden rastrearse en las páginas de la revista cuatro recursos diferentes entre sí para participar del fervor bélico. El primero se basa en la consideración de que ciertas voces de autoridad podían dar cuenta de lo que estaba sucediendo y dar indicios para comprender el conflicto. En este sentido, se destacan dos artículos. Por un lado, uno firmado por Luis V. Varela, descrito como «Magistrado Argentino». Varela, nacido en Montevideo y reconocido con el cargo de Juez en la Corte Suprema de Justicia de Argentina, firmaba un texto titulado «La Guerra inicua. Mackinley [sic] ante el derecho. La constitución y las leyes violadas».41 El texto, quizás publicado unos días antes en otro soporte —está fechado el 24 de abril de 1898— revisa algunos aspectos de la constitución de Estados Unidos y de la toma de decisiones de William McKinley. Luego de algunos argumentos basados en consideraciones de derecho internacional y jurisprudencia, el texto asume un tono de marcada defensa a España, que concluye con una sentencia: «la causa de España es hoy la causa de la humanidad». Además de darle lugar a la firma de un magistrado, el otro texto que interpela a quien se considera una voz de autoridad se reproduce con el título «La guerra hispano-yankee. Opinión de autoridad». Con este título se introducen unas notas del «teniente coronel de ingenieros de España y especialista en asuntos militares, señor Jenaro Alas». La estrategia de intervención se describía con las siguientes palabras: «tomamos los siguientes párrafos, que creemos serán de interés para lo que sigue en desarrollo de los acontecimientos de la guerra hispano-yankee». El comentario parte de la idea de que «los Estados Unidos carecen de tradiciones militares»; posteriormente señala errores del país del Norte a la hora de diseñar las estrategias de guerra, pero también las limitaciones en España. La conclusión es que la guerra es un conflicto entre buques.42 En el contexto del conflicto, Jenaro Alas realizó varias presentaciones en el Consejo de Ministros de España que eran seguidas por la prensa española y americana con atención; ocupaba, además el rol de PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. 47 La Ilustración Sud-Americana, nº 130, 16 de mayo de 1898, 190. 43 Puede verse, por ejemplo: el Heraldo de Madrid, Madrid, 9 de mayo de 1898; La correspondencia militar, Madrid, 10 de mayo de 1898. Sobre el rol de Jenaro Alas en el contexto de la guerra puede consultarse: Cristóbal Robles Muñoz, La política exterior de España: Política mediterránea, occidental y de paz (1899-1905), tomo 1 (Madrid: Consejo Superior de Investigaciones Científicas, 2006), 55. 44 Rafael Sánchez Lizardi, Viaje a España (París: Garnier, 1889). Fragmentos reproducidos en La Ilustración Sud- Americana, nº 131, 1 de junio de 1898, 210. 45 Carlos Lix Klett fue un activo representante de Argentina en lo que respecta a las relaciones comerciales con Estados Unidos. Sus crónicas de viajes y recorridos por el país del Norte se publicaban en distintos periódicos de Buenos Aires en el cambio del siglo XIX al XX. Varios de estos textos fueron recogidos en su libro Carlos Lix Klett, Estudios sobre producción, comercio, finanzas e intereses generales de la República Argentina (Buenos Aires: Tailhade y Rosselli, 1900). Se encuentran fragmentos de sus crónicas en La Ilustración Sud-Americana, nº 131, 1 de junio de 1898, 206; nº 135, 1 de agosto de 1898, 282; nº 137, 1 de septiembre de 1898, 323. 46 Rafael Puig y Valls, Viaje á América: Estados Unidos, Exposición Universal de Chicago, México, Cuba y Puerto Rico (Barcelona: Tipolitografía de Luis Tasso, 1894). Fragmentos reproducidos en La Ilustración Sud-Americana, nº 129, 1 de mayo de 1898, 169. 45 Carlos Lix Klett fue un activo representante de Argentina en lo que respecta a las relaciones comerciales con Estados Unidos. Sus crónicas de viajes y recorridos por el país del Norte se publicaban en distintos periódicos de Buenos Aires en el cambio del siglo XIX al XX. Varios de estos textos fueron recogidos en su libro Carlos Lix Klett, Estudios sobre producción, comercio, finanzas e intereses generales de la República Argentina (Buenos Aires: Tailhade y Rosselli, 1900). Se encuentran fragmentos de sus crónicas en La Ilustración Sud-Americana, nº 131, 1 de junio de 1898, 206; nº 135, 1 de agosto de 1898, 282; nº 137, 1 de septiembre de 1898, 323. 46 Rafael Puig y Valls Viaje á América: Estados Unidos Exposición Universal de Chicago México Cuba y Puerto Rico 46 Rafael Puig y Valls, Viaje á América: Estados Unidos, Exposición Universal de Chicago, México, Cuba y Puerto Rico (Barcelona: Tipolitografía de Luis Tasso, 1894). Fragmentos reproducidos en La Ilustración Sud-Americana, nº 129, 1 de mayo de 1898, 169. 43 Puede verse, por ejemplo: el Heraldo de Madrid, Madrid, 9 de mayo de 1898; La correspondencia militar, Madrid, 10 de mayo de 1898. Sobre el rol de Jenaro Alas en el contexto de la guerra puede consultarse: Cristóbal Robles Muñoz, La política exterior de España: Política mediterránea, occidental y de paz (1899-1905), tomo 1 (Madrid: Consejo Superior de Investigaciones Científicas, 2006), 55. 44 Rafael Sánchez Lizardi, Viaje a España (París: Garnier, 1889). Fragmentos reproducidos en La Ilustración Sud- Americana, nº 131, 1 de junio de 1898, 210. el Sánchez Lizardi, Viaje a España (París: Garnier, 1889). Fragmentos reproducidos en La Ilustración Sud- a, nº 131, 1 de junio de 1898, 210. La Ilustración Sud-Americana: opinión americana, relatos premonitorios y conferencias LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS diputado por Sanctis Spiritus, Santa Clara, Cuba.43 Es decir, en este caso, la voz de autoridad estaba legitimada en un doble rol: militar y conocedor de la situación cubana. Una segunda forma de dar cuenta del clima bélico que se puede rastrear en las páginas de la revista es el de publicar fragmentos de libros sobre asuntos de guerra de décadas anteriores, o relatos de viajes a Estados Unidos y a España publicados en los años previos al conflicto bélico. Se encuentran en distintos números de 1898, por ejemplo, fragmentos breves del viaje a España de Rafael Sánchez Lizardi,44 los comentarios de Carlos Lix Klett acerca de su gira por Estados Unidos —visitas a universidades, fábricas y establecimientos agrícolas45—, y fragmentos del viaje del español Rafael Puig y Valls por Estados Unidos.46 La reproducción de estos textos apunta a mostrar impresiones de corte sociológico sobre las dos naciones en conflicto. De alguna manera, se sugiere que la disputa entre estas dos naciones estaba inscripta en sus propias dinámicas internas. Se publicaron fragmentos que, además, subrayan la diversidad de características de España y Estados Unidos, que pueden sintetizarse en la oposición entre tradición —España como síntesis del Viejo Continente— y modernidad —Estados Unidos como nación pujante del continente americano—. Junto con los relatos de travesías, se publicaban otros textos producidos con anterioridad, pero que, en consideración de los redactores de La Ilustración Sud-Americana, revestían una «indiscutida actualidad» para pensar en las dinámicas de la guerra y la paz. Es el caso de unas páginas de autoría de Juan Bautista Alberdi descritas como «nacidas al calor de la contienda franco- prusiana» que bajo el título «La Guerra Moderna» se publican in extenso.47 PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS El tercer recurso que se puede rastrear en la revista coincide con una práctica que devino bastante usual en estos años: la impresión de conferencias signadas por la coyuntura que se pronunciaban en ámbitos de sociabilidad reconocidos, como academias, círculos literarios y ateneos. 49 La Ilustración Sud-Americana, nº 130, 16 de mayo de 1898, 183-189. 50 La Ilustración Sud-Americana, nº 132, 16 de junio de 1898. El comentario de Calixto Oyuela se encuentra en: 244; la conferencia de del Solar se reproduce en: 245-250. 51 Alberto Del Solar, “La Doctrina Monroe y la América Latina”, Conferencia leída en el Ateneo el 20 de junio de 1898 (Buenos Aires: Imprenta, Tipografía y Encuadernación Jacobo Peuser, 1898). 48 Un caso clave que constata esta dinámica es el de la Sociedad Científica Argentina y la publicación de las conferencias allí pronunciadas en su revista, Anales de la Sociedad Científica Argentina. Como ejemplo, puede consultarse una conferencia que evaluó el escenario internacional luego de la guerra de 1898 y los Tratados de París pronunciada por Eduardo L. Holmberg y publicada bajo el título: “De siglo a siglo”, Anales de la Sociedad Científica Argentina 51 (1901): 51-60. La Ilustración Sud-Americana: opinión americana, relatos premonitorios y conferencias En general, se trataba de reproducciones de alocuciones ocurridas en ámbitos asociados a las publicaciones que los difundían en letra impresa.48 La Ilustración Sud-Americana no era el órgano de un ámbito de sociabilidad concreto y, pese a que varios de sus redactores y fundadores eran de origen ibérico, no se presentaba como publicación de la comunidad étnica española. Así, en el contexto de 1898, optó por reproducir varias de estas intervenciones de intelectuales pronunciadas en distintos cenáculos. Por un lado, bajo el título «El 2 de mayo en el “Victoria”» se replicaron de manera total los ya referidos discursos de Sáenz Peña, Groussac y Tarnassi sin ningún tipo de acápite, aclaración o comentario. Estas voces parecían haber asumido un efecto casi oracular en el despliegue de la guerra. La apuesta de los redactores puede notarse en una reorganización del orden en el que publicaron las intervenciones, que alteran las que señalan las crónicas de los periódicos y el programa de la función —Saénz Peña, Groussac y Tarnassi—; en este caso, se publicó en primer lugar el discurso de Groussac, en segundo lugar el poema de Tarnassi y, por último, la conferencia de Roque Sáenz Peña.49 Apenas dos números después, bajo el título «En el Ateneo» se encuentra un comentario introductorio de Calixto Oyuela, ferviente defensor de España, para presentar otra de las conferencias que tuvo marcada repercusión en los meses de la guerra. La misma llevó el título «La Doctrina Monroe y la América Latina» y fue pronunciada por Alberto del Solar el 20 de junio de 1898 en el Ateneo.50 La conferencia de del Solar, figura de la vida letrada chilena, se refería a la violación ejercida por el «coloso norteamericano», y a los derechos de la «desgraciada España» sobre sus últimas posesiones.51 A su vez, argumentaba que «nuestra madre común» no pretendía violentar las intenciones de Cuba de ser independiente. Despuntaba en esta apreciación PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS un argumento interesante; el conferencista destacaba: «se puede ser buen hijo de la América emancipada […] y admirar, al mismo tiempo, el brío, la hidalguía, el heroísmo hispanos».52 La conferencia había, de alguna manera, devuelto un lugar a Cuba en el escenario de la guerra. La Ilustración Sud-Americana: opinión americana, relatos premonitorios y conferencias Una Cuba que no aparecía casi referida como «hermana» en otros discursos pronunciados por voces intelectuales de América Latina.53 Estas declaraciones destrababan, de este modo, la tensión España/Estados Unidos y sumaban nuevas preguntas sobre la independencia cubana. Los ecos de la conferencia y el entusiasmo que le había generado a Oyuela se expresó en la fórmula: «falta ahora que sea impresa en folleto». De hecho, fue publicada en este formato y devino una referencia usual en textos que han estudiado en el largo plazo el rol de la Doctrina Monroe en las decisiones de política exterior norteamericana.54 La cuarta forma que la revista dinamizó fue explicitada a comienzos de mayo con estas palabras: «en nuestras columnas anotamos algunos de los brillantes artículos de escritores de ambas orillas del Plata y hemos de continuar agrupando los que en lo sucesivo se escriban, como antecedentes históricos de la cruenta guerra que se inicia en los postreros años del siglo».55 La cuarta forma que la revista dinamizó fue explicitada a comienzos de mayo con estas palabras: «en nuestras columnas anotamos algunos de los brillantes artículos de escritores de ambas orillas del Plata y hemos de continuar agrupando los que en lo sucesivo se escriban, como antecedentes históricos de la cruenta guerra que se inicia en los postreros años del siglo».55 La propuesta de tomar escritos de otros diarios y organizar las voces de lo que solían denominar «opinión sud-americana» puede verse puesta en acción en varias páginas de la revista en las que se reprodujeron textos publicados por medios de prensa de distintas latitudes. Destaca en este sentido la apuesta desplegada en el número 129. Bajo el título general «La actualidad. Por España (La opinión Sud-Americana)», se encuentran reproducciones de fragmentos de textos misceláneos como los siguientes: «La cuestión palpitante», con firma de Julio Herrera Obes (con la aclaración «Ex Presidente de la República Oriental del Uruguay», una vez más se utilizaba aquí el recurso de la voz autorizada), «Oda a España», un poema de Calixto Oyuela (debajo de su nombre se lee «Argentino»), que circuló ampliamente en el contexto de la guerra; y varios textos de periódicos: un fragmento firmado por Sr. Ariel J. 52 Alberto Del Solar, “La Doctrina Monroe”, 57. 53 Para una mirada general sobre este tema puede verse Carolina López, “Los intelectuales argentinos frente a la independencia cubana de 1898: último bastión imperialista y nuevo status colonial”, Araucaria. Revista Iberoamericana de Filosofía, Política y Humanidades, nº 26 (2011): 3-25. 54 Véase, por ejemplo, Dexter Perkins, Historia de la Doctrina Monroe (Buenos Aires: Eudeba, 196), 260. 55 La Ilustración Sud-Americana, nº 129, 1 de mayo de 1898, 162. Para una mirada general sobre este tema puede verse Carolina López, “Los intelectuales argentinos frente a la pendencia cubana de 1898: último bastión imperialista y nuevo status colonial”, Araucaria. Revista Iberoamericana osofía, Política y Humanidades, nº 26 (2011): 3-25. 4 Véase, por ejemplo, Dexter Perkins, Historia de la Doctrina Monroe (Buenos Aires: Eudeba, 196), 260. 5 La Ilustración Sud-Americana, nº 129, 1 de mayo de 1898, 162. La Ilustración Sud-Americana: opinión americana, relatos premonitorios y conferencias Pérez que aclara entre paréntesis «De La Razón de Montevideo»; otro con el título «España», con la indicación «De Tribuna de Buenos Aires»; párrafos titulados «Brigantaggio», provenientes de «La Patria degli Italiani, Buenos Aires»; «La guerra hispanoamericana», con la referencia «De El Tiempo de PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS Buenos Aires»; «La Madre Patria», referido como un fragmento proveniente de «De El Bien de Montevideo»; «Por España» proveniente «De El Porvenir de Santiago de Chile»; «España y Estados Unidos» procedente de «De El Heraldo de Valparaíso». Esta composición miscelánica no está acompañada de ninguna aclaración, introducción o jerarquía clara. No se aclara la fecha de publicación de los fragmentos y no siempre cuentan con firma. Sin embargo, hay un énfasis en destacar los nombres de los periódicos y las ciudades de origen de los mismos. La apuesta parecía intentar ofrecer una especie de coro americano que podía revelar, al menos, dos cuestiones: por un lado, la importancia que se le daba desde la revista a los diarios a la hora de cubrir las noticias de actualidad de la guerra; por otro, la centralidad de esta puesta en escena por una revista ilustrada de presentar fragmentos inconexos entre sí a priori, pero considerarlos representativos de la «opinión sud-americana». Esta puede ser la explicación del énfasis a la hora de mostrar diversas voces de Buenos Aires —diarios étnicos y periódicos de tirada nacional—, Santiago de Chile, Valparaíso y Montevideo. En suma, La Ilustración Sud-Americana optó por diferentes modalidades de expresión en el contexto de la guerra. En sus páginas convivieron conferencias de coyuntura, relatos de viaje publicados en el último cuarto del siglo XIX, artículos de voces de autoridad, textos generales sobre la guerra escritos por intelectuales destacados de América Latina, y fragmentos de notas periodísticas. Todo ello esbozaba una composición que, lejos de mostrar unidad, daba cuenta de la amplitud de voces que podían sumar información e ideas para pensar la guerra y las identidades en disputa en su contexto. 56 Puede consultarse un índice realizado con exhaustividad y una caracterización de la revista en Gregoria Celada Domínguez y Rita Giacalone, “Revista de Derecho, Historia y Letras (1898-1923). Estudio e índice general”, Iushistoria, nº 4 (2007): 1-144. Revista de Derecho, Historia y Letras: imperialismo, expansionismo y Derecho Internacional Entre 1898 apareció el primer número de la Revista de Derecho Historia y Letras, fundada y dirigida por Estanislao Zeballos y pensada como una publicación de aparición mensual.56 En su prospecto se indicaba: 56 Puede consultarse un índice realizado con exhaustividad y una caracterización de la revista en Gregoria Celada Domínguez y Rita Giacalone, “Revista de Derecho, Historia y Letras (1898-1923). Estudio e índice general”, Iushistoria, nº 4 (2007): 1-144. PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS la acción de la Revista será materia de derecho, crítica y científica […] Los estudios históricos están incorporados a las grandes escuelas jurídicas, su enseñanza es virtud fundadora […] La crónica tiene para sus páginas un interés secundario, pero felizmente empieza la literatura histórica en la República y en América el período de la crítica y la filosofía […] No será extraño a este plan el estímulo de la cultura literaria.57 Si estas eran sus intenciones iniciales, para comienzos del siglo XX, la revista estaba posicionada como un órgano para intervenir sobre temas ligados al Derecho Internacional y los vínculos entre naciones.58 Temas de geopolítica, relaciones, tratados y convenciones internacionales, fueron incorporados con ampliado interés en la revista en forma de artículo erudito. El despliegue de la guerra, la firma del Tratado de París y sus efectos en territorios americanos, coincidieron con los años iniciales de la revista. Para entonces, los intereses de Estanislao Zeballos en política internacional y su trayectoria diplomática eran conocidos en el continente americano y en el europeo.59 La posibilidad de comandar la revista se convirtió, entonces, en un ejercicio de curaduría abierto a posibilidades para su director, que contaba con la posibilidad de detectar y seleccionar contenidos para presentarlos en las páginas de la publicación. Encuentro que en las páginas de la Revista de Derecho, Historia y Letras se desplegaron cuatro modalidades de intervención respecto de la guerra. La primera opción se encuentra en el tomo inaugural de la revista y hace uso, en sintonía con lo reseñado para La Ilustración Sud-Americana, de la voz de autoridad militar. Se encuentra allí un artículo titulado «Las matemáticas del desastre», de Enrique Howard (presentado como «el comodoro Howard») con un tono que combina consideraciones técnicas y un llamamiento a aunar las fuerzas hispanoamericanas. 57 Estanislao Zeballos, “Prospecto”, Revista de Derecho. Historia y Letras 1 (1898): 6. 58 Melisa Deciancio, “Puentes para pensar lo internacional en los albores del siglo XX: La Revista Argentina de Ciencia Política (1910-1928) y la Revista de Historia, Derecho y Letras (1898-1923) en las relaciones internacionales de Argentina”, Ciclos en la Historia, la economía y la sociedad 26, nº 47 (2018): 1-15; Camila Bueno Grejo, “A construção da identidade internacional argentina nas páginas da Revista de Derecho, Historia y Letras”, Antiteses 10, nº 19 (2017): 64-87; Enrique Shaw, “Una mirada particular de las relaciones entre América-nos y europeos, entre 1898 y 1910”, Estudios, nº 18 (2006): 131-145. 59 Sobre Zeballos pueden consultarse Roberto Etchepareborda, Zeballos y la política exterior argentina (Buenos Aires: Pleamar, 1982). 57 Estanislao Zeballos, “Prospecto”, Revista de Derecho. Historia y Letras 1 (1898): 6. Sobre Zeballos pueden consultarse Roberto Etchepareborda, Zeballos y la política exterior argentina (Buenos Aires: mar, 1982). Revista de Derecho, Historia y Letras: imperialismo, expansionismo y Derecho Internacional En este caso, no se trata de la reproducción de un texto originalmente publicado en otro lugar. Zeballos señala que solicitó a un mando militar un comentario sobre los episodios bélicos. El texto PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS propone un análisis de las potencialidades y límites de la marina española y ofrece sentencias a tono con las discusiones de derecho internacional contemporáneas. Se señala, por ejemplo: la guerra, cuya probabilidad no era un misterio para nadie, dado el alcance doctrinario y práctico de Monroe sobre proteccionismo americano, se inicia entre España y Estados Unidos, y al más negado en la materia no se le hubiera escapado que el éxito estaba librado al poder naval de una y otra potencia […] Huérfana y aislada la armada española, sin el robusto sostén de una cabeza dirigente que le inculcara administración y disciplina, pierde al instante su vigor e iniciativa, se sostiene con languidez, gira acobardada e ineficaz dentro del elemento limitado de su acción, lo recorre con embarazosa lentitud, restringe y contrae las operaciones que le hubieran dado prestigio y vida, y viene, al fin, a morir de inacción y parálisis. Tenía que suceder: estaba escrito.60 44 ISSN: 1510-5024 (papel) - 2301-1629 (en línea) eo, No 9, Junio 2021, pp. 23-58 Al darle voz a una figura que ocupó a lo largo de su trayectoria cargos de Comodoro, Vicealmirante y Capitán de fragata, parece que la apuesta de Zeballos era mostrar un análisis especializado de la contienda en términos de estrategia militar.61 La segunda modalidad que se puso en acción en las páginas de la Revista de Derecho, Historia y Letras fue la traducción de artículos de periódicos y revistas que, generados en otras geografías, fueron considerados de interés para comprender el fenómeno de la guerra. Mientras que, en algunos casos se trata de traducciones de textos asociados directamente con la guerra, en otros, Zeballos seleccionó intervenciones que revestían, desde su perspectiva, interés. Este es el caso de los fragmentos comentados de una entrevista a Cecil Rhodes. En nota al pie, el director aclara que, enterado por el telégrafo de esta entrevista, decidía dar a conocer a los lectores estas opiniones. 61 De hecho, además de publicarse en la Revista de Derecho Historia y Letras, el mismo fue reproducido en Boletín del Centro Naval 16 (1898): 59-60, con una breve introducción que hace referencia a la Revista de Derecho, Historia y Letras y a las palabras elogiosas que le dedicó Zeballos a Enrique Howard. Enrique Howard, “Las matemáticas del desastre”, Revista de Derecho. Historia y Letras 1 (1898): 244. 62 Sobre Cecil Rhodhes puede verse Paul Maylam, The Cult of Rhodes. Remembering an Imperialist in Africa (Claremont: South Africa David Philip, 2005). 63 Pueden verse las consideraciones sobre el impacto de las intervenciones de Rhodes en la prensa en Edmund Garrett, A memoir (London: Cook, 1909). Se reproducen allí en anexo una entrevista que Garrett (escritor, periodista y miembro del Parlamento del Cabo de Buena Esperanza) le realizó e Rhodes en marzo de 1898, páginas 222-227. 64 Cecil Rhodes, “Los americanos en Sur América (traducido del “New York Herald por R. Pérez”)”, Revista de Derecho Historia y Letras 3 (1899): 450-452. 70 Al publicar el segundo artículo de Bryce, Zeballos aclara: “el Editor de Harper’s Magazine cree que interesará a los lectores americanos conocer cómo se aprecian estos asuntos en Europa por los que han estudiado la situación de los Estados Unidos; y aunque no se me oculta cuan delicado es el encargo, correspondo a la invitación”, Revista de Derecho, Historia y Letras 2 (1898): 458. Traducido para la Revista de Derecho, Historia y Letras, por Domingo de Vívaro; Segundo artículo publicado en The Harper’s Magazine, Traducido para la Revista de Derecho, Historia y Letras, por D. de V.), Revista de Derecho, Historia y Letras 2 (1898): 308-320 y 457-474. 65 El artículo original se tituló: “The policy of annexation for America”, The Forum 29 (1897): 385-395. 68 James Bryce, “Nueva política exterior americana” (Primer artículo publicado en The Forum de Nueva York. Traducido para la Revista de Derecho, Historia y Letras, por Domingo de Vívaro; Segundo artículo publicado en The Harper’s Magazine, Traducido para la Revista de Derecho, Historia y Letras, por D. de V.), Revista de Derecho, Historia y Letras 2 (1898): 308 320 y 457 474 69 Sobre el ideario de Bryce puede verse: Héctor Domínguez, James Bryce y los fundamentos intelectuales del internacionalismo liberal (1864-1922) (Madrid: Centro de Estudios Políticos y Constitucionales, 2018). Agradezco, 7 James Bryce, The American Commonwealth (London/New York: Macmillan, 1889). 69 Sobre el ideario de Bryce puede verse: Héctor Domínguez, James Bryce y los fundamentos intelectuales del internacionalismo liberal (1864-1922) (Madrid: Centro de Estudios Políticos y Constitucionales, 2018). Agradezco, por su parte, a Héctor Domínguez el acceso a los textos originales de Bryce que fueron traducidos en la Revista de or su parte, a Héctor Domínguez el acceso a los textos originales de Bryce que fueron traducidos en la Revista Derecho, Historia y Letras. 8 James Bryce, “Nueva política exterior americana” (Primer artículo publicado en The Forum de Nueva Yo raducido para la Revista de Derecho, Historia y Letras, por Domingo de Vívaro; Segundo artículo publicado en 6 “Some thoughts on the policy of United States”, Harper’s New Monthly Magazine, 1 de junio de 1898, 609-6 Harper’s Magazine, Traducido para la Revista de Derecho, Historia y Letras, por D. de V.), Revista de Derecho, Histor etras 2 (1898): 308-320 y 457-474. g p y ( ) 66 “Some thoughts on the policy of United States”, Harper’s New Monthly Magazine, 1 de junio de 1898, 609-618. 67 James Bryce, The American Commonwealth (London/New York: Macmillan, 1889). g p y , ( ) 66 “Some thoughts on the policy of United States”, Harper’s New Monthly Magazine, 1 de junio de 1898, 609-618. 67 J B Th A i C l h (L d /N Y k M ill 1889) Revista de Derecho, Historia y Letras: imperialismo, expansionismo y Derecho Internacional LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS estos textos de Rhodes o Bryce, y mostrar en las páginas de la revista dos polos de un debate tácito entre defensores y detractores del imperialismo. Otra modalidad que se ve en la revista se advierte en dos textos de opinión con firma. Consisten en reflexiones sobre Estados Unidos, España y América Latina que, si bien no hacen referencia explícita a la guerra, comparten el clima de evaluación acerca de las tensiones entre nuevo y viejo continente, sintetizadas en expresiones que oponen valores sajones y valores latinos. El primero se titula «Situación y futuro de la América española». Se trata de unas observaciones que «en forma de carta», según aclara la nota al pie, le hacía llegar Paulino Alfonso desde Santiago de Chile (con fecha 21 de junio de 1898) a Estanislao Zeballos. Alfonso aprovechaba esta epístola a Zeballos para hacer un balance sobre lo que acaecía en la América de habla hispana, y comparaba su situación con Estados Unidos. Sus declaraciones, en tanto figura de la vida cultural chilena, ponían los acentos en una lectura pesimista sobre América Latina: «el espectáculo que se ofrece al espíritu medianamente observador y estudioso, al echar una mirada sobre el conjunto de la América española, no es por cierto halagüeño. Juzgo exactísima la idea de que la civilización hispano-americana está retardada».71 A esta realidad, contraponía lo «realizado en la gran república norteamericana. Es que allí, fuera de las condiciones naturales y especialmente propicias de la situación geográfica, de los mares y los climas, de las tierras y los ríos, hubo alguna educación y hubo alguna libertad». La epístola presenta las tensiones entre una tierra de libertades y un sistema político en funciones, y una región de caudillos y políticas facciosas demasiado atenta al ingrediente popular. Alfonso era un conocedor de las dinámicas norteamericanas, tempranamente había recorrido el país del Norte oficiando de secretario de su padre en la I Conferencia Panamericana en Washington; a su vez, las cuestiones de arbitraje internacional estaban entre sus temas de interés como jurista.72 El tono de Alfonso refleja un clima de opinión que en los cenáculos intelectuales de Santiago estaba bastante extendido. 71 Paulino Alfonso, “Situación y futuro de la América española (A propósito del Prospecto de la Revista de Derecho, Historia y Letras)”, Revista de Derecho, Historia y Letras 1 (1898): 530. 72 Para referencias biográficas puede verse: Don Paulino Alfonso,1862-1923. Homenaje a su memoria (Santiago de Chile: Talleres Gráficos San Rafael, 1928). 73 Enrique Rodríguez Mendoza, Ante la decadencia, Conferencia leída en el Ateneo de Santiago (Santiago de Chile: Imprenta Moderna, 1899). Revista de Derecho, Historia y Letras: imperialismo, expansionismo y Derecho Internacional En el texto, Rhodes realizaba consideraciones sobre cómo Estados Unidos contaba con todo lo necesario para avanzar sobre Sud América, comparando esta situación con la de Inglaterra respecto de Sudáfrica; de hecho, hacía prácticamente un llamamiento a que Estados Unidos avanzara determinadamente sobre el control del resto del continente. Seleccionar y exponer las opiniones de Rhodes, descripto como «el renombrado e infatigable promotor y agitador PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS de las posesiones inglesas al sur de África», una controvertida figura en el contexto de imperialismo, no parecía una elección inocente.62 El buen tino de Zeballos al reseñar la entrevista y usar algunas citas textuales de la misma, y otras traducidas, puede verse constatado por las repercusiones que esa entrevista y otras con contenido similar tuvieron durante esos años en la prensa internacional.63 El texto fue publicado con el título «Los americanos en Sur América» y se señalaba que era una traducción del New York Herald, realizada por R. Pérez.64 45 ISSN: 1510-5024 (papel) - 2301-1629 (en línea) Humanidades: revista de la Universidad de Montevideo, No 9, Junio 2021, pp. 23-58 Si elegir la figura de Cecil Rhodes para opinar sobre cuestiones de imperialismo y geopolítica muestra a un Zeballos atento al clima internacional, no es menos interesante su decisión de dar espacio en las páginas de la revista a la traducción de dos textos de James Bryce que habían sido publicados en The Forum de Nueva York,65 y en Harper’s New Monthly Magazine.66 Bryce era ya una figura reputada en tanto autor de The American Commonwealth (1889)67 y una voz autorizada en tanto conocedor de Estados Unidos, de su constitución y de las dinámicas de política interna y exterior. Por su parte, era un detractor de la política expansionista británica que condujo a la guerra anglo-boer. Sus dos textos fueron traducidos bajo el mismo título «Nueva política exterior norteamericana»,68 y se señala en la nota al pie del primero que las traducciones fueron realizadas por Domingo de Vivero.69 Puede atribuirse a Zeballos —y a sus conexiones70— la lucidez para dar con PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. Revista de Derecho, Historia y Letras: imperialismo, expansionismo y Derecho Internacional Sus observaciones positivas sobre Estados Unidos, de hecho, son coincidentes con las que Enrique Rodríguez Mendoza pronunció en 1899 durante una conferencia resonante en el Ateneo de Santiago.73 PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS La segunda contribución que aporta reflexiones generales propone ya desde su título, «Anglosajones y latinos», la contraposición señalada en el párrafo anterior, y está firmada por Felipe Senillosa, en Génova, el 1 de enero de 1900. Terminada ya la guerra y firmados los Tratados de París, el texto hace hincapié en «la decadencia heroica de España» y subraya cómo «los Estados Unidos progresan rápidamente (gozando) de mayor libertad, de más orden, de más vitalidad moral», todas características que «han motivado la idea de la superioridad de la raza».74 Senillosa, un miembro destacado de esta familia, que combinaba sus negocios agrícolas con su interés por el espiritismo, ponderaba, como parte de sus hermanos y primos, las virtudes comerciales y emprendedoras de los norteamericanos y veía en el país del Norte un modelo,75 a tono con las consideraciones apenas reseñadas de Paulino Alfonso. Zeballos, al mando indiscutido de la revista, parece haber captado el rol central de la prensa en el contexto de la guerra, como se puede constatar en las traducciones seleccionadas de entrevistas y artículos de opinión. En un sentido complementario, en la revista se encuentra un artículo que hace específicamente foco en este fenómeno. Se trata de una contribución firmada por Julio Carrié, que devino una especie de corresponsal sui generis del estado de situación en Estados Unidos. Bajo el título «La cuestión Filipinas y la opinión americana»,76 Carrié se ocupó de mostrar una discusión que se estaba dando a ritmo cotidiano en la prensa y entre los hombres de la política sobre el expansionismo norteamericano y la anexión de Filipinas. El artículo reviste interés porque el autor se ocupa de comentar cómo en dos medios de prensa se estaba desplegando el mencionado debate. Es decir, Carrié oficiaba como un lector de la prensa norteamericana que comentaba, a su vez, a lectores argentinos qué estaba sucediendo en los periódicos en el contexto del fervor bélico. Centraba su atención en el New York Times y el New York World. 4 Felipe Senillosa, “Anglosajones y latinos”, Revista de Derecho, Historia y Letras 6 (1900): 227-233. 75 Sobre Felipe Senillosa puede verse: Roy Hora y Leandro Losada, Una Familia de la elite argentina: los Senillosa, 1810-1930 (Buenos Aires: Prometeo Libros, 2015), 127-128. Julio Carrié, “La cuestión Filipinas y la opinión americana”, Revista de Derecho, Historia y Letras 3 (1899): 99-101. 74 Felipe Senillosa, “Anglosajones y latinos”, Revista de Derecho, Historia y Letras 6 (1900): 227-233. 75 Sobre Felipe Senillosa puede verse: Roy Hora y Leandro Losada, Una Familia de la elite argentina: los Senillosa, 1810-1930 (Buenos Aires: Prometeo Libros, 2015), 127-128. 76 Julio Carrié “La cuestión Filipinas y la opinión americana” Revista de Derecho Historia y Letras 3 (1899): 99 101 p , g j y , , y ( ) 75 Sobre Felipe Senillosa puede verse: Roy Hora y Leandro Losada, Una Familia de la elite argentina: los Senillosa, 1810-1930 (Buenos Aires: Prometeo Libros, 2015), 127-128. 76 Julio Carrié, “La cuestión Filipinas y la opinión americana”, Revista de Derecho, Historia y Letras 3 (1899): 99-101. Revista de Derecho, Historia y Letras: imperialismo, expansionismo y Derecho Internacional Subrayaba que en el debate se utilizaba el concepto de «imperialismo» y el vocablo «expansión» para describir fenómenos que se ponderaban con distintos acentos. La sorpresa del corresponsal a la hora de cubrir la discusión candente en la prensa le da una tonalidad fresca, casi coyuntural, que es notablemente diferente a la que en general se encuentra en las páginas de la revista. Apenas un años después, el autor de esta nota tradujo al español Gobierno y administración de los Estados Unidos, de Benjamin PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS Harrison.77 Sumaba así su pluma a la lista corta de los conocedores argentinos de la política y el pensamiento norteamericano. Harrison.77 Sumaba así su pluma a la lista corta de los conocedores argentinos de la política y el pensamiento norteamericano. 77 Benjamin Harrison, Gobierno y administración de los Estados Unidos (Buenos Aires: Editorial M. Biedma, 1900). La traducción de la obra fue realizada por Julio Carrié de la Universidad de Buenos Aires. 78 La Ilustración Sud-Americana, nº 129, 1 de mayo de 1898, 167. 78 La Ilustración Sud-Americana, nº 129, 1 de mayo de 1898, 167. Benjamin Harrison, Gobierno y administración de los Estados Unidos (Buenos Aires: Editorial M. Biedma, 1900 traducción de la obra fue realizada por Julio Carrié de la Universidad de Buenos Aires. Benjamin Harrison, Gobierno y administración de los Estados Unidos (Buenos Aires: Editorial M. Biedma, 1900 traducción de la obra fue realizada por Julio Carrié de la Universidad de Buenos Aires. Consideraciones finales Los redactores de una de las revistas aquí analizadas, La Ilustración Sud- Americana, señalaban durante la guerra: En tales circunstancias, extraordinarias y solemnes, los que escribimos para el público y creemos que la prensa periódica tiene una misión más alta que la de ser simple receptáculo de noticias y telegramas, muchas veces falsos y absurdos, faltaríamos a nuestro deber, si por debilidad, conveniencia o hipocresía esquivásemos el dar nuestra opinión franca y categórica sobre las causas del conflicto, y la parte a quien incumbe su responsabilidad tremenda.78 Esta reflexión es un indicio para analizar cómo los conductores de las publicaciones periódicas estaban discutiendo al calor de la coyuntura qué se debía hacer durante la guerra en diarios y revistas. La centralidad de los periódicos en la circulación de información pasó a ser, en sí misma, un motivo de discusión en este contexto. Las revistas, por su parte, tal como aquí se argumentó, desplegaron distintas modalidades para dar cuenta de la «cuestión palpitante» y ofrecer reflexiones que trascendieran el ritmo de los acontecimientos. Las posibilidades de las que dispusieron fueron varias y las opciones por unas u otras tenían que ver, seguramente, con sus propios formatos. Una revista como La Ilustración Sud-Americana debía exponer cada quince días la información que consideraba pertinente para dar cuenta de la guerra. La multiplicación de recursos que se utilizaron y superpusieron demuestra que en la experiencia se podían combinar algunas estrategias de la prensa periódica con otras de las revistas culturales. La variedad de opciones así lo demuestra: diarios de viajes de años previos, relatos de cronistas azarosos, poemas y odas a España, voces legitimadas por ser de padres fundadores —Alberdi— y voces PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS expertas militares —como la de Jenaro Alas—, y conferencias realizadas en teatros y ateneos, desfilaron en sus páginas junto a collages de fragmentos de periódicos sudamericanos. Quedaba clara la apuesta, como se señalaba en la columna citada del 1 de mayo: todos los interesados en el debate público pasarían a ocuparse de la guerra. La revista La Biblioteca, por su parte, incluso en su número final, dio cuenta, de la mano de Groussac, de que la guerra no era un asunto para obviar. Consideraciones finales LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS a diarios y revistas norteamericanos y europeos, devinieron recursos para intervenir en los debates y polémicas sobre la guerra, pero sin utilizar textos que versaran explícitamente sobre las dinámicas del conflicto. En suma, en estas revistas, diferentes en sus intenciones y formatos, es posible advertir las diversas modulaciones que, desde un sector de la vida cultural argentina, se ensayaron para tratar de cubrir una serie de acontecimientos internacionales del periodo estudiado. Las estrategias utilizadas para pensar «lo internacional» buscaron suplir la falta de información y de plumas especializadas y expertas mediante diferentes tipos de escritos e intervenciones. Esto demuestra, por un lado, un alto grado de creatividad intelectual; por otro, revela las limitaciones con que contaban este tipo de revistas para ofrecer intervenciones propias sobre contextos como el bélico. Y muestra, a su vez, la eficacia de los recursos de reproducción, reiteración, selección de fragmentos, y otro tipo de operaciones de apropiación de contenidos generados en otras latitudes y para otros soportes.79 En este clima, las traducciones realizadas para la Revista de Derecho, Historia y Letras con reconocimiento de los nombres de los traductores marcaron, claramente, una diferencia de paradigma que debe ser explorado de manera más sistemática. Propongo, por último, dos consideraciones para pensar desde las revistas, pero más allá de ellas. La primera tiene que ver con la tendencia sistemática de dejar de lado los conflictos y querellas entre naciones americanas, para pasar a dar espesor a un conflicto de escala mayor. Los redactores de La Ilustración Sud-Americana así lo notaron en una de las columnas aquí citadas. En trabajos anteriores postulé que en torno a 1898 los repertorios de ideas e imágenes que surgieron al calor de la guerra en América Latina pueden organizarse en los siguientes grupos: 1. impresiones sobre la lucha entre yanquismo y latinidad; 2. lecturas favorables sobre España y condenatorias de Estados Unidos basadas en principios del Derecho Internacional; 3. propuestas de nuevos horizontes para América Latina más allá de la tensión Estados Unidos- España como polos de definición identitaria; 4. proyectos renovadores para pensar un futuro compartido de la comunidad hispanoamericana. Estas tendencias asumieron sus propias dinámicas, con intensidades variables, en las revistas analizadas. Consideraciones finales Si hasta el momento el interés principal sobre asuntos internacionales que había tenido espacio en la revista era la cuestión de los límites con Chile, el conflicto de 1898 demostraba que era hora de pensar más allá de la región sudamericana para dar cuenta de un escenario geopolítico amplio y cargado de tensiones. La voz de Groussac, amplificada por la crónica de Darío, había dado la pauta para pensar esas tensiones en términos de conflicto cultural y apostado por dejar claro que ya no había espacios para criticar a España. Había llegado la hora de filiarse con la dadora de la latinidad para América y enfrentarse a Estados Unidos y su avance territorial, pero también cultural. El gesto de Groussac de publicar su propio discurso en el Teatro de la Victoria da cuenta de su perspicacia para notar que su voz había devenido una autoridad para pensar el conflicto en clave culturalista. El impacto de Del Plata al Niágara le había dado la pauta de que era una voz legitimada para pensar en las tensiones del continente y acusar el avance de Calibán. Se definía así un conjunto de sentidos comunes sobre yanquismo y latinidad en el ambiente intelectual porteño, que sería un foco de irradiación de estas ideas al resto de la comunidad letrada hispanoamericana. Humanidades: revista de la Universidad de Montevideo, No 9, Junio 2021, pp. 23-58 Mientras que en el emblemático año 1898 La Biblioteca llegaba a su fin por un conflicto entre su director y autoridades ministeriales argentinas, la Revista de Derecho Historia y Letras, se presentaba en sociedad como una empresa renovadora de la vida intelectual americana. Con Zeballos comandando esta empresa editorial, las intenciones de dar cuenta de las dinámicas internacionales quedaban explicitadas desde la fundación de la misma; no dudaba en desplegar sus saberes y contactos con figuras de la cultura y la diplomacia. Este hecho otorgó acentos particulares a las modalidades adoptadas en la Revista de Derecho, Historia y Letras en los años comprendidos entre 1898 y 1900. La sucesión de traducciones realizadas particularmente para la revista —y firmadas—, la exposición de conocimientos sobre Derecho Internacional, la interpelación a autoridades militares, la selección de nombres resonantes europeos —como Rhodes y Bryce—, y latinoamericanos —como Paulino Alfonso, Felipe Senillosa y Julio Carrié—, las referencias explícitas PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. 79 Algunas consideraciones para problematizar estos temas se encuentran en Hernán Pas, “Prensa y literatura en el siglo XIX: nuevas lecturas alrededor de un “viejo” problema”, Ponencia presentada en VI Congreso Internacional de Letras, 2014. Consideraciones finales Ahora bien, me interesa puntualizar que los llamamientos a la confraternidad entre naciones latinas de América parecían dejar de lado PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS de manera rotunda las experiencias de los independentistas de Cuba, y las situaciones específicas de Filipinas, Guam y Puerto Rico en la contienda para pensar estas tensiones geopolíticas. En este sentido, voces como la de Alberto del Solar, conferencista del Ateneo recuperado en las páginas de La Ilustración Sud-Americana, que apuntaba que las naciones hermanas de Cuba debían atender a sus demandas, eran más bien disonantes en el escenario general. Puede que esta falta de presencia en la agenda general y de las revistas tuviera que ver con la predominancia de los repertorios del latinoamericanismo, el antimperialismo latinoamericano y el anti-yankismo como repertorios de fuerte impacto cultural, pero de escasa repercusión en las políticas internacionales. La última reflexión que propongo se vincula con el tipo de voces intelectuales y las identidades en disputa relevadas en este artículo, al analizar las modalidades de intervención que asumieron las revistas en el contexto de la guerra. En el marco de actos públicos abundaron las liturgias y referencias de carácter nacional. Por ejemplo, en el evento del Teatro de la Victoria se entonó el Himno Nacional Argentino y la Marcha Real Española antes de la conferencia de Sáenz Peña; se escuchó la Marsellesa y un Potpurrí de aires españoles «Maiquez» antes de la conferencia de Groussac; sonó la Marcha Real Italiana y la Jota de «La Dolores» antes de que Tarnassi leyera su oda; y se cerró el acto con la Marcha de Cádiz.80 Los cronistas del evento hacen referencia a la proliferación de banderas dentro del recinto. Sin embargo, los repertorios que se esbozaron al calor del conflicto apuntaban a identidades regionales, atlánticas o bien a las que hoy denominaríamos transnacionales. Por su parte, varias de las figuras claves mencionadas aquí eran difícilmente reconocidas como figuras de una sola nación, ejemplarmente Groussac y Darío, que contaban con un aire de patriotas transnacionales —uso la expresión del título de un libro de Núñez Seixas solamente como imagen—, que los habilitaba a explotar los filones del hispanoamericanismo y el latinoamericanismo más allá de los corsets identitarios nacionales. 80 Se encuentra el “Programa de la Función” en el folleto ya citado de las conferencias. 52 ISSN: 1510-5024 (papel) - 2301-1629 (en línea) Revistas sobre las que versa el artículo La Biblioteca (Buenos Aires). La Ilustración Sudamericana (Buenos Aires). Revista de Derecho, Historia y Letras (Buenos Aires). Otras revistas consultadas Almanaque Sud-Americano (Buenos Aires). Anales de la Sociedad Científica Argentina (Buenos Aires). Caras y Caretas (Buenos Aires). El sol del domingo (Buenos Aires). La España Moderna (Madrid). Revista Brazileira (Río de Janeiro). Revue Hispanique (París). 81 Véase Anne-Marie Thiesse, La creación de las identidades nacionales. Europa: siglos XVIII-XX (Madrid: Éz 2010), 65. Humanidades: revista de la Universidad de Montevideo, No 9, Junio 2021, pp. 23-58 ISSN: 1510-5024 (papel) - 2301-1629 (en línea) 81 Véase Anne-Marie Thiesse, La creación de las identidades nacionales. Europa: siglos XVIII-XX (Madrid: Ézaro, 2010), 65. Consideraciones finales Estanislao Zeballos, por su parte, era una figura diplomática reconocida más allá de Argentina, y sus obras circulaban y se reconocían como aportes a temas de Derecho Internacional y convenios entre naciones. Los redactores de La Ilustración Sud-Americana, cuyos nombres propios no son tan distinguibles en cuanto a su peso intelectual, apostaban a dejar de lado las querellas entre naciones e intentaban conformar una dimensión sudamericana de la vida cultural. En suma, en el contexto Humanidades: revista de la Universidad de Montevideo, No 9, Junio 2021, pp. 23-58 PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. LA BIBLIOTECA, LA ILUSTRACIÓN SUD-AMERICANA Y REVISTA DE DERECHO, HISTORIA Y LETRAS, ENTRE LA «CUESTIÓN PALPITANTE» Y LAS ENCRUCIJADAS IDENTITARIAS de la guerra de 1898, se puede ver en acción a una cantidad de actores que, lejos de estar condicionados por intereses patrióticos y nacionales, parecían dispuestos a pensar en los problemas de orden geopolítico por los canales de lo que Anne-Marie Thiesse ha denominado «cosmopolitismo intelectual», gesto característico de los letrados que establecían relaciones con sus pares de otras latitudes en el marco de los procesos de consolidación de identidades nacionales.81 Revistas sobre las que versa el artículo PAULA BRUNO - REVISTAS DE BUENOS AIRES DURANTE LA GUERRA DE 1898. 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Recommendations for designing genetic test reports to be understood by patients and non-specialists
European journal of human genetics
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Abstract Patients and non-specialist healthcare professionals are increasingly expected to understand and interpret the results of genetic or genomic testing. These results are currently reported using a variety of templates, containing different amounts, levels, and layouts of information. We set out to establish a set of recommendations for communicating genetic test results to non-expert readers. We employed a qualitative-descriptive study design with user-centred design principles, including a mixture of in-person semi-structured interviews and online questionnaires with patients, healthcare professionals and the general public. The resulting recommendations and example template include providing at-a-glance comprehension of what the test results mean for the patient; suggested next steps; and details of further information and support. Separation and inclusion of technical methodological details enhances non-specialists’ understanding, while retaining important information for specialists and the patients’ records. The recommendations address the high-level needs of patients and their non- specialist clinicians when receiving genetic test results. These recommendations provide a solid foundation for the major content and structure of reports, and we recommend further engagement with patients and clinicians to tailor reports to specific types of test and results. European Journal of Human Genetics (2020) 28:885–895 https://doi.org/10.1038/s41431-020-0579-y European Journal of Human Genetics (2020) 28:885–895 https://doi.org/10.1038/s41431-020-0579-y ARTICLE ARTICLE ARTICLE Recommendations for designing genetic test reports to be understood by patients and non-specialists George D. Farmer 1,2 ●Harry Gray 1,3,4 ●Gemma Chandratillake5,6 ●F Lucy Raymond5,7 ● Alexandra L J Freeman 1 George D. Farmer 1,2 ●Harry Gray 1,3,4 ●Gemma Chandratillake5,6 ●F Lucy Raymond5,7 Alexandra L. J. Freeman 1 Received: 13 September 2019 / Revised: 12 December 2019 / Accepted: 22 January 2020 / Published online: 5 February 2020 © The Author(s) 2020. This article is published with open access * George D. Farmer george.farmer@manchester.ac.uk Introduction Genetic and genomic testing is predicted to have a greatly increased role in healthcare [1]. In practice this means that such testing is increasingly likely to be ordered, and the results interpreted, by non-specialist clinicians involved in routine patient care. Under the broader directives of shared decision-making and less paternalistic medicine, patients themselves also have an increasing desire to receive and understand detailed information about their unique genetic make-up and its consequences for their lives. Genetic test reports, therefore, need to be able to deliver the results and their implications clearly and unambiguously to those who have no training in genetics. Supplementary information The online version of this article (https:// doi.org/10.1038/s41431-020-0579-y) contains supplementary material, which is available to authorized users. Supplementary information The online version of this article (https:// doi.org/10.1038/s41431-020-0579-y) contains supplementary material, which is available to authorized users. * George D. Farmer george.farmer@manchester.ac.uk * George D. Farmer george.farmer@manchester.ac.uk 1 Winton Centre for Risk & Evidence Communication, University of Cambridge, Cambridge CB3 0WA, UK 1 Winton Centre for Risk & Evidence Communication, University of Cambridge, Cambridge CB3 0WA, UK 2 Division of Neuroscience & Experimental Psychology, University of Manchester, Manchester M13 9PL, UK 2 Division of Neuroscience & Experimental Psychology, University of Manchester, Manchester M13 9PL, UK 3 Leverhulme Research Centre for Forensic Science, University of Dundee, Dundee DD1 4HN, UK This is no small challenge. Even in the simple case of genetic testing in clinical healthcare there are multiple domains of uncertainty surrounding the result itself (e.g. testing error) and what it actually means (e.g. variant sig- nificance). These challenges are exacerbated as more genomic regions are tested, since a greater quantity of errors will be induced (even for a low testing error-rate) and more variants will be of ‘uncertain significance’ as more clinically uncertain loci of the genome are considered. 4 MRC Biostatistics Unit, University of Cambridge, Cambridge CB2 0SR, UK 5 East Midlands & East of England NHS Genomic Medicine Service, Cambridge CB2 0QQ, UK 6 Institute of Continuing Education, University of Cambridge, Cambridge CB23 8AQ, UK 7 Department of Medical Genetics, Cambridge Institute for Medical Research, Cambridge CB2 0XY, UK 886 G. D. Farmer et al. This point has been recognised for some time now by leading national governing bodies for genetics. Introduction As a result, guidelines for genetic test reporting have been released by the American College of Medical Genetics and Genomics (ACMG) [2], European Society of Human Genetics (ESHG) [3], and Association for Clinical Genetic Science (ACGS) [4]. These guidelines are primarily concerned with reporting the technical details associated with the testing procedure and result interpretation. Importantly, they all vary in their recommendations [5] and interpretation (see O’Daniel et al. [6] for a US example) and are perpetually revised as testing methodologies and the clinical relevance of genetic variations become better understood. This results in the need for extremely clear communication of exactly what was tested and what the result means in every single report so that non-specialists (and specialists alike) can readily interpret reports without requiring contextual knowledge of the reporting landscape at the time of testing and from the location in which the report was generated. The requirement for clear communication (including to non- experts) is stated in the guidelines for all of the organisa- tions listed above but isn’t explicitly elaborated on any further. In fact, the practical guidance for ensuring this clear communication of results is acknowledged to be minimal. also looked at reporting from a patient perspective, identi- fying that with the complex results of whole-genome sequencing, some supportive interpretation information was necessary to communicate potential clinical relevance to their patient. They also appreciated that active clinical guidance was given so that they could confidently convey this to the patient at the time of reading. The challenge for reports is to provide information that is seen as relevant for all parties, while being concise enough to not deter patients. A second issue is the communication of the uncertainties inherent in genetic tests and their results. These can be classified into two kinds: genetic risk (the estimated chance that a person will experience a given consequence) and testing limitations (the chance that estimate is inaccurate). Perceptions of genetic risk information have been shown to be affected by individuals’ preconceptions of testing [12], which can have its roots in familial and cultural back- grounds [8]. While managing testing expectations is the role of the genetic counsellor, there is considerable evidence in the literature concerning risk communication strategies that could be used to build a robust report template. A com- prehensive review on this topic is provided in Lautenbach et al. Introduction [13], which pools together the literature for genetic risk communication with that of general medical risk. It highlights important messages that are echoed throughout the risk communication world: there is no one-size-fits-all approach and so multiple methods of communication should be presented, positive and negative framing of risk should both be given in order to avoid framing bias, and pictographs can be used as effective methods of commu- nicating percentages. Shaer et al. [14] empirically demon- strated that visual presentations of genetic variant information (regarding pathogenicity and clinical impor- tance) to non-experts showed improved comprehension and perceived comprehension when compared with traditional tabular design formats. Adding the uncertainty resulting from limitations to the testing accuracy compounds this complex landscape of risk information but is necessary. Both Dorschner et al. [15] and Kelman et al. [16] have reported clinicians as wanting access to false positive and false negative rates of tests for themselves and patients, although it is uncertain from the literature whether patients would find this kind of information understandable and useful, and it is seldom mentioned in current reports. A hi d h i h d f i i f i ifi i Review of existing knowledge Example reports were collected to illustrate a wide variety of approaches, including some from direct-to- consumer genetic testing. Examples came from existing genetic test reports and guidelines in current usage in the UK, US and Europe to assess the range of different styles of design, wording and length employed. These included ACGS, ACMG, and ESHG guidelines and sample reports [3, 22], as well as suggested reports in the literature from Scheuner et al. [23], Dorschner et al. [15], Williams et al. [11], and report examples from GeneDX, 23andme, Part- ners Healthcare, and Lineagen. One theme with relatively little available evidence in the literature is the graphic design and layout of reports. This is interesting because there is a stark contrast between the visual appearance of reports that have been developed by, for example, direct-to-consumer (DTC) genetic testing companies compared with those suggested in the academic literature; the former tend to make more use of colour and graphical elements to separate sections of their reports when compared with the latter. It is likely that these private companies have conducted testing with their users that shows preference for graphics, but that that data is proprietary. More data needs to be gathered on this topic in the public domain when shifting focus towards patients as recipients, since it may directly increase comprehension [14]. The interviews were divided into two phases. Initially we asked about participants’ experience with genetic reports in their personal and professional capacities. In the second phase we sought participants’ views on the example reports addressing a number of prepared topics including the overall visual impression and appropriateness of the length, the level of the language and ease of understanding, the ‘actionability’ of the information given, and what degree of trust the report engendered. In addition, we sought com- ments from participants on a prototype outline based on our review of the literature, which was iteratively updated based on comments made in interviews. Interviews were conducted by a combination of one or more of three authors (HG, AF & GF) and notes were taken during the interview. Audio was recorded for all interviews. We adopted a descriptive qualitative approach [20] with elements of a thematic analysis. Any comments from the recordings that could form the basis of a recommendation for a laboratory test report were transcribed. These com- ments were then aggregated for all participants and an initial code applied. Review of existing knowledge Efforts have been made to assess the communication of results in reports from a variety of testing contexts for both clinicians and patients, leading to the development of communication guidelines and suggested report formats (e.g. Lubin et al. [7] and Haga et al. [8]). There are a few common themes. First, authors have commented on the relevance of information contained within reports. Joseph et al. [9] noted the discrepancies between the information that genetic counsellors provided to patients versus the information that the patients indicated caring most about. Given that it is experts in genetics who have traditionally decided the content and format of reports, we identified this as likely to be an ongoing issue to be addressed. Stuckey et al. [10] found that parents in their study continually searched for relevant information and resources for their child’s genetic condition, but that current reporting didn’t meet that need and led parents to extensive online searching, with some admitting reticence relating to the information that they found, and at least one saying that she scared herself. Not all patients want more information, though: Joseph et al. [9] described how one patient didn’t want to know about information regarding the test, genetics, or risk since they were factors beyond their control—only the outcome was seen as meaningful. Williams et al. [11] A third theme is the need for citations of scientific evi- dence from which conclusions in the report derive, and available resources for further information. In focus groups, Cutting et al. [17] found clinicians wanted citations to the academic literature that supports the interpretations stated in the report. Johnson et al. [18], taking a more patient-centric approach, found interviewees also wished to see additional information about genomic testing, as well as the actions of others in similar situations. 887 Recommendations for designing genetic test reports to be understood by patients and non-specialists Finally, unsurprisingly, the language used throughout the report has been seen as critical to users’ understanding. Studies have shown that non-experts prefer simpler lan- guage [9, 11], with technical genetic terms creating confu- sion and reluctance to even try to understand the content [10]. Aside from this negative perception of technical lan- guage, it has also been shown that there is a positive per- ception of ‘simpler’ language. Lewis et al. [19] showed that simpler language facilitated understanding and confidence to talk about results. Semi-structured interviews: results We identified thirteen key recommendations, shown in Table 1 (See Supplementary Materials for full list). Four of these were made unanimously in all the interviews (where n = 9). Review of existing knowledge Codes were then summarised into themes. In applying a descriptive qualitative approach we sought to produce a record of suggestions with minimal interpretation. In light of the limitations identified in this review of the literature, and the lack of specific official guidance, we undertook qualitative research with an objective of pro- viding recommendations for making the content and struc- ture of a genetic test report more accessible to patients and non-specialist clinicians. To achieve this we undertook a qualitative-descriptive study [20] aimed at eliciting the requirements that our target audiences had. In particular we adopted the user-centred design principle of involving end- users in the design process and iterating based on their feedback [21]. We started with semi-structured interviews to elicit major themes, and followed this up with online testing in a larger sample to verify our findings and elicit further recommendations. Semi-structured interviews: methods Use plain language Structure & appearance Use colour to make things clear and easy to read 6 Colours help with understanding and appearance of document Structure & appearance Keep reports as short and simple as possible 6 Avoid dense blocks of text and lengthy reports as much as possible Structure & appearance Don’t dilute the main message 5 Don’t intersperse key messages with genetics explainers or technical details Comm. style Provide patients with all information 5 Patients should receive all of the information resulting from the test including technical details Structure & appearance Present result in neutral terms 5 Don’t use ‘positive’ or ‘negative’, or colour-code results. Aim rather for a statement of fact. The recommendations in Table 1 fall broadly into three groupings. The first of these is communication style which captures a desire for plain language, the use of lay terms and the avoidance of jargon where possible. Interviewees also expressed a desire for a slightly more personal tone, for example, using ‘your’ rather than ‘the proband’s’. The content grouping addresses the key content that patients and non-specialist clinicians seek in a report. The three main sections are: ‘What the result means’, ‘What actions should be taken’, and ‘where can further infor- mation and support be found’. The first section addresses the interpretation of the test result, including its implica- tions for diagnosis and prognosis. The second section concerns actions to be taken, such as genetic counselling, whether testing of relatives is recommended, and acces- sing treatment. The final section concerns the provision of sources of further information and support. These include links to information about any diagnosed conditions, information about genetic counselling, information about communicating results to family and employers, and information on support that might be available, in parti- cular peer support from other people similarly affected. Providing trusted links to further information helps guide patients towards trustworthy online information; patients are otherwise likely to search for online guidance them- selves, which can lead to distressing and misleading information. The structure and appearance grouping relates to the design of the document. This is important not because it is aesthetically pleasing but because it helps readers under- stand the content of the document. Clear section headings convey a useful hierarchy of information, documents that flow simply from top to bottom without large dense blocks of text avoid overwhelming readers. Semi-structured interviews: methods This work was approved by the University of Cambridge Psychology Research Ethics Committee. In August 2017, semi-structured interviews lasting ~90 min were conducted with a convenience sample (n = 9, 7 female, mean age 41, SD = 12). Participants had varying relationships to genetic testing. Four had experience as a patient, and five as health professionals. Two participants additionally had experience as patient advocates. Participants were interviewed about their experience with genetic testing results and then asked to comment on existing examples of genetic reports. The first unanimous recommendation concerns the fact that existing genetic test reports are very difficult, if not impossible, for patients and non-specialist clinicians to understand. The main action required is the use of plain language and the avoidance of jargon. The second concerns the appearance and structure of the document itself. The clarity and flow of section headings can greatly enhance the ease of reading and comprehension. Recommendation three was to make the result of the test prominent in the report. This means that the result of the test should stand out, and G. D. Farmer et al. 888 Table 1 A summary of recommendations from the semi-structured interviews that were made independently by a majority of interviewees (n ≥5). A full table of all recommendations is in the Supplementary Materials. Grouping Recommendation n Detail Comm. style Make reports easier for non-specialists to understand 9 Use layman’s terms, avoid jargon, most reports are incomprehensible even to (non-specialist) medical professionals Structure & appearance Consider the structure and appearance of the document 9 The structure and appearance of the document affect understanding, and ease of reading Structure & appearance Make the result prominent 9 The result of the test should stand out and be easily found within the document Structure & appearance Keep technical test details separate 9 Put technical details such as test methodology into a separate section Content Provide an ‘actions to be taken’ section 8 Include a section of recommendations and concrete next steps Content Provide sources of further information and support 8 Provide sources of authoritative information, especially on the condition, communicating the result to others and obtaining support including genetic counselling and peer support Content Provide a ‘what this result means’ section 7 Explain what the implications of the result are (diagnosis, risks, treatment, family) Content Ensure the result wording is unambiguous 6 Make the result as unambiguous as possible. Semi-structured interviews: methods Use plain language Structure & appearance Use colour to make things clear and easy to read 6 Colours help with understanding and appearance of document Structure & appearance Keep reports as short and simple as possible 6 Avoid dense blocks of text and lengthy reports as much as possible Structure & appearance Don’t dilute the main message 5 Don’t intersperse key messages with genetics explainers or technical details Comm. style Provide patients with all information 5 Patients should receive all of the information resulting from the test including technical details Structure & appearance Present result in neutral terms 5 Don’t use ‘positive’ or ‘negative’, or colour-code results. Aim rather for a statement of fact. preferably be the first thing the eye is drawn to. The final unanimous recommendation was to separate the technical methodological details of the genetic test conducted from the main message to be communicated to non-specialist audiences. Table 1 A summary of recommendations from the semi-structured interviews that were made independently by a majority of interviewees (n ≥5). A full table of all recommendations is in the Supplementary Materials. Grouping Recommendation n Detail Comm. style Make reports easier for non-specialists to understand 9 Use layman’s terms, avoid jargon, most reports are incomprehensible even to (non-specialist) medical professionals Structure & appearance Consider the structure and appearance of the document 9 The structure and appearance of the document affect understanding, and ease of reading Structure & appearance Make the result prominent 9 The result of the test should stand out and be easily found within the document Structure & appearance Keep technical test details separate 9 Put technical details such as test methodology into a separate section Content Provide an ‘actions to be taken’ section 8 Include a section of recommendations and concrete next steps Content Provide sources of further information and support 8 Provide sources of authoritative information, especially on the condition, communicating the result to others and obtaining support including genetic counselling and peer support Content Provide a ‘what this result means’ section 7 Explain what the implications of the result are (diagnosis, risks, treatment, family) Content Ensure the result wording is unambiguous 6 Make the result as unambiguous as possible. Online survey: methods participants rated the reports favourably. This was true across all of the features we probed, and was broadly similar for patients, clinicians, and the general public. Participants’ Likert ratings of the graphic prototype are shown in Fig. 1. The same pattern in ratings was observed for the text pro- totype (see Supplementary Materials). We generated two prototype designs (see Supplementary Materials) incorporating the unanimous recommendations from the interviews, but differing in the tone and the amount of text they contained, and the presence or absence of a graphic to convey risk. The difference between the reports was designed to elicit comments on the extent that graphic design features were desirable and acceptable. The reports contained a pathogenic result for a fictitious condition ‘Brendt Syndrome’ causing an increased risk in bowel cancer. Page 1 of the report addressed the issues raised in the interviews, and page 2 contained technical and metho- dological details of the test. yp pp y Table 2 lists 26 themes summarising a total of 478 initially coded comments. There were four broad groupings of the themes. For the content themes, respondents mainly wanted more information and detail on the topics that were already present in the report. In particular, they wanted more information on actionable next steps they could take. Respondents also wanted more information about the syn- drome itself. Respondents were in favour of using graphics to explain and contextualise risk. Finally, respondents were sensitive to ambiguity in the phrasing of the result, leading to comments like ‘do I have it or not?’ and questions around whether retesting would be needed. With these prototype templates in place we sought fur- ther feedback by asking participants to complete an online survey rating different aspects of the reports. We recruited 28 patients (26 female, mean age 41, SD = 8), 29 non- specialist clinicians (25 female, mean age 43, SD = 12) and 49 members of the general public (35 female, mean age 33, SD = 10) to view our prototype reports and answer ques- tions about them online. The patient group was recruited by advertising via support groups and advocacy organisations. Eighty-nine percent of these participants reported that they or a family member had received a genetic test report. The general public were recruited from prolific.ac, an online participant pool. Twenty percent of these participants reported having received the results of a genetic test. Online survey: methods In the clinician group, 86% reported some professional experience of receiving genetic test reports. On the design group of themes, the overwhelming response was that people appreciated a document that was simple to look at and navigate. This means a document that flows from top to bottom without columns or text boxes. It also means clearly delineating sections and avoiding dense blocks of text. The use of colour to differentiate sections of the document was appreciated, but less so in the commu- nication of the result. Respondents from all three groups identified the grey box containing the result in one of the reports as ‘foreboding’ and ‘sinister’. The grouping of communication style themes reflected a desire for lay terms and avoiding jargon. Many respondents identified that the first page was easy to understand and the second (technical page) was very difficult to understand. Many respondents also identified that despite this they would still like the technical details included in the report. Of the two reports people preferred the tone of the ‘text’ version as it was felt to be more personal and less ‘brusque’. Participants rated the following statements on a seven- point Likert scale: ‘I understand the results’; ‘I understand the actions [the patient/I] could take’; ‘I understand how the risk of developing cancer has changed’; ‘I would trust the results are correct’; ‘The language used is appropriate’; ‘The appearance of the report (colours, design) feels appropriate’; ‘I would want to see the technical information on Page 2’. Free text fields in the survey also allowed participants to comment on the forms under the following categories: Questions you would still have; Trust in the result; Appearance and structure of the report; Ease of finding information; and Ease of comprehension. As with the interviews, these data were analysed using a qualitative description approach [20] with an abbreviated thematic analysis. We first coded each comment and then classified these into themes. Participants rated the following statements on a seven- point Likert scale: ‘I understand the results’; ‘I understand the actions [the patient/I] could take’; ‘I understand how the risk of developing cancer has changed’; ‘I would trust the results are correct’; ‘The language used is appropriate’; ‘The appearance of the report (colours, design) feels appropriate’; ‘I would want to see the technical information on Page 2’. Semi-structured interviews: methods These issues are par- ticularly important given the sometimes immensely emotive and stressful nature of obtaining and making sense of a genetic test result. The position of technical information concerning var- iants and test methodology is important to consider. It was clear that this information should be contained in a patient-facing report, but that it should be clearly sepa- rated in order to allow patients and non-specialists to extract the key content they are looking for quickly and unambiguously. Many patients also reiterated that even a well-designed report should certainly not take the place of a face-to-face meeting in which the results are communicated by a healthcare professional. Recommendations for designing genetic test reports to be understood by patients and non-specialists 889 Online survey: methods On the final grouping of trust, respondents commented on the professional appearance of the document and the fact that it was sourced by the UK’s National Health Service (NHS). These comments reflect the fact that appearing authoritative inspires trust. By contrast, some participants determined their trust in the results by assessing the statis- tical claims in the document. For example, respondents identified the test sensitivity (99%) as a reason to trust the result. Others found reason not to trust the result because the technical section stated the variant had not previously been found in that laboratory. Free text fields in the survey also allowed participants to comment on the forms under the following categories: Questions you would still have; Trust in the result; Appearance and structure of the report; Ease of finding information; and Ease of comprehension. As with the interviews, these data were analysed using a qualitative description approach [20] with an abbreviated thematic analysis. We first coded each comment and then classified these into themes. Overall recommendations Ratings for the prototype with a risk depiction and more graphic design features, were broadly similar to those for the prototype with more text. For both, the large majority of Table 3 shows a summary of recommendations for the high- level content and structure of genetic reports suitable for 890 G. D. Farmer et al. Clinicians Patients Public Understand Actions Risk Trust Language Appearance Technical −3 −2 −1 0 +1 +2 +3 −3 −2 −1 0 +1 +2 +3 −3 −2 −1 0 +1 +2 +3 0.0 0.2 0.4 0.6 0.0 0.2 0.4 0.6 0.0 0.2 0.4 0.6 0.0 0.2 0.4 0.6 0.0 0.2 0.4 0.6 0.0 0.2 0.4 0.6 0.0 0.2 0.4 0.6 Answer Proportion Fig. 1 Participants’ ratings of the ‘graphic’ prototype. The x axis spans −3 (Completely disagree) to +3 (Completely agree) via 0 (Neither agree nor disagree). Ratings for the other prototype follow the same pattern and are included in the Supplementary Materials. being piloted by the Patient Information Forum, and which aims to help patients identify information they can trust. being piloted by the Patient Information Forum, and which aims to help patients identify information they can trust. These recommendations are in principle compliant with ISO15189 [24]. However, report providers should check that any final report they develop is compliant with the appropriate standards and regulations. being piloted by the Patient Information Forum, and which aims to help patients identify information they can trust. patients and non-specialists that came out of our work. We emphasise that such reports are not designed to replace a face-to-face consultation between a patient and a healthcare professional. These reports may improve the experience of consultations and should be given to a patient as a take- home, not sent before a consultation. These recommendations are in principle compliant with ISO15189 [24]. However, report providers should check that any final report they develop is compliant with the appropriate standards and regulations. These recommendations are in principle compliant with ISO15189 [24]. However, report providers should check that any final report they develop is compliant with the appropriate standards and regulations. Figure 2 shows an example report applying some of the recommendations. Much of the more detailed information that participants wanted from reports pertains to condition-, test- or result-specific scenarios. Overall recommendations We therefore, as an over- arching recommendation, suggest that the following should be seen as a solid foundation upon which further con- sultation with stakeholders can help identify the additional detail that would be necessary (e.g. identifying appropriate peer support groups, or testing how results should be wor- ded for very different types of condition such as autism, Huntington’s or raised cancer risks). When providing information about support groups, it will be important that these have passed some form of quality check. One potential standard in the UK is Quality Mark currently Discussion There is clearly a need for an empirically-tested template for genetic or genomic results that communicates equally well to specialist and non-specialist clinicians and patients. A review of the literature and existing reports suggested that none currently existed, and that there were clear gaps in our knowledge of what information, language and graphical design was required to construct this. We have developed a set of recommendations and an example template that contains the major elements most important to the patients, clinicians and specialists who Recommendations for designing genetic test reports to be understood by patients and non-specialists 891 Table 2 Summary of themes derived from online comments and the number of endorsements from each participant group. Group Theme Clinicians (n = 29) Patients (n = 28) Public (n = 49) Content Would want more information about topics addressed in report (family implications, screening, treatment, next steps) 20 17 39 Content The use of simple diagrams and figures is helpful. Avoid unfamiliar designs 11 13 15 Content Want more detailed information about syndrome/condition (e.g. prognosis, prevalence) 5 6 9 Content Helpful to include separated technical section 5 5 4 Content Ambiguous wording of result unhelpful 4 0 2 Content Would like more information about the test statistics (sensitivity, specificity etc.) 4 1 1 Content Technical section may cause fear or confusion 3 1 0 Content Include glossary to help with technical terms 0 2 0 Content Unhelpful to include technical section 2 0 0 Content Inclusion of patient details useful 0 1 0 Design Clearly labelled sections, white space, avoiding columns, and avoiding dense blocks of text, all help with comprehension 24 22 45 Design The appropriate use of colour helps delineate sections, but avoid it in communicating test result 12 12 26 Design Prominence of result helpful 2 4 2 Design Prominence of result alarming/stark 2 0 3 Design Use large enough font 1 0 1 Comm. Style Be concise and clear, with a personal tone, but avoid brevity at the expense of important detail. 14 14 10 Comm. Style Patient section easy to understand, technical section is difficult 11 6 7 Comm. Style Technical details difficult to understand 6 5 12 Comm. Style Use lay language and avoid jargon 4 6 4 Comm. Discussion Style Make clear who the audience is for the technical section 2 0 0 Trust Trust of result based on technical aspects of test 7 4 5 Trust Trust result because sourced by NHS 4 0 12 Trust Trust of result based on appearance of document 6 0 4 Trust Trust if confirmed by another test 1 1 2 Trust Trust result because signed 1 0 2 Trust Trust based on comprehension of report 0 0 2 om online comments and the number of endorsements from each participant group. implementing recommended practice (raised in Lautenbach et al. [13]), and our report uses favourable graphical design while remaining appropriate (highlighted in Shaer et al. [14], and apparent in direct-to-consumer reports). Finally, we included necessary technical information and limitations (as raised in Dorschner et al. [15] and Kelman et al. [16]). receive reports. There was generally broad consensus within each group as to what was important to them, and we believe the recommendations can be implemented without compromising the information required by each audience. It should be noted that there was a bias toward female volunteers in our sample, and although males did take part at all stages, any future work implementing these recom- mendations would benefit from a more equal balance. This work, however, concentrated on the design of the report template rather than the specific wording that it would carry. There are many different types of genetic or genomic test result—from diagnostic testing for patients with a condition for which they are seeking a genetic explanation, to incidental findings from a genomic test with no relevant family history, and even polygenic risk scores. This study also does not address whether or how to com- municate more complex issues such as finding variants of Our recommendations echo many findings from the existing literature. In particular, we have created a ‘patient friendly report’ [8] which has shown with appropriate lan- guage that non-specialists perceive that they understand results and know which actions they can take, addressing concerns in Stuckey et al. [10], Williams et al. [11], and Joseph et al. [9]. Risk was effectively communicated by G. D. Farmer et al. 892 Table 3 The main recommendations on the design of genetic reports that came out of this study. Discussion Recommendation Detail Use lay language wherever possible Avoid technical terms and words that can be interpreted differently by people with different backgrounds or expectations (such as ‘positive’ and ‘negative’). Don’t let brevity lead to ambiguity (e.g. ‘consistent with’) – test your wording with a lay audience to see what they would understand by it. Employ simple design considerations such as white space, colour and clearly labelled sections Good design can enhance trust, ease of comprehension, and lead to reduction in stress. Single column text was preferred for ease of reading. Avoid embellishments, except important logos to show the provenance of results. Use the layout: Result > What it means > Actions > More support > Technical info. The order of information in the report is important, and this layout was universally liked. Providing details of where to find more information and support from others is important to help steer patients through the online world, and to give them social support. Support groups should be accredited if included in a report. The appropriate use of graphics is helpful Graphics can help people understand numbers, or put risks into context. Unambiguous result Make the wording of the result as unambiguous as possible, or if the result is inherently ambiguous, explain the source of ambiguity. Use a neutral and factual presentation of the result Don’t use colour-coding or language to indicate whether a result is ‘good news’ or ‘bad news’ – that interpretation could be different for different people. Use a personal tone in communication style Address the report to the patient, using the second person (‘your…’) not the third person (‘the patient’s…’). Clinicians don’t mind reading this style, and it makes it better for the patient. Separate, but include, the technical methodological details of the test Patients want to know the technical details at least as much as clinicians, and including them makes the report useful for the patient’s records - particularly if they move to a new healthcare provider – and for treatment plans. But clearly label sections that are not necessary for the patient to understand so that they don’t worry that they don’t. Communicate absolute pre- and post- risks with population for comparison. Include both framings. If a test increases or decreases the chance of something, it is important to put those risk changes in absolute terms and in the context of the general population. Recommendations for designing genetic test reports to be understood by patients and non-specialists Recommendations for designing genetic test reports to be understood by patients and non-specialists 893 Design, colour, clear labelling and single column text make form easy to read Patient's and sample details on the top of each page Neutral, factual and unambiguous wording of result Clear non-technical language, addressed to the patient Graphics to help put numbers into context. Risks given with appropriate comparison and both positive & negative framing Full technical details provided, clearly marked as not necessary for the patient to read Sections on first page presented in order: Result – What it Means – Actions – Support Fig. 2 An illustration of the recommendations in action. N.B. The NHS logo does not imply endorsement or funding of this work by the National Health Service. Fig. 2 An illustration of the recommendations in action. N.B. The NHS logo does not imply endorsement or funding of this work by the National Health Service. Fig. 2 An illustration of the recommendations in action. N.B. The NHS logo does not imply endorsement or funding of this work by the National Health Service. provides a methodology for tailoring the framework to more specific testing scenarios. unknown significance or when re-analysis of a sample leads to new findings. Each of these types of testing scenario will demand quite different wordings and explanations. For example, some will be attempting to convey a possible increased risk of an event with several layers of uncertainty (purely aleatoric uncertainty which can be portrayed as a probability; epistemic uncertainty about the strength and quality of the evidence surrounding the influence of the gene variant on future risk; uncertainty related to the sen- sitivity and specificity of the test etc.), each needing quite a different approach. This would be different from, say, a carrier testing result where the gene variant has very high penetrance and is well characterised so the communication is mainly about the chances of inheritance in future children. The process of iteratively engaging with recipients of reports to produce this template and recommendations, is in itself analogous to shared decision-making, a concept becoming increasingly familiar in medicine. It involves a dialogue with patients to find out what is important to them and come to a mutually-agreed solution that serves their needs for clear and accurate information. Discussion What is the chance of someone in the general population compared with someone with this known genetic make-up? Frame it both positively and negatively (how many people will this happen to, and how many won’t it happen to, out of 100?) Don’t include information that is unnecessary to understanding the key message Although it’s tempting to attempt explain inheritance or genetics to give background to the information being provided, that is unnecessary for people to understand the result. Leave that to face-to-face genetic counselling and additional patient information leaflets. Include the patient’s details and the context of the test on each page Ensure the patient’s details, indication for testing, circulation and contacts of the laboratory are at the top of every page to allow instant checking that this is the ‘right report’ and who a patient can contact. The indication for testing will also help give context for interpretation for both patient and clinician. Recommendations for designing genetic test reports to be understood by patients and non-specialists Providing patients, and their non-specialist healthcare providers, with accurate and unambiguous test results arms them with the information that they need to be able to take part in shared decision-making. Where the results do not need any further action, these forms should be able to give clear reassurance without the need for input from genetic specialists. When the results have implications that need full discussion and decision-making, they can help ensure that the patient is referred correctly to a suitably qualified pro- fessional and give them the basic information they need to take part in shared decision-making and informed consent during that consultation. To ascertain how best to illustrate and explain results in the many complex reporting scenarios beyond the scope of this research, it will be critical that the content and formats of reports are carefully co-designed with their target audiences. Following the design of this generic template, we continued with development of wording and further design refinements, starting with templates for carrier testing, and when formally assessed against report forms in current clinical use proved clearer and more actionable for patients (see Recchia et al.) [25]. This article provides a framework for effective communication of genetic test results. In addition, Recchia et al. [25] In genetics, the unit of care is often the family rather than just the individual, it is really important therefore that genetic information be conveyed to other family members accurately and having the patient understand the information makes this much more likely to happen. Indeed a number of interviewees identified that facilitating G. D. Farmer et al. 894 communication with others would be a particularly desir- able benefit of a well-designed report. Although it is worth noting that patients do not always wish to communicate results to family members raising difficult privacy and consent issues (see section 4 in a recent report by the Joint Committee on Genomics in Medicine [26] for a discussion). 3. Matthijs G, Souche E, Alders M, Corveleyn A, Eck S, Feenstra I, et al. Guidelines for diagnostic next-generation sequencing. Eur J Hum Genet. 2016;24:2–5. 4. Smith K, Martindale J, Wallis Y, Bown N, Leo N, Creswell L, et al. General genetic laboratory reporting recommendations. Birmingham: Association for Clinical Genetic Science: 2015. 5. Tack V, Dufraing K, Deans ZC, Van Krieken HJ, Dequeker EMC. The ins and outs of molecular pathology reporting. Virchows Arch 2017;471:199–207. Compliance with ethical standards 12. Kaufman DJ, Bollinger JM, Dvoskin RL, Scott JA. Risky busi- ness: risk perception and the use of medical services among customers of DTC personal genetic testing. J Genet Couns. 2012;21:413–22. Conflict of interest None of the authors have conflicts of interest to report. GC owns shares in companies related to genetics (Personalis, Petagene), as does her husband (Sophia Genetics). 13. Lautenbach DM, Christensen KD, Sparks JA, Green RC. Com- municating genetic risk information for common disorders in the era of genomic medicine. Annu Rev Genomics Hum Genet. 2013;14:491–513. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 14. Shaer O, Nov O, Okerlund J, Balestra M, Stowell E, Ascher L, et al. Informing the design of direct-to-consumer interactive per- sonal genomics reports. J Med Internet Res. 2015;17:e146. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. 15. Dorschner MO, Amendola LM, Shirts BH, Kiedrowski L, Salama J, Gordon AS, et al. Refining the structure and content of clinical genomic reports. Am J Med Genet C Semin Med Genet. 2014;0:85–92. 16. Kelman A, Robinson CO, Cochin E, Ahluwalia NJ, Braverman J, Chiauzzi E, et al. Communicating laboratory test results for rheumatoid factor: What do patients and physicians want? Patient Preference Adherence. 2016;10:2501–17. 17. Cutting E, Banchero M, Beitelshees AL, Cimino JJ, Del FiolG, Gurses AP, et al. User-centered design of multi-gene sequencing panel reports for clinicians. J Biomed Inform. 2016;63:1–10. 18. Johnson KJ, Schahl KA, Sinicrope PS, Mcallister TM, Mccormick JB, Ruckman LE, et al. The “ Genomic Novel “ and “ Priority Mapping Tool “: using empathic design to develop innovative patient-centered decision-making tools for the genomic testing experience. Recommendations for designing genetic test reports to be understood by patients and non-specialists By working across multiple audiences, and hand-in- hand with those who need to implement any reports in actual clinical practice, we have sought to develop a practical and useful product which is flexible enough to be able to carry test results of many kinds, and will lead to better-informed decision-making. We also hope to have demonstrated more generally that involving the target audience in the design of a communication is an efficient and valuable way to ensure effective communication of important medical information. 6. O’Daniel JM, McLaughlin HM, Amendola LM, Bale SJ, Berg JS, Bick D, et al. A survey of current practices for genomic sequen- cing test interpretation and reporting processes in US laboratories. Genet Med. 2017;19:575–82. 7. Lubin IM, McGovern MM, Gibson Z, Gross SJ, Lyon E, Pagon RA, et al. Clinician perspectives about molecular genetic testing for heritable conditions and development of a clinician-friendly laboratory report. J Mol Diagnostics. 2009;11:162–71. 8. Haga SB, Mills R, Pollak KI, Rehder C, Buchanan AH, Lipkus IM, et al. Developing patient-friendly genetic and genomic test reports: formats to promote patient engagement and under- standing. Genome Med. 2014;6:1–11. Acknowledgements This work was carried out using the core funding of the Winton Centre for Risk & Evidence Communication at the University of Cambridge which is provided by a philanthropic dona- tion from the David & Claudia Harding Foundation. We thank Zsófia Szlamka for help with participant recruitment. Graphic design of the report template was by Alison Norden. We would also like to thank all the participants who kindly took part, and the organisations that helped advertise the study. GF was also supported by the Wellcome Institu- tional Strategic Support Fund award [204796/Z/16/Z]. HG was sup- ported by Wellcome Trust PhD Studentship [105362/Z/14/Z]. 9. Joseph G, Pasick RJ, Schillinger D, Luce J, Guerra C, Ka J, et al. Information mismatch: cancer risk counseling with diverse underserved patients. J Genet Couns. 2017;26:1090–104. 10. Stuckey H, Williams JL, Fan AL, Rahm AK, Green J, Feldman L, et al. Enhancing genomic laboratory reports from the patients’ view: a qualitative analysis. Am J Med Genet, Part A. 2015;167:2238–4223. 11. Williams JL, Rahm AK, Stuckey H, Green J, Feldman L, Zallen DT, et al. Enhancing genomic laboratory reports: a qualitative analysis of provider review. Am J Med Genet, Part A. 2016;170:1134–41. Compliance with ethical standards J Genet Disor Genet Rep. 2015;5:1–7. 21. Norman DA, Draper SW. User centered system design: new perspectives on human-computer interaction. Hillsdale, NJ: Erl- baum; 1986. 20. Sandelowski M. Whatever happened to qualitative description? Res Nurs Health. 2000;23:334–40. Recommendations for designing genetic test reports to be understood by patients and non-specialists 26. Royal College of Physicians, Royal College of Pathologists, British Society for Genetic Medicine. Consent and con- fidentiality in genomic medicine: guidance on the use of genetic and genomic information in the clinic. 3rd ed. London: Royal College of Physicians, Royal College of Pathologists, British Society for Genetic Medicine; 2019. (Report of the Joint Committee on Genomics in Medicine). 22. Rehm HL, Bale SJ, Bayrak-Toydemir P, Berg JS, Brown KK, Deignan JL, et al. ACMG clinical laboratory standards for next- generation sequencing. Genet Med. 2013;15:733–47. 23. Scheuner MT, Orlando Edelen M, Hilborne LH, Lubin IM, the members of the RAND Molecular Genetic Test Report Advisory Board. Effective communication of molecular genetic test results to primary care providers. Genet Med. 2013;15:444–9. 20. Sandelowski M. Whatever happened to qualitative description? Res Nurs Health. 2000;23:334–40. 21. Norman DA, Draper SW. User centered system design: new perspectives on human-computer interaction. Hillsdale, NJ: Erl- baum; 1986. 20. Sandelowski M. Whatever happened to qualitative description? Res Nurs Health. 2000;23:334–40. 21. Norman DA, Draper SW. User centered system design: new perspectives on human-computer interaction. Hillsdale, NJ: Erl- baum; 1986. 22. Rehm HL, Bale SJ, Bayrak-Toydemir P, Berg JS, Brown KK, Deignan JL, et al. ACMG clinical laboratory standards for next- generation sequencing. Genet Med. 2013;15:733–47. 23. Scheuner MT, Orlando Edelen M, Hilborne LH, Lubin IM, the members of the RAND Molecular Genetic Test Report Advisory Board. Effective communication of molecular genetic test results to primary care providers. Genet Med. 2013;15:444–9. References 1. Davies SC. Annual report of the Chief Medical Officer 2016, generation genome. London: Department of Health: 2017. 2. Richards S, Aziz N, Bick D, Das S, Gastier-Foster J, Bick D, et al. Standards and guidelines for the interpretation of sequence var- iants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17:405–23. 19. Lewis KL, Hooker GW, Connors PD, Hyams TC, Wright MF, Caldwell S, et al. Participant use and communication of findings from exome sequencing: a mixed methods study: use and com- munication of findings from exome sequencing HHS Public Access. Genet Med. 2016;18:577–83. Recommendations for designing genetic test reports to be understood by patients and non-specialists 895 24. British Standards Institution. ISO 15189:2012. Medical laboratories - requirements for quality and competence. London: BSI; 2012. 25. Recchia G, Chiappi A, Chandratillake G, Raymond L, Freeman A. Creating genetic reports that are understood by non-specialists: a case study. Genetics Med. 2019. https://doi.org/10.1038/s41436- 019-0663-2. 22. Rehm HL, Bale SJ, Bayrak-Toydemir P, Berg JS, Brown KK, Deignan JL, et al. ACMG clinical laboratory standards for next- generation sequencing. Genet Med. 2013;15:733–47. 26. Royal College of Physicians, Royal College of Pathologists, British Society for Genetic Medicine. Consent and con- fidentiality in genomic medicine: guidance on the use of genetic and genomic information in the clinic. 3rd ed. London: Royal College of Physicians, Royal College of Pathologists, British Society for Genetic Medicine; 2019. (Report of the Joint Committee on Genomics in Medicine). 23. Scheuner MT, Orlando Edelen M, Hilborne LH, Lubin IM, the members of the RAND Molecular Genetic Test Report Advisory Board. Effective communication of molecular genetic test results to primary care providers. Genet Med. 2013;15:444–9.
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A Signal to Noise Optimization Algorithm for Speckle Interferometry Applications
Applied mechanics and materials
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Online: 2008-07-11 Online: 2008-07-11 Applied Mechanics and Materials ISSN: 1662-7482, Vols. 13-14, pp 29-38 doi:10.4028/www.scientific.net/AMM.13-14.29 © 2008 The Author(s). Published by Trans Tech Publications Ltd, Switzerland. Applied Mechanics and Materials Keywords: speckle – interferometry – shearography – correlation – signal to noise ratio Abstract. Optical Full Field Techniques (OFFT) are more and more utilized by mechanical laboratories. Among these methods, interferometry techniques (mainly composed of Speckle/Grating Interferometry or Speckle/Grating Shearography) are more difficult to use in a mechanical lab context, because of their sensitivity to external vibrations (except shearography), and because of the global lack of optical culture of mechanical engineers. Speckle-based methods are of great practical interest for the users, but their signal to noise ratio (SNR) is affected by the rigid body motion of the specimen. Here, the speckle decorrelation is minimized at local scale directly using the SNR. First, a shearography experiment is modeled to characterize the recorrelation procedure for a rigid body motion, a constant strain map and finally a high degree of localization. The mean noise level is found to be 6 times higher than a fully-correlated phase map for a 1 pixel speckle size. Last, a first application to a single-ply fabric composite lamina is shown. Resulting strain maps are of high quality with a very low spatial resolution (4 pixels). The local bending / global tension coupling effect is clearly put in evidence. This article is an open access article under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0) Introduction Optical Full Field Techniques (OFFT) are more and more utilized by mechanical laboratories. Among these methods, the kinematics measurements are of great interest. They can be classified in two main groups: geometric methods and interferometric methods. The former is composed of Grid Method (or Moiré geometry) and Digital Image Correlation among others; the latter of grating and speckle interferometry or grating and speckle shearography. The first group is easier to develop and use, but its sensitivity is poor. In so far, it is particularly useful to large displacements applications (metal forming [1]) or to damage monitoring (crack propagation [2]). The second group is more difficult to use in a mechanical lab context, because of its sensitivity to external vibrations (except shearography), and because of the global lack of optical culture of mechanical engineers and scientists. Among these methods, because they need few surface preparing (at worst, a white diffusive paint), speckle-based methods are of great practical interest for the users. But, their signal to noise ratio (SNR) is poor compared to grating interferometry or shearography. This signal to noise ratio has been investigated by many authors and a theoretical as well a practical approach shows that the SNR is mainly function of the speckle correlation [3]. If the speckles are fully correlated, the noise is minimum. Then, the noise level can be addressed in this ideal case [4]. But, when the speckles are decorrelated, the noise level is maximum, i.e. the signal has completely vanished. The main contribution to the speckle decorrelation is the deformation outside the field of view, in other words the rigid body motion. A first strategy consists in applying Advances in Experimental Mechanics VI 30 small-enough load step in order to keep the correlation from one step to the other [5]. In many cases, a simple global translation of the phase maps before subtraction is enough to recover the signal [6]. But if the speckle size is small, as recommended by many authors, and if the displacement field is disturbed, a simple global translation is not enough and a local translation has to be defined and used. small-enough load step in order to keep the correlation from one step to the other [5]. In many cases, a simple global translation of the phase maps before subtraction is enough to recover the signal [6]. Introduction But if the speckle size is small, as recommended by many authors, and if the displacement field is disturbed, a simple global translation is not enough and a local translation has to be defined and used. A first idea consists in combining the speckle interferometry and the so-called “speckle photography” technique [7]. This latter is an image correlation technique. The latest development of this approach was presented by Svanbro [8]. The correlation coefficient was written using complex intensity maps, i.e. including the phase maps. However, the method seems to present some drawbacks in case of high fringe densities. Moreover it requires the acquisition of an additional intensity map, which increases the complexity of the procedure For a simple out-of-plane interferometry set-up, this should be negligible, but a complete shearography in-plane and out-of- plane procedure [9] already needs 24 images at most for a 3 arms set-up, with a classical 4-steps temporal phase extraction, and 56 images for a 4 arms set-up, with the Windowed Fourier Transform algorithm proposed by Surrel [10]. Obviously, the global trend is to simplify this apparatus by adding some automated capabilities, and adding one more operation is unwanted for the seek of simplicity and time-saving. In addition, image correlation is not well adapted to very small speckles (i.e. close to the pixel size). We propose here a new procedure directly dealing with the phase maps and optimizing the SNR at the local level. A shearography set-up is modeled, and the recorrelation principle tested on different cases (translation, constant strain, high density fringes). Mean maximum decorrelation error is quantified. Then, a first practical application shows some results in the case of a single-ply fabric laminate. Principle Neither the optical set-up and or phase extraction technique will be considered in this section. The discussion should be independent of either. The only requirement is that an initial phase map be subtracted from a final phase map, giving a low-frequency information, as it is generally in the case for mechanical strain/displacement fields. Consequently, this work should be well adapted to speckle shearography or speckle interferometry. The section is centered on the description of the SNR optimization procedure, as it is shown Fig. 1. First, the initial phase map ini is divided into small regions of interest (ROI), typically 32×32 pixels, centered around the co-ordinates {i,j}. In the final phase map fin, ROI of the same size, centered around {i+i,j+j} are subtracted to the initial sub-map.        ini j i, fin δj + j δi, + i j i, Φ Φ = δj δi, ΔΦ  (1) The off-set vector {i,j} varies within a certain range, corresponding to the expected translation, usually {±5, ±5} pixels. The signal to noise ratio for a given ROI position and a given offset vector can be calculated. As these are phase maps, a phase jump is possible, changing dramatically the map frequency signature. To eliminate this difficulty, the sine of the phase difference is considered. It is split in two components, using a simple FFT algorithm:  the low-frequency one,     LF j i, δj δi, ΔΦ sin , considered as a possible mechanical signal,  the low-frequency one,     LF j i, δj δi, ΔΦ sin , considered as a possible mechanical signal,    the high-frequency one,     HF j i, δj δi, ΔΦ sin , considered as noise.  the high-frequency one,     HF j i, δj δi, ΔΦ sin , considered as noise. The signal, denoted S(i, j)LF, can be defined either by the dynamic range of the low-frequency component or by the mean value of the low frequency component. In some cases, these two data sets can be close to zero, and may be replaced by a constant. The noise is defined by the standard Applied Mechanics and Materials Vols. 13-14 31 deviation of the high-frequency component. Then, it is possible to calculate a SNR for each off-set {i,j}. deviation of the high-frequency component. Principle Then, it is possible to calculate a SNR for each off-set {i,j}.             HF j i, LF j i, δj δi, ΔΦ STD δj δi, S = δj δi, SNR sin (2             HF j i, LF j i, δj δi, ΔΦ STD δj δi, S = δj δi, SNR sin             HF j i, LF j i, δj δi, ΔΦ STD δj δi, S = δj δi, SNR sin (2) The frequency filter has to be adjusted arbitrarily. In the following, it will be taken to 1/5th of the sub-map size. A SNR map can be drawn considering any combination of the off-set vector. The optimum translation corresponds to the vector giving the highest SNR. Last, entire displacement maps according to x and y directions are calculated. These maps can be processed as any other displacement maps. In particular, even in optimal conditions these maps are of good quality, a median filter can be useful to remove incoherent points. Then, the local displacement maps are taken into account in the phase subtraction procedure to calculate the optimum global phase difference map. No sub-pixel capability is addressed yet. This should be done in the future if necessary, but it needs some precautions in the interpolation of speckle phases, because of the randomness characteristic of a speckle chart. In particular, the lack of continuity between phase speckles prohibits any interpolation algorithm, either in direct or Fourier space. Furthermore, it would increase the time processing. Figure 1. Flow chart of recorrelation procedure. Figure 1. Flow chart of recorrelation procedure. Figure 1. Flow chart of recorrelation procedure. As in image correlation techniques, many parameters require fine tune adjustment: the ROI size, the off-set range, the displacement grid step, the low/high frequency threshold... Optimization of these parameters should be addressed carefully in the future. In the mean time, some simple Advances in Experimental Mechanics VI 32 considerations can be used: note that the ROI should be adapted to the expected gradient within the field in order to have sufficiently localized information. But if the ROI is too small, the signal / noise discrimination, i.e. the extraction of the mechanical information becomes impossible. Principle Here, the ROI size is set to 32×32 pixels2, as it is commonly used in classical digital image correlation techniques. Note that no assumption is made in the nature of the displacement (strain) fields . Usually, these fields are continuous, but discontinuities could occur (cracks, bolt joining, contacts ...). The knowledge of the user should be introduced as a tool to verify the quality of the fields, and an incoherent displacement should be removed. Here, the choice of no prior knowledge on the displacement (strain) field is justified by the generality of the approach. Last, the procedure does not consider the possibility of a rigid body rotation. This should dramatically decrease the SNR ratio. This point would have to be addressed if necessary. Simulated loading procedure Simulated conditions are chosen to be close to common practice in laboratories [9]. Speckle size is a priori close to the pixel size, thus its standard deviation is chosen to be 1 pixel. Even if in common practice, the shearing distance is usually higher, we use here a small value of 3 pixels, close to the most recent developments in our laboratory. In this particular case, speckles are fully decorrelated in the Michelson interferometer. The pixel size is set to 40 µm. Last, illumination source is assumed to be a doubled YAG Laser, so the wavelengh  = 532 nm, and the illumination direction  = /4. Last, a random phase noise of 1/100 fringe is added to the signal. The covered field of view is 500 pixels (20 mm). Speckle simulation principle The speckle simulation used here is based on previous work by Equis [11]. A randomly distributed complex amplitude field is generated. Then, the speckle size is adjusted using a Gaussian low-pass filter. Because we aim to apply this approach to speckle shearography, the amplitudes are combined as it would be in a classical Michelson interferometer. If the shearing distance is lower than the speckle size, the initial and final phase map should be correlated, then results should be different than speckle interferometry results. Last, phases are averaged over a certain area, simulating the pixel recording. A simulated mechanical loading is applied to the field. It acts in two directions: first, points are moved, leading to the basic decorrelation process. Second, the phase is changed according to the sensitivity vector used. We consider here a single source under a shearography set-up, with a sensitivity given by:       x w δ θ + λ + x u δ θ λ = j i, Δφ i i           cos 1 4π sin 4π (3 (3) where  is the illumination wavelength,  is the angle between illumination and observation directions, i is the shearing distance, ∂u / ∂x and ∂w / ∂x being the kinematics changes of the surface, respectively one in-plane strain, and one out-of-plane rotation. Now, the phase map between the two latter situations can be easily calculated as the angle of the difference of the two complex amplitudes. Rigid body translation A simple translation is applied to the speckle map. It can be easily verified that standard deviation on the phase difference increases as a function of the translation, and that a complete Applied Mechanics and Materials Vols. 13-14 33 decorrelation is achieved when the translation is 3 times the standard deviation of the speckle (blue cross, Fig. 2). Re-correlation procedure in applied for each case. Due to the specific nature of the test (the signal is uniformly equal to zero), the inverse of the standard deviation is taken as a signature of the signal to noise ratio. decorrelation is achieved when the translation is 3 times the standard deviation of the speckle (blue cross, Fig. 2). Re-correlation procedure in applied for each case. Due to the specific nature of the test (the signal is uniformly equal to zero), the inverse of the standard deviation is taken as a signature of the signal to noise ratio. g g Results show the efficiency of the re-correlation procedure (red circles, Fig. 2). Maximum errors are obtained for translations of (n+0.5) pixels. The maximum error value corresponds to the 0.5 pixel translation without recorrelation technique. Considering an equally probable occurrence of each translations in a given map, the mean error is 0.39 rad i.e. 6/100 fringe. Another test was performed with a larger speckle size. The trends remained the same, but the maximum is decreased corresponding again to the (n+0.5) pixel translation. For a speckle 3 times larger, the mean noise level is 2.2 times smaller. Figure 2. Decorrelation as a function of the translation between 2 steps. Figure 2. Decorrelation as a function of the translation between 2 steps. 1st and 2nd order displacement gradient A simple tensile test with data corresponding to the previous case is performed with a constant 5.10-3 µm/m longitudinal strain. On the left boundary, the displacement is null, and increases linearly up to 3 pixels (120 µm) on the right boundary. Results are shown in Fig. 3. The interest of such recorrelation technique is outlined Fig. 3a and 3b: with a simple classical subtraction, complete decorrelation is achieved on the right side. In this case of a global translation of -1 pixel, partial decorrelation takes place on both sides, with a maximum noise level of 18.8/100e fringe on the left boundary and 29.2/100e fringe on the right boundary. In fact, it is impossible to find a global translation to minimize the noise at all points, hence a local procedure is necessary. Fig. 3c shows that most of the estimated displacements are reasonable. Apparent noise varies within the map as described before and is minimum for a n-pixels displacement. Some salt and pepper noise remains for (n+0.5) displacement. A simple post-treatment could find and remove these points. Here, no post-treatment is applied in order to point out the effect of the correlation procedure alone. Last, it could be important to test the efficiency of the method in case of a strong strain gradient, i.e. with a high fringe density. A sine-wave strain is simulated in order to reach large enough displacements to have decorrelation and sufficient strain gradient to have 5 pixels/fringe for a correlation window of 32 pixels. Again, the efficiency of the recorrelation procedure is evident (Fig. 4). Advances in Experimental Mechanics VI 34 a) b) c) Figure 3. Original and recorrelated phase difference for a 3.103 µm/m homogeneous strain (in rad). Figure 3. Original and recorrelated phase difference for a 3.103 µm/m homogeneous strain (in rad). a) b) Figure 4. Original and recorrelated phase difference for a sine-wave strain field (in rad). igure 4. Original and recorrelated phase difference for a sine-wave strain field (in rad). Applied Mechanics and Materials Vols. 13-14 35 Specimen The specimen is a T700S/M10 12K plain-weave carbon fabric (48192, Hexcel Corporation), having the waviness (hy/2a) of 0.0078, as already used by Lee [12]. The single ply composite is cured using the procedure recommended by the supplier. The size of one unit cell is about 8 × 8 mm2 and the inspecting zone contains six unit cells. Optical set-up Phase maps have been recorded using a 4-beam portable shearography set-up. It is composed of an 110mW Diode-Pumped Solid-State (DPSS) laser with a wavelength of 532 nm, 5 optical fibres, a Michelson interferometer, a CCD camera and a PC. The principles of this method based on different illumination beams to obtain the displacements derivatives have been already presented by Lee [12] or Bounda [9]. The set-up presented here uses four illumination beams, two symmetrical in the horizontal plane and two symmetrical in the vertical plane. The experimental procedure consists in recording 8 phase maps (4 illumination beams, and 2 shearing directions) before and after loading. In such a configuration, ESPSI allows a direct measurement of the displacement derivatives maps on the surface, and consequently strains and out-of-plane rotations. The sensitivity can be adjusted by tuning the shear distance i, avoiding the fringe collapse due to strain concentration. In the following experiments, the system parameters are an illumination angle = /4 for all the illumination beams and the shearing distance i = 200 µm for x and y directions. These last values are obtained by using the grid method [13]. First results Phase maps are recorded at 50 N increments, from 200 to 450 N. Here, we only focus on the tensile direction maps, either the -/4, or + /4 illumination angle. The difference of the two is proportional to the strain xx, and the sum to the surface out-of-plane slope ∂w / ∂x. If we consider the 200 N load case and the 300 N load case, the difference shows some local decorrelation (Fig. 5a). Then, the proposed recorrelation procedure is applied (Fig. 5b). Some quality assessments were necessary: erroneous displacements can be easily isolated by comparing the displacement maps corresponding to each of the two phase maps. Then, displacement values are removed and replaced by an interpolated value. Last, salt and pepper noise were partially removed using a statistically- based procedure detecting the 10% least probable points. 36 Advances in Experimental Mechanics VI Advances in Experimental Mechanics VI 36 a) b) Figure 5. Real experimental map on a fabric specimen phase difference without (a) and with recorrelation procedure (b). a) b) a) b) Figure 5. Real experimental map on a fabric specimen phase difference without (a) and with recorrelation procedure (b). Conclusion The weak point of speckle techniques is their spatially random wavefront, and consequently a possible decorrelation of the speckles. In a classical mechanical context, rigid body motion is a very common problem. Then, it is absolutely necessary to address this point when using a speckle-based technique. Some solutions exist already, such as adding intermediate states or speckle correlation, but these solutions don't focus on the basic problem, which is the optimization of signal to noise ratio. In this article, we developed a simple way maximizing this data locally. A simulation was used to study some basic properties. Error level due to remaining decorrelation is completely known, and an error propagation model can be used on good basis. Last, a first example on a real experimental map is presented. Results show good trends, even without the use of an additional low-pass filter, except for a salt and pepper filter. Mechanical interpretation Finally, it is possible to calculate the strain component xx and the slope ∂w / ∂x. Fig. 6 shows raw results, the spatial resolution remains 4 pixels. Any classical post-processing should be applied to decrease the apparent noise. In this particular case, speckle size is lower than 1 pixel, leading to a rather bad situation. From a mechanical perspective, results are quite classical, and correspond to previous results from Lee [12]: the weaves transverse to the loading direction are strongly deformed, and the longitudinal ones show a smaller strain (the fabric structure is represented schematically by the yellow lines). This effect can be explained by the coupling between global tension and local flexure effect. Here in particular, the shape indicates a misalignment between the longitudinal weaves and the loading direction. A better inspection of the specimen reveals that it contains some waviness in the plane of the specimen itself, probably due to the manipulation of the prepreg. Applied Mechanics and Materials Vols. 13-14 37 a) b) Figure 6. Strain (a) and wrapped slope (b) for a 100 N load step. a) b) Figure 6. Strain (a) and wrapped slope (b) for a 100 N load step. b) Figure 6. Strain (a) and wrapped slope (b) for a 100 N load step. Future work This paper presented a first attempt at localized optimization of signal to noise ratio in phase difference methods. Consequently, there are still many points to be developed. In particular, the use of multiple internal parameters (filter cut-off, ROI size) is an important drawback of the method, and should be clarified in the future. More theoretical points should be addressed to further decrease the noise, especially in case of low speckle size: first, some mechanical considerations such as displacement continuity should permit to avoid aberrant points, and give a better sub-pixel estimation of displacement maps. Then, noise sub-pixel interpolation of speckle phase will have to be developed to better re-correlate the phase maps. Advances in Experimental Mechanics VI 38 The authors would like to thank Y. Zhou, Msc, and Dr. C. Fournier from Hubert Curien Laboratory of the University of Saint-Etienne for their collaboration to this work. The authors would like to thank Y. Zhou, Msc, and Dr. C. Fournier from Hubert Curien Laboratory of the University of Saint-Etienne for their collaboration to this work. [13] Surrel Y., Moiré and grid methods in optics, SPIE 2342, pp. 213-220 (1994). Bibliography [1] Brunet M., Touchal S., Morestin F., Numerical and experimental analysis of necking in 3D. Sheet forming prosesses using damage variable, Advanced Methods in Materials Processing Defects, M. Predeleanu & P.Gilormini, Elsevier Science BV (1997) p. 205 -214. [2] Rethore J., Roux S., Hild F., Noise-robust stress intensity factor determination from kinematic field measurements. Engineering Fracture Mechanics. Available online 3 May 2007 (Science Direct). [3] Bruno L., Pagnotta L., Poggialini A., Laser speckle decorrelation in NDT, Optics and Lasers in Engineering, 34 (2000) p. 55-65. [4] Cordero R.R., Molimard J., Martinez A., Labbe F., Uncertainty analysis of temporal phase- stepping algorithms for interferometry, Optics Communications, 275 (2007) p. 144–155. [5] Davila A., Huntley J.M., Kaufmann G. H., Kerr D., High-speed dynamic speckle interferometry: phase errors due to intensity, velocity, and speckle decorrelation, Appl Opt., 44, 19 (2005) p. 3954- 3962 [6] Hung Y.Y., Wang J.Q., Hovanesian J. D., Technique for Compensating Excessive Rigid Body Motion in Nondestructive Testing of Large Structures Using Shearography, Optics and Lasers in Engineering, 26 (1997) p. 249-250. [7] Martınez-Celorio R.A., Barrientos B., Sanchez-Marın F.J., Lopez L.M., Rayas J.A., Out-of- plane displacement measurement by electronic speckle pattern interferometry in presence of large in-plane displacement, Optics Communications, 208 (2002) p. 17–24. [8] Svanbro A, DSPI: complex amplitude correlation for large in-plane compensations and phase evaluation, Proceedings of Speckle'06, SPIE vol. 6341 (2006) p. 63410I-1~5.. [9] Molimard J., Bounda D., Vautrin A., Quantitative strain and slope evaluation on a double lap joint tensile test using ESPSI, Proceedings of Speckle'06, SPIE vol. 6341 (2006) p. 63412R-1~6. [10] Surrel Y., Design of algorithms for phase measurements by the use of phase stepping, Applied Optics 35, 1 (1996) p. 51– 60. [11] Equis S., Jacquot P., Simulation of Speckle Complex Amplitude : Advocating the Linear Model, Proceedings of Speckle'06, SPIE vol. 6341 (2006) p. 634136-1~6. [12] Lee J.R., Molimard J., Vautrin A., Surrel Y., Digital phase-shifting grating shearography for experimental analysis of fabric composites under tension, Composites: Part A, 35 (2004) p. 849– 859. [13] Surrel Y., Moiré and grid methods in optics, SPIE 2342, pp. 213-220 (1994)
https://openalex.org/W2924811773
https://link.springer.com/content/pdf/10.1007/s11325-019-01810-w.pdf
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Self-reported sleepiness in the context of fitness-to-drive
Sleep & breathing
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cc-by
4,028
Abstract There was no difference in the ESS [8 (8) vs 8 (8) points; p = 0.289] or the SSS [2 (2) vs 2 (2) points; p = 0.320] between the two occasions, although seven patients (5.7%) changed their scores from Bsleepy^ to Bnon-sleepy^ and four patients (3.3%) from Bnon-sleepy^ to Bsleepy^. Results One hundred twenty-two subjects were studied (age 59.4 years (15.2); 72 males; BMI 32.1 kg/m2 (8.3), driving licence held for 25.2 years (20.6) (n = 94); collar size 42.7 cm (5.0)). There was no difference in the ESS [8 (8) vs 8 (8) points; p = 0.289] or the SSS [2 (2) vs 2 (2) points; p = 0.320] between the two occasions, although seven patients (5.7%) changed their scores from Bsleepy^ to Bnon-sleepy^ and four patients (3.3%) from Bnon-sleepy^ to Bsleepy^. Conclusion Providing patients with information about the risk of driving in the context of sleepiness does not significantly change how they score their symptoms using self-administered questionnaires; only about 9.0% of the patients had inconsistent results. Keywords Excessive daytime sleepiness . Epworth scale . Driving . Sleep Apnoea Self-reported sleepiness in the context of fitness-to-drive Aanuolupo Ayeni1 & Gurpreet Singh Beghal1 & Martino F Pengo1,2,3 & Nimish Shah1,4 & Joerg Steier1,2 Received: 5 August 2018 /Revised: 4 February 2019 /Accepted: 19 February 2019 # The Author(s) 2019 /Published online: 19 March 2019 Received: 5 August 2018 /Revised: 4 February 2019 /Accepted: 19 February 2019 # The Author(s) 2019 /Published online: 19 March 2019 Aanuolupo Ayeni and Gurpreet Singh Beghal are joint first authors of this paper. https://doi.org/10.1007/s11325-019-01810-w Sleep and Breathing (2019) 23:1227–1232 https://doi.org/10.1007/s11325-019-01810-w Sleep and Breathing (2019) 23:1227–1232 SLEEP BREATHING PHYSIOLOGY AND DISORDERS • ORIGINAL ARTICLE Abstract Background Excessive daytime sleepiness (EDS) is a contributing factor to road traffic accidents. It is commonly assessed using self-administered questionnaires. These assessments are important information when discussing with the Driver and Vehicle Licensing Agency (DVLA) about fitness-to-drive. We hypothesised that patients may be confounded in their assessments after being informed about these potential implications. Patients and methods This was a prospective single-centre study. Patients attending clinics for sleep-disordered breathing were asked to fill in the Epworth Sleepiness Scale (ESS) and the Stanford Sleepiness Scale (SSS). Following their consultation, patients were informed about EDS in the context of driving and that the DVLA might request information based on their self- assessed sleepiness. They were then asked to complete the same questionnaires again. Parameters recorded included age, gender, body mass index (BMI), driving licence holder, and collar size. An ESS score above 10 points was defined as EDS. Patients and methods This was a prospective single-centre study. Patients attending clinics for sleep-disordered breathing were asked to fill in the Epworth Sleepiness Scale (ESS) and the Stanford Sleepiness Scale (SSS). Following their consultation, patients were informed about EDS in the context of driving and that the DVLA might request information based on their self- assessed sleepiness. They were then asked to complete the same questionnaires again. Parameters recorded included age, gender, body mass index (BMI), driving licence holder, and collar size. An ESS score above 10 points was defined as EDS. Results One hundred twenty-two subjects were studied (age 59.4 years (15.2); 72 males; BMI 32.1 kg/m2 (8.3), driving licence held for 25.2 years (20.6) (n = 94); collar size 42.7 cm (5.0)). There was no difference in the ESS [8 (8) vs 8 (8) points; p = 0.289] or the SSS [2 (2) vs 2 (2) points; p = 0.320] between the two occasions, although seven patients (5.7%) changed their scores from Bsleepy^ to Bnon-sleepy^ and four patients (3.3%) from Bnon-sleepy^ to Bsleepy^. Results One hundred twenty-two subjects were studied (age 59.4 years (15.2); 72 males; BMI 32.1 kg/m2 (8.3), driving licence held for 25.2 years (20.6) (n = 94); collar size 42.7 cm (5.0)). * Joerg Steier joerg.steier@gstt.nhs.uk Introduction and it affects quality of life; it results in reduced reaction time, vigilance, alertness, concentration and, subsequently, results in an impaired ability to successfully carry out attention-based activities [3]. EDS while driving is increasingly being recognised as a cause of road traffic accidents (RTA) [4], and approximately 20% of RTAs in the UK are caused by EDS and driver fatigue [5]. Excessive daytime sleepiness (EDS) is one of the cardinal symptoms of patients presenting to sleep laboratories as many patients with EDS suffer from obstructive sleep apnoea syn- drome (OSAS), obesity hypoventilation syndrome (OHS), narcolepsy or idiopathic hypersomnia [1, 2]. Severe EDS im- pacts on behavioural, physiological and cognitive functioning, It is difficult to objectively assess EDS, but questionnaires like the Epworth Sleepiness Scale (ESS) or the Stanford Sleepiness Scale (SSS) are widely used in clinical practice to subjectively quantify and determine whether an individual is suffering from EDS [1, 2]. However, it is important to recognise that information contained in these tools might be used by the Driver and Vehicle Licensing Agency (DVLA) when deciding on the fitness to drive; drivers who suffer with conditions caus- ing EDS need to cease driving and inform the DVLA [6]. Aanuolupo Ayeni and Gurpreet Singh Beghal are joint first authors of this paper. * Joerg Steier joerg.steier@gstt.nhs.uk 1 Lane Fox Respiratory Unit / Sleep Disorders Centre, Guy’s and St Thomas’ NHS Foundation Trust, Westminster Bridge Road, London SE1 7EH, UK 2 Faculty of Life Sciences and Medicine, King’s College London, London, UK 3 Sleep Disorder Centre, Department of Cardiovascular, Neural and Metabolic Sciences, IRCCS Istituto Auxologico Italiano, Milan, Italy 4 Jaslok Hospital and Research Centre, Mumbai, India Aanuolupo Ayeni and Gurpreet Singh Beghal are joint first authors of this paper. * Joerg Steier joerg.steier@gstt.nhs.uk 1 Lane Fox Respiratory Unit / Sleep Disorders Centre, Guy’s and St Thomas’ NHS Foundation Trust, Westminster Bridge Road, London SE1 7EH, UK However, patients might offer a biased view when self- reporting their symptoms by using tools like the ESS, if they know about possible implications regarding their driving li- cence. We hypothesised that patients might change how they score on the ESS after they are made aware of official guid- ance regarding fitness-to-drive. Introduction 2 Faculty of Life Sciences and Medicine, King’s College London, London, UK 3 Sleep Disorder Centre, Department of Cardiovascular, Neural and Metabolic Sciences, IRCCS Istituto Auxologico Italiano, Milan, Italy 4 Jaslok Hospital and Research Centre, Mumbai, India 1228 Sleep Breath (2019) 23:1227–1232 Patients and methods If an individual has any condition that affects their abil- ity to drive, which lasts longer than three months, they must inform the DVLA. The DVLA then considers whether sleepiness influences the ability to drive when reviewing these cases. [7] Epworth sleepiness scale The ESS is a questionnaire with eight items to measure sever- ity of daytime sleepiness; respondents report their likelihood of falling asleep in situations using a 4-point Likert scale (‘0’ not at all to ‘3’ highly likely). Responses are summed and higher scores indicate greater sleepiness, the minimum total score is 0 and the highest ‘24’ points; scores of more than 10 points suggest excessive daytime sleepiness [8]. Stanford Sleepiness Scale – Aged > 18 years – Both genders The SSS is a self-rated 7-point scale of equal intervals to quan- tify the symptom [9]. The scale is a one-item questionnaire measuring levels of sleepiness throughout the day. It is generally used to track alertness at different hours of the day and ranges from ‘feeling active and vital; alert, wide awake’ (1) to ‘Asleep’ (x) and is widely used to assess the effects of sleep deprivation. A score of more than 3 points at any time when the respondent should be feeling alert indicates serious sleep debt [10]. – Literate and holding capacity – Fluent English speaker. In addition, patients were excluded if they met any of the below exclusion criteria: – Aged <18 years – Unable to read or write, illiterate – Non-English speakers Inclusion and exclusion criteria The following inclusion criteria were assessed prior to inclu- sion into the analysis: – Patient referred to the sleep centre Compliance with ethical standards This was a prospective study conducted at a clinic for sleep- disordered breathing at a tertiary university hospital between June 2017 and July 2017 (registration number: GSTT/2017/ 7478). Patients were informed and consented prior to their clinic appointment, and adults aged 18 years and above were included. Patients were addressed following initial identifica- tion by the direct clinical care team. Funding was provided via the King’s Undergraduate Research Fellowship 2017, King’s College London, UK (A.A.). The authors have no conflict of interest related to the content of the manuscript. All appointments were timed to be 15–30 min slots. The 1st questionnaires were filled in prior to and the 2nd question- naires following these slots. All patients completed the entire assessment within a 60-min time frame. Outcome parameters – Not holding capacity – Acute illness or delirium. The primary outcome of this study was the change in the ESS (ΔESS, points). The secondary outcome parameters were the change in the SSS (ΔSSS, points), factors associated with a change in the ESS (sleepiness, driving licence holder, RTA) and patients who changed from ‘sleepy’ (ESS > 10) to ‘non- sleepy’ self-assessment (n). Short protocol Parameters recorded included age, gender, ethnicity, body mass index (BMI), past medical history, drug history, aller- gies, driving licence holder (in years), number of previous road traffic accidents (RTA), smoking (in pack years), alcohol consumption (in units/week), use of illegal highs/illicit sub- stances and collar size. Sample size calculation The sample size was calculated based on the change in the ESS. Expecting a change in the ESS of two or more points in the total score with an alpha of 5%, a power of 90%, and an approximate standard deviation of the mean difference of the paired observation of three points, a total sample size of 122 patients would need to be included [11]. Following previous experience with the dropout of patients who were assessed in similar scenarios [10], we expected that an additional 5–10% would need to be recruited. Finally, we addressed 138 pa- tients, 14 did not meet inclusion criteria and 2 patients did The first ESS and the SSS were filled in prior to the con- sultation with the sleep physician. Following the consultation, patients were informed about the risk of EDS and driving, and about official DVLA guidance. It was pointed out that their self-assessed sleepiness might provide information for the DVLA to decide about their fitness-to-drive. Prior to complet- ing a second ESS and the SSS, patients were read the follow- ing statement: 1229 Sleep Breath (2019) 23:1227–1232 not complete the assessment; a complete dataset was included for analysis in 122 patients (Fig. 1). not complete the assessment; a complete dataset was included for analysis in 122 patients (Fig. 1). complete the assessment (Fig. 1). A total of 122 patients were included in the data analysis (72 males, age 59.4 years (15.2), BMI 32.1 kg/m2 (8.3), collar size 42.7 cm (5.0), smoking (n = 20): pack years 15 (10–33); ex-smoking (n = 49): pack years 20 (10–40); alcohol (n-62): 16 (4.75–40) units per week). Female participants were slightly younger, smaller and lighter, although their BMI was about the same as that of male partic- ipants; men smoked more and had a larger neck circumference (Table 1). Ninety-four patients held a driving licence for 25.2 years (20.6), and their age was 59.6 years (13.9). The longer patients held their driving licence, the older they were (r = 0.387, p < 0.001). There was a negative correlation be- tween their age and the ESS (r = −0.334, p < 0.001), as well as between the duration of holding a driving licence and the ESS (r = −0.363, p < 0.001). Statistical analysis The statistical analysis was conducted using SPSS V23.0 (IBM Corp, Armonk, NY/USA). The Shapiro-Wilk normality test was used to assess data distribution. Results from the ESS, the SSS and subgroups of patients based on EDS (ESS > 10), and whether they held a driving licence were compared using the Wilcoxon signed ranks t test. ESS and SSS scores are presented as median (interquartile range) and age, gender, BMI, driving licence duration, smoking history and collar size are presented as mean (SD). We finally performed a multiple linear regression analysis to identify correlations (r) between the independent predictors (age, gender, BMI, driving licence duration and collar size) with the dependent variable (ESS). A p value <0.05 was assumed to represent statistical significance. Sleep-related breathing disorders were present in 53.0% of the patients (patients were naïve to continuous positive airway pressure, CPAP, at the time of assess- ment), 28.0% of participants had neuromuscular disorders with associated chest wall disease (NMD/CWD), 17.0% had obstructive airway disorders, hypersomnias in 14.0%, sleep movement disorders were present in 1.0%, parasomnias in 1.0% and 18.0% of participants were clas- sified as ‘other’ (this group consisted of patients with depression, anxiety, cardiac comorbidity, seizures, diabe- tes, hypothyroidism or unexplained syncopes). 65.6% of Demographics One hundred thirty-eight patients were screened for the study, but 14 patients did not meet the eligibility criteria and 2 did not ENROLLMENT ALLOCATION ANALYSIS Assessed for eligibility (n = 138) Total (n = 122) 2nd assessment ESS and SSS (n=122) Did not complete 2nd assessment (n =2) Declined (n = 1) Other reasons (n =1) 1st assessment ESS and SSS (n=124) Not meeting inclusion criteria (n =14) Fig. 1 Modified CONSORT diagram. Fourteen patients were excluded, as they were unable to communicate sufficiently in English Fig. 1 Modified CONSORT diagram. Fourteen patients were excluded, as they were unable to communicate sufficiently in English Fig. 1 Modified CONSORT diagram. Fourteen patients were excluded, as they were unable to communicate sufficiently in English ENROLLMENT Not meeting inclusion criteria (n =14) 1st assessment ESS and SSS (n=124) ALLOCATION Did not complete 2nd assessment (n =2) Declined (n = 1) Other reasons (n =1) Total (n = 122) Sleep Breath (2019) 23:1227–1232 1230 Table 1 Demographics of the studied cohort of patients Parameter Total (n = 122) Female (n = 50) Male (n = 72) Age (years) 59.4 (15.2) 57.6 (17.6) 60.8 (13.9) Height (m) 1.70 (0.12) 1.59 (0.09) 1.77 (0.08) Weight (kg) 92.5 (28.8) 78.8 (23.8) 101.6 (28.3) BMI (kg/m2) 32.1 (8.3) 31.0 (8.6) 32.6 (8.3) Neck circumference (cm) 42.7 (5.0) 40.1 (5.0) 44.0 (4.5) Smoking status (never, n/pack years) n = 61/25.7 (25.7) n = 34/19.3 (17.6) n = 27/30.0 (29.0) Table 1 Demographics of the studied cohort of patients more than 2 points (16.4%). For the total cohort, the change in the ESS from baseline was −0.11 (2.67) points, and for the SSS, the change was −0.04 (0.42) points. patients identified as White British, 4.9% Black British, 6.6% Black African, 6.6% Black Caribbean and 16.4% classified as other. Thirty-seven patients reported having an allergy but no patient reported any previous RTAs; three patients reported the use of legal highs/illegal substances. A stepwise multiple linear regression analysis to predict the average ESS scores included age, gender, BMI, driving licence duration, smoking pack years and collar size as independent variables (alpha = 0.05). Driving licence hold- er duration (p = 0.009) and age (p = 0.008) were identified as independent predictors of the ESS, while gender (p = 0.990), BMI (p = 0.697), smoking history (p = 0.95) and collar size (p = 0.812) were excluded from the model. Demographics About 17% of the variability of the ESS score were accounted for by this model (adjusted R2 = 0.171, F(6,112) = 5.043, p < 0.05). Sleepiness assessments There was no significant difference in the ESS [8 (8) vs 8 (8) points; p = 0.289] (Fig. 2) or the SSS [2 (2.25) vs 2 (2) points; p = 0.320] between first and second scores (Fig. 3). A total of 39 sleepy and 83 non-sleepy patients were identified, based on the ESS cutoff (> 10 points). There was no change between the scores in the ESS (p = 0.430) or the SSS (p = 0.830) based on subgroup analysis (sleepy/non-sleepy) either. A total of seven pa- tients (5.7%) changed their scores from Bsleepy^ to Bnon- sleepy,^ and four patients (3.3%) changed from non- sleepy to sleepy during their second assessments. A total of 35 patients reported significant changes in the ESS scores of more than 1 point (28.7%) and 20 patients by Discussion Patients attending sleep clinics who are provided with infor- mation about sleepiness and fitness-to-drive provide largely reproducible information when reporting their symptoms using standard tools like the Epworth or the Stanford Fig. 2 Box-Whisker plot for the Epworth Sleepiness Scale (ESS), first (ESS1) vs second score (ESS2), p = 0.289 Fig. 2 Box-Whisker plot for the Epworth Sleepiness Scale (ESS), first (ESS1) vs second score (ESS2), p = 0.289 Fig. 2 Box-Whisker plot for the Epworth Sleepiness Scale (ESS), first (ESS1) vs second score (ESS2), p = 0.289 1231 Sleep Breath (2019) 23:1227–1232 Sleep Breath (2019) 23:1227–1232 Fig. 3 Box-Whisker plot for the Stanford Sleepiness Scale (SSS), first (SSS1) vs second (SSS2) scores, p = 0.320 Sleep Breath (2019) 23:1227 1232 1231 Fig. 3 Box-Whisker plot for the Stanford Sleepiness Scale (SSS), first (SSS1) vs second (SSS2) scores, p = 0.320 Fig. 3 Box-Whisker plot for the Stanford Sleepiness Scale (SSS), first (SSS1) vs second (SSS2) scores, p = 0.320 information about the DVLA requirements. This rejects the idea that patients display significant and unconscious bias when self-reporting sleepiness [12], an observation that proves valuable in terms of assessing patient conditions asso- ciated with sleepiness, and when assessing fitness-to-drive. Although about 1/6 patients scored higher than what would be the expected minimally clinical important difference in the ESS [13], this only changed the ESS score in a relevant way in about 5% of the cases (non-sleepy to sleepy), which is about the expected level for random findings and errors for confi- dence intervals. Sleepiness Scale. Despite high intra-individually variability, only 9.0% of the patients change the Epworth Sleepiness Scale score in a relevant way, while 5.7% of patients re- consider and change their score from sleepy to non-sleepy. How long someone held their driving licence and how old they are accounted for about 17% of the observed variability in the Epworth Sleepiness Scale scores. There was no signif- icant difference in the results that was related to subscores or other factors like RTA or duration of driving licence. Clinical significance Self-reported questionnaires for the assessment of sleepiness are commonly used in sleep laboratories, and previous studies have identified internal consistency, reliability and validity of the ESS [11]. Current DVLA guidelines advocate that those who suffer from conditions resulting in excessive daytime sleepiness need to inform the DVLA and information regard- ing a patient’s condition may then be requested from medical health professionals [7]. Conclusion 1. Pagel J (2009) Excessive daytime sleepiness. Am Fam Physician 79(5):391–396 Self-assessment tools for sleepiness provide more robust and reproducible information than expected. Only in a minority of patients, there is a relevant change in the scores following provision of information about fitness- to-drive and clinicians should be encouraged to inform patients about potential implications prior to filling in the Epworth Sleepiness Scale. 2. Slater G, Pengo MF, Kosky C, Steier J (2013) Obesity as an inde- pendent predictor of subjective excessive daytime sleepiness. Respir Med 107(2):305–309 p 3. Roth T (2015) Effects of excessive daytime sleepiness and fatigue on overall health and cognitive function. J Clin Psychiatry 76(9): e1145 4. Nabi H, Guéguen A, Chiron M, Lafont S, Zins M, Lagarde E (2006) Awareness of driving while sleepy and road traffic acci- dents: prospective study in GAZEL cohort. BMJ 333(7558):75 Acknowledgments All the authors have contributed to the design, analysis and manuscript writing and all the authors have acknowledged the final version. Dr. Steier’s contribution was partially supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. 5. de Mello MT, Narciso FV, Tufik S, Paiva T, Spence DW, Bahammam AS, Verster JC, Pandi-Perumal SR (2013) Sleep dis- orders as a cause of motor vehicle collisions. Int J Prev Med 4(3): 246–257 6. Assessing fitness to drive: a guide for medical professionals - GOV.UK. 04/08/2017]; Available from: http://www.gov.uk/dvla/ fitnesstodrive 7. Tiredness can kill: advice for drivers. 17/09/2017]; Available from: https://www.gov.uk/government/uploads/system/uploads/ attachment_data/file/503534/INF159_150216.pdf Funding Data acquisition (A.A.) was supported by the King’s Undergraduate Research Fellowship 2017, King’s College London, UK. 8. Johns MW (1991) A new method for measuring daytime sleepi- ness: the Epworth sleepiness scale. Sleep 14(6):540–545 Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Limitations While efforts were made to standardise the length of time between patients completing their first ESS and being interviewed for the second ESS, this was not always possible. Although all assessments finished within a 30–60 min period, as such, these differences may account for some variation in how patients reported their sleepiness. Prior patient knowl- edge regarding the ESS and DVLA criteria may have further influenced results, as some patients may have previously heard about the DVLA. This may have also been influenced by the variation in duration of diagnoses, patient knowledge and expertise and lack of inclusion of additional information, e.g. about the educational background, which may have con- founded results. Furthermore, the study did not include objec- tive measures of sleepiness and relied solely upon subjective and self-reported measurements that are the current standard for sleep clinics. It may therefore prove beneficial to include other objective measures to further evaluate the impact on patients’ sleepiness and compare objective and subjective measures of sleepiness assessment. In this study, the Epworth Sleepiness Scale was chosen as it is a widely used tool within sleep centres and acces- sible to patients with sleep disorders. In the context of fitness-to-drive, the Epworth Sleepiness Scale is referred to when writing medical reports for the Drivers and Vehicles Licensing Agency (DVLA) in the UK. However, the ESS typically does not respond to changes in sleepiness quickly. The Stanford Sleepiness Scale is more suitable to pick up ad hoc changes in alertness, as it can be used to assess hourly daytime alertness. The results from this study support the general validity when using results from the patients’ ESS, as the information provided is not significantly influenced by additional Sleep Breath (2019) 23:1227–1232 1232 References Compliance with ethical standards 9. Hoddes E, Zarcone V, and Dement W (1972) Development and use of Stanford sleepiness scale (SSS). In Psychophysiology. Cambridge Univ Press, New York Conflict of interest The authors declare that they have no conflict of interest. g 10. Herscovitch J, Broughton R (1981) Sensitivity of the Stanford sleepiness scale to the effects of cumulative partial sleep deprivation and recovery oversleeping. Sleep 4(1):83–91 Local clinical governance approval board (Guy’s and St Thomas’ NHS Foundation Trust) All procedures performed in studies involving hu- man participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical stan- dards (registration number: GSTT/2017/7478). 11. Spira AP, Beaudreau SA, Stone KL, Kezirian EJ, Lui LY, Redline S, Ancoli-Israel S, Ensrud K, Stewart A, for the Osteoporotic Fractures in Men Study (2012) Reliability and validity of the Pittsburgh sleep quality index and the Epworth Sleepiness Scale in older men. J Gerontol A Biol Sci Med Sci 67A(4):433–439 12. Nickerson RS (1998) Confirmation bias: a ubiquitous phenomenon in many guises. Rev Gen Psychol 2(2):175–220 Informed consent Informed consent was obtained from all individual participants included in the study. 13. Patel S, Kon SSC, Nolan CM, Barker RE, Simonds AK, Morrell MJ, Man WD (2018) The epworth sleepiness scale: minimum clin- ically important difference in obstructive sleep apnea. Am J Respir Crit Care Med 197(7):961–963 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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The fossil bivalve Angulus benedeni benedeni: a potential seasonally resolved stable isotope-based climate archive to investigate Pliocene temperatures in the southern North Sea basin Nina M.A. Wichern1, Niels J. de Winter2,3, Andy L.A. Johnson4, Stijn Goolaerts5, Frank Wesselingh2,6, Maartje F. Hamers2, Pim Kaskes3, Philippe Claeys3, Martin Ziegler2 5 1Institute of Geology and Paleontology, Westfälische Wilhelms-Universität, Münster, 48149, Germany 2Department of Earth Sciences, Utrecht University, Utrecht, 3584 CB, the Netherlands 3Analytical, Environmental, and Geochemistry Research Group, Department of Chemistry, Vrije Universiteit Brussel, Brussels, 1050, Belgium 4School of Environmental Sciences, University of Derby, Derby, DE22 1GB, UK 10 5Department of Palaeontology, Royal Belgian Institute of Natural Sciences, Brussels, 1000, Belgium 6Naturalis Biodiversity Center, Leiden, 2333 CR, the Netherlands Correspondence to: Nina M.A. Wichern (nwichern@uni-muenster.de) https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. Correspondence to: Nina M.A. Wichern (nwichern@uni-muenster.de) y y y Department of Palaeontology, Royal Belgian Institute of Natural Sciences, Brussels, 1000, Belgium Naturalis Biodiversity Center, Leiden, 2333 CR, the Netherlands y j g Maartje F. Hamers2, Pim Kaskes3, Philippe Claeys3, Martin Ziegler2 5 1Institute of Geology and Paleontology, Westfälische Wilhelms-Universität, Münster, 48149, Germany 2Department of Earth Sciences, Utrecht University, Utrecht, 3584 CB, the Netherlands 3Analytical, Environmental, and Geochemistry Research Group, Department of Chemistry, Vrije Universiteit Brussel, Brussels, 1050, Belgium 4School of Environmental Sciences, University of Derby, Derby, DE22 1GB, UK 10 5Department of Palaeontology, Royal Belgian Institute of Natural Sciences, Brussels, 1000, Belgium 6Naturalis Biodiversity Center, Leiden, 2333 CR, the Netherlands g chool of Environmental Sciences, University of Derby, Derby, DE22 1GB, UK https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. 15 Abstract. Obtaining temperature data from the mid-Piacenzian warm period (ca. 3 Ma, Pliocene epoch) is a key factor in outlining the impact of projected anthropogenic climate change. The mid-Piacenzian warm period was a high-CO2 world with a paleogeography similar to modern times. The time interval has been used to validate and improve climate model retrodictions, which in turn enables assessing the predictive strength of these models. Validating climate models requires a Abstract. Obtaining temperature data from the mid-Piacenzian warm period (ca. 3 Ma, Pliocene epoch) is a key factor in outlining the impact of projected anthropogenic climate change. The mid-Piacenzian warm period was a high-CO2 world with a paleogeography similar to modern times. The time interval has been used to validate and improve climate model retrodictions, which in turn enables assessing the predictive strength of these models. Validating climate models requires a large array of robust proxy data. Here, we increase the potential of this proxy database by showing that the extinct tellinid 20 bivalve Angulus benedeni benedeni can be used for stable isotope-based temperature reconstructions. This species is found in the Pliocene sediments of the southern North Sea basin. Oxygen isotope and carbonate clumped isotope measurements on the shell of A. benedeni benedeni resulted in a mean annual temperature reconstruction of 13.5±3.8°C. This is 2.5°C warmer than today, 3.5°C warmer than the pre-industrial North Sea, and in line with global Pliocene temperature estimates of +2-4°C large array of robust proxy data. Here, we increase the potential of this proxy database by showing that the extinct tellinid 20 bivalve Angulus benedeni benedeni can be used for stable isotope-based temperature reconstructions. This species is found in the Pliocene sediments of the southern North Sea basin. Oxygen isotope and carbonate clumped isotope measurements on the shell of A. benedeni benedeni resulted in a mean annual temperature reconstruction of 13.5±3.8°C. This is 2.5°C warmer than today, 3.5°C warmer than the pre-industrial North Sea, and in line with global Pliocene temperature estimates of +2-4°C compared to the pre-industrial climate. Limited amounts of clumped isotope data hindered determining summer and winter 25 temperatures, but the oxygen isotope record shows that the growth band spacing of A. benedeni benedeni allows for sampling at a resolution of 2-3 months. The fossil bivalve Angulus benedeni benedeni: a potential seasonally resolved stable isotope-based climate archive to investigate Pliocene temperatures in the southern North Sea basin , , y , j , g , Maartje F. Hamers2, Pim Kaskes3, Philippe Claeys3, Martin Ziegler2 5 1Institute of Geology and Paleontology, Westfälische Wilhelms-Universität, Münster, 48149, Germany 2Department of Earth Sciences, Utrecht University, Utrecht, 3584 CB, the Netherlands 3Analytical, Environmental, and Geochemistry Research Group, Department of Chemistry, Vrije Universiteit Brussel, Brussels, 1050, Belgium 10 1 1 1 Introduction 35 Direct comparisons between the mPWP and our future climate are not appropriate, however. The mPWP was an equilibrated rather than a transient climate (e.g., Salzmann et 50 al. 2009), and some palaeogeographical features—such as seaways configurations—that have changed since could have had a significant impact on regional climate (Hill 2015). Validating climate models requires large, high-quality datasets. An example is the PRISM project (Dowsett et al. 2010; 2016), which is a compilation of data on paleogeography, land and sea temperatures, vegetation cover, land and sea ice, and ), p p g g p y, p , g , , soil type for the mPWP. Assessing the suitability of bivalve species that have not yet been used for this purpose is a way to 55 potentially increase this database. To this goal, we investigated specimens of the extinct bivalve species Angulus benedeni benedeni (Nyst and Westendorp 1839) that were collected from the mid-Piacenzian sediments of the southern North Sea basin. Investigating its potential use as a climate archive was done by means of oxygen isotope and carbonate clumped isotope analyses. In addition, electron backscatter diffraction in combination with light microscopy, X-ray diffraction, and micro-X-ray fluorescence, was used to analyse microstructures and assess preservation. The results indicate that A. benedeni 60 benedeni’s shell contain multiannual records. Sampling these at a sub-annual resolution allows for the reconstruction of the soil type for the mPWP. Assessing the suitability of bivalve species that have not yet been used for this purpose is a way to 55 potentially increase this database. To this goal, we investigated specimens of the extinct bivalve species Angulus benedeni benedeni (Nyst and Westendorp 1839) that were collected from the mid-Piacenzian sediments of the southern North Sea basin. Investigating its potential use as a climate archive was done by means of oxygen isotope and carbonate clumped isotope analyses. In addition, electron backscatter diffraction in combination with light microscopy, X-ray diffraction, and soil type for the mPWP. Assessing the suitability of bivalve species that have not yet been used for this purpose is a way to 55 potentially increase this database. To this goal, we investigated specimens of the extinct bivalve species Angulus benedeni benedeni (Nyst and Westendorp 1839) that were collected from the mid-Piacenzian sediments of the southern North Sea basin. Investigating its potential use as a climate archive was done by means of oxygen isotope and carbonate clumped isotope analyses. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. 1 Introduction 35 During the Pliocene-Pleistocene transition, global climate changed from a “coolhouse” state to the icehouse state (Westerhold et al. 2020). One specific period in the late Pliocene, the mid-Piacenzian warm period (mPWP—3.264-3.025 Ma; Dowsett et al. 2010), is a period of interest in light of our current warming climate. During the mPWP, global average temperatures were 2-4°C warmer than today (Ravelo et al. 2004; Haywood and Valdes 2004; Dowsett et al. 2010; 2012; 1 Introduction 35 During the Pliocene-Pleistocene transition, global climate changed from a “coolhouse” state to the icehouse state (Westerhold et al. 2020). One specific period in the late Pliocene, the mid-Piacenzian warm period (mPWP—3.264-3.025 Ma; Dowsett et al. 2010), is a period of interest in light of our current warming climate. During the mPWP, global average temperatures were 2-4°C warmer than today (Ravelo et al. 2004; Haywood and Valdes 2004; Dowsett et al. 2010; 2012; The species could live for up to a decade, and therefore has the potential to be used for multiannual seasonality reconstructions. The pristine nature of the aragonitic shell material was verified through electron backscatter diffraction analysis (EBSD), and backed by light microscopy, X-ray diffraction, and X-ray fluorescence. The various microstructures as obtained from the EBSD maps have been described, and they provide a template of pristine A. 30 benedeni benedeni material to which potentially altered shells may be compared. The bivalve A. benedeni benedeni is suitable for high resolution isotope-based paleoclimatic reconstruction and it can be used to unravel the marine conditions in the Pliocene southern North Sea basin at a seasonal scale, yielding enhanced insights into imminent western European climate conditions. various microstructures as obtained from the EBSD maps have been described, and they provide a template of pristine A. 30 benedeni benedeni material to which potentially altered shells may be compared. The bivalve A. benedeni benedeni is suitable for high resolution isotope-based paleoclimatic reconstruction and it can be used to unravel the marine conditions in the Pliocene southern North Sea basin at a seasonal scale, yielding enhanced insights into imminent western European climate conditions. 2 2 1 Introduction 35 Haywood et al. 2013; 2020), atmospheric CO2 concentrations were 340-380 ppm (360 ppm on average—Raymo et al. 1996; 40 Kürschner et al. 1996; Pagani et al. 2010; de la Vega et al. 2020), and sea level was 10-30 m higher (Ravelo et al. 2004; Naish and Wilson 2009; Dowsett et al. 2010). The cause of warming during this period is still debated. Increased CO2 levels and ocean circulation changes, or a combination thereof, are the dominant hypotheses (e.g., Raymo et al. 1996; Mudelsee and Raymo 2005; Dowsett et al. 2009; De Schepper et al. 2014). These oceanic circulation changes were brought on by the Haywood et al. 2013; 2020), atmospheric CO2 concentrations were 340-380 ppm (360 ppm on average—Raymo et al. 1996; 40 Kürschner et al. 1996; Pagani et al. 2010; de la Vega et al. 2020), and sea level was 10-30 m higher (Ravelo et al. 2004; Naish and Wilson 2009; Dowsett et al. 2010). The cause of warming during this period is still debated. Increased CO2 levels and ocean circulation changes, or a combination thereof, are the dominant hypotheses (e.g., Raymo et al. 1996; Mudelsee and Raymo 2005; Dowsett et al. 2009; De Schepper et al. 2014). These oceanic circulation changes were brought on by the closing, restriction, or through-flow change of several seaways (respectively, the Isthmus of Panama, the Indonesian Seaway, 45 and the Bering Strait), resulting in the redirection of heat flows (e.g., Mudelsee and Raymo 2005; De Schepper et al. 2014; Horikawa et al. 2015). The estimated warming during the mPWP is similar to what has been predicted for the end of this century according to the moderate and high emission RCP scenarios (RCP4.5: average of 2°C warming, RCP6.0: average of 2.5°C warming, RCP 8.5: average of 4°C warming; Collins et al. 2013). Direct comparisons between the mPWP and our closing, restriction, or through-flow change of several seaways (respectively, the Isthmus of Panama, the Indonesian Seaway, 45 and the Bering Strait), resulting in the redirection of heat flows (e.g., Mudelsee and Raymo 2005; De Schepper et al. 2014; Horikawa et al. 2015). The estimated warming during the mPWP is similar to what has been predicted for the end of this century according to the moderate and high emission RCP scenarios (RCP4.5: average of 2°C warming, RCP6.0: average of 2.5°C warming, RCP 8.5: average of 4°C warming; Collins et al. 2013). https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. 2.1 Seasonality records from bivalve shells 65 Reconstructions of past high-CO2 worlds deliver crucial insights into the processes, rates, and outcomes that are associated with anthropogenic climate change. The knowledge we have of these past climate modes, however, is often limited to long- term climate records such as deep-sea benthic oxygen isotope curves (e.g., Westerhold et al. 2020). This long-term record bias limits our understanding of climate impact on shallow marine and terrestrial ecosystems. This is especially relevant as short-term events such as heatwaves and extreme precipitation have already been observed to have increased in frequency 70 and are predicted to become even more common in the future (Seneviratne et al. in Press). Under a warming climate, seasonality is expected to intensify as well. Climate models predict an enhanced seasonal contrast in both temperature and precipitation (Seneviratne et al. in Press). Moreover, seasonality encompasses the most important periodicity in climate and is therefore crucial to our understanding of past and future climates (Mitchell 1976; Huybers and Curry 2006; von der Heydt et al. 2021). Many proxies provide insight into mean annual temperatures, but these data do not capture the range of 75 temperatures that a region experiences. Ontogenetic profiles from bivalves have the potential to record climate variability at the sub-annual scale. Bivalve shells can be suitable archives for seasonality reconstructions for several reasons. Long-lived species record multiple seasons as they live for several years—up to 500 years in certain species (Butler et al. 2013)—and can record down to sub-daily environmental conditions in their growth increments (e.g., Schöne et al. 2002). They are abundant in the fossil record across all latitudes (Moss et al. 2016 and references therein). Through sclerochronologic 80 analysis of bivalve shells, the growth patterns can be used to infer life history (e.g., Sato 1999; Palmer et al. 2021). Many bivalves occupy the shallow marine zone. This zone is less well-represented than the open ocean in climate records (e.g., de Winter et al. 2020 and references therein). Shallow marine and coastal records can also be used to connect terrestrial and marine records, as they collect input from both realms (Crampton-Flood et al. 2020). Most bivalve species precipitate isotopically in near equilibrium with water and are relatively little affected by so-called “vital effects” that skew the 85 temperature calibration (Weiner and Dove 2003; Huyghe et al. 2022). 2.1 Seasonality records from bivalve shells 65 Bivalve shells can be suitable archives for seasonality reconstructions for several reasons. Long-lived species record multiple seasons as they live for several years—up to 500 years in certain species (Butler et al. 2013)—and can record down to sub-daily environmental conditions in their growth increments (e.g., Schöne et al. 2002). They are abundant in the fossil record across all latitudes (Moss et al. 2016 and references therein). Through sclerochronologic 80 analysis of bivalve shells, the growth patterns can be used to infer life history (e.g., Sato 1999; Palmer et al. 2021). Many bivalves occupy the shallow marine zone. This zone is less well-represented than the open ocean in climate records (e.g., de Winter et al. 2020 and references therein). Shallow marine and coastal records can also be used to connect terrestrial and marine records, as they collect input from both realms (Crampton-Flood et al. 2020). Most bivalve species precipitate isotopically in near equilibrium with water and are relatively little affected by so-called “vital effects” that skew the 85 temperature calibration (Weiner and Dove 2003; Huyghe et al. 2022). 2.1 Seasonality records from bivalve shells 65 Reconstructions of past high-CO2 worlds deliver crucial insights into the processes, rates, and outcomes that are associated with anthropogenic climate change. The knowledge we have of these past climate modes, however, is often limited to long- term climate records such as deep-sea benthic oxygen isotope curves (e.g., Westerhold et al. 2020). This long-term record bias limits our understanding of climate impact on shallow marine and terrestrial ecosystems. This is especially relevant as short-term events such as heatwaves and extreme precipitation have already been observed to have increased in frequency 70 and are predicted to become even more common in the future (Seneviratne et al. in Press). Under a warming climate, seasonality is expected to intensify as well. Climate models predict an enhanced seasonal contrast in both temperature and precipitation (Seneviratne et al. in Press). Moreover, seasonality encompasses the most important periodicity in climate and is therefore crucial to our understanding of past and future climates (Mitchell 1976; Huybers and Curry 2006; von der Heydt short-term events such as heatwaves and extreme precipitation have already been observed to have increased in frequency 70 and are predicted to become even more common in the future (Seneviratne et al. in Press). Under a warming climate, seasonality is expected to intensify as well. Climate models predict an enhanced seasonal contrast in both temperature and precipitation (Seneviratne et al. in Press). Moreover, seasonality encompasses the most important periodicity in climate and is therefore crucial to our understanding of past and future climates (Mitchell 1976; Huybers and Curry 2006; von der Heydt et al. 2021). Many proxies provide insight into mean annual temperatures, but these data do not capture the range of 75 temperatures that a region experiences. Ontogenetic profiles from bivalves have the potential to record climate variability at the sub-annual scale. Bivalve shells can be suitable archives for seasonality reconstructions for several reasons. Long-lived species record multiple seasons as they live for several years—up to 500 years in certain species (Butler et al. 2013)—and can record down to sub-daily environmental conditions in their growth increments (e.g., Schöne et al. 2002). They are et al. 2021). Many proxies provide insight into mean annual temperatures, but these data do not capture the range of 75 temperatures that a region experiences. Ontogenetic profiles from bivalves have the potential to record climate variability at the sub-annual scale. 1 Introduction 35 In addition, electron backscatter diffraction in combination with light microscopy, X-ray diffraction, and micro-X-ray fluorescence, was used to analyse microstructures and assess preservation. The results indicate that A. benedeni 60 benedeni’s shell contain multiannual records. Sampling these at a sub-annual resolution allows for the reconstruction of the average temperature and seasonality of its shallow marine habitat, showing that A. benedeni benedeni can be a valuable addition to the mid-Piacenzian climate archive of western Europe. micro-X-ray fluorescence, was used to analyse microstructures and assess preservation. The results indicate that A. benedeni 60 benedeni’s shell contain multiannual records. Sampling these at a sub-annual resolution allows for the reconstruction of the average temperature and seasonality of its shallow marine habitat, showing that A. benedeni benedeni can be a valuable addition to the mid-Piacenzian climate archive of western Europe. micro-X-ray fluorescence, was used to analyse microstructures and assess preservation. The results indicate that A. benedeni 60 benedeni’s shell contain multiannual records. Sampling these at a sub-annual resolution allows for the reconstruction of the average temperature and seasonality of its shallow marine habitat, showing that A. benedeni benedeni can be a valuable addition to the mid-Piacenzian climate archive of western Europe. 60 3 3 Some studies indicate generally cool winter conditions, with only slightly higher summer temperatures and a slightly larger seasonal range compared to today (Vignols et al. 2019). Other studies suggest summer temperatures and a slightly larger seasonal range compared to today (Vignols et al. 2019). Other studies suggest cool winter conditions similar to today with significant warming during summer, yielding significantly larger seasonal 110 temperature ranges of up to 17°C (Raffi et al. 1985; Johnson et al. 2009; Valentine et al. 2011; Johnson et al. 2022). Yet another range of studies suggest strong warming in both summer and winter (Strauch 1968; Head 1997; 1998). Finally, some suggest a decreased seasonality compared to today (Wood et al. 1993; O’Dea and Okamura 2000; Knowles et al. 2009). Clumped isotope data from bivalves could constrain seasonal temperature range, as it enables a high-resolution, absolute temperature reconstruction that does not rely on estimates of oceanic isotopic composition (see also sect. 3.5.3). 115 cool winter conditions similar to today with significant warming during summer, yielding significantly larger seasonal 110 temperature ranges of up to 17°C (Raffi et al. 1985; Johnson et al. 2009; Valentine et al. 2011; Johnson et al. 2022). Yet another range of studies suggest strong warming in both summer and winter (Strauch 1968; Head 1997; 1998). Finally, some suggest a decreased seasonality compared to today (Wood et al. 1993; O’Dea and Okamura 2000; Knowles et al. 2009). Clumped isotope data from bivalves could constrain seasonal temperature range, as it enables a high-resolution, absolute temperature reconstruction that does not rely on estimates of oceanic isotopic composition (see also sect. 3.5.3). 115 2.2 The southern North Sea basin The southern North Sea basin (SNSB) has already warmed by 1-1.3°C since the late 1800s (from a mean annual average temperature of ca.10°C to 11°C), with the majority of the warming having taken place since the late 1980s (Schöne et al. 2004; Mackenzie and Schiedek 2007; Belkin 2009; Emeis et al. 2015; Quante and Colijn 2016). Within the North Sea the 90 rate of warming varies regionally, ranging from 0.2°C/decade in the north to up to 0.5°C/decade in the south since the 1980s The southern North Sea basin (SNSB) has already warmed by 1-1.3°C since the late 1800s (from a mean annual average temperature of ca.10°C to 11°C), with the majority of the warming having taken place since the late 1980s (Schöne et al. ( ) y y ( g temperature of ca.10°C to 11°C), with the majority of the warming having taken place since the late 1980s (Schöne et al. 2004; Mackenzie and Schiedek 2007; Belkin 2009; Emeis et al. 2015; Quante and Colijn 2016). Within the North Sea the 90 rate of warming varies regionally, ranging from 0.2°C/decade in the north to up to 0.5°C/decade in the south since the 1980s (Quante and Colijn 2016). North Sea ecosystems are already affected by these increasing temperatures (Quante and Colijn 2016). Besides its response to recent climate change, it is also an important region to understand in the context of past climate 2004; Mackenzie and Schiedek 2007; Belkin 2009; Emeis et al. 2015; Quante and Colijn 2016). Within the North Sea the 90 rate of warming varies regionally, ranging from 0.2°C/decade in the north to up to 0.5°C/decade in the south since the 1980s (Quante and Colijn 2016). North Sea ecosystems are already affected by these increasing temperatures (Quante and Colijn 2016). Besides its response to recent climate change, it is also an important region to understand in the context of past climate Besides its response to recent climate change, it is also an important region to understand in the context of past climate change through its connection to the North Atlantic. Proxy data indicate strong warming in the North Atlantic region during 95 4 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. the mid-Piacenzian, with global mean sea-surface temperature (SST) anomalies of up to +4-7°C instead of the global +2-4°C (Dowsett et al. 2012; 2013). Modelling studies have often failed to reproduce this enhanced sensitivity (Dowsett et al. 2013), although recent studies have obtained a better reconciliation of proxy and model data (Haywood et al. 2020). The North Atlantic is sensitive to changes in the Atlantic Meridional Overturning Circulation (AMOC) (Chen and Tung 2018). The the mid-Piacenzian, with global mean sea-surface temperature (SST) anomalies of up to +4-7°C instead of the global +2-4°C (Dowsett et al. 2012; 2013). Modelling studies have often failed to reproduce this enhanced sensitivity (Dowsett et al. 2013), although recent studies have obtained a better reconciliation of proxy and model data (Haywood et al. 2020). The North Atlantic is sensitive to changes in the Atlantic Meridional Overturning Circulation (AMOC) (Chen and Tung 2018). The Gulf Stream, a component of the AMOC, extends north-eastward as the North Atlantic Current (NAC). This NAC water, 100 which retains the warm and nutrient-rich signature of the Gulf Stream, enters the North Sea via the northern side and via the English Channel (Winther and Johannessen 2006). During the mPWP, only the northern route existed, as modern-day France and England were connected by the Weald-Arrtois land bridge to the south (e.g., Gibbard and Lewin 2016). Understanding the mid-Piacenzian North Sea climate could provide indirect evidence on North Atlantic circulation modes. Insights in 100 Gulf Stream, a component of the AMOC, extends north-eastward as the North Atlantic Current (NAC). This NAC water, 100 which retains the warm and nutrient-rich signature of the Gulf Stream, enters the North Sea via the northern side and via the English Channel (Winther and Johannessen 2006). During the mPWP, only the northern route existed, as modern-day France and England were connected by the Weald-Arrtois land bridge to the south (e.g., Gibbard and Lewin 2016). Understanding the mid-Piacenzian North Sea climate could provide indirect evidence on North Atlantic circulation modes. Insights in seasonality in particular could help us understand the distribution of heat throughout the year and how that relates to oceanic 105 and atmospheric circulation patterns. However, previous research into the SNSB climate and seasonality during the Pliocene, based on a range of biogenic proxies, has produced conflicting results. 2.3 Geological context: the Lillo Formation Three specimens of Angulus benedeni benedeni (SG-125, SG-126, and SG-127), were collected in 2013 from a shell bed in the top of Oorderen Member of the Pliocene Lillo Formation. The exact location was the temporary outcrop for the construction of the Deurganck Dock Lock (now Kieldrecht Lock; 51°16′44″N 4°14′52″E) in the port of Antwerp area, northern Belgium (Fig. 1a, b). Photos of the collecting locality with an in-situ specimen of A. benedeni benedeni can be 120 found in Deckers et al. (2020)’s Fig. 6. The marine Lillo Formation (De Meuter and Laga 1976) was deposited during the late Zanclean to the Piacenzian (ca. 3.7-2.7 Ma) in the SNSB (Fig. 1c). The Oorderen Member as introduced by De Meuter 120 5 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. and Laga (1976) is a greyish fine-grained shell-rich unit containing numerous mollusc shells, both dispersed in a g ( 9 ) g y g g , p Figure 1: Location of the modern North Sea basin and the stratigraphic level from which the A. benedeni benedeni specimens originated. (a) Overview map of the location of Belgium and the present-day extent of the North Sea within northwest Europe. The dark rectangle indicates the location of map (b). Adapted after Louwye et al. (2020). (b) The geographical extent of the Lillo Formation in Belgium, after Louwye et al. (2020), and the location of the city of Antwerp. (c) Lithological column of the Lillo Formation and its members with their chronostratigraphic position, after Louwye et al. (2020). The approximate stratigraphic position of the A. benedeni benedeni specimens is shaded in grey. Figure 1: Location of the modern North Sea basin and the stratigraphic level from which the A. benedeni benedeni specimens 125 originated. (a) Overview map of the location of Belgium and the present-day extent of the North Sea within northwest Europe. The dark rectangle indicates the location of map (b). Adapted after Louwye et al. (2020). (b) The geographical extent of the Lillo Formation in Belgium, after Louwye et al. (2020), and the location of the city of Antwerp. (c) Lithological column of the Lillo Formation and its members with their chronostratigraphic position, after Louwye et al. (2020). The approximate stratigraphic position of the A. benedeni benedeni specimens is shaded in grey. 130 Figure 1: Location of the modern North Sea basin and the stratigraphic level from which the A. benedeni benedeni specimens 125 originated. (a) Overview map of the location of Belgium and the present-day extent of the North Sea within northwest Europe. The dark rectangle indicates the location of map (b). Adapted after Louwye et al. (2020). (b) The geographical extent of the Lillo Formation in Belgium, after Louwye et al. (2020), and the location of the city of Antwerp. (c) Lithological column of the Lillo Formation and its members with their chronostratigraphic position, after Louwye et al. (2020). The approximate stratigraphic position of the A benedeni benedeni specimens is shaded in grey. 130 Figure 1: Location of the modern North Sea basin and the stratigraphic level from which the A. benedeni benedeni specimens 125 originated. Dinoflagellate assemblages point to a warming trend within the Oorderen Member, with warm- temperate conditions in the upper part from which the specimens were collected (Louwye et al. 2004; De Schepper et al. 2009). 145 Biostratigraphically, the Oorderen Member lies within planktonic foraminifera biozone NPF16, benthic foraminifera biozone B12, otolith zone 19, and benthic mollusc BM22B biozone (Vandenberghe and Louwye 2020). The Oorderen Member has previously been interpreted as having been deposited during the mPWP (Louwye and De Schepper 2010; Valentine et al. 2011), and Louwye et al. (2020) place the Oorderen at ca. 3.71-3.15 Ma. This places its top, from which the specimens were collected, within the 3.264-3.025 Ma mid Piacenzian Warm Period (mPWP) as defined by Dowsett et al. (2010). 150 2009). 145 Biostratigraphically, the Oorderen Member lies within planktonic foraminifera biozone NPF16, benthic foraminifera biozone B12, otolith zone 19, and benthic mollusc BM22B biozone (Vandenberghe and Louwye 2020). The Oorderen Member has previously been interpreted as having been deposited during the mPWP (Louwye and De Schepper 2010; Valentine et al. 2011), and Louwye et al. (2020) place the Oorderen at ca. 3.71-3.15 Ma. This places its top, from which the specimens were collected, within the 3.264-3.025 Ma mid Piacenzian Warm Period (mPWP) as defined by Dowsett et al. (2010). 150 2.4 Angulus benedeni benedeni Angulus benedeni benedeni (Nyst and Westendorp 1839) (family Tellinidae) is one of the characteristic molluscs of the Oorderen Member of the Pliocene Lillo Formation in Belgium, with an acme in the clayey upper part of the member (Marquet 2005; Marquet and Herman 2009). It is the youngest member of an extinct North Sea basin lineage starting in the late Eocene, with this subspecies emerging in the Pliocene (Marquet 2005; Moerdijk et al. 2010). It has been found in 155 Belgium, the Netherlands, and the southeastern United Kingdom (Marquet 2005). Angulus benedeni benedeni has not been used in palaeoclimatological research before. Therefore, its potential as a climate archive is unknown, as is its life history: the maximum age it could reach, its growth rate and how this rate changed throughout its life, the amount of time represented in one growth increment, and possible periods of growth cessation. (a) Overview map of the location of Belgium and the present-day extent of the North Sea within northwest Europe. The dark rectangle indicates the location of map (b). Adapted after Louwye et al. (2020). (b) The geographical extent of the Lillo Formation in Belgium, after Louwye et al. (2020), and the location of the city of Antwerp. (c) Lithological column of the Lillo Formation and its members with their chronostratigraphic position, after Louwye et al. (2020). The approximate stratigraphic position of the A. benedeni benedeni specimens is shaded in grey. 130 glauconiferous quartz sand matrix as well as arranged into a number of cm to dm thick shell beds. Three intervals characterised by different sedimentary structures (respectively: throughs and storm beds, predominantly homogenised sand, predominantly bioturbated clayey sand) and mollusc fauna composition (respectively frequent occurrence of: Atrina fragilis pos o o e spec e s s s aded g ey 30 glauconiferous quartz sand matrix as well as arranged into a number of cm to dm thick shell beds. Three intervals characterised by different sedimentary structures (respectively: throughs and storm beds, predominantly homogenised sand, predominantly bioturbated clayey sand) and mollusc fauna composition (respectively frequent occurrence of: Atrina fragilis glauconiferous quartz sand matrix as well as arranged into a number of cm to dm thick shell beds. Three intervals characterised by different sedimentary structures (respectively: throughs and storm beds, predominantly homogenised sand, predominantly bioturbated clayey sand) and mollusc fauna composition (respectively frequent occurrence of: Atrina fragilis 6 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. kalloensis, Cultellus cultellatus, Angulus benedeni benedeni), and separated by shell beds that experienced load coasting, can be recognised all over the Port of Antwerp area, namely the Atrina level, the Cultellus interval and the Angulus interval (see 135 e.g., Vervoenen, Herman, and Van Waes 1995; Marquet and Herman 2009). The specimens have been collected from the upper bioturbated Angulus clayey sand interval. The Oorderen Member has been interpreted to represent a neritic environment (Louwye et al. 2004; De Schepper et al. 2009). Marquet (2004) estimated a water depth of 35-45 m based on bivalve depth ranges, but the sedimentology of the Oorderen suggests a shallower depth. The member contains storm beds, cross-stratification, tidal structures, and evidence of periods of emergence (Louwye et al. 2004; Louwye et al. 2020). We 140 therefore estimate a paleo water depth of 20 m. The increasing amount of clay towards the top of the Oorderen has been interpreted to reflect a transition from calm water to a more high-energy tidal environment with clayey tidal lag deposits (Louwye et al. 2004). Dinoflagellate assemblages point to a warming trend within the Oorderen Member, with warm- temperate conditions in the upper part from which the specimens were collected (Louwye et al. 2004; De Schepper et al. cross-stratification, tidal structures, and evidence of periods of emergence (Louwye et al. 2004; Louwye et al. 2020). We 140 therefore estimate a paleo water depth of 20 m. The increasing amount of clay towards the top of the Oorderen has been interpreted to reflect a transition from calm water to a more high-energy tidal environment with clayey tidal lag deposits (Louwye et al. 2004). Dinoflagellate assemblages point to a warming trend within the Oorderen Member, with warm- temperate conditions in the upper part from which the specimens were collected (Louwye et al. 2004; De Schepper et al. cross-stratification, tidal structures, and evidence of periods of emergence (Louwye et al. 2004; Louwye et al. 2020). We 140 therefore estimate a paleo water depth of 20 m. The increasing amount of clay towards the top of the Oorderen has been interpreted to reflect a transition from calm water to a more high-energy tidal environment with clayey tidal lag deposits (Louwye et al. 2004). 3.2 X-ray diffraction To reconstruct accurate growth temperatures through clumped isotope analysis, we had to To reconstruct accurate growth temperatures through clumped isotope analysis, we had to verify that the original aragonitic mineralogy was preserved. To check for diagenetic remineralization of original aragonite into calcite, powder X-ray 170 diffraction (XRD) analysis of a representative section of one A. benedeni benedeni specimen (SG-125) was carried out on a Bruker D8 Advance diffractometer at Utrecht University that had been calibrated with a corundum crystal. The sample was ground to < 10μm in a Retsch McCrone mill with zirconium oxide grinding elements prior to front-loading it into a PMMA sample holder (diameter: 25 mm; depth: 1 mm). The instrument settings are given in table 1. The A. benedeni benedeni sample holder (diameter: 25 mm; depth: 1 mm). The instrument settings are given in table 1. The A. benedeni benedeni pattern was compared to XRD spectra of pure aragonite and calcite samples. The calcite spectrum was recovered from the 175 RRUFF database (Lafuente et al. 2015; RRUFF ID: R040070) and consisted of a powdered sample of a single calcite mineral from Pryor Mountain, Big Horn Country, Montana (USA) measured for X-Ray Diffraction at the University of Arizona Mineral Museum. For the pure aragonite spectrum, 1.12 g of shell from a common cockle (Cerastoderma edule) collected at the North Sea coast near Hoek van Holland (51°59’14” N, 4°05’56” E) was prepared and measured under the same conditions as the fossil samples. XRD spectra of specimen SG-125 as well as the aragonite spectrum are available in 180 the supplementary materials (see data availability). pattern was compared to XRD spectra of pure aragonite and calcite samples. The calcite spectrum was recovered from the 175 RRUFF database (Lafuente et al. 2015; RRUFF ID: R040070) and consisted of a powdered sample of a single calcite mineral from Pryor Mountain, Big Horn Country, Montana (USA) measured for X-Ray Diffraction at the University of Arizona Mineral Museum. For the pure aragonite spectrum, 1.12 g of shell from a common cockle (Cerastoderma edule) collected at the North Sea coast near Hoek van Holland (51°59’14” N, 4°05’56” E) was prepared and measured under the same conditions as the fossil samples XRD spectra of specimen SG 125 as well as the aragonite spectrum are available in 180 Table 1. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. 0.3 µm aluminium oxide. These thick sections were viewed under a KEYENCE VHX-5000 digital microscope at x250 and 165 x1000 magnification. Light microscopy composite images were then made with the Fiji stitching ImageJ plugin (Preibisch et al. 2009). These composites were used to count the growth increments. 0.3 µm aluminium oxide. These thick sections were viewed under a KEYENCE VHX-5000 digital microscope at x250 and 165 x1000 magnification. Light microscopy composite images were then made with the Fiji stitching ImageJ plugin (Preibisch et al. 2009). These composites were used to count the growth increments. 3.2 X-ray diffraction X-ray diffraction measurement settings Instrument setting Value Voltage [kV] 40 Ampere [mA] 40 Radiation [Å] 1.5418 (CuKα) Divergence slit [mm] 0.165 Primary soller slit [°] 2.5 Secondary soller slit [°] not present Measuring range [°2θ] 15-70 Step-size [°2θ] 0.02 Counting time [s] 0.85 Sample rotation [rpm] 15 3.1 Light microscopy 7 Light microscopy was employed on all specimens to visually check for potential diagenetic alteration, to analyse the macrostructures of A. benedeni benedeni’s shell, and to count growth increments. Two specimens were partially or fully embedded in Araldite 2020 epoxy and high-polish thick (approximately 5 mm) sections were made, using a final polish of 7 This setting allows for enough time to reach the Time of Stable Reproducibility and provides the optimal balance between longer measurement time 195 (resulting in better defined XRF spectra) and higher sample sizes (see discussion in de Winter et al. 2017b). Spectra were quantified using on-line calibration with the matrix-matched standard BAS-CRM393 (Bureau of Analyzed Samples, Middlesborough, UK) in the M4 software (following de Winter et al. 2017b). After quantification, the trace element results were calibrated using an off-line calibration constructed using a set of matrix-matched carbonate standards (CCH-1; enough time to reach the Time of Stable Reproducibility and provides the optimal balance between longer measurement time 195 (resulting in better defined XRF spectra) and higher sample sizes (see discussion in de Winter et al. 2017b). Spectra were quantified using on-line calibration with the matrix-matched standard BAS-CRM393 (Bureau of Analyzed Samples, Middlesborough, UK) in the M4 software (following de Winter et al. 2017b). After quantification, the trace element results were calibrated using an off-line calibration constructed using a set of matrix-matched carbonate standards (CCH-1; Université de Liège, Belgium, COQ-1; US Geological Survey, Denver, CO, USA, CRM393, CRM512, CRM513, 200 ECRM782; Bureau of Analyzed Samples, Middlesborough, UK; and SRM-1d; National Institute of Standards and Technology, Gaithersburg, MD, USA; see e.g. de Winter et al. 2021). All major and trace element data can be found in the supplementary materials (see data availability). Université de Liège, Belgium, COQ-1; US Geological Survey, Denver, CO, USA, CRM393, CRM512, CRM513, 200 ECRM782; Bureau of Analyzed Samples, Middlesborough, UK; and SRM-1d; National Institute of Standards and Technology, Gaithersburg, MD, USA; see e.g. de Winter et al. 2021). All major and trace element data can be found in the supplementary materials (see data availability). 3.3 Micro-X-ray fluorescence For additional screening of diagenetic alteration, concentrations of major and trace elements (including Fe, Mn and Sr) in three specimen of A. benedeni benedeni (SG-125, SG-126, SG-127) were determined using non-destructive, energy- 185 185 8 3.4 Electron backscatter diffraction To determine which microstructures are present in A. benedeni benedeni and to identify the potential occurrences of minor, 205 localised diagenetic calcite that was not detected through XRD or XRF analysis, electronic backscatter diffraction (EBSD) was carried out on thin sections (ca. 40 µm) of all three specimens. EBSD involves the diffraction of electrons, generated by a SEM-based electron beam, off the crystal planes of a sample, forming a diffraction pattern related to the crystal lattice parameters. This allows for the precise determination of crystal orientation, shape, and size (e.g. Cusack 2016). EBSD To determine which microstructures are present in A. benedeni benedeni and to identify the potential occurrences of minor, 205 localised diagenetic calcite that was not detected through XRD or XRF analysis, electronic backscatter diffraction (EBSD) was carried out on thin sections (ca. 40 µm) of all three specimens. EBSD involves the diffraction of electrons, generated by a SEM-based electron beam, off the crystal planes of a sample, forming a diffraction pattern related to the crystal lattice parameters. This allows for the precise determination of crystal orientation, shape, and size (e.g. Cusack 2016). EBSD analysis was carried out using an Oxford Instruments Symmetry EBSD detector attached to a Zeiss Gemini 450 SEM at 210 Utrecht University. Thin sections of the samples were mechanically polished with 0.3 µm aluminium oxide suspension and finished with chemical Syton® polish. The thin sections were covered with a thin (several nm) carbon coating to keep charge build-up during the measurements at a minimum. Beam and map acquisition settings (voltage, beam current, dwell time, step size, high/low vacuum) were varied to obtain the optimal results for each map; the acquisition settings for the map shown analysis was carried out using an Oxford Instruments Symmetry EBSD detector attached to a Zeiss Gemini 450 SEM at 210 Utrecht University. Thin sections of the samples were mechanically polished with 0.3 µm aluminium oxide suspension and finished with chemical Syton® polish. The thin sections were covered with a thin (several nm) carbon coating to keep charge build-up during the measurements at a minimum. Beam and map acquisition settings (voltage, beam current, dwell time, step size, high/low vacuum) were varied to obtain the optimal results for each map; the acquisition settings for the map shown analysis was carried out using an Oxford Instruments Symmetry EBSD detector attached to a Zeiss Gemini 450 SEM at 210 Utrecht University. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. dispersive micro-X-ray fluorescence (µXRF) analysis. A benchtop Bruker M4 Tornado µXRF scanner was used for this, available at the Analytical, Environmental, and Geochemistry Research Group (AMGC) of the Vrije Universiteit Brussel (Brussels, Belgium). The Bruker M4 Tornado is equipped with a 30 W Rh anode metal-ceramic X-ray tube operated at 50 kV and 600 µA. A polycapillary lens focuses an X-ray beam on the polished sample surface on a spot with a diameter of 25 dispersive micro-X-ray fluorescence (µXRF) analysis. A benchtop Bruker M4 Tornado µXRF scanner was used for this, available at the Analytical, Environmental, and Geochemistry Research Group (AMGC) of the Vrije Universiteit Brussel (Brussels, Belgium). The Bruker M4 Tornado is equipped with a 30 W Rh anode metal-ceramic X-ray tube operated at 50 kV and 600 µA. A polycapillary lens focuses an X-ray beam on the polished sample surface on a spot with a diameter of 25 µm (calibrated for Mo kα radiation). Fluorescing X-ray radiation is detected using two 30 mm2 silicon drift detectors with a 190 spectral resolution of 135 eV (calibrated for Mn-kα radiation). The system is operated under near-vacuum conditions (20 mbar) to allow lower energy radiation of lighter elements (e.g., Mg) to be measured. More details on the µXRF measurement setup and acquisition are provided in de Winter and Claeys (2017) and Kaskes et al. (2021). X-ray spectra were produced using a spatial resolution of 25 um and a dwell time of 60 seconds This setting allows for µm (calibrated for Mo kα radiation). Fluorescing X-ray radiation is detected using two 30 mm2 silicon drift detectors with a 190 spectral resolution of 135 eV (calibrated for Mn-kα radiation). The system is operated under near-vacuum conditions (20 mbar) to allow lower energy radiation of lighter elements (e.g., Mg) to be measured. More details on the µXRF measurement setup and acquisition are provided in de Winter and Claeys (2017) and Kaskes et al. (2021). X-ray spectra were produced using a spatial resolution of 25 um and a dwell time of 60 seconds. This setting allows for 190 X-ray spectra were produced using a spatial resolution of 25 um and a dwell time of 60 seconds. 3.5.1 Sample preparation Samples of approximately 100-300 μg were taken from the outer surface of two specimens (SG-126 and SG-127) by drilling 225 into it from the exterior along commarginal paths. A Dremel model 225 hand-held drill was used, which was equipped with a tungsten carbide drill bit of 0.8 mm in diameter (Fig. A1). During sampling, care was taken not to drill too deep as to not drill into the inner shell layers. The distance from the umbo to the sample track at the axis of maximum growth was measured in planar view with digital callipers (Fig. A1). The entire shell height of specimen SG-126 was sampled (Fig. A1 a). A total of 55 samples was taken from specimen SG-126 and a total of 30 samples was taken from specimen SG-127 (Fig. 230 A1 b). The sampling resolution was 0.10 to 1.07 mm (mean: 0.48 mm, 1σ: 0.24 mm; no significant difference between the two specimens, p>0.05). a). A total of 55 samples was taken from specimen SG-126 and a total of 30 samples was taken from specimen SG-127 (Fig. 230 A1 b). The sampling resolution was 0.10 to 1.07 mm (mean: 0.48 mm, 1σ: 0.24 mm; no significant difference between the two specimens, p>0.05). a). A total of 55 samples was taken from specimen SG-126 and a total of 30 samples was taken from specimen SG-127 (Fig. 230 A1 b). The sampling resolution was 0.10 to 1.07 mm (mean: 0.48 mm, 1σ: 0.24 mm; no significant difference between the two specimens, p>0.05). 3.5.2 Oxygen isotope mass spectrometry Drilled samples from specimen SG-126 were first analysed for carbonate oxygen isotopes (δ18Oc) on a Thermo Scientific GasBench II gas preparation system coupled to a Thermo Scientific MAT 253 isotope ratio mass spectrometer. 50-100 μg of 235 powdered sample material was weighed on a Sartorius microbalance and placed in glass vials that were sealed off with barrier septa. 50-100 μg of two types of in-house standards, Naxos marble and Kiel carbonate, was weighed and placed in vials as well. Their accepted values are given in Table A1. Each sample was then flushed with helium for 5 minutes to remove atmospheric air. The samples were reacted with 103% phosphoric acid at 70°C. The produced CO2 gas was led through a series of cleaning vessels, consisting of a first water trap, a gas chromatograph, and a second water trap. It then 240 entered the mass spectrometer and masses 44, 45, and 46 were measured using a LIDI (long-integration dual-inlet) workflow (Hu et al. 2014). A Naxos standard was measured approximately every ten samples during the run. From the bivalve samples, every tenth sample was weighed in duplicate. These duplicates were measured at the end of the run. The carbon isotope data (δ13Cc) are reported only when it is relevant for the reproducibility of the analyses. All stable isotope GasBench data can be found in the supplementary materials (see data availability). Sample δ18Oc data were corrected for intensity- 245 dependent fractionation using a linear regression between δ18ONAXOS and mass 44 intensity (Fig. A2). No significant change in the Naxos standard values over time (also called “drift”) was observed during the measurement. The δ18Oc and δ13Cc data are reported relative to VPDB. Drilled samples from specimen SG-126 were first analysed for carbonate oxygen isotopes Drilled samples from specimen SG-126 were first analysed for carbonate oxygen isotopes (δ18Oc) on a Thermo Scientific GasBench II gas preparation system coupled to a Thermo Scientific MAT 253 isotope ratio mass spectrometer. 50-100 μg of 235 powdered sample material was weighed on a Sartorius microbalance and placed in glass vials that were sealed off with barrier septa. 50-100 μg of two types of in-house standards, Naxos marble and Kiel carbonate, was weighed and placed in vials as well. Their accepted values are given in Table A1. Each sample was then flushed with helium for 5 minutes to remove atmospheric air. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. grain boundary threshold was set to 10°. Contoured pole figures were plotted to visualise the main crystallite orientations (Fig. 5f). For these pole figures, multiples of uniform density (MUD) values were calculated. The MUD value is an 220 indication of the degree of clustering of the poles relative to a random distribution: the higher the MUD, the stronger the co- orientation (or preferred orientation) of the crystals. 3.4 Electron backscatter diffraction Thin sections of the samples were mechanically polished with 0.3 µm aluminium oxide suspension and finished with chemical Syton® polish. The thin sections were covered with a thin (several nm) carbon coating to keep charge build-up during the measurements at a minimum. Beam and map acquisition settings (voltage, beam current, dwell time, step size, high/low vacuum) were varied to obtain the optimal results for each map; the acquisition settings for the map shown here are indicated in Fig. 5. Data processing was done in the Oxford Instruments AZtecCrystal software. Data clean-up 215 consisted of wild spike removal followed by filling in unclassified areas using an iterative nearest neighbour procedure from eight down to six nearest neighbours. The map presented here had 90% indexing after this step. The raw data of this map, as well as additional maps that are not presented here, can be found in the supplementary materials (see data availability). The here are indicated in Fig. 5. Data processing was done in the Oxford Instruments AZtecCrystal software. Data clean-up 215 consisted of wild spike removal followed by filling in unclassified areas using an iterative nearest neighbour procedure from eight down to six nearest neighbours. The map presented here had 90% indexing after this step. The raw data of this map, as well as additional maps that are not presented here, can be found in the supplementary materials (see data availability). The 9 3.5.2 Oxygen isotope mass spectrometry The samples were reacted with 103% phosphoric acid at 70°C. The produced CO2 gas was led GasBench II gas preparation system coupled to a Thermo Scientific MAT 253 isotope ratio mass spectrometer. 50-100 μg of 235 powdered sample material was weighed on a Sartorius microbalance and placed in glass vials that were sealed off with barrier septa. 50-100 μg of two types of in-house standards, Naxos marble and Kiel carbonate, was weighed and placed in vials as well. Their accepted values are given in Table A1. Each sample was then flushed with helium for 5 minutes to remove atmospheric air. The samples were reacted with 103% phosphoric acid at 70°C. The produced CO2 gas was led through a series of cleaning vessels, consisting of a first water trap, a gas chromatograph, and a second water trap. It then 240 entered the mass spectrometer and masses 44, 45, and 46 were measured using a LIDI (long-integration dual-inlet) workflow (Hu et al. 2014). A Naxos standard was measured approximately every ten samples during the run. From the bivalve samples, every tenth sample was weighed in duplicate. These duplicates were measured at the end of the run. The carbon isotope data (δ13Cc) are reported only when it is relevant for the reproducibility of the analyses. All stable isotope GasBench through a series of cleaning vessels, consisting of a first water trap, a gas chromatograph, and a second water trap. It then 240 entered the mass spectrometer and masses 44, 45, and 46 were measured using a LIDI (long-integration dual-inlet) workflow (Hu et al. 2014). A Naxos standard was measured approximately every ten samples during the run. From the bivalve samples, every tenth sample was weighed in duplicate. These duplicates were measured at the end of the run. The carbon isotope data (δ13Cc) are reported only when it is relevant for the reproducibility of the analyses. All stable isotope GasBench data can be found in the supplementary materials (see data availability). Sample δ18Oc data were corrected for intensity- 245 dependent fractionation using a linear regression between δ18ONAXOS and mass 44 intensity (Fig. A2). No significant change in the Naxos standard values over time (also called “drift”) was observed during the measurement. The δ18Oc and δ13Cc data are reported relative to VPDB. data can be found in the supplementary materials (see data availability). https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. 3.5.3 Clumped isotopes mass spectrometry Due to the near-equilibrium precipitation of bivalves, we can use clumped isotope analysis to reconstruct seasonal water 250 temperatures from their carbonate shells (Huyghe et al. 2022). Clumped isotope analysis on carbonate involves analysing the abundance of 18O-13C bonds and has several advantages compared to conventional oxygen stable isotope (δ18Oc) temperature reconstruction. The deviation of the occurrence of these heavy 18O-13C bonds from the expected stochastic distribution is thermodynamically determined and thus independent of the isotopic composition of oxygen of the seawater (δ18Osw) and carbon of the DIC (δ13C) (e.g., Ghosh et al. 2006; Schauble et al. 2006). This deviation, termed Δ47, is given in per mil and 255 defined as follows: carbon of the DIC (δ13C) (e.g., Ghosh et al. 2006; Schauble et al. 2006). This deviation, termed Δ47, is given in per mil and 255 defined as follows: ∆47 = [( 𝑅47 𝑅47∗−1) −( 𝑅46 𝑅46∗−1) −( 𝑅45 𝑅45∗−1)] × 1000 (1) (1) in which Rn is the abundance ratio of isotopic mass n over mass 44 (the most abundant mass, formed by 12C16O16O) in the 260 sample relative to a standard, and Rn* is the theoretical abundance ratio of mass n over mass 44 in the sample relative to a standard if that sample were to have a stochastic distribution (Eiler 2007). Since the value of Δ47 depends only on formation temperature, it allows precise temperature reconstruction without knowledge of δ18Osw, which is required for the more traditional palaeothermometer based only on the δ18O of shells (δ18Oc). This independence from δ18Osw is especially in which Rn is the abundance ratio of isotopic mass n over mass 44 (the most abundant mass, formed by 12C16O16O) in the 260 sample relative to a standard, and Rn* is the theoretical abundance ratio of mass n over mass 44 in the sample relative to a standard if that sample were to have a stochastic distribution (Eiler 2007). Since the value of Δ47 depends only on formation temperature, it allows precise temperature reconstruction without knowledge of δ18Osw, which is required for the more traditional palaeothermometer based only on the δ18O of shells (δ18Oc). This independence from δ18Osw is especially important for the mid-Piacenzian Warm Period, as the size of the ice sheets—which store light 16O and thus drive up the 265 δ18O of the oceans—is not well constrained for this period (e.g., Dowsett et al. 2016). 3.5.2 Oxygen isotope mass spectrometry Sample δ18Oc data were corrected for intensity- 245 dependent fractionation using a linear regression between δ18ONAXOS and mass 44 intensity (Fig. A2). No significant change in the Naxos standard values over time (also called “drift”) was observed during the measurement. The δ18Oc and δ13Cc data are reported relative to VPDB. 10 3.5.3 Clumped isotopes mass spectrometry It is also relevant for the shallow- marine waters in which bivalves often live, as the δ18Osw can be influenced by changing influxes in isotopically light river water (e.g., Schöne et al. 2004; Chauvaud et al. 2005; Johnson et al. 2009) Clumped isotope values (Δ47) were analysed using a Kiel IV carbonate device coupled to a Thermo Scientific MAT 253 Plus important for the mid-Piacenzian Warm Period, as the size of the ice sheets—which store light 16O and thus drive up the 265 δ18O of the oceans—is not well constrained for this period (e.g., Dowsett et al. 2016). It is also relevant for the shallow- marine waters in which bivalves often live, as the δ18Osw can be influenced by changing influxes in isotopically light river water (e.g., Schöne et al. 2004; Chauvaud et al. 2005; Johnson et al. 2009) Clumped isotope values (Δ47) were analysed using a Kiel IV carbonate device coupled to a Thermo Scientific MAT 253 Plus Clumped isotope values (Δ47) were analysed using a Kiel IV carbonate device coupled to a Thermo Scientific MAT 253 Plus isotope ratio mass spectrometer (available at Utrecht University) with a LIDI workflow (Hu et al. 2014; Müller et al. 2017) 270 and following the protocol described by Meckler et al. (2014). For specimen SG-126, analysis was focussed on samples with δ18Oc values in the ranges 2.0-3.3‰ (close to the maximum) and 0.4-1.2‰ (close to the minimum), based on the previously measured samples (sect. 3.5.2). From specimen SG-127, all samples were analysed. “Sample” here refers to the total amount of powder drilled from a single sampling track (Fig. A1), whereas “aliquot”, “measurement”, or “datapoint” refers to a small isotope ratio mass spectrometer (available at Utrecht University) with a LIDI workflow (Hu et al. 2014; Müller et al. 2017) 270 and following the protocol described by Meckler et al. (2014). For specimen SG-126, analysis was focussed on samples with δ18Oc values in the ranges 2.0-3.3‰ (close to the maximum) and 0.4-1.2‰ (close to the minimum), based on the previously measured samples (sect. 3.5.2). From specimen SG-127, all samples were analysed. “Sample” here refers to the total amount of powder drilled from a single sampling track (Fig. A1), whereas “aliquot”, “measurement”, or “datapoint” refers to a small amount of powder taken from a sample and measured for stable isotopes. Multiple aliquots were measured from each 275 sample. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. Merck and IAEA-C2 were not involved in data processing (treated as samples) and used to assess long-term measuring uncertainty. Merck and IAEA-C2 were not involved in data processing (treated as samples) and used to assess long-term measuring uncertainty. traps (-170°C) and a Porapak trap (-50°C). It then entered the mass spectrometer and masses 44-49 were measured against a 285 reference gas of known composition (δ18O = −4.67‰, δ13C = −2.82‰). A negative baseline correction proportional to the mass 44 intensity was performed for all masses. The calculated δ values were then corrected for 17O through the method presented in Brand et al. (2010). The Δ47 values were calculated and averaged over the 40 pulses as per the LIDI system. Clumped results were corrected for drift through bracketing with ETH-3 standards. An empirical transfer function (ETF) was traps (-170°C) and a Porapak trap (-50°C). It then entered the mass spectrometer and masses 44-49 were measured against a 285 reference gas of known composition (δ18O = −4.67‰, δ13C = −2.82‰). A negative baseline correction proportional to the mass 44 intensity was performed for all masses. The calculated δ values were then corrected for 17O through the method presented in Brand et al. (2010). The Δ47 values were calculated and averaged over the 40 pulses as per the LIDI system. Clumped results were corrected for drift through bracketing with ETH-3 standards. An empirical transfer function (ETF) was constructed by regressing the raw ETH-1, ETH-2, and ETH-3 values over their accepted Δ47 values (Bernasconi et al. 2021) 290 and using the resulting linear regression line to transfer the sample Δ47 values to the I-CDES90°C reference frame (Fig. A3). For the ETF, 403 ETH standards from multiple runs measured over the span of several weeks were used for linear regression (53 ETH-1, 64 ETH-2, 286 ETH-3; Fig. A4). No acid fractionation correction was necessary even though the samples were reacted at 70°C, as this offset is already incorporated into the new values of the ETH standards in the I-CDES reference constructed by regressing the raw ETH-1, ETH-2, and ETH-3 values over their accepted Δ47 values (Bernasconi et al. 2021) 290 and using the resulting linear regression line to transfer the sample Δ47 values to the I-CDES90°C reference frame (Fig. A3). 3.5.3 Clumped isotopes mass spectrometry 75-95 μg of powdered material was weighed with a Mettler Toledo microbalance or a Sartorius microbalance and placed in glass vials. Similarly weighed carbonate standards were measured in an approximately one-to-one ratio to the standards in each run (22 samples, 24 standards; Kocken et al. 2019). These standards were two control standards—Merck CaCO3 (synthetic; product code 1.02059.0050) and IAEA-C2 (Bavarian travertine)—as well as ETH-1, ETH-2, and ETH-3 amount of powder taken from a sample and measured for stable isotopes. Multiple aliquots were measured from each 275 sample. 75-95 μg of powdered material was weighed with a Mettler Toledo microbalance or a Sartorius microbalance and placed in glass vials. Similarly weighed carbonate standards were measured in an approximately one-to-one ratio to the standards in each run (22 samples, 24 standards; Kocken et al. 2019). These standards were two control standards—Merck CaCO3 (synthetic; product code 1.02059.0050) and IAEA-C2 (Bavarian travertine)—as well as ETH-1, ETH-2, and ETH-3 (Bernasconi et al. 2018; 2021). Their composition is given in Table A1. The ETH standards, which have varying Δ47, δ18Oc, 280 and δ13C compositions, were used to transfer the sample Δ47 values to the I-CDES reference frame (Bernasconi et al. 2021). (Bernasconi et al. 2018; 2021). Their composition is given in Table A1. The ETH standards, which have varying Δ47, δ18Oc, 280 and δ13C compositions, were used to transfer the sample Δ47 values to the I-CDES reference frame (Bernasconi et al. 2021). 11 For the ETF, 403 ETH standards from multiple runs measured over the span of several weeks were used for linear regression (53 ETH-1, 64 ETH-2, 286 ETH-3; Fig. A4). No acid fractionation correction was necessary even though the samples were reacted at 70°C, as this offset is already incorporated into the new values of the ETH standards in the I-CDES reference frame (Bernasconi et al. 2021). 295 The δ18Oc and δ13C data of the same measurements were corrected through a 15-point running average to eliminate long- term trends present within the δ18Oc and δ13C raw data. Mass-dependent fractionation occurred in some samples, likely due to the loss of a fraction of the CO2 through leakage. This is evident from a correlation between δ18Oc and δ13C (these runs are marked in Fig. A4). The δ18Oc and δ13C data from these runs were not used for the running average correction nor were they used in the δ18Oc records of A. benedeni benedeni, as their isotopic composition no longer reflected the original signal. 300 As Δ47 is calculated as the deviation from a stochastic distribution for a given δ18Oc and δ13C composition, it was not influenced by this fractionation. The final δ18Oc and δ13C values were reported relative to VPDB. Samples that showed a strong drift during the 40 LIDI measurement pulses, had a low intensity, a high standard deviation, or showed signs of contamination were deemed unreliable and were removed from the dataset. The intensity cut-off was <9.0 V. The cut-off for they used in the δ18Oc records of A. benedeni benedeni, as their isotopic composition no longer reflected the original signal. 300 As Δ47 is calculated as the deviation from a stochastic distribution for a given δ18Oc and δ13C composition, it was not influenced by this fractionation. The final δ18Oc and δ13C values were reported relative to VPDB. Samples that showed a strong drift during the 40 LIDI measurement pulses, had a low intensity, a high standard deviation, or showed signs of contamination were deemed unreliable and were removed from the dataset. The intensity cut-off was <9.0 V. The cut-off for standard deviations in Δ47 data within the 40 measurement pulses was >0.10‰ for both standards and samples. 305 Contamination evident from increased intensity on the mass 49 cup, was quantified using the 49 parameter—the ratio between mass 49 and mass 44 intensities—and a cut-off of >0.1 was used for standards, and >0.2 for samples. All standards used for correction, as well as the IAEA-C2 and Merck values and the samples, can be found in the supplementary materials (see data availability). After removing erroneous samples, 103 sample measurements remained. standard deviations in Δ47 data within the 40 measurement pulses was >0.10‰ for both standards and samples. 305 Contamination evident from increased intensity on the mass 49 cup, was quantified using the 49 parameter—the ratio between mass 49 and mass 44 intensities—and a cut-off of >0.1 was used for standards, and >0.2 for samples. All standards used for correction, as well as the IAEA-C2 and Merck values and the samples, can be found in the supplementary materials (see data availability). After removing erroneous samples, 103 sample measurements remained. To obtain warm vs cold datasets from the clumped isotope data, the samples were grouped based on their δ18Oc values, with 310 low values corresponding to warm temperatures and vice versa. The Δ47 data from the same aliquots were then averaged to obtain warm and cold average temperatures. As individual Δ47 measurements have a large uncertainty, Δ47 datapoints cannot be split into meaningful “warm” and “cold” groups directly. The choice of a cut-off value for warm and cold δ18Oc values is a trade-off between confidence and seasonality: A cut-off close to the average δ18Oc value results in a higher sample size and a narrower confidence interval for the resulting temperature reconstruction. Such a cut-off, however, also results in averaging 315 To obtain warm vs cold datasets from the clumped isotope data, the samples were grouped based on their δ18Oc values, with 310 low values corresponding to warm temperatures and vice versa. The Δ47 data from the same aliquots were then averaged to obtain warm and cold average temperatures. As individual Δ47 measurements have a large uncertainty, Δ47 datapoints cannot be split into meaningful “warm” and “cold” groups directly. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. samples from a large part of the year and thus a dampening of the seasonality range. For a cut-off that only includes the lowest and highest δ18Oc values, the opposite is true. To find the best compromise between confidence and seasonality, the average warm and cold Δ47 values and their associated 95% confidence levels were plotted for a range of δ18Oc-based cut- offs (see also Fig. 9). A Student’s t-test was used to determine whether cold and warm datasets were statistically different at a 95% confidence level. Instead of looking at each A. benedeni benedeni specimen individually, δ18Oc data from both 0 specimens were combined in order to improve the statistics. samples from a large part of the year and thus a dampening of the seasonality range. For a cut-off that only includes the lowest and highest δ18Oc values, the opposite is true. To find the best compromise between confidence and seasonality, the average warm and cold Δ47 values and their associated 95% confidence levels were plotted for a range of δ18Oc-based cut- offs (see also Fig. 9). A Student’s t-test was used to determine whether cold and warm datasets were statistically different at a 95% confidence level. Instead of looking at each A. benedeni benedeni specimen individually, δ18Oc data from both specimens were combined in order to improve the statistics. 320 3.6 Palaeotemperature reconstruction (1999): 𝛿18𝑂𝑠𝑤 [‰VSMOW] = 𝛿18𝑂𝑐 [‰VPDB] − 20.6−𝑇[℃] 4.34 + 0.2 (3) 340 𝛿18𝑂𝑐 [‰VPDB] − 20.6−𝑇[℃] 4.34 + 0.2 (3) 𝛿18𝑂𝑠𝑤 [‰VSMOW] = 𝛿18𝑂𝑐 [‰VPDB] − 20.6−𝑇[℃] 4.34 + 0.2 (3) (3) 340 This average δ18Osw, the average Δ47-based temperature, and minimum and maximum δ18Oc values were then re-inserted into this equation to obtain δ18Oc-based summer and winter temperatures. This was done as there were not enough clumped isotope data to reconstruct Δ47-based summer and winter temperatures (see section 4.4.3 and 4.4.4). This average δ18Osw, the average Δ47-based temperature, and minimum and maximum δ18Oc values were then re-inserted into this equation to obtain δ18Oc-based summer and winter temperatures. This was done as there were not enough clumped isotope data to reconstruct Δ47-based summer and winter temperatures (see section 4.4.3 and 4.4.4). The choice of a cut-off value for warm and cold δ18Oc values is a trade-off between confidence and seasonality: A cut-off close to the average δ18Oc value results in a higher sample size and fid i l f h l i i S h ff h l l i i 315 a narrower confidence interval for the resulting temperature reconstruction. Such a cut-off, however, also results in averaging 315 12 3.6 Palaeotemperature reconstruction Δ47 values were converted to temperatures using the temperature transfer function of Meinicke et al. (2020; 2021): Δ47 values were converted to temperatures using the temperature transfer function of Meinicke et Δ47 values were converted to temperatures using the temperature transfer function of Meinicke et al. (2020; 2021): 𝛥 47[‰ ICDES90°𝐶] = 0.0397 ± 0.0011 ∗106 ∗𝑇−2[𝐾] + 0.1518 ± 0.0128 (2) 𝛥 47[‰ ICDES90°𝐶] = 0.0397 ± 0.0011 ∗106 ∗𝑇−2[𝐾] + 0.1518 ± 0.0128 325 (2) (2) This temperature transfer function is based on foraminifera and therefore more suited to the relatively low temperature range in which bivalves live. Since this function for Δ47 is not linear, errors associated with the Δ47 data were propagated using a Monte Carlo simulation (N = 105) of uncertainty in slope and intercept of the temperature transfer function as well as ( ) y p p p measurement uncertainty on Δ47 values assuming a normal uncertainty distribution. The standard deviation, standard error, 330 and 95% confidence level were then calculated from this normally distributed simulated temperature dataset. All calculations can be found in the supplementary materials (see code availability). Uncertainties on temperatures were calculated after temperature conversion. Since the temperature transfer function for clumped isotopes is not linear, this introduces a bias, specifically towards warmer temperatures. Therefore, the temperature means were calculated from averaging the Δ47 values measurement uncertainty on Δ47 values assuming a normal uncertainty distribution. The standard deviation, standard error, 330 and 95% confidence level were then calculated from this normally distributed simulated temperature dataset. All calculations can be found in the supplementary materials (see code availability). Uncertainties on temperatures were calculated after temperature conversion. Since the temperature transfer function for clumped isotopes is not linear, this introduces a bias, specifically towards warmer temperatures. Therefore, the temperature means were calculated from averaging the Δ47 values instead, and the Monte Carlo-generated errors were transferred to these mean temperatures. 335 The δ18O of the seawater (δ18Osw) was calculated from Δ47-based temperatures and δ18Oc data using the temperature transfer function for aragonite of Grossman and Ku (1986), modified by Dettman et al. (1999): The δ18O of the seawater (δ18Osw) was calculated from Δ47-based temperatures and δ18Oc data using the temperature transfer function for aragonite of Grossman and Ku (1986), modified by Dettman et al. 3.7 Growth models To gain insights into the nature of the growth of A. benedeni benedeni, two types of growth models were fitted to the growth 345 data of specimen SG-126: The Von Bertalanffy growth function (VBGF), a logarithmic model, and the Gompertz equation, a To gain insights into the nature of the growth of A. benedeni benedeni, two types of growth models were fitted to the growth 345 data of specimen SG-126: The Von Bertalanffy growth function (VBGF), a logarithmic model, and the Gompertz equation, a 13 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. logistic model. Both are widely used for estimating and analysing growth in modern species (e.g., Lee et al. 2020). The built- in nls() function in R (R Core Team 2021) was used to fit the data to both growth functions (see code availability). This function calculates the nonlinear least-squares estimates of the parameters of a nonlinear model that is defined by the user. logistic model. Both are widely used for estimating and analysing growth in modern species (e.g., Lee et al. 2020). The built- in nls() function in R (R Core Team 2021) was used to fit the data to both growth functions (see code availability). This function calculates the nonlinear least-squares estimates of the parameters of a nonlinear model that is defined by the user. The VBGF and the Gompertz equations are restrictive in terms of how much their shape can change, and much more 350 sophisticated approaches are available today (Lee et al. 2020). However, as this study has only one specimen available for growth model fitting. and since it concerns a fossil species, we believe that this simple approach is appropriate. The VBGF and the Gompertz equations are restrictive in terms of how much their shape can change, and much more 350 sophisticated approaches are available today (Lee et al. 2020). However, as this study has only one specimen available for growth model fitting. and since it concerns a fossil species, we believe that this simple approach is appropriate. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. following Bieler et al. (2014), where M+ and M- denote layers external and internal relative to the pallial myostracum (M). following Bieler et al. (2014), where M+ and M- denote layers external and internal relative to the pallial myostracum (M). g ( ), y p y ( ) Figure 2: Digital microscopy images of shell layers in the A. benedeni benedeni shells. Layers are marked 1-4, growth increments are indicated with dark blue dotted lines, dog = direction of growth. (a) section of SG-127 indicating all four layers, magnification of x250. 1: layer M+2 with crescent-shaped growth increments. 2: layer M+1 with low-angle growth increments and prism-like crystals parallel to the layer thickness. 3: layer M-1 with horizontal, parallel growth increments. 4: layer M-2, dark brown with horizontal, parallel growth increments (difficult to see in image a due to a similar tone to the background). (b) section of SG-126 showing a part of layer M+2 in more detail, magnification of x500. (c) section of SG-126 showing a part of layer M-2 at a better contrast, magnification of x250. (d) schematic overview of the four shell layers and their positions relative to each other and the pallial myostracum (M). In brackets is the equivalent of the layer name in the terminology of Popov (1986). Figure 2: Digital microscopy images of shell layers in the A. benedeni benedeni shells. Layers are marked 1-4, growth increments 360 are indicated with dark blue dotted lines, dog = direction of growth. (a) section of SG-127 indicating all four layers, magnification of x250. 1: layer M+2 with crescent-shaped growth increments. 2: layer M+1 with low-angle growth increments and prism-like crystals parallel to the layer thickness. 3: layer M-1 with horizontal, parallel growth increments. 4: layer M-2, dark brown with horizontal, parallel growth increments (difficult to see in image a due to a similar tone to the background). (b) section of SG-126 showing a part of layer M+2 in more detail, magnification of x500. (c) section of SG-126 showing a part of layer M-2 at a better 365 contrast, magnification of x250. (d) schematic overview of the four shell layers and their positions relative to each other and the pallial myostracum (M). 4.1.1 Structural analysis 355 The material of all three specimens looks pristine: the narrow increments are sharp and clearly visible, and there are no signs of dissolution or recrystallization (Fig. 2a-c). Four layers are visible in each specimen (Fig. 2a, d). These layers are named 14 14 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. Their equivalent to another common naming scheme (e.g., Popov 1986; 2014; Milano et al. 2017) is shown in Fig. 2d. Starting from the outer surface, these four layers are: 1) a thin (ca. 100 μm) white-to-grey coloured layer with crescent- shaped growth increments, termed M+2; 2) a ca. 200 μm thick layer (measured at the thickest point, halfway along the shell 370 height) with low-angle growth increments, a white-beige colour, and a prism-like structure that is oriented parallel to the layer thickness, termed M+1; 3) a thick (ca. 1000 μm) layer with clear growth increments parallel to the layer boundaries and a white-beige colour, termed M-1; and 4) a thin (ca. 50 μm) brown-coloured layer with horizontal, parallel growth increments, termed M-2. Layer M+2 extends from the ventral margin to near the hinge, where it thins and ultimately disappears. Layer M+1 thickens toward the ventral margin. Layer M-1 forms most of the hinge. It thins toward the ventral 375 margin. Layer M-2 covers the inside of the shell. It starts at the umbo and extends to the ventral margin. disappears. Layer M+1 thickens toward the ventral margin. Layer M-1 forms most of the hinge. It thins toward the ventral 375 margin. Layer M-2 covers the inside of the shell. It starts at the umbo and extends to the ventral margin. In brackets is the equivalent of the layer name in the terminology of Popov (1986). 15 4.2 X-ray based analyses X-ray diffraction analysis (XRD) and micro-X-ray fluorescence (μXRF) point analysis indicate that the shells of A. benedeni benedeni were not diagenetically altered. XRD analysis revealed that the shell of A. benedeni benedeni consists of 100% 390 original aragonite, as its spectrum is identical to that of pure aragonite (Fig. 4a). Micro-X-ray fluorescence (μXRF) point analyses following the methodology in de Winter et al. (2017b) supports this interpretation, as the specimen is high in Sr, while low in Fe and Mn (Fig. 4b). Higher concentrations of Fe and Mn and lower concentrations of Sr in fossil carbonates are generally associated with diagenetic alteration (e.g., partial recrystallisation) of the carbonate (e.g., Brand and Veizer benedeni were not diagenetically altered. XRD analysis revealed that the shell of A. benedeni benedeni consists of 100% 390 original aragonite, as its spectrum is identical to that of pure aragonite (Fig. 4a). Micro-X-ray fluorescence (μXRF) point analyses following the methodology in de Winter et al. (2017b) supports this interpretation, as the specimen is high in Sr, while low in Fe and Mn (Fig. 4b). Higher concentrations of Fe and Mn and lower concentrations of Sr in fossil carbonates are generally associated with diagenetic alteration (e.g., partial recrystallisation) of the carbonate (e.g., Brand and Veizer 1980). Pore waters in many common burial environments (especially in shallow marine successions) become more reducing 395 over time due to the decay of buried organic matter in the absence of oxygen (Calvert and Pedersen 1993). These conditions tend to remobilise Mn and Fe, which are fixed in oxides under surface conditions, resulting in high Fe and Mn and low Sr concentrations compared to those in seawater. These distinct pore water concentrations are captured in recrystallised carbonates during diagenesis, causing high Mn and Fe concentrations and low Sr concentrations to serve as indicators for burial alteration (Al-Aasm and Veizer 1986; Hendry et al. 1995). The absence of high Fe and Mn concentrations and 400 burial alteration (Al-Aasm and Veizer 1986; Hendry et al. 1995). The absence of high Fe and Mn concentrations and 400 presence of high Sr suggests that this process has not taken place in this specimen. Figure 4: X-ray based analysis results to assess diagenetic alteration in the specimens. (a) X-ray diffraction analysis of A. benedeni benedeni compared with patterns of pure aragonite and calcite, indicating an aragonitic composition of the shell. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 3: Growth lines in the A. benedeni benedeni specimens. (a) Counted growth lines in specimen SG-126, 115 in total. (b) 385 Counted growth lines in specimen SG-127, 131 in total. (c) Schematic figure of the shell indicating where the growth lines were counted. Figure 3: Growth lines in the A. benedeni benedeni specimens. (a) Counted growth lines in specimen SG-126, 115 in total. (b) 385 Counted growth lines in specimen SG-127, 131 in total. (c) Schematic figure of the shell indicating where the growth lines were counted. Figure 3: Growth lines in the A. benedeni benedeni specimens. (a) Counted growth lines in specimen SG-126, 115 in total. (b) 385 Counted growth lines in specimen SG-127, 131 in total. (c) Schematic figure of the shell indicating where the growth lines were counted. 4.1.2 Growth increments In specimen SG-126, a maximum of 115 growth lines was counted in layer M-1 at the hinge (Fig. 3a, c). In specimen SG- 127, a maximum of 131 growth lines was counted just ventral of the hinge (Fig. 3b, c), also in layer M-1. The growth lines were not all clearly visible, and in some areas in the shell, less growth lines could be discerned. As it was deemed easier to 380 miss some growth lines due to the limited resolution than to overestimate the number, the microscope images with the In specimen SG-126, a maximum of 115 growth lines was counted in layer M-1 at the hinge (Fig. 3a, c). In specimen SG- 127, a maximum of 131 growth lines was counted just ventral of the hinge (Fig. 3b, c), also in layer M-1. The growth lines In specimen SG-126, a maximum of 115 growth lines was counted in layer M-1 at the hinge (Fig. 3a, c). In specimen SG- 127, a maximum of 131 growth lines was counted just ventral of the hinge (Fig. 3b, c), also in layer M-1. The growth lines were not all clearly visible, and in some areas in the shell, less growth lines could be discerned. As it was deemed easier to 380 miss some growth lines due to the limited resolution than to overestimate the number, the microscope images with the maximum counted number of growth lines are shown here, as they are thought to be the best estimate of the actual number of growth lines. were not all clearly visible, and in some areas in the shell, less growth lines could be discerned. As it was deemed easier to 380 miss some growth lines due to the limited resolution than to overestimate the number, the microscope images with the maximum counted number of growth lines are shown here, as they are thought to be the best estimate of the actual number of growth lines. 380 16 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. 17 17 4.3.1 Diagenesis assessment Electron backscatter diffraction (EBSD) analysis shows no evidence of diagenetic alteration in the shells. In the EBSD maps, 410 0 to 0.2% of the area was classified as calcite. Upon inspection, these “calcite” grains are actually wild spikes that were subsequently removed in the data clean-up (Fig. B1). There was no local secondary mineral growth visible in any map (e.g., Fig. 5b-e), which would have presented itself as larger crystals (“blocky calcite”) that do not follow the surrounding structures (Cusack 2016; Casella et al. 2017). The pole figures show a clear preferred orientation (Fig. 5f), which is indicative of original growth structures (Casella et al. 2017). 415 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. 4.3.2 Grain size and orientation The aragonitic shell material is very fine grained and its crystals show a strong preferred orientation as well as twinning. Most grains have an area of a few μm2 or smaller (Fig. 5g). Layer M-1 has a narrower grain size distribution than the outer and inner outer layers, with no grains larger than 25 µm2 present in the analysed section. The multiples of uniform density (MUD) values indicate a strong preferred orientation (Fig. 5f). Layer M-1 has an increased MUD of 104.07 compared to ca. 420 40 for the other two layers, indicating a stronger preferred orientation (Fig. 5f). This is mainly due to the strong preferred orientation of the 100 axis, while the 001 and 010 axes show a weaker preferred orientation compared to the other layers. The pole plots show double maxima of the 001 and 010 axes, which rotate around the 100 axes (M+2 and M+1, Fig. 5f). This is observed in layers M+2 and M+1. In layer M-1, the 001 and 010 axes form a girdle around the 100 axis rather than (MUD) values indicate a strong preferred orientation (Fig. 5f). Layer M-1 has an increased MUD of 104.07 compared to ca. 420 40 for the other two layers, indicating a stronger preferred orientation (Fig. 5f). This is mainly due to the strong preferred orientation of the 100 axis, while the 001 and 010 axes show a weaker preferred orientation compared to the other layers. The pole plots show double maxima of the 001 and 010 axes, which rotate around the 100 axes (M+2 and M+1, Fig. 5f). This is observed in layers M+2 and M+1. In layer M-1, the 001 and 010 axes form a girdle around the 100 axis rather than two distinct maxima (M-1, Fig. 5f). The rotational 100 axis is generally oriented parallel to the growth direction. This is 425 especially evident from the lamellae in layer M-1 of Fig. 5b-e. The angle of ca. 64° between the 001 and 010 axes is characteristic of aragonite polysynthetic (110) twinning (e.g., Griesshaber et al. 2013). Calculated twinning boundaries indicate that the two dominant orientations, as indicated by the IPF colour coding, in layers M+2 and M-1 represent twins (Fig. 5i). In layer M+1, the two dominant orientations that make up the lamellae are not a result of twinning. 4.2 X-ray based analyses (b) Violin plot showing the results of the X-ray fluorescence analysis of A. benedeni benedeni for the elements Fe, Mn, and Sr. The “violin” shape 405 depicts the kernel density function for the elemental concentration, plotted vertically and mirrored. The specimen is high in Sr and low in Fe and Mn. Figure 4: X-ray based analysis results to assess diagenetic alteration in the specimens. (a) X-ray diffraction analysis of A. benedeni benedeni compared with patterns of pure aragonite and calcite, indicating an aragonitic composition of the shell. (b) Violin plot showing the results of the X-ray fluorescence analysis of A. benedeni benedeni for the elements Fe, Mn, and Sr. The “violin” shape 405 depicts the kernel density function for the elemental concentration, plotted vertically and mirrored. The specimen is high in Sr and low in Fe and Mn. 18 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 5: EBSD map of specimen SG-127. Map acquisition specifics: 20 kV, high vacuum, 0.2 μm step size. (a) Band contrast map with scale, orientation, growth lines, direction of growth (dog), and shell layers M+2, M+1, and M-1, separated by striped lines. (b) EBSD IPF X map, showing grain orientations parallel to the X0 axis. Unit cells of aragonite with the most common orientations are shown, the colour of which corresponds to the colour coding of the map. (c) EBSD IPF Y map showing grain orientations parallel to the Y0 axis, with unit cells. (d) EBSD IPF Z map showing grain orientations parallel to the Z0 axis, with unit cells. (e) Inverse pole figure (IPF) colour key for the EBSD orientation maps. (f) Contoured pole figures for the three main crystallographic directions and the MUD, for the different shell layers. (g) Grain size distribution chart for the entire map and the different shell layers. Note the logarithmic y-axis. (h) Schematic figure indicating the approximate location of the map on the shell. (i) Shell layers from map IPF Z zoomed in and with added grain (black) and twinning (yellow) boundaries. 4.3.3 Microstructures Within the EBSD maps, several different structures are observed (Fig. 6). Layer M+2 shows bundles of crystals that diverge from the centre of the layer and show overlap in a scale-like pattern (Fig. 6a). Layer M+1 shows two sets of crystal orientations that are oriented perpendicular to the growth lines and that alternate and interfinger, similar to a zebra pattern (Fig. 6b). Layer M-1 shows various structures: In the hinge region, a hatched pattern is visible (Fig. 6c), In other parts of M- 1, elongated, columnar crystals with their long side oriented parallel to the growth direction are observed (Fig. 6d). Around this prismatic structure, there are small grains with no clear preferred shape orientation (Fig. 6d). Within the EBSD maps, several different structures are observed (Fig. 6). Layer M+2 shows bundles of crystals that diverge from the centre of the layer and show overlap in a scale-like pattern (Fig. 6a). Layer M+1 shows two sets of crystal orientations that are oriented perpendicular to the growth lines and that alternate and interfinger, similar to a zebra pattern 445 (Fig. 6b). Layer M-1 shows various structures: In the hinge region, a hatched pattern is visible (Fig. 6c), In other parts of M- 1, elongated, columnar crystals with their long side oriented parallel to the growth direction are observed (Fig. 6d). Around this prismatic structure, there are small grains with no clear preferred shape orientation (Fig. 6d). orientations that are oriented perpendicular to the growth lines and that alternate and interfinger, similar to a zebra pattern 445 (Fig. 6b). Layer M-1 shows various structures: In the hinge region, a hatched pattern is visible (Fig. 6c), In other parts of M- 1, elongated, columnar crystals with their long side oriented parallel to the growth direction are observed (Fig. 6d). Around this prismatic structure, there are small grains with no clear preferred shape orientation (Fig. 6d). orientations that are oriented perpendicular to the growth lines and that alternate and interfinger, similar to a zebra pattern 445 (Fig. 6b). Layer M-1 shows various structures: In the hinge region, a hatched pattern is visible (Fig. 6c), In other parts of M- 1, elongated, columnar crystals with their long side oriented parallel to the growth direction are observed (Fig. 6d). Around this prismatic structure, there are small grains with no clear preferred shape orientation (Fig. 6d). 4.3.2 Grain size and orientation Instead, each of the two orientations (orange and purple) consists of their own set of 430 twins (light and dark orange and purple, respectively; Fig. 5i). the lamellae are not a result of twinning. Instead, each of the two orientations (orange and purple) consists of their own set of 430 twins (light and dark orange and purple, respectively; Fig. 5i). 19 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. 20 4.4.1 Oxygen isotopes 450 4.4.1 Oxygen isotopes 450 The δ18Oc data of specimens reveal sinusoidal records that range between +0.3 and +3.3‰ (Fig. 7a, b) and suggest multi- annual growth, with δ18Oc peaks corresponding to winters and valleys corresponding to summers. Specimen SG-126 shows at least 8-9 years of growth (Fig. 7a). The first and last years of this specimen may not have been recovered during sampling, as the first and last few millimetres were not sampled. In addition, the sampling resolution was likely too coarse to capture yg p The δ18Oc data of specimens reveal sinusoidal records that range between +0.3 and +3.3‰ (Fig. 7a, b) and suggest multi- annual growth, with δ18Oc peaks corresponding to winters and valleys corresponding to summers. Specimen SG-126 shows at least 8-9 years of growth (Fig. 7a). The first and last years of this specimen may not have been recovered during sampling, as the first and last few millimetres were not sampled. In addition, the sampling resolution was likely too coarse to capture The δ18Oc data of specimens reveal sinusoidal records that range between +0.3 and +3.3‰ (Fig. 7a, b) and suggest multi- annual growth, with δ18Oc peaks corresponding to winters and valleys corresponding to summers. Specimen SG-126 shows at least 8-9 years of growth (Fig. 7a). The first and last years of this specimen may not have been recovered during sampling, as the first and last few millimetres were not sampled. In addition, the sampling resolution was likely too coarse to capture all years in the ontogenetically oldest section of the shell, as growth increments generally become thinner with age. In SG- 455 126, high δ18Oc values generally correspond to darker bands and vice versa, which suggests that the dark-light couplets represent the winter and summer seasons. The δ18Oc variability decreases near the ventral margin of the shell. The sampled section of SG-127 shows six cycles in δ18Oc values (Fig. 7b). Contrary to SG-126, the relationship between δ18Oc value and shell colour is not as straightforward. The dark-light alternation on SG-127 is not as consistent as the pattern seen on SG-126. There are many very thin light and dark bands on SG-127, and many of the samples likely contain material 460 from both light and dark bands (Fig. 7b; not all indicated on Fig. 7b as the exact location relative to the samples was not determined). There were not enough duplicate δ18Oc measurements for SG-127, so the value of 0.15‰ was also applied to this specimen. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 7: δ18Oc records of the A. benedeni benedeni shells, plotted relative to the distance from the umbo. (a) Record of specimen SG-126, consisting of 55 samples each measured 1 to 3 times. (b) Record of specimen SG-127, consisting of 30 samples. The average was calculated from the different measurements of a single sample. If there was just one measurement, that value was taken. One and two standard deviations are plotted around the average. The standard deviation was calculated as described in sect. 4.4.1 (0.15‰). In the background, the approximate colour of the growth band on the outer surface of the shell is indicated (see also Fig. A1). The warm and cold δ18Oc ‘bins’ are marked in red and blue bands. These bins encompass all datapoints below 0.9‰ and above 2.4‰. See also section 3.4.3 and Fig. 9. 475 The external reproducibility of the standards, however, does not reflect the reproducibility of the samples based on duplicate measurements. The samples from each sampling track were not homogeneous, as they likely contain multiple thin growth 480 increments and were not homogenised after drilling. Therefore, different aliquots from the same sample sometimes resulted in quite large differences when measured, in both the GasBench and the MAT 253 PLUS analyses. Instead of using the external reproducibility on the Naxos standard, the uncertainty on the samples was determined as follows: for each sample of which multiple aliquots were analysed, the standard deviation of these aliquot results was taken, and all these standard The external reproducibility of the standards, however, does not reflect the reproducibility of the samples based on duplicate measurements. The samples from each sampling track were not homogeneous, as they likely contain multiple thin growth 480 increments and were not homogenised after drilling. Therefore, different aliquots from the same sample sometimes resulted in quite large differences when measured, in both the GasBench and the MAT 253 PLUS analyses. Instead of using the external reproducibility on the Naxos standard, the uncertainty on the samples was determined as follows: for each sample of which multiple aliquots were analysed, the standard deviation of these aliquot results was taken, and all these standard deviations were averaged. To this goal, δ18Oc data from GasBench and MAT 253 PLUS analyses were combined. This 485 resulted in an uncertainty of 0.15‰ for SG-126. 4.4.1 Oxygen isotopes 450 The internal reproducibility of conventional stable isotope analyses was 0.04‰ (1σ) for δ13C and 0.06‰ (1σ) for δ18Oc. External reproducibility was 0.05‰ for δ13C and 0.07‰ for δ18Oc based on repeated measurements of the Naxos standard. seen on SG-126. There are many very thin light and dark bands on SG-127, and many of the samples likely contain material 460 from both light and dark bands (Fig. 7b; not all indicated on Fig. 7b as the exact location relative to the samples was not determined). The internal reproducibility of conventional stable isotope analyses was 0.04‰ (1σ) for δ13C and 0.06‰ (1σ) for δ18Oc. External reproducibility was 0.05‰ for δ13C and 0.07‰ for δ18Oc based on repeated measurements of the Naxos standard. The Kiel carbonate standard showed external reproducibility of 0.09‰ for δ13C and 0.06‰ for δ18Oc. 465 The Kiel carbonate standard showed external reproducibility of 0.09‰ for δ13C and 0.06‰ for δ18Oc. 465 21 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 6: Different shell structures identified in the EBSD maps. (a) Overlapping scale-like structure in layer M+2. (b) Alternating zebra-like patterns in layer M+1. (c) Hatched pattern in layer M-1 at the hinge of the shell. (d) Rectangular, columnar crystals with their length oriented perpendicular to the growth lines in layer M-1. There are also areas in this layer (e.g., top right in panel d) without a clear grain shape structure. (e) Schematic figure of the shell indicating the approximate location of the maps. Figure 6: Different shell structures identified in the EBSD maps. (a) Overlapping scale-like structure in layer M+2. (b) Alternating zebra-like patterns in layer M+1. (c) Hatched pattern in layer M-1 at the hinge of the shell. (d) Rectangular, columnar crystals with their length oriented perpendicular to the growth lines in layer M-1. There are also areas in this layer (e.g., top right in panel d) without a clear grain shape structure. (e) Schematic figure of the shell indicating the approximate location of the maps. 470 22 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. 23 4.4.2 Growth curves Growth model fitting suggests a maximum size of A. benedeni benedeni of 34-52 mm. A growth curve has been reconstructed for the specimen using the δ18Oc record of specimen SG-126 (Fig. 8a-b). This was not done for specimen SG- 490 127 as only a small section of the entire shell was sampled. Figure 8 also shows the fitted Von Bertalanffy and Gompertz growth models. The VBGF has the form of Eq. 4, where H is the body size of the specimen at time t (here: shell height measured as distance from umbo), Hasymp is the asymptotic height (i.e., the average theoretical maximum shell height), k is the growth coefficient, t is the time (here: in years), and t0 is the theoretical time where H equals 0. Growth model fitting suggests a maximum size of A. benedeni benedeni of 34-52 mm. A growth curve has been 495 𝐻(𝑡) = 𝐻𝑎𝑠𝑦𝑚𝑝(1 −𝑒−𝑘(𝑡−𝑡0)) (4) Each parameter, its associated standard error (SE), and p-value were calculated. Hasymp, k, and t0 were estimated to be 52.35±7.64 SE (p<0.05), 0.09±0.02 SE (p<0.05), and 0.03±0.16 SE (p>>0.05) respectively. The interpretation of growth coefficient k is problematic, and besides having a very large standard error and low confidence, t0 is merely a mathematical 500 Each parameter, its associated standard error (SE), and p-value were calculated. Hasymp, k, and t0 were estimated to be 52.35±7.64 SE (p<0.05), 0.09±0.02 SE (p<0.05), and 0.03±0.16 SE (p>>0.05) respectively. The interpretation of growth coefficient k is problematic, and besides having a very large standard error and low confidence, t0 is merely a mathematical 500 artefact that does not have any biological meaning (Lee et al. 2020). Hasymp is the estimated average maximum shell height in millimetres that A. benedeni benedeni would have reached. The Gompertz equation has the form of Eq. 5: Each parameter, its associated standard error (SE), and p-value were calculated. Hasymp, k, and t0 were estimated to be 52.35±7.64 SE (p<0.05), 0.09±0.02 SE (p<0.05), and 0.03±0.16 SE (p>>0.05) respectively. The interpretation of growth Each parameter, its associated standard error (SE), and p-value were calculated. Hasymp, k, and t0 were estimated to be 52.35±7.64 SE (p<0.05), 0.09±0.02 SE (p<0.05), and 0.03±0.16 SE (p>>0.05) respectively. The interpretation of growth p p y p 52.35±7.64 SE (p<0.05), 0.09±0.02 SE (p<0.05), and 0.03±0.16 SE (p>>0.05) respectively. 4.4.2 Growth curves The interpretation of growth coefficient k is problematic, and besides having a very large standard error and low confidence, t0 is merely a mathematical 500 artefact that does not have any biological meaning (Lee et al. 2020). Hasymp is the estimated average maximum shell height in millimetres that A. benedeni benedeni would have reached. The Gompertz equation has the form of Eq. 5: coefficient k is problematic, and besides having a very large standard error and low confidence, t0 is merely a mathematical 500 artefact that does not have any biological meaning (Lee et al. 2020). Hasymp is the estimated average maximum shell height in millimetres that A. benedeni benedeni would have reached. The Gompertz equation has the form of Eq. 5: 𝐻(𝑡) = 𝐻𝑎𝑠𝑦𝑚𝑝𝑒−𝑏𝑒−𝑐𝑡 (5) 505 505 24 4.4.3 Δ47 and palaeotemperature 515 A mean annual temperature (MAT) of 13.5±3.8°C has been calculated through averaging all Δ47 values and converting this average to a single temperature, with the 95%CL determined through Monte Carlo error propagation. Δ47-based summer and winter temperatures could not be determined due to a limited amount of data. The summer and winter data were compiled by selecting samples with respectively low and high δ18Oc values from both specimens, and subsequently averaging the A mean annual temperature (MAT) of 13.5±3.8°C has been calculated through averaging all Δ47 values and converting this average to a single temperature, with the 95%CL determined through Monte Carlo error propagation. Δ47-based summer and winter temperatures could not be determined due to a limited amount of data. The summer and winter data were compiled by selecting samples with respectively low and high δ18Oc values from both specimens, and subsequently averaging the associated Δ47 values (Fig. 9, see also section 3.4.3). However, Fig. 9 shows that to obtain statistically sound summer and 520 winter temperatures from A. benedeni benedeni, more clumped isotope measurements are required than analysed here. The δ18Oc cut-offs for <0.9‰ and >2.4‰ are highlighted to illustrate this. At these cut-off points, the number of datapoints (N=20 and N=21, respectively) is large enough to bring down the 95% confidence level (CL) range somewhat, but the two datasets are not statistically different (p>0.05, Student’s t-test), both due to the wide range included in these cut-offs and the associated Δ47 values (Fig. 9, see also section 3.4.3). However, Fig. 9 shows that to obtain statistically sound summer and 520 winter temperatures from A. benedeni benedeni, more clumped isotope measurements are required than analysed here. The δ18Oc cut-offs for <0.9‰ and >2.4‰ are highlighted to illustrate this. At these cut-off points, the number of datapoints (N=20 and N=21, respectively) is large enough to bring down the 95% confidence level (CL) range somewhat, but the two datasets are not statistically different (p>0.05, Student’s t-test), both due to the wide range included in these cut-offs and the large 95%CL range. Increasing the number of clumped isotope measurements decreases the large errors and allows for 525 making the cut-offs narrower so that they better represent the seasonal extremes. That this approach works, is exemplified by the MAT of 13.5±3.8°C. Due to the large number of measurements (N=103), the error on this temperature has been reduced to a reasonable level. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. where H is again the shell height at time t, Hasymp is the average theoretical maximum shell height, and b and c are coefficients. Hasymp, b, and c were estimated to be 34.11±1.98 SE (p<0.05), 2.58±0.14 SE (p<0.05), and 0.29±0.03 SE (p<0.05), respectively. The Gompertz model thus suggests a shorter average maximum shell height of 34.11 mm. The standard error on the residuals is 0.51 for the VBGF and 0.63 for the Gompertz equation. The R2 value for both fits is 0.99, but as both functions are non-linear, this is not a reliable indicator of goodness-of-fit (e.g., Spiess and Neumeyer 2010). 510 but as both functions are non-linear, this is not a reliable indicator of goodness-of-fit (e.g., Spi 0 510 Figure 8: Growth curve for specimen SG-126. (a) Growth curve constructed by inserting the counted years versus distance-from- umbo datapoints into two different fitting algorithms: the asymptotic Von Bertalanffy growth function and the logistic Gompertz function. (b) δ18Oc record with the inferred growth years marked by lines connecting to plot (a). Figure 8: Growth curve for specimen SG-126. (a) Growth curve constructed by inserting the counted years versus distance-from- umbo datapoints into two different fitting algorithms: the asymptotic Von Bertalanffy growth function and the logistic Gompertz function. (b) δ18Oc record with the inferred growth years marked by lines connecting to plot (a). 25 4.4.4 δ18Osw, δ18Oc, and temperature 4.4.4 δ18Osw, δ18Oc, and temperature The average δ18Osw is 0.10±0.88‰ VSMOW (95%CL), based on the average Δ47-based temperature of 13.5±3.8°C and the 540 average δ18Oc of both shells combined (1.53±0.015 VSMOW, 95%CL). The Grossman and Ku (1986) equation was applied to the highest and lowest δ18Oc values and the mean δ18Osw to obtain new winter and summer temperatures (Fig. 10a). To obtain these highest and lowest δ18Oc values, the 3 lowest (0.30, 0.42, 0.43‰) and highest (3.07, 3.16, 3.29‰) datapoints were averaged. This resulted in summer and winter temperatures of 18.5±3.9°C and 6.4±3.9°C (95%CL, Fig. 10b). 545 Figure 10: δ18Oc-based summer and winter temperatures. (a) Schematic illustrating how the δ18Oc-based temperatures have been calculated and what values were used. All values after ± are 95%CLs. b. Mean δ18Oc-based summer (red circle) and winter (blue diamond) temperatures and their 95%CL. In the background, the Δ47-based mean temperature and confidence interval is shown (grey band). The δ18Oc values used are the average of the lowest and highest 3 datapoints. Figure 10: δ18Oc-based summer and winter temperatures. (a) Schematic illustrating how the δ18Oc-based temperatures have been calculated and what values were used. All values after ± are 95%CLs. b. Mean δ18Oc-based summer (red circle) and winter (blue diamond) temperatures and their 95%CL. In the background, the Δ47-based mean temperature and confidence interval is shown (grey band). The δ18Oc values used are the average of the lowest and highest 3 datapoints. 5.1 Diagenesis No signs of diagenesis of the shells were found through micro-X-ray fluorescence (μXRF), X-ray diffraction (XRD), light microscopy, and electron backscatter diffraction (EBSD) analyses. The μXRF analyses indicated that the shell material was high in Sr and low in Fe and Mn, these latter two elements are regularly used to pinpoint diagenesis (Fig. 2b; de Winter & No signs of diagenesis of the shells were found through micro-X-ray fluorescence (μXRF), X-ray diffraction (XRD), light microscopy, and electron backscatter diffraction (EBSD) analyses. The μXRF analyses indicated that the shell material was high in Sr and low in Fe and Mn, these latter two elements are regularly used to pinpoint diagenesis (Fig. 2b; de Winter & microscopy, and electron backscatter diffraction (EBSD) analyses. The μXRF analyses indicated that the shell material was high in Sr and low in Fe and Mn, these latter two elements are regularly used to pinpoint diagenesis (Fig. 2b; de Winter & Claeys, 2016). XRD and EBSD analysis indicated no presence of calcite, to which the metastable aragonite alters as it 555 undergoes diagenesis (e.g., Al-Aasm and Veizer 1986; Casella et al. 2017). The XRD pattern of specimen SG-125 consisted of 100% aragonite, although it must be noted that this specimen was not analysed for stable isotopes. No blocky calcite was observed in the EBSD images of any of the three specimens (Cusack 2016; Casella et al. 2017). This alone does not preclude diagenetic alteration, as calcite is not formed in the earliest stages of diagenesis (Cochran et al. 2010; Marcano et al. 2015; Claeys, 2016). XRD and EBSD analysis indicated no presence of calcite, to which the metastable aragonite alters as it 555 undergoes diagenesis (e.g., Al-Aasm and Veizer 1986; Casella et al. 2017). The XRD pattern of specimen SG-125 consisted of 100% aragonite, although it must be noted that this specimen was not analysed for stable isotopes. No blocky calcite was observed in the EBSD images of any of the three specimens (Cusack 2016; Casella et al. 2017). This alone does not preclude diagenetic alteration, as calcite is not formed in the earliest stages of diagenesis (Cochran et al. 2010; Marcano et al. 2015; Ritter et al. 2017) and there is not necessarily a difference in grain size (due to the presence of larger, secondary grains) 560 between pristine and altered aragonite (Casella et al. 2017). https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. 4.4.3 Δ47 and palaeotemperature 515 The same is possible for seasonal temperatures as long as the dataset is large enough. The external reproducibility for the clumped isotope analyses was 38 ppm for IAEA-C2 (n=23) and 60 ppm for Merck (n=15). The external reproducibility of IAEA-C2 was used as the standard deviation on Δ47 for the samples, as it is closer in 530 composition to the samples than Merck (Fig. A4). After corrections, most of the Δ47 data range between 0.55 and 0.70‰ for both specimens (Fig. A5). 26 Figure 9: Overview of averages (round and diamond symbols) and 95% confidence intervals (dashed lines) for different sizes of grouping summer and winter Δ47 aliquots. Sizes of groups increase towards the middle of the plot. The larger symbols with vertical 535 error bars highlight the issue of having insufficient aliquot measurements. The bin sizes are small, and so they should come close to the true (non-averaged) seasonality. However, the number of aliquots is reduced to ca. 20 at this point. This translates to large errors, making these cold and warm averages statistically indistinguishable from each other. Figure 9: Overview of averages (round and diamond symbols) and 95% confidence intervals (dashed lines) for different sizes of grouping summer and winter Δ47 aliquots. Sizes of groups increase towards the middle of the plot. The larger symbols with vertical 535 error bars highlight the issue of having insufficient aliquot measurements. The bin sizes are small, and so they should come close to the true (non-averaged) seasonality. However, the number of aliquots is reduced to ca. 20 at this point. This translates to large errors, making these cold and warm averages statistically indistinguishable from each other. 26 2022) and from the early Pliocene Ramsholt Member of and the unconsolidated and fine-grained nature of the strata, this evidence suggests that no significant diagenetic alteration 570 took place. The isotope values presented here therefore record the formation temperature of the biogenic aragonite, assuming equilibrium fractionation. Previous studies have indicated good preservation for bivalves from the Oorderen Member (Valentine et al. 2011; Johnson et al. 2022), from other members of the Lillo Formation (Johnson et al. 2022) and from the early Pliocene Ramsholt Member of the Coralline Crag Formation (Johnson et al. 2009; Vignols et al. 2019). Pliocene shells from the North Sea basin are 575 expected to yield decent temperature results by geochemical analysis, as long as preservation is not obviously suspected (e.g., in the Sudbourne Member of the Coralline Crag Formation in southeast England, whose aragonitic shells have nearly all been dissolved; Balson et al. 1993). the Coralline Crag Formation (Johnson et al. 2009; Vignols et al. 2019). Pliocene shells from the North Sea basin are 575 expected to yield decent temperature results by geochemical analysis, as long as preservation is not obviously suspected (e.g., in the Sudbourne Member of the Coralline Crag Formation in southeast England, whose aragonitic shells have nearly all been dissolved; Balson et al. 1993). the Coralline Crag Formation (Johnson et al. 2009; Vignols et al. 2019). Pliocene shells from the North Sea basin are 575 expected to yield decent temperature results by geochemical analysis, as long as preservation is not obviously suspected (e.g., in the Sudbourne Member of the Coralline Crag Formation in southeast England, whose aragonitic shells have nearly all been dissolved; Balson et al. 1993). 5.1 Diagenesis Further analysis of the EBSD maps did not, however, produce 27 5.2 Angulus benedeni benedeni shell structure The shell of A. benedeni benedeni consists of four shell layers, of which the outer three show different macrostructures that 580 are also observed in other tellinid bivalves. Three of the four shell layers can be assigned to a specific bivalve shell structure through comparison with the structures described in the review paper of Popov (2014). The fourth layer, M-2, was not captured on any EBSD map. The bundled, diverging crystals in layer M+2 (Fig. 6a) have been interpreted as a compound composite prismatic structure (Popov 2014). The zebra-like pattern observed in layer M+1 (Fig. 6b) has been interpreted as a crossed lamellar structure (Popov 2014; Crippa et al. 2020). Layer M-1 shows various structures: the hatched pattern 585 observed in the hinge region (Fig. 6c) is interpreted as a complex crossed lamellar structure (Popov 2014; Crippa et al. 2020). The column-like crystals (Fig. 6d) have been interpreted as a prismatic structure (Popov 2014). The small crystals with no clear preferred orientation that surround this prismatic structure (Fig. 6d) were interpreted as a homogeneous structure (Popov 2014). However, it might also be a different structure that cannot be analysed at this resolution due to the very fine-grained nature of the aragonite. 590 Aragonite polysynthetic (110) twinning—indicated by a 64° misorientation angle between the 001 and 010 axes—is frequently observed in bivalves (Kobayashi and Akai 1994; Griesshaber et al. 2013; Crippa et al. 2020) and other molluscs (Schoeppler et al. 2019). It can contribute to rapid shell growth as it is more efficient at filling up space than non-twinned growth (Schoeppler et al. 2019). This twinning is observed in all analysed layers of A. benedeni benedeni. As the double 001 very fine-grained nature of the aragonite. 590 Aragonite polysynthetic (110) twinning—indicated by a 64° misorientation angle between the 001 and 010 axes—is frequently observed in bivalves (Kobayashi and Akai 1994; Griesshaber et al. 2013; Crippa et al. 2020) and other molluscs (Schoeppler et al. 2019). It can contribute to rapid shell growth as it is more efficient at filling up space than non-twinned growth (Schoeppler et al. 2019). This twinning is observed in all analysed layers of A. benedeni benedeni. As the double 001 very fine-grained nature of the aragonite. 590 Aragonite polysynthetic (110) twinning—indicated by a 64° misorientation angle between the 001 and 010 axes—is frequently observed in bivalves (Kobayashi and Akai 1994; Griesshaber et al. 2013; Crippa et al. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. any evidence for diagenetic alteration. Microstructures that are similar to those found in modern Tellinidae were observed in the EBSD maps (Popov 2014; see also section 5.2), suggesting that these are original structures. In altered aragonitic bivalves, the structures as observed from EBSD maps are more homogenous and have a lower MUD compared to pristine material (Casella et al. 2017; study carried out on Arctica islandica). The absolute MUD values cannot be compared with 565 different studies due to different measurement settings. However, comparison with maps of pristine versus altered aragonite in Casella et al. (2017) shows that the altered material in Casella et al. (2017) is much more homogeneous and randomised (i.e., low preferred orientation of the grains) than observed in this study. Even the homogeneous structure in A. benedeni benedeni (Fig. 6d) appears less chaotic than altered aragonite (Casella et al. 2017). Combined with the shallow burial depth any evidence for diagenetic alteration. Microstructures that are similar to those found in modern Tellinidae were observed in the EBSD maps (Popov 2014; see also section 5.2), suggesting that these are original structures. In altered aragonitic bivalves, the structures as observed from EBSD maps are more homogenous and have a lower MUD compared to pristine material (Casella et al. 2017; study carried out on Arctica islandica). The absolute MUD values cannot be compared with 565 different studies due to different measurement settings. However, comparison with maps of pristine versus altered aragonite in Casella et al. (2017) shows that the altered material in Casella et al. (2017) is much more homogeneous and randomised (i.e., low preferred orientation of the grains) than observed in this study. Even the homogeneous structure in A. benedeni benedeni (Fig. 6d) appears less chaotic than altered aragonite (Casella et al. 2017). Combined with the shallow burial depth 565 and the unconsolidated and fine-grained nature of the strata, this evidence suggests that no significant diagenetic alteration 570 took place. The isotope values presented here therefore record the formation temperature of the biogenic aragonite, assuming equilibrium fractionation. Previous studies have indicated good preservation for bivalves from the Oorderen Member (Valentine et al. 2011; Johnson et al. 2022), from other members of the Lillo Formation (Johnson et al. As this study documents one of the first EBSD analyses done on fossil molluscs, some suggestions for future research exploring this method include 1) comparing the microstructures of multiple shell layers between species through geological time; 2) producing maps at higher resolution—step size of 0.1 or even 0.05 μm—to better capture very fine-grained microstructures; 3) analysing larger numbers of A. benedeni benedeni specimens to characterise the microstructural variation present within species; and 4) analysing A. benedeni benedeni specimens that have 615 d di i d i h h l h This study supports previous investigations in highlighting the utility of EBSD for analysing shell structures in bivalves (e.g., 610 Cusack 2016; Checa et al. 2019; Crippa et al. 2020). As this study documents one of the first EBSD analyses done on fossil molluscs, some suggestions for future research exploring this method include 1) comparing the microstructures of multiple shell layers between species through geological time; 2) producing maps at higher resolution—step size of 0.1 or even 0.05 μm—to better capture very fine-grained microstructures; 3) analysing larger numbers of A. benedeni benedeni specimens to characterise the microstructural variation present within species; and 4) analysing A. benedeni benedeni specimens that have 615 undergone diagenesis to determine how that alters the structures. characterise the microstructural variation present within species; and 4) analysing A. benedeni benedeni specimens that have 615 undergone diagenesis to determine how that alters the structures. characterise the microstructural variation present within species; and 4) analysing A. benedeni benedeni specimens that have 615 undergone diagenesis to determine how that alters the structures. The M-1 layer in Tellinidae is usually homogeneous, as it looks to be here in some sections. Angulus benedeni benedeni appears to be very similar in structure to the closely related A. nysti. The latter has an M-1 layer that is complex crossed lamellar with Tellinidae generally have three layers, sometimes with sub-layers present. These three layers as described by Popov (2014) 600 correspond to what is called here M+2 (outer in Popov 2014), M+1 (middle in Popov 2014), and M-1 (inner in Popov 2014); the brown M-2 layer we observed in A. benedeni benedeni is not mentioned separately, and is perhaps part of M-1. The M-1 layer in Tellinidae is usually homogeneous, as it looks to be here in some sections. Angulus benedeni benedeni appears to be very similar in structure to the closely related A. nysti. The latter has an M-1 layer that is complex crossed lamellar with interlayers of prisms and an M+1 layer that is crossed lamellar (Popov 2014). Various differences are observed as well: layer 605 M+2 of A. nysti is fibrous prismatic with megaprisms rather than compound composite prismatic. Furthermore, in A. nysti the prisms diverge from the top rather than from the centre of this layer, as in A. benedeni benedeni (Popov 2014). Layer M- 2 was not studied with EBSD here, but light microscopy suggests that it might be structurally similar to the neighbouring M- 1 layer. interlayers of prisms and an M+1 layer that is crossed lamellar (Popov 2014). Various differences are observed as well: layer 605 M+2 of A. nysti is fibrous prismatic with megaprisms rather than compound composite prismatic. Furthermore, in A. nysti the prisms diverge from the top rather than from the centre of this layer, as in A. benedeni benedeni (Popov 2014). Layer M- 2 was not studied with EBSD here, but light microscopy suggests that it might be structurally similar to the neighbouring M- 1 layer. This study supports previous investigations in highlighting the utility of EBSD for analysing shell structures in bivalves (e.g., 610 Cusack 2016; Checa et al. 2019; Crippa et al. 2020). 5.2 Angulus benedeni benedeni shell structure 2020) and other molluscs (Schoeppler et al. 2019). It can contribute to rapid shell growth as it is more efficient at filling up space than non-twinned growth (Schoeppler et al. 2019). This twinning is observed in all analysed layers of A. benedeni benedeni. As the double 001 28 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. and 010 maxima are much stronger in the external layers M+2 and M+1 than in the internal layer M-1, twinning may be 595 more significant in these two layers. This may, in turn, be linked to more rapid growth. The described microstructures—compound composite prismatic, crossed lamellar, complex crossed lamellar, prismatic, and homogeneous—were all previously described in other tellinid genera such as Macoma, Peronidia, and the lucinid Megaxinus (Popov 2014). However, there is a large variation in structural composition within the Tellinidae family (Popov 2014). and 010 maxima are much stronger in the external layers M+2 and M+1 than in the internal layer M-1, twinning may be 595 more significant in these two layers. This may, in turn, be linked to more rapid growth. and 010 maxima are much stronger in the external layers M+2 and M+1 than in the internal layer M-1, twinning may be 595 more significant in these two layers. This may, in turn, be linked to more rapid growth. The described microstructures—compound composite prismatic, crossed lamellar, complex crossed lamellar, prismatic, and homogeneous—were all previously described in other tellinid genera such as Macoma, Peronidia, and the lucinid Megaxinus (Popov 2014). However, there is a large variation in structural composition within the Tellinidae family (Popov 2014). Tellinidae generally have three layers, sometimes with sub-layers present. These three layers as described by Popov (2014) 600 correspond to what is called here M+2 (outer in Popov 2014), M+1 (middle in Popov 2014), and M-1 (inner in Popov 2014); the brown M-2 layer we observed in A. benedeni benedeni is not mentioned separately, and is perhaps part of M-1. The M-1 layer in Tellinidae is usually homogeneous, as it looks to be here in some sections. Angulus benedeni benedeni appears to be very similar in structure to the closely related A. nysti. The latter has an M-1 layer that is complex crossed lamellar with Tellinidae generally have three layers, sometimes with sub-layers present. These three layers as described by Popov (2014) 600 correspond to what is called here M+2 (outer in Popov 2014), M+1 (middle in Popov 2014), and M-1 (inner in Popov 2014); the brown M-2 layer we observed in A. benedeni benedeni is not mentioned separately, and is perhaps part of M-1. If it is briefly assumed that most of SG-126’s growth years have been captured and using the maximum amount of growth lines counted here (115), approximately 13 growth lines per year are recorded. This corresponds to an interval of approximately 29 days, which matches the periodicity 650 of the monthly synodic lunar-tidal cycle. A crude estimate of the minimum and maximum period is possible based on conservative estimates for the upper and lower limits for the number of counted growth lines (~75 to 150; see section 3.2.3; Fig. 3) and years recorded (~7 to 13, depending on how many early growth years are missing and how much aliasing is present in the ontogenetically oldest part of the shell). These estimates yield a range of a periodicity of 17 days (7 years, 150 growth lines) to 63 days (75 growth lines, 13 years). Both growth lines and years are more likely to be over- rather than 655 underestimated, as their recordings are both limited by either the microscopy or the sampling resolution. Therefore, a 17-day periodicity, which requires a maximum age of 7, is unlikely, making the biweekly tidal cycle an unlikely external forcing candidate. Within this range, the monthly tidal cycle seems the most plausible external forcing for the growth bands in A. benedeni benedeni. It is also possible that the formation of growth lines is driven by aperiodic or quasi-periodic processes such as storms—which would skew the correlation between growth years and growth lines—or is a result of an internally 660 Bivalves can form growth increments on a variety of timescales and their growth rhythm can be influenced by solar and lunar cycles (e.g., Tran et al. 2011). The number of growth lines counted in A. benedeni benedeni were divided by the 645 inferred age of the specimen to determine its growth rhythm. The sampled intervals span several years—around nine years for SG-126, which represents most of the shell height, and around six years for SG-127, which represents only around 1/3rd of the shell height. In both shells, 100+ growth lines have been counted. If it is briefly assumed that most of SG-126’s growth years have been captured and using the maximum amount of growth lines counted here (115), approximately 13 lunar cycles (e.g., Tran et al. 2011). The number of growth lines counted in A. benedeni benedeni were divided by the 645 inferred age of the specimen to determine its growth rhythm. The sampled intervals span several years—around nine years for SG-126, which represents most of the shell height, and around six years for SG-127, which represents only around 1/3rd of the shell height. In both shells, 100+ growth lines have been counted. If it is briefly assumed that most of SG-126’s growth years have been captured and using the maximum amount of growth lines counted here (115), approximately 13 growth lines per year are recorded. This corresponds to an interval of approximately 29 days, which matches the periodicity 650 of the monthly synodic lunar-tidal cycle. A crude estimate of the minimum and maximum period is possible based on conservative estimates for the upper and lower limits for the number of counted growth lines (~75 to 150; see section 3.2.3; Fig. 3) and years recorded (~7 to 13, depending on how many early growth years are missing and how much aliasing is present in the ontogenetically oldest part of the shell). These estimates yield a range of a periodicity of 17 days (7 years, 150 growth lines per year are recorded. This corresponds to an interval of approximately 29 days, which matches the periodicity 650 of the monthly synodic lunar-tidal cycle. A crude estimate of the minimum and maximum period is possible based on conservative estimates for the upper and lower limits for the number of counted growth lines (~75 to 150; see section 3.2.3; Fig. 3) and years recorded (~7 to 13, depending on how many early growth years are missing and how much aliasing is present in the ontogenetically oldest part of the shell). These estimates yield a range of a periodicity of 17 days (7 years, 150 growth lines) to 63 days (75 growth lines, 13 years). Both growth lines and years are more likely to be over- rather than 655 underestimated, as their recordings are both limited by either the microscopy or the sampling resolution. Therefore, a 17-day periodicity, which requires a maximum age of 7, is unlikely, making the biweekly tidal cycle an unlikely external forcing candidate. Within this range, the monthly tidal cycle seems the most plausible external forcing for the growth bands in A. benedeni benedeni. 5.3 Angulus benedeni benedeni growth history Angulus benedeni benedeni experienced slower growth in winter, could live for up to a decade or longer (Fig. 7, 8), could reach lengths of 34-52 mm, and likely formed monthly growth increments. The maximum (winter) δ18Oc values decrease, reach lengths of 34-52 mm, and likely formed monthly growth increments. The maximum (winter) δ18Oc values decrease, while the minimum (summer) values stay similar throughout the oxygen isotope record of specimen SG-126. This amplitude 620 reduction may reflect growth breaks during the colder months. This is supported by the correlation of high δ18Oc values with dark growth bands on the shell’s exterior. Darker bands in bivalves often represent high organic matter content related to decreased mineralisation rates (Lutz and Rhoads 1980, cited in Carré et al. 2005). The ontogenetic growth rate decline that is often seen in bivalves (e.g., McConnaughey and Gillikin 2008) is not apparent from the growth curve of specimen SG-126. while the minimum (summer) values stay similar throughout the oxygen isotope record of specimen SG-126. This amplitude 620 reduction may reflect growth breaks during the colder months. This is supported by the correlation of high δ18Oc values with dark growth bands on the shell’s exterior. Darker bands in bivalves often represent high organic matter content related to decreased mineralisation rates (Lutz and Rhoads 1980, cited in Carré et al. 2005). The ontogenetic growth rate decline that is often seen in bivalves (e.g., McConnaughey and Gillikin 2008) is not apparent from the growth curve of specimen SG-126. The growth curve only becomes slightly less steep in the last few measured years, and there is no marked increase in the 625 wavelength of the δ18Oc record (Fig. 8). 29 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. Growth modelling has provided a preliminary range of maximum shell height for A. benedeni benedeni. Both the VBGF and the Gompertz equation show a good fit with the growth data of Shell SG-126, indicated by the similar standard errors of the residuals (0.51 and 0.63 mm, respectively). The data do not appear to show the inflection point that is present in the logistic Gompertz function but not in the logarithmic VBGF. The exponential growth on the left-hand side of such an inflection is 630 suitable for the first stages of growth in bivalves, when they are still in the larval stage (Urban 2002). Since SG-126 is an adult shell, and since it is likely that not the entire life history was sampled (the first few millimetres of growth may have been missed, see Fig. A1), the larval growth stage is not represented. The second major difference between the VBGF and Gompertz function but not in the logarithmic VBGF. The exponential growth on the left-hand side of such an inflection is 630 suitable for the first stages of growth in bivalves, when they are still in the larval stage (Urban 2002). Since SG-126 is an adult shell, and since it is likely that not the entire life history was sampled (the first few millimetres of growth may have been missed, see Fig. A1), the larval growth stage is not represented. The second major difference between the VBGF and the Gompertz equation is their estimation of Hasymp, the average theoretical maximum shell height. The VBGF yields a Gompertz function but not in the logarithmic VBGF. The exponential growth on the left-hand side of such an inflection is 630 suitable for the first stages of growth in bivalves, when they are still in the larval stage (Urban 2002). Since SG-126 is an adult shell, and since it is likely that not the entire life history was sampled (the first few millimetres of growth may have been missed, see Fig. A1), the larval growth stage is not represented. The second major difference between the VBGF and the Gompertz equation is their estimation of Hasymp, the average theoretical maximum shell height. The VBGF yields a 630 maximum shell height estimate of around 52 mm, while the Gompertz equation gives an estimate of around 34 mm. Due to 635 the nature of the respective models, the VBGF tends to overestimate the maximum shell height, while the Gompertz equation tends to underestimate it (Urban 2002), so these two estimates yield a plausible range for the maximum shell height of A. benedeni benedeni. The heights of specimens SG-126 and SG-127 (30-36 mm) are close to the Hasymp of the Gompertz model, and the other specimens in the collection of the Royal Belgian Insitute of Natural Sciences generally do not exceed maximum shell height estimate of around 52 mm, while the Gompertz equation gives an estimate of around 34 mm. Due to 635 the nature of the respective models, the VBGF tends to overestimate the maximum shell height, while the Gompertz equation tends to underestimate it (Urban 2002), so these two estimates yield a plausible range for the maximum shell height of A. benedeni benedeni. The heights of specimens SG-126 and SG-127 (30-36 mm) are close to the Hasymp of the Gompertz model, and the other specimens in the collection of the Royal Belgian Insitute of Natural Sciences generally do not exceed 40 mm in length. It should be noted that Hasymp represents the average theoretical maximum shell height, and individual 640 shells can grow larger. The shape of the VBGF and Gompertz curves suggest that specimen SG-126 had not yet reached its maximum shell height. The fact that growth had not yet significantly slowed at around 8-9 years suggests that A. benedeni benedeni may have lived significantly longer, recording environmental variability on a scale of seasons to decades. Bivalves can form growth increments on a variety of timescales and their growth rhythm can be influenced by solar and Bivalves can form growth increments on a variety of timescales and their growth rhythm can be influenced by solar and lunar cycles (e.g., Tran et al. 2011). The number of growth lines counted in A. benedeni benedeni were divided by the 645 inferred age of the specimen to determine its growth rhythm. The sampled intervals span several years—around nine years for SG-126, which represents most of the shell height, and around six years for SG-127, which represents only around 1/3rd of the shell height. In both shells, 100+ growth lines have been counted. 5.4 Angulus benedeni benedeni as a climate archive 670 5.4 Angulus benedeni benedeni as a climate archive 670 Angulus benedeni benedeni shows promise as an archive for high-resolution climate reconstruction. It can live for up to a decade or longer and enables the reconstruction of multiannual climate records at a seasonal resolution. Both δ18Oc and Δ47 analyses can be successfully applied to this species, and with a larger dataset, it is possible to obtain clumped isotope-based summer and winter temperatures. This species is especially suited for reconstructions of climate in the Pliocene of the North Angulus benedeni benedeni shows promise as an archive for high-resolution climate reconstruction. It can live for up to a decade or longer and enables the reconstruction of multiannual climate records at a seasonal resolution. Both δ18Oc and Δ47 analyses can be successfully applied to this species, and with a larger dataset, it is possible to obtain clumped isotope-based summer and winter temperatures. This species is especially suited for reconstructions of climate in the Pliocene of the North Sea, where it is common (De Meuter and Laga 1976). However, it dates back to the Late Eocene (Marquet et al. 2008) and 675 can thus be used to reconstruct older climates as well, given that these older specimens are well-preserved. Its relatively thin shell in combination with its long lifespan makes it more challenging to sample at high temporal resolution. This first palaeoclimatological study on A. benedeni benedeni highlights the opportunities for using this species as a climate archive. These are not limited to stable isotope analysis, but may extend to, for example, minor and trace elemental analyses by e.g. extended μXRF analyses and Laser-Ablation ICP-MS profiles that can provide information about short-term changes in the 680 extended μXRF analyses and Laser-Ablation ICP-MS profiles, that can provide information about short-term changes in the 680 North Sea living environment during the Pliocene (e.g. de Winter et al. 2017a). extended μXRF analyses and Laser-Ablation ICP-MS profiles, that can provide information about short-term changes in the 680 North Sea living environment during the Pliocene (e.g. de Winter et al. 2017a). extended μXRF analyses and Laser-Ablation ICP-MS profiles, that can provide information about short-term changes in the 680 North Sea living environment during the Pliocene (e.g. de Winter et al. 2017a). 665 dominant than the other, not as individual growth lines as observed here. Finally, many bivalves form growth increments on 665 much smaller timescales as well (e.g., (semi-)diurnal or circatidal; Judd, Wilkinson, and Ivany 2018 and references therein). We cannot confidently rule out the presence of such ultradian cyclicity in A. benedeni benedeni, and more complete records from multiple specimens may be needed to further characterise and statistically solidify the periodicity in growth lines of A. benedeni benedeni. It is also possible that the formation of growth lines is driven by aperiodic or quasi-periodic processes growth lines) to 63 days (75 growth lines, 13 years). Both growth lines and years are more likely to be over- rather than 655 underestimated, as their recordings are both limited by either the microscopy or the sampling resolution. Therefore, a 17-day periodicity, which requires a maximum age of 7, is unlikely, making the biweekly tidal cycle an unlikely external forcing candidate. Within this range, the monthly tidal cycle seems the most plausible external forcing for the growth bands in A. benedeni benedeni. It is also possible that the formation of growth lines is driven by aperiodic or quasi-periodic processes such as storms—which would skew the correlation between growth years and growth lines—or is a result of an internally 660 30 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. controlled rhythm rather than being externally forced. Internal forcing is a strong possibility, as there is little difference between the two spring-neap cycles within a monthly cycle (Kvale 2006). If tides exerted influence, the fortnightly spring- neap cycle would be expected to be present as well due to its larger amplitude. Monthly lunar cycles have been observed in bivalves (Pannella and MacClintock 1968), but these are present as bundled spring-neap tide pairs with one being more controlled rhythm rather than being externally forced. Internal forcing is a strong possibility, as there is little difference between the two spring-neap cycles within a monthly cycle (Kvale 2006). If tides exerted influence, the fortnightly spring- neap cycle would be expected to be present as well due to its larger amplitude. Monthly lunar cycles have been observed in bivalves (Pannella and MacClintock 1968), but these are present as bundled spring-neap tide pairs with one being more controlled rhythm rather than being externally forced. Internal forcing is a strong possibility, as there is little difference between the two spring-neap cycles within a monthly cycle (Kvale 2006). If tides exerted influence, the fortnightly spring- neap cycle would be expected to be present as well due to its larger amplitude. Monthly lunar cycles have been observed in bivalves (Pannella and MacClintock 1968), but these are present as bundled spring-neap tide pairs with one being more dominant than the other, not as individual growth lines as observed here. Finally, many bivalves form growth increments on 665 much smaller timescales as well (e.g., (semi-)diurnal or circatidal; Judd, Wilkinson, and Ivany 2018 and references therein). We cannot confidently rule out the presence of such ultradian cyclicity in A. benedeni benedeni, and more complete records from multiple specimens may be needed to further characterise and statistically solidify the periodicity in growth lines of A. benedeni benedeni. dominant than the other, not as individual growth lines as observed here. Finally, many bivalves form growth increments on 665 much smaller timescales as well (e.g., (semi-)diurnal or circatidal; Judd, Wilkinson, and Ivany 2018 and references therein). We cannot confidently rule out the presence of such ultradian cyclicity in A. benedeni benedeni, and more complete records from multiple specimens may be needed to further characterise and statistically solidify the periodicity in growth lines of A. benedeni benedeni. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. ranges and indicative of a greater sensitivity (Dowsett et al. 2012; Haywood et al. 2020). The recent increase of 1-1.3°C in North Sea temperatures since the late 1800s (Schöne et al. 2004; Mackenzie and Schiedek 2007; Belkin 2009; Emeis et al. ranges and indicative of a greater sensitivity (Dowsett et al. 2012; Haywood et al. 2020). The recent increase of 1 1.3 C in North Sea temperatures since the late 1800s (Schöne et al. 2004; Mackenzie and Schiedek 2007; Belkin 2009; Emeis et al. 2015; Quante and Colijn 2016) is similar to the observed increase in global temperatures of 0.9-1.3°C (Gillett et al. 2021). 695 However, most of the warming in the North Sea took place since the 1980s (e.g., Emeis et al. 2015), while the steep warming trend on a global scale took off a few decades earlier, around 1960 (Gillett et al. 2021). Very recent warming in the North Sea thus appears to have been faster than the global average (e.g., Belkin 2009; Quante and Colijn 2016). The current climate is transient, not stable, so a direct comparison is not possible. However, both Pliocene and modern data suggest that we might see near-future warming in the North Sea that exceeds the global average trend. 700 2015; Quante and Colijn 2016) is similar to the observed increase in global temperatures of 0.9-1.3°C (Gillett et al. 2021). 695 However, most of the warming in the North Sea took place since the 1980s (e.g., Emeis et al. 2015), while the steep warming trend on a global scale took off a few decades earlier, around 1960 (Gillett et al. 2021). Very recent warming in the North Sea thus appears to have been faster than the global average (e.g., Belkin 2009; Quante and Colijn 2016). The current climate is transient, not stable, so a direct comparison is not possible. However, both Pliocene and modern data suggest that 695 we might see near-future warming in the North Sea that exceeds the global average trend. 700 This warming was likely not evenly distributed throughout the year during the Pliocene. The δ18Oc-based winter and summer temperatures show a seasonal range of 12.1°C, from 6.4±3.9°C in winter to 18.5±3.9°C in summer (Fig. 10b). This is higher than the modern range of ca. 7-9°C, from 7-8°C in winter to 15-16°C in summer (Kooij et al. 2016). The actual Pliocene range might have been larger, as the calculated range is likely an underestimation due to reasons pertaining to the g g g g This warming was likely not evenly distributed throughout the year during the Pliocene. The δ18Oc-based winter and summer temperatures show a seasonal range of 12.1°C, from 6.4±3.9°C in winter to 18.5±3.9°C in summer (Fig. 10b). This is higher than the modern range of ca. 7-9°C, from 7-8°C in winter to 15-16°C in summer (Kooij et al. 2016). The actual Pliocene range might have been larger, as the calculated range is likely an underestimation due to reasons pertaining to the methodology as well as to the bivalve-archive and its living environment. Firstly, as the sampling resolution was limited, 705 some of the highest and lowest δ18Oc values may not have been analysed. Secondly, short growth breaks during winter, as suggested by the dark bands present on the exterior shell surface, may have prevented the coldest temperatures from being recorded in A. benedeni benedeni. Thirdly, summer stratification during deposition of the lower Oorderen Member at 40-50 m water depth was found to be a likely possibility by previous research on bivalves from this member (Valentine et al. 2011; methodology as well as to the bivalve-archive and its living environment. Firstly, as the sampling resolution was limited, 705 some of the highest and lowest δ18Oc values may not have been analysed. Secondly, short growth breaks during winter, as suggested by the dark bands present on the exterior shell surface, may have prevented the coldest temperatures from being recorded in A. benedeni benedeni. Thirdly, summer stratification during deposition of the lower Oorderen Member at 40-50 m water depth was found to be a likely possibility by previous research on bivalves from this member (Valentine et al. 2011; Johnson et al. 2022). Previous research on the somewhat older Coralline Crag Formation also found evidence for summer 710 stratification in the early Pliocene (Jenkins and Houghton 1987; Johnson et al. 2009). When vertical mixing is limited, the summer heat is trapped in the surface layer of the water while the bottom waters remain a cooler temperature. This would lead to underestimation of SSTs in benthic organisms such as bivalves. The upper Oorderen interval from which this study’s specimens were collected is interpreted to have been shallower and more strongly influenced by tidal currents than the lower Johnson et al. 5.5 Clumped and oxygen isotope based palaeotemperatures The reconstructed mean annual temperature (MAT) of 13.5±3.8°C based on clumped isotope thermometry is 3.5°C higher than that of the pre-industrial North Sea (Mackenzie and Schiedek 2007; Emeis et al. 2015). Our temperature reconstructions The reconstructed mean annual temperature (MAT) of 13.5±3.8 C based on clumped isotope thermometry is 3.5 C higher than that of the pre-industrial North Sea (Mackenzie and Schiedek 2007; Emeis et al. 2015). Our temperature reconstructions for the mid-Piacenzian Warm Period (mPWP) in the North Sea are similar to a previous estimate of ca. 13°C for the North 685 Sea from the somewhat older shallow marine Coralline Crag Formation in southeast England (Dowsett et al. 2012). In addition, modelled anomalies for the mPWP North Sea area of around +3.5°C are in close agreement (Haywood et al. 2020). Compared to estimates for the global mPWP, a 3.5°C warming is on the higher end of the range given by proxy data (+2- 4°C, sea surface temperature, Dowsett et al. 2012) and models (+1.7-5.2°C, surface air temperature, Haywood et al. 2020). It for the mid-Piacenzian Warm Period (mPWP) in the North Sea are similar to a previous estimate of ca. 13°C for the North 685 Sea from the somewhat older shallow marine Coralline Crag Formation in southeast England (Dowsett et al. 2012). In addition, modelled anomalies for the mPWP North Sea area of around +3.5°C are in close agreement (Haywood et al. 2020). Compared to estimates for the global mPWP, a 3.5°C warming is on the higher end of the range given by proxy data (+2- 4°C, sea surface temperature, Dowsett et al. 2012) and models (+1.7-5.2°C, surface air temperature, Haywood et al. 2020). It is higher than the modelled average SST warming of +2.8°C (Haywood et al. 2020). The North Sea in the Pliocene appears 690 to have a somewhat heightened sensitivity to increased CO2 concentrations compared to the global average. The North Atlantic showed a much stronger warming of +4-7°C during the mPWP, well outside the global average proxy and model 31 2022). Previous research on the somewhat older Coralline Crag Formation also found evidence for summer 710 stratification in the early Pliocene (Jenkins and Houghton 1987; Johnson et al. 2009). When vertical mixing is limited, the summer heat is trapped in the surface layer of the water while the bottom waters remain a cooler temperature. This would lead to underestimation of SSTs in benthic organisms such as bivalves. The upper Oorderen interval from which this study’s specimens were collected is interpreted to have been shallower and more strongly influenced by tidal currents than the lower Oorderen interval from which the shells of Valentine et al. (2011) and Johnson et al. (2022) were collected (Louwye et al. 715 2004). Still, as the water depth is not well constrained and the thermocline depth is unknown, summer stratification cannot be ruled out for our interval. Finally, the above temperatures assume a constant δ18Osw. A δ18Osw that varies throughout the year can either dampen or amplify the δ18Oc signal. A dampening corresponds to a positive correlation between δ18Osw and temperature, i.e., higher δ18Osw values in summer, when temperatures are high and δ18Oc is low, and vice versa. Such a signal Oorderen interval from which the shells of Valentine et al. (2011) and Johnson et al. (2022) were collected (Louwye et al. 715 2004). Still, as the water depth is not well constrained and the thermocline depth is unknown, summer stratification cannot be ruled out for our interval. Finally, the above temperatures assume a constant δ18Osw. A δ18Osw that varies throughout the year can either dampen or amplify the δ18Oc signal. A dampening corresponds to a positive correlation between δ18Osw and temperature, i.e., higher δ18Osw values in summer, when temperatures are high and δ18Oc is low, and vice versa. Such a signal can be caused by enhanced evaporation in the summer, and enhanced precipitation and runoff in the winter/spring. This 720 pattern is expected in a mid-latitude settings such as the North Sea, and a strong positive correlation between δ18Osw and temperature is observed in the eastern North Sea today (Ullmann et al. 2010). These four factors—limited sampling resolution, possible growth slow-down in winter, possible stratification in summer, and possible dampening due to δ18Osw fluctuations—all contribute to a potential underestimation of the actual seasonality. can be caused by enhanced evaporation in the summer, and enhanced precipitation and runoff in the winter/spring. A lower primary production has been observed in the past decades, which is partly attributed to warming (Capuzzo et al. 2018). A disturbance of the base of the food web can affect all higher trophic levels and ultimately fisheries. Fish are already affected as well: a of the base of the food web can affect all higher trophic levels and ultimately fisheries. Fish are already affected as well: a decrease in body size due to rising temperatures was demonstrated by Baudron et al. (2014). While the overall temperature 735 in the North Sea basin has increased, there is no robust evidence that temperatures are rising faster in summer than in winter or vice versa (Quante and Colijn 2016). In the neighbouring Baltic Sea, winter temperatures have been rising faster than summer temperatures over the past few decades (Rutgersson et al. 2014), and asymmetric winter-warming has been predicted for the North Sea area by climate models under RCP scenarios 4.5-8.5 (Quante and Colijn 2016 and references decrease in body size due to rising temperatures was demonstrated by Baudron et al. (2014). While the overall temperature 735 in the North Sea basin has increased, there is no robust evidence that temperatures are rising faster in summer than in winter or vice versa (Quante and Colijn 2016). In the neighbouring Baltic Sea, winter temperatures have been rising faster than summer temperatures over the past few decades (Rutgersson et al. 2014), and asymmetric winter-warming has been predicted for the North Sea area by climate models under RCP scenarios 4.5-8.5 (Quante and Colijn 2016 and references therein). As the mPWP should not be a one-to-one comparison for modern transient changes, this is not a direct discrepancy. 740 Rather, we should use mPWP data to strengthen climate model predictions. A large part of paleotemperature data in general, and thus of those used for model boundary conditions, reflect an annual average (although some are biased towards one growing season, e.g., TEX86 from the Pliocene North Sea; Dearing Crampton-Flood et al. 2020). As such, an important part of the climate system is often missing. Adding explicit summer and winter data has the potential to significantly enrich these validation datasets In turn this will lead to more robust predictions for the near future 745 validation datasets. In turn, this will lead to more robust predictions for the near future. 745 This 720 pattern is expected in a mid-latitude settings such as the North Sea, and a strong positive correlation between δ18Osw and temperature is observed in the eastern North Sea today (Ullmann et al. 2010). These four factors—limited sampling resolution, possible growth slow-down in winter, possible stratification in summer, and possible dampening due to δ18Osw fluctuations—all contribute to a potential underestimation of the actual seasonality. The mPWP seasonal temperature range in the North Sea was likely larger than today, and potentially significantly larger. 725 How much larger remains to be determined from additional clumped isotope data, which can solve the δ18Osw-dampening The mPWP seasonal temperature range in the North Sea was likely larger than today, and potentially significantly larger. 725 How much larger remains to be determined from additional clumped isotope data, which can solve the δ18Osw-dampening 32 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. issue. The increase in range is attributed to higher summer temperatures and somewhat colder winter temperatures compared to today. This summer-only warming has been observed by previous studies of the Pliocene SNSB (Raffi et al. 1985; Johnson et al. 2009; Valentine et al. 2011; Johnson et al. 2022). In the context of a warmer global climate, this suggests a reduced heat transport in winter in order to attenuate the overall warming. Such a reduction in heat transport could be related 730 to a change in Gulf Stream intensity, translated to the North Sea via the North Atlantic Current (Valentine et al. 2011). issue. The increase in range is attributed to higher summer temperatures and somewhat colder winter temperatures compared to today. This summer-only warming has been observed by previous studies of the Pliocene SNSB (Raffi et al. 1985; Johnson et al. 2009; Valentine et al. 2011; Johnson et al. 2022). In the context of a warmer global climate, this suggests a reduced heat transport in winter in order to attenuate the overall warming. Such a reduction in heat transport could be related 730 to a change in Gulf Stream intensity, translated to the North Sea via the North Atlantic Current (Valentine et al. 2011). Rising temperatures in the modern North Sea have already taken their toll on regional ecosystems. A lower primary production has been observed in the past decades, which is partly attributed to warming (Capuzzo et al. 2018). A disturbance of the base of the food web can affect all higher trophic levels and ultimately fisheries. Fish are already affected as well: a issue. The increase in range is attributed to higher summer temperatures and somewhat colder winter temperatures compared to today. This summer-only warming has been observed by previous studies of the Pliocene SNSB (Raffi et al. 1985; Johnson et al. 2009; Valentine et al. 2011; Johnson et al. 2022). In the context of a warmer global climate, this suggests a reduced heat transport in winter in order to attenuate the overall warming. Such a reduction in heat transport could be related 730 to a change in Gulf Stream intensity, translated to the North Sea via the North Atlantic Current (Valentine et al. 2011). Rising temperatures in the modern North Sea have already taken their toll on regional ecosystems. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. summer and winter temperatures suggest a larger seasonal range than today, caused by warmer summers but similarly cool winters. This is in line with several other bivalve-based studies that have found cool winters in the Pliocene North Sea basin. 760 760 6 Conclusions 1. Angulus benedeni benedeni’s macro-and micro shell-structures have been described in detail. Four different macrostructures have been observed. In three of these, three different microstructures were recognized. From the outer to the inner layer, these structures have been identified as complex prismatic, crossed lamellar, and complex crossed lamellar with prismatic interlayers and possibly homogeneous sections. This is comparable to what has been observed in other tellinid 750 bivalves. 1. Angulus benedeni benedeni’s macro-and micro shell-structures have been described in detail. Four different macrostructures have been observed. In three of these, three different microstructures were recognized. From the outer to the inner layer, these structures have been identified as complex prismatic, crossed lamellar, and complex crossed lamellar with prismatic interlayers and possibly homogeneous sections. This is comparable to what has been observed in other tellinid 750 bivalves. 2. Based on petrography and δ18Oc measurements, we can state that A. benedeni benedeni life’s span was up to a decade or more, and it likely formed monthly growth increments. It might have experienced slower growth during winter, as characterised by darker growth bands. 2. Based on petrography and δ18Oc measurements, we can state that A. benedeni benedeni life’s span was up to a decade or more, and it likely formed monthly growth increments. It might have experienced slower growth during winter, as characterised by darker growth bands. 3. Angulus benedeni benedeni shows promise as a climate archive, as demonstrated by the first successful isotope 755 analyses performed on this species. Microsampled oxygen isotope records reveal a characteristic sinusoidal pattern from which summer and winter intervals could be discerned. Clumped isotope analysis revealed that the mean annual temperature in the mid-Piacenzian southern North Sea basin was 13.5±3.8°C, 3.5°C warmer than the pre-industrial average. Preliminary 3. Angulus benedeni benedeni shows promise as a climate archive, as demonstrated by the first successful isotope 755 analyses performed on this species. Microsampled oxygen isotope records reveal a characteristic sinusoidal pattern from which summer and winter intervals could be discerned. Clumped isotope analysis revealed that the mean annual temperature in the mid-Piacenzian southern North Sea basin was 13.5±3.8°C, 3.5°C warmer than the pre-industrial average. Preliminary 33 Appendices Figure A1: Sampling procedure of the shells for stable isotope analysis. (a) Sample tracks on specimen SG-126, indicated by dotted lines. 55 in total, each fifth sample track has been labelled. The figure shows the sampled half (top) and that same half prior to 5 sampling (bottom). (b) Sample tracks on specimen SG-127, indicated by dotted lines. 30 in total. This specimen was for a large part covered by epoxy, so only a small section at the upper surface could be sampled. Standard Use δ18O [‰ VPDB] δ13C [‰ VPDB] Δ47 [‰ I-CDES90] Naxos marble Oxygen isotopes (ThermoScientific MAT 253) -6.83 2.08 - Kiel carbonate Oxygen isotopes -16.14 -35.64 - Figure A1: Sampling procedure of the shells for stable isotope analysis. (a) Sample tracks on specimen SG-126, indicated by dotted lines. 55 in total, each fifth sample track has been labelled. The figure shows the sampled half (top) and that same half prior to 65 sampling (bottom). (b) Sample tracks on specimen SG-127, indicated by dotted lines. 30 in total. This specimen was for a large part covered by epoxy, so only a small section at the upper surface could be sampled. Standard Use δ18O [‰ VPDB] δ13C [‰ VPDB] Δ47 [‰ I-CDES90] Naxos marble Oxygen isotopes (ThermoScientific MAT 253) -6.83 2.08 - Kiel carbonate Oxygen isotopes (ThermoScientific MAT 253) -16.14 -35.64 - ETH-3 Clumped isotopes (ThermoScientific MAT 253 Plus) -1.78 1.71 0.6132 34 ETH-2 Clumped isotopes (ThermoScientific MAT 253 Plus) -18.69 -10.17 0.2085 ETH-1 Clumped isotopes (ThermoScientific MAT 253 Plus) -2.19 2.02 0.2052 IAEA-C2 Clumped isotopes (ThermoScientific MAT 253 Plus) -9.00 -8.25 0.6409 Merck Clumped isotopes (ThermoScientific MAT 253 Plus) -15.51 -42.21 0.5135 Table A1: Accepted values for stable isotope measurements, both oxygen and clumped. Figure A2: Linear correlation between the Naxos δ18O data (11 standards measured) and the mass 44 intensity that was used to correct the δ18O data. ETH-2 Clumped isotopes (ThermoScientific MAT 253 Plus) -18.69 -10.17 0.2085 ETH-1 Clumped isotopes (ThermoScientific MAT 253 Plus) -2.19 2.02 0.2052 IAEA-C2 Clumped isotopes (ThermoScientific MAT 253 Plus) -9.00 -8.25 0.6409 Merck Clumped isotopes (ThermoScientific MAT 253 Plus) -15.51 -42.21 0.5135 Table A1: Accepted values for stable isotope measurements, both oxygen and clumped. Figure A2: Linear correlation between the Naxos δ18O data (11 standards measured) and the mass 44 intensity that was used to 70 correct the δ18O data. ETH-2 Clumped isotopes (ThermoScientific MAT 253 Plus) -18.69 -10.17 0.2085 ETH-1 Clumped isotopes (ThermoScientific MAT 253 Plus) -2.19 2.02 0.2052 IAEA-C2 Clumped isotopes (ThermoScientific MAT 253 Plus) -9.00 -8.25 0.6409 Merck Clumped isotopes (ThermoScientific MAT 253 Plus) -15.51 -42.21 0.5135 Table A1: Accepted values for stable isotope measurements, both oxygen and clumped. Table A1: Accepted values for stable isotope measurements, both oxygen and clumped. Figure A2: Linear correlation between the Naxos δ18O data (11 standards measured) and the mass 44 intensity that was used to 770 Figure A2: Linear correlation between the Naxos δ18O data (11 standards measured) and the mass 44 intensity that was used to 770 correct the δ18O data. n between the Naxos δ18O data (11 standards measured) and the mass 44 intensity that was used to Figure A2: Linear correlation between the Naxos δ18O data (11 standards measured) and the mass 44 intensity that was used to 770 correct the δ18O data. 35 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure A3: Empirical transfer function (ETF) constructed from a linear regression between accepted and measured Δ47 values from standards ETH-1, ETH-2, and ETH-3. This function was used to correct all samples and standards and transfer them to the I-CDES90C reference frame. 775 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure A3: Empirical transfer f nction (ETF) constr cted from a linear regression bet een accepted and meas red Δ values Figure A3: Empirical transfer function (ETF) constructed from a linear regression between accepted and measured Δ47 values from standards ETH-1, ETH-2, and ETH-3. This function was used to correct all samples and standards and transfer them to the I-CDES90C reference frame. 75 36 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. 37 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. 38 Figure A4: corrected Δ47 data for external (ETH-3, ETH-2, ETH-1) and internal (IAEA-C2, Merck) standards. (a)-(c): ETH-3 Δ47, δ18O, and δ13O data. (d)-(f): ETH-2 Δ47, δ18O, and δ13O data. (g)-(i): ETH-1 Δ47, δ18O, and δ13O data. (j)-(l): IAEA-C2 Δ47, δ18O, and 0 δ13O data. (m)-(o): Merck Δ47, δ18O, and δ13O data. Dates corresponding to the run numbers can be found in the supplementary materials. δ18O and δ13O measurements affected by mass-dependent fractionation are marked as red open circles (see sect. 3.5.3). https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure A4: corrected Δ47 data for external (ETH-3, ETH-2, ETH-1) and internal (IAEA-C2, Merck) standards. (a)-(c): ETH-3 Δ47, δ18O, and δ13O data. (d)-(f): ETH-2 Δ47, δ18O, and δ13O data. (g)-(i): ETH-1 Δ47, δ18O, and δ13O data. (j)-(l): IAEA-C2 Δ47, δ18O, and 780 δ13O data. (m)-(o): Merck Δ47, δ18O, and δ13O data. Dates corresponding to the run numbers can be found in the supplementary materials. δ18O and δ13O measurements affected by mass-dependent fractionation are marked as red open circles (see sect. 3.5.3). 780 39 Figure A5: Δ47 data for specimens SG-126 and SG-127, plotted against the run number as counted in the Utrecht University stable isotope laboratory, to show the (lack of) drifting of values over time. Dates corresponding to the run numbers can be found in the 785 supplementary materials. https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure A5: Δ47 data for specimens SG-126 and SG-127, plotted against the run number as counted in the Utrecht University stable Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. Competing interests The authors declare that there are no competing interests involved in this research. Data availability 800 Oxygen isotope, clumped isotope, X-ray fluorescence, X-ray diffraction, and electron backscatter diffraction data are available at the PANGAEA data repository (LINK PENDING; SUBMITTED ON 19/09/2022). Author contribution NJW designed the study and carried out X-ray diffraction measurements. NMAW carried out stable isotope measurements, analysed all data and wrote the original manuscript. ALAJ assisted with sclerochronogy and ecological and environmental 805 interpretations. SG provided the shell material and carried out the palaeoenvironmental interpretation of the Oorderen Formation. FW carried out the taxonomical analysis of the shells and assisted with ecological interpretation. PK and PC carried out micro-X-ray fluorescence measurements and analysis. MFH carried out electron backscatter diffraction analyses and assisted with their interpretation. MZ supervised the project and aided in data analysis. All authors contributed to interpreting the compiled data and editing the manuscript. 810 Code availability All code used was written in R. Codes for Monte Carlo error propagation (Benedeni_Monte_Carlo.R) and growth modelling (Benedeni_Growth_Model.R) are available at the Github repository at https://github.com/NMAWichern/Benedeni_benedeni. All code used was written in R. Codes for Monte Carlo error propagation (Benedeni_Monte_Carlo.R) and growth modelling (Benedeni_Growth_Model.R) are available at the Github repository at https://github.com/NMAWichern/Benedeni_benedeni. All code used was written in R. Codes for Monte Carlo error propagation (Benedeni_Monte_Car (Benedeni_Growth_Model.R) are available at the Github repository at https://github.com/NMAW https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure A5: Δ47 data for specimens SG-126 and SG-127, plotted against the run number as counted in the Utrecht University stable isotope laboratory, to show the (lack of) drifting of values over time. Dates corresponding to the run numbers can be found in the 785 supplementary materials. 40 https://doi.org/10.5194/egusphere-2022-951 Preprint. Discussion started: 12 October 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure B1: EBSD map of specimen SG-125 indicating the absence secondary calcite crystals. Map acquisition specifics: 10kV voltage, 1 μm step size. Measured at low vacuum due to excessive charging. Classification: 54.6% aragonite, 45.2% unclassified, 790 0.2% calcite. The map consists of the band contrast overlain by the classified pixels, which are either aragonite (blue) or calcite (red). M+2, M+1, and M-1 correspond to the shell layers as described in sect. 4.1.1. No clean-up was done on this map. The pixels identified as calcite do not show any of the characteristics of actual secondary calcite (large crystals compared to the very fine- grained aragonite, breaking up the aragonitic structure), are always single pixels, and are removed through AZtecCrystal’s wild spike removal algorithm. Several dust shadows are also seen in this image; these areas are unclassified. There are also several 795 scratches present on the surface. Figure B1: EBSD map of specimen SG-125 indicating voltage, 1 μm step size. Measured at low vacuum due 0.2% calcite. The map consists of the band contrast ov Figure B1: EBSD map of specimen SG-125 indicating the absence secondary calcite crysta Figure B1: EBSD map of specimen SG-125 indicating the absence secondary calcite crystals. Map acquisition specifics: 10kV voltage, 1 μm step size. Measured at low vacuum due to excessive charging. Classification: 54.6% aragonite, 45.2% unclassified, 790 0.2% calcite. The map consists of the band contrast overlain by the classified pixels, which are either aragonite (blue) or calcite (red). M+2, M+1, and M-1 correspond to the shell layers as described in sect. 4.1.1. No clean-up was done on this map. The pixels identified as calcite do not show any of the characteristics of actual secondary calcite (large crystals compared to the very fine- grained aragonite, breaking up the aragonitic structure), are always single pixels, and are removed through AZtecCrystal’s wild spike removal algorithm. Several dust shadows are also seen in this image; these areas are unclassified. There are also several 795 scratches present on the surface. 41 References Al-Aasm, I. S. and Veizer, J.: Diagenetic stabilization of aragonite and low-Mg calcite: I, Trace elements in rudists, SEPM JSR, 56, 138–153, https://doi.org/10.1306/212F88A5-2B24-11D7-8648000102C1865D, 1986. Balson, P. S., Mathers, S. J., and Zalasiewicz, J. 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CC BY 4.0 License. 1219–1236, https://doi.org/10.1306/212F7BB7-2B24-11D7-8648000102C1865D, 1980. 850 Brand, W. A., Assonov, S. S., and Coplen, T. B.: Correction for the 17O interference in δ (13C) measurements when analyzing CO2 with stable isotope mass spectrometry (IUPAC Technical Report), Pure Appl. Chem., 82, 1719–1733, https://doi.org/10.1351/PAC-REP-09-01-05, 2010. Acknowledgements The authors gratefully acknowledge the following people for their help with this work: Leonard Bik (Utrecht University) made the polished thin sections for microscopy and EBSD work. Desmond Eefting and Arnold van Dijk (Utrecht University) 815 provided technical assistance during isotope analyses. Lucas Lourens (Utrecht University) proof-read the original version of this manuscript. Maarten Zeylmans (Utrecht University) helped produce high-resolution scans of cross sections of the specimens. Pim Kaskes is supported by a Research Foundation Flanders (FWO) PhD Fellowship (11E6621N). 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The Howard League and liberal colonial penality in mid‐20th‐century Britain: The death penalty in Palestine and the Kenya Emergency
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Link to external publisher version https://doi.org/10.1111/hojo.12513 Link to external publisher version https://doi.org/10.1111/hojo.12513 The Howard League and liberal colonial penality in mid-20th-century Britain: the death penalty in Palestine and the Kenya Emergency Lizzie Seal, Roger Ball Lizzie Seal, Roger Ball Licence This work is made available under the CC BY 4.0 licence and should only be used in accordance with that licence. For more information on the specific terms, consult the repository record for this item. Document Version Published version Citation for this work (American Psychological Association 7th edition) Seal, L., & Ball, R. (2023). The Howard League and liberal colonial penality in mid-20th-century Britain: the death penalty in Palestine and the Kenya Emergency (Version 1). University of Sussex. https://hdl.handle.net/10779/uos.23491688.v1 Published in The Howard Journal of Criminal Justice O R I G I NA L A RT I C L E O R I G I NA L A RT I C L E The Howard League and liberal colonial penality in mid-20th-century Britain: The death penalty in Palestine and the Kenya Emergency Lizzie Seal1 Roger Ball2 1Lizzie Seal is Professor of Criminology, University of Sussex 2Roger Ball is Research Fellow, University of the West of England 1Lizzie Seal is Professor of Criminology, University of Sussex 2Roger Ball is Research Fellow, University of the West of England Received: 6 January 2022 Accepted: 12 July 2022 DOI: 10.1111/hojo.12513 Received: 6 January 2022 Accepted: 12 July 2022 DOI: 10.1111/hojo.12513 DOI: 10.1111/hojo.12513 Abstract Abstract This article analyses the Howard League’s campaigning against the death penalty in mid-20th-century British colonies. It examines two case studies: the Howard League’s campaign to limit the death penalty in the Palestine Mandate in the 1930s and their silence on mass executions during the Kenya Emergency in the 1950s. Drawing on Ben-Natan’s (2021) concept of the dual penal regime, we argue the Howard League concentrated its intervention in ordinary penal regimes and demarcated emergency penal regimes as outside its sphere of interest and influence. Consequently, it was silent on the penal excess of colonial authorities during periods of counter- insurgency. Criminology as a discipline largely shares this demarcation of the penal measures associated with colonial wars, militarism and states of emergency as beyond its purview. Inclusion of these aspects of colonial penality into the criminological narrative highlights the significance of colonialism and colonial ways of thinking to penal liberalism. Howard J. Crim. Justice. 2023;62:149–166. wileyonlinelibrary.com/journal/hojo 149 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2023 The Authors. The Howard Journal of Crime and Justice published by Howard League and John Wiley & Sons Ltd. Copyright and reuse: py g This work was downloaded from Sussex Research Open (SRO). This document is made available in line with publisher policy and may differ from the published version. Please cite the published version where possible. Copyright and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners unless otherwise stated. For more information on this work, SRO or to report an issue, you can contact the repository administrators at sro@sussex.ac.uk. Discover more of the University’s research at https://sussex.figshare.com/ 1 INTRODUCTION On 2 June 1930, the Howard League for Penal Reform’s Colonial Subcommittee held its first meeting.1 At this time, the Howard League had a keen interest in international penal reform. The establishment of this subcommittee followed the opening of the Howard League’s Interna- tional Bureau in Geneva a year previously, which had a special focus on prison conditions and aimed to get penal reform onto the agenda of the League of Nations.2 The purpose of the Colo- nial Subcommittee was to gain an overview of penal administration in the colonies of the British Empire and to intervene to bring about improvements and modernisation. The subcommittee was argued to be necessary ‘especially in regard to the treatment of the natives’.3 In keeping with the Howard League’s priorities, the Colonial Subcommittee intended to maintain a focus on prison conditions, especially those for young people.4 The work of the Colonial Subcommittee also involved intervention in relation to capital pun- ishment. Their stance on the colonies was that the death penalty should be abolished, but they were willing to intervene to ameliorate its use in lieu of full abolition. In 1932, in response to the imposition of 60 death sentences in Kenya for the murder of someone believed to be a witch, the Colonial Subcommittee resolved to approach the Colonial Office to suggest revising the law to enable discretion in sentencing and the use of alternatives to capital punishment, which would bring the law in Kenya in line with the Indian Penal Code.5 At this time, the death penalty in Britain was mandatory for murder so the Indian Penal Code was more liberal on this issue than the law in the metropole. This article analyses the Howard League’s campaigning in relation to the death penalty in mid- 20th-century British colonies. It does so through two case studies: their campaigning to limit the death penalty in the Palestine Mandate in the 1930s and their silence on mass executions dur- ing the Kenya Emergency in the 1950s. The majority of the Howard League’s work in the colonies concerned prisons but the death penalty is also instructive as an example through which to under- stand its involvement with colonial penality. 150 THE HOWARD JOURNAL OF CRIME AND JUSTICE THE HOWARD JOURNAL OF CRIME AND JUSTICE 150 Correspondence Funding information British Academy, Grant/Award Number: BA/IC3/100170 K E Y WO R D S colonialism, death penalty, Howard League, Kenya Emergency, Palestine, penal liberalism This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2023 The Authors. The Howard Journal of Crime and Justice published by Howard League and John Wiley & Sons Ltd. Howard J. Crim. Justice. 2023;62:149–166. wileyonlinelibrary.com/journal/hojo 149 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2023 The Authors. The Howard Journal of Crime and Justice published by Howard League and John Wiley & Sons Ltd. civilians by military courts, summary proceedings, and harsh punishments’ (Ben-Natan, 2021, p.742). Emergency powers remove restrictions on military action without the need for martial law (Thomas, 2018). Ben-Natan argues that colonial authorities do not acknowledge that penal emergency regimes are part of the penal system. civilians by military courts, summary proceedings, and harsh punishments’ (Ben-Natan, 2021, p.742). Emergency powers remove restrictions on military action without the need for martial law (Thomas, 2018). Ben-Natan argues that colonial authorities do not acknowledge that penal emergency regimes are part of the penal system. We adopt this concept of the dual penal regime to discuss the Howard League and the death penalty in both Palestine and Kenya. Their intervention in Palestine concentrated only on the ordinary penal regime, not the emergency one. Their very limited attention to 1950s Kenya sug- gests the executions that took place in the emergency penal regime were understood to be beyond their remit. The Howard League was created in 1921 via a merger of the Howard Association and the Penal Reform League. It has been described as a classic example of an ‘insider’ organisation with close connections to government, particularly the Home Office and the Prison Commission, and having a liberal, rather than radical, orientation. The Howard League was (and is) the most influential penal reform group in England and Wales. It had a relatively small membership, which skewed middle to upper middle class, and a metropolitan orientation (Ryan, 2003). Ryan (2003) notes that policymaking was undertaken by members of the Executive Committee and its subcommittees as experts, who frequently wielded influence informally in addition to sitting on committees and advisory councils linked to government. A leading proponent of penal liberalism, the Howard League emphasised the need for ratio- nality, expertise and a scientific approach in penal policymaking and practice. Penal intervention should be directed towards rehabilitation. The death penalty was perceived as contrary to this penal modernism and a relic of barbarism. The League adopted abolition of the death penalty as one of its campaigning priorities in 1923 and the National Council for the Abolition of the Death Penalty (NCADP) was founded in 1925. This was a separate organisation from the Howard League but was based in the same building and shared key members (it merged with the League in 1948). 1 INTRODUCTION As Dubber (2018, p.7) argues, capital punishment is ‘the sharpest point of the sharp end of the stick of state penal power’, which shows the distinc- tion between the ‘abstract threat of penal violence’ and ‘the infliction of that threatened violence on a particular person’. As we shall discuss, the civilian death penalty was not the sharpest end of the stick of colonial penal power. Penality is bound up with the legitimacy of state power, but this power operated differently in colonial territories and protectorates than in the liberal state (Ben-Natan, 2021). We draw on Ben-Natan’s (2021) analysis of the ‘dual penal regime’ in Palestine to contextualise the discussion of the Howard League’s campaigning activities. She adapts Dubber’s (2018) con- cept of the dual penal state, according to which liberal penal governance rests on the duality of the penal law and penal police. Dubber (2018) argues that these two paradigms coexist but are in opposition, creating a paradox. The penal law has an egalitarian identification, whereby crime is the violation of one person’s autonomy by another. The penal police is hierarchical and crime is an offence against the state’s sovereignty. Empire is more openly illiberal than the liberal state and colonial penality needs to be understood differently from the national dual penal state. There is no pretence in colonial settings that ‘penal law’ applies equally to everyone (Ben-Natan, 2021). In particular, racialised enemy populations – those perceived as a threat to colonial rule – are tar- geted by colonial law. The dual penal regime encapsulates the separate penal regime established by use of emergency powers that ran (and runs) in parallel to the ordinary penal regime. Emer- gency statutes ‘feature broad criminalization of political and military resistance, prosecution of THE HOWARD JOURNAL OF CRIME AND JUSTICE 151 stories about the development of modern Western penology omit analysis of penal colonialism and of penal mixing between the metropole and the colonies. In relation to management studies, Frenkel & Shenhav (2006) employ Latour (1993) to describe this process of writing out non-Western influences and developments as ‘purification’. The story of mid-20th-century Britain’s penal culture and practice looks very different if it is narrated through colonial settings, particularly emergency penal regimes. The mass executions carried out under emergency powers during colonial dirty wars in Kenya and Malaya are sharply divergent from the declining use of capital punishment in Britain itself after the Second World War. stories about the development of modern Western penology omit analysis of penal colonialism and of penal mixing between the metropole and the colonies. In relation to management studies, Frenkel & Shenhav (2006) employ Latour (1993) to describe this process of writing out non-Western influences and developments as ‘purification’. The story of mid-20th-century Britain’s penal culture and practice looks very different if it is narrated through colonial settings, particularly emergency penal regimes. The mass executions carried out under emergency powers during colonial dirty wars in Kenya and Malaya are sharply divergent from the declining use of capital punishment in Britain itself after the Second World War. p p This article contributes to burgeoning work that incorporates the significance of colonialism, and the legacies of Empire, into criminological analysis. As Agozino (2003, 2004) argues, Western criminology has paid scant attention to the harms of slavery and colonialism even though these harms dwarf much of what it chooses to focus on in terms of effects and legacy. For Agozino, criminology’s silence about colonialism is an effect of colonialism. A parallel can be drawn with interpretations of British penality in the mid-20th century. The story of the development of penal welfarism in Britain is portrayed as commitment to the use of rehabilitative measures as consti- tuting progress, underpinned by faith in practitioners and experts to bring about a correctionalist agenda (Garland, 2001). This story does not incorporate British colonial penality, whatever its character, correctionalist or brutal. King (2017) argues that contemporary colonial reason hides the legacies of colonialism. Construing the counter-insurgency in 1930s Palestine or the Kenya Emergency as separate from British penal practice and culture accepts colonial reason. Dimou (2021) draws on Quijano’s (2000, 2007) concept of coloniality to analyse the colonial foundations of criminology. Coloniality shapes ways of being, interacting and perceiving and is constitutive of modernity, forming its ‘dark side’. Penal modernism, which as Brown (2002) highlights incorporates penal excess, is inevitably colonial. British criminology should examine this coloniality because the British Empire was the most extensive empire in history and coercive networks were essential to its maintenance (Sher- man, 2009). Despite a growing body of criminological work on the significance of colonialism to crime and punishment and a well-developed historiography of penality in different colonial settings, British criminology remains largely unaware of, and uninterested in, the penality of the British Empire and its legacies (Moore, 2020). Following Brown (2002), we argue that penal excess is a constituent part of penal modernity and not a departure from it; penal coloniality must be included in the analysis to fully understand this point. We discuss penal coloniality and the use of emergency penal regimes to contextualise our examination of the Howard League’s campaigning on the colonial death penalty, but our analysis is not of the development of penal coloniality and emergency penal regimes per se. The campaign for the abolition of capital punishment in Britain gathered speed in the 1930s and at the same time the Howard League and NCADP undertook limited campaigning in relation to the colonial death penalty. The Howard League’s focus on the ordinary penal regime was consistent with the priorities of liberal colonial penality, which is defined further below. Part of the significance of examining both the Howard League’s intervention and lack of intervention in relation to the colonial death penalty is that the organisation’s assumptions about what counted as the appropriate sphere of influence for British penal reform are replicated by British criminology as a discipline in terms of the con- trolling regimes that it counts as penal. As highlighted by the criminology of war, critical analysis of state-based violence, while not completely absent, has not been included in criminology’s ‘core set of concerns’ (Walklate & McGarry, 2015, p.5). War and states of emergency entail increased use of social regulation, punishment, ideological control and techniques of surveillance – all of which require penal regimes (Jamieson, 2016). Similarly to Ben-Natan (2021), Jamieson (2016) argues that war and occupation are underpinned by the ‘convergence of normal and exceptional penal practice’ (p.xxi). Colonialism and counter-insurgency during the era of decolonisation relied on violence and militarism. Dirty wars entailed ‘highly demonstrative acts of collective violence that [were] designed to compel popular compliance’ and which primarily targeted civilian populations (Thomas, 2018, p.505). The wars, militarism and states of emergency linked to colonialism should fall within crimi- nology’s purview because they entail forms of penal control which, as Ben-Natan (2021) argues, are denied as part of the penal system by authorities. Criminologists should ask how attention to colonial penality potentially modifies understanding of penality in the metropole. Canonical THE HOWARD JOURNAL OF CRIME AND JUSTICE 152 the Howard League’s colonial and international work more generally, rather than only on capital punishment. This extension was to gain a fuller understanding of the organisation’s perspective and actions related to colonial penality. The Howard League gave us permission to read and photo- graph uncatalogued records held at their London offices. These contained some extra documents, but many duplications of records held in the MRC. the Howard League’s colonial and international work more generally, rather than only on capital punishment. This extension was to gain a fuller understanding of the organisation’s perspective and actions related to colonial penality. The Howard League gave us permission to read and photo- graph uncatalogued records held at their London offices. These contained some extra documents, but many duplications of records held in the MRC. Meeting minutes constituted important sources for this research but have limitations. As a genre, minutes frequently document consensus, downplaying debate, disagreement and conflict (Wolfe, 2006). The minutes kept by the Howard League and NCADP can be understood as ‘action oriented’ (Wolfe, 2006), meaning that they focused on future actions and responsibilities rather than recording all aspects of the discussion that took place. They are indispensable sources of decisions taken and action points decided on but do not represent debate over, or dissent from, these decisions. The archival sources were photographed and compiled into folders, and annotated catalogues were created. Notes were made on the basis that a particular item had a relationship to the death penalty and British colonies, international and colonial work, relationships with other NGOs and the Colonial Office or intersections of these categories. Notes for items of particular interest were highlighted. For the case study on Palestine, we also drew on news articles from the 1930s iden- tified from searches of the digital archives of The Times and Manchester Guardian. The more extensive historiography of the Kenya Emergency meant we consulted secondary works for how the press reported this conflict. As part of the research, we constructed a prosopography of the relevant decision-making com- mittees of the Howard League and NCADP in the period of interest. Prosopography is ‘collective biography, describing the external features of a population group’ (Verboven, Carlier & Dumolyn, 2007, p.39). In both organisations, it was the Executive Committee and subcommittees that ran things, determined policy and facilitated interaction with the outside world. 2 SOURCES AND METHODS This article is developed from a research strand of a larger project entitled Reforming British Law and Policy on the Global Death Penalty (BA/IC3/100170). The strand was an analysis of historical and contemporary British-based NGO campaigns that oppose(d) the death penalty worldwide. The sources for the research into the international campaigning activities of the Howard League and NCADP in relation to the death penalty were meeting minutes, correspondence, reports and copies of the journal Penal Reformer held in the Modern Records Centre (MRC), University of Warwick for the period 1921–1965. The MRC is a repository for archives of trade unions, employ- ers’ organisations, small political parties and pressure groups. We extended the data collection to THE HOWARD JOURNAL OF CRIME AND JUSTICE 153 3 LIBERAL COLONIAL PENALITY Liberal colonial penality transplanted the tenets of penal liberalism and penal modernism to penal administration in colonial settings. It was not the only form of colonial penality, but it was the one that cohered with the priorities of the Howard League as a penal reform organisation. Penal liber- alism refers to the belief that government should respond to crime and punishment ‘in ways that, above all, seek to preserve “civilised values”’ and was espoused (and enacted) by politicians, senior civil servants, penal reformers and academic criminologists (Loader, 2006, p.563). The mid-20th- century advocates of penal liberalism comprised a ‘closely networked world of colleagues and friends’ who moved between academia, the civil service and penal reform organisations (Loader, 2006, p.563). Loader (2006) situates the heyday of these ‘platonic guardians’ in Britain as the 1950s and 1960s but his description also applies to the earlier part of the mid-20th century. Penal liberal- ism entailed a commitment to rehabilitation, which was perceived as both a humane way to treat people and one grounded in expertise and scientific principles. As such, rehabilitative approaches were consistent with the ‘wider civilizing purpose’ of liberal government (Loader, 2006, p.565). In Loader’s (2006) assessment, penal liberalism was underpinned by the ‘liberalism of fear’, which is driven by the fear of violence – from individuals who would commit violent crime and the state’s potentially violent response. Penal liberalism opposed bodily punishment, like cor- poral and capital punishment. Liberal colonial penality evinced less interest in, or fear of, the violence of colonial government but it shared the guiding assumption that such violence should be constrained in the ordinary penal regime. It also prioritised rehabilitation and decent prison conditions as constituting a civilised approach. We do not argue that penal reformers such as the Howard League approved of, or contributed to, the repression enacted in emergency penal regimes, but they did not organisationally oppose it. In accepting the emergency penal regime as a separate sphere beyond their interest, they tacitly accepted its legitimacy. Penal liberalism’s language of civilisation was shared by, and entirely consistent with, colonial penality, whether liberal or otherwise. Brown (2004) analyses the significance of different forms of liberalism to British imperialism, distinguishing between orthodox and authoritarian liberalism. Orthodox liberalism’s ‘narrative of uplift’ was based on the notion that British colonial governance would advance colonised populations up the civilisational ladder, making them ready for self- governance. Ordinary members could attend an AGM, but otherwise had no formal input into the organisations’ policies and cam- paigning activities. Ryan’s (1978) description of the Howard League in the 1970s as ‘a small, well connected London based elite with no adequate democratic structures’ (p.86) applied also prior to the 1970s. Rose (1961) argues that the Howard League’s membership did not want to play a role in formulating policy; they wanted ‘a body of experts who press upon the authorities an attitude of mind with which the membership in general agree’ (p.264). The Executive Committees of the Howard League and NCADP comprised people who had backgrounds in Quakerism, suffragism, conscientious objection and political affiliations with the Fabian Society, the Labour Party and the Independent Labour Party. Legal experience, whether as lawyers, judges or, for women, justices of the peace, was a common denominator. Women were strongly represented, with ten out of 17 leading members of NCADP being female. The Howard League’s Colonial Subcommittee in the 1930s included members with military back- grounds in British colonies and experience of colonial administration. For example, there was a former Inspector General of Prisons, Bengal and a former Senior Commissioner, British East Africa Protectorate (later Kenya). The Howard League’s post-war Executive Committee contin- ued to be drawn from people with experience as magistrates, judges, lawyers or barristers, as well as academic criminologists. Connections to the Labour Party remained, with Labour MPs such as John Paton and Charles Hale having membership. The majority of leading members of the Howard League and NCADP were from upper-middle-class backgrounds, with very few from the working class. No people of colour sat on the committees of either organisation. 154 THE HOWARD JOURNAL OF CRIME AND JUSTICE 3 LIBERAL COLONIAL PENALITY 154 as blowing Indian mutineers from canon had no metropolitan equivalent. Local populations were viewed as inherently different from white British people, making differential punishment accept- able (Wagner, 2016). A racial divide between white people and people of colour existed across the Empire and the use of excessive violence reinforced hierarchies of race (Wagner, 2016; Wiener, 2009). Exceptionality and the use of criminal law to target certain racial groups did not require a state of emergency but was part of the ordinary penal regime in colonies (Brown, 2004; McClure, 2020). Public displays of colonial violence were assertions of sovereign power and a means of governance (Anderson, 2015; Wagner, 2016). g g When public hanging was abolished in Britain in 1868, the amendment was sent to colonies excluding India but not all complied (Anderson, 2015). In the early 20th century, execution in many African states took place in public, although this changed to mainly in private by the 1920s and 1930s (Hynd, 2008). Incidents of public execution occurred in other colonies in the 1930s. In 1934, the Howard League’s members’ journal Penal Reformer noted that NCADP had asked Samuel Hoare, Secretary of State for India, to inquire into the circumstances in which two men were hanged before a large crowd in Sind.6 In 1935, the journal recorded that Hoare had declined to forbid public execution in India on the grounds it was ‘necessary for the preservation of law and order’. The editorial commented that legal terrorism ‘begets terrorism’.7 This example demon- strates that the Howard League was willing to be critical of colonial penality and to highlight the repressive use of penal violence when it happened under the ordinary penal regime. From the 1920s onwards, colonial penality embraced discourses of reform, emphasising mod- ernisation and becoming more bureaucratic. This shift paralleled changes in the administration of colonialism itself (Hynd, 2015). In the 1930s, the purpose of colonial imprisonment was redefined away from violent punishment towards rehabilitation via scientific means (Bruce-Lockhart, 2017; Hynd, 2015). There was a growing commitment to penal welfarism and emerging networks of professional knowledge in relation to colonial imprisonment (Bruce-Lockhart, 2017; Hynd, 2015). This professionalisation did not mean coercive violence disappeared from colonial prison systems and the use of corporal punishment against Black African prisoners was sometimes interpreted as a type of ‘civilising violence’ (Hynd, 2015). 3 LIBERAL COLONIAL PENALITY Authoritarian liberalism’s ‘narrative of debasement’ saw colonised populations as having no capacity for civilisation. British colonial rule would secure their well-being but would not act as a transition to self-governance. Common to both discourses was belief in colonialism’s ‘civilising mission’. Brown (2004) argues that ultimately both forms of liberalism rested on the necessity of repression to secure colonial governance. Penal networks were ‘a key nexus of colonial authority’ through which colonial states sought to maintain order and ‘civilise’ the population (Hynd, 2011, p.432). There was, however, a tension between the violence of colonial rule and its civilising claims (Bruce-Lockhart, 2017; Hynd, 2015). Imprisonment and fines were the most commonly used penalties, but as Hynd (2011) asserts: ‘the apex of the colonial state’s penal response remained corporeal – and particularly capital – punishment’ (p.442). Executions were deployed as a deterrent to uphold colonial order and to underline its authority. In the 19th century, capital punishment took different forms across the British Empire, which often varied with practices in the metropole. Execution was used to pun- ish ‘ordinary’ criminals but also to quell resistance to imperial rule, sometimes under martial law (Anderson, 2015). Practices such as displaying corpses had ended in Britain and spectacles such THE HOWARD JOURNAL OF CRIME AND JUSTICE 155 4 THE NEW CRIMINAL CODE FOR PALESTINE British forces conquered Palestine in 1917 during the First World War. Prior to that, it was part of the Ottoman Empire. From 1922 until the founding of Israel in 1948, Britain ruled Palestine under a League of Nations mandate having previously held it under military occupation. In 1929 there were riots and attacks on Jewish communities across Palestine by Palestinian Arabs. The Shaw Commission (1930) was established to examine the riots and what caused them. Its report recommended replacing the Ottoman Penal Code as it had ‘inadequate provisions about the law of homicide’ (Bentwich, 1938, p.72). Under the Ottoman Code, murder was only a capital offence with evidence of premeditation, which had a technical meaning of having been planned at least 24 hours before the murder took place (Bentwich, 1934). Most rioters could not be charged with this and were not eligible for the death penalty. The other part of the rationale for the new criminal code was to bring the law in Palestine into line with ‘English models’ (Bentwich, 1938, p.72). Norman Bentwich, the Attorney General in Palestine through the 1920s and until 1931, joined the Colonial Subcommittee in 1933. He argued in an article for the Howard Journal that the new Palestinian Criminal Code, proposed in 1933, recognised ‘a more scientific statement of the Law should be enacted’ (Bentwich, 1934, p.61). However, he noted ‘two major innovations in the pro- posed code call for anxious consideration, because in both cases they run counter both to popular feeling and to modern principles of penology’ (p.61). These innovations were to make the death penalty mandatory for murder and introduce flogging as a judicial penalty. Bentwich argued that both Muslim and Jewish people in Palestine were opposed to capital punishment and that corporal punishment did not exist under the Ottoman Penal Code. The proposals were culturally inappro- priate; in a later article Bentwich (1938) described the ‘severity’ of the mandatory death penalty as ‘altogether alien to Arab morality’ (p.73). The extension of the death penalty and introduction of flogging ran counter to penal modernity and induced penal anxiety; in this case, the British colonial authorities would violate principles of rationality, science and civilisation by deploying archaic bodily punishment. 2019; Thomas & Thompson, 2014). Article 22 of the League of Nations’ Covenant established the mandate system, which was based on this principle of tutelage by colonial powers, and modernised and legitimised colonialism as something which could be ‘humane’ (Gopal, 2019). Colonial tutelage was not incompatible with the promotion of social justice and the protection of minorities, but this was based on paternalism rather than the notion that different groups had inherent rights (Thomas & Thompson, 2014). There was resistance to the imposition of the mandates and the emergence of an international anticolonial movement (Gopal, 2019). Capital punishment remained in use, as it did in Britain, with rates of the execution of non-politically motivated criminals not necessarily higher than in the metropole (see Campbell, 2015; Hynd, 2012). The establishment of the Howard League’s Colonial Subcommittee in 1930 was emblematic of reformist developments, as was its work with the Society of Friends and League of Nations Union to successfully persuade the League of Nations to adopt a set of global minimum standards for prisoners in 1934 (Bruce-Lockhart, 2017). Consistent with the priorities of penal welfarism, provision for women and young people was of particular interest to colonial penal reform, as was the development of probation systems (Hynd, 2011, 2015). However, the ‘civilising mission’ of liberal colonial penality conflicted with ‘the imperatives of indirect rule’ (Hynd, 2012, p.89). Colo- nial violence and repression remained routine, increasing at times when colonial authority was challenged or opposed. As Hynd (2011) argues: ‘colonial legal regimes ultimately remained depen- dent upon the threat and application of state-sanctioned violence’ (p.447). Excessive violence was ‘intrinsic to the colonial encounter’ and 20th-century massacres of civilians such as at Jallianwala Bagh in Amritsar in 1919 functioned as a form of punishment, recalling the mass public executions in India of the 19th century (Wagner, 2016, p.189). Penal anxiety that colonialism could mean the imposition of uncivilised forms of punishment was at the heart of the Colonial Subcommittee’s intervention to revise the law relating to the death penalty and corporal punishment in Palestine. In the interwar period, the justification for colonialism was one of tutelage to societies that were supposedly not ready for self-rule (Gopal, THE HOWARD JOURNAL OF CRIME AND JUSTICE 156 The Howard League’s involvement in Palestine extended beyond objection to the New Draft Penal Code to penal welfarist concerns such as the development of probation and separate provision for young people. Demonstrating this interest, the Howard Journal published an article in 1935 by Margaret Nixon (1935), Welfare Inspector for the Palestine Government, about incarcer- ated women and girls in Palestine. As part of her role, Nixon inspected the women’s prison and lock ups, and superintended the girls’ reformatory home. Her article evinces the tenets of liberal penal colonial penality and the assumption that the imposition of British colonialism meant a superior way of doing things. Reflecting gendered principles of rehabilitation, women prisoners worked in the prison’s laundry, kitchens and garden to gain suitable domestic skills. A distinct system for young people was an important aspect of penal welfarism and a girls’ reformatory was established, where previously girls had been incarcerated with women. Nixon also explained that a system of probation was growing. The Howard League supported the extension of the use of probation in Palestine up until the Second World War via representation on the Standing Advisory Committee on Penal Administration, which in 1943 became the Advisory Committee on the Treatment of Offenders in the Colonies.13 A Revised Criminal Law Bill was published in Palestine in 1936, which restricted the death penalty to certain types of murder: premeditated, in the course of another crime, to escape pun- ishment or for the murder of someone’s own father, mother, grandmother or grandfather. The Revised Bill did not include flogging. Bentwich (1938) noted there had been local opposition to the previous Criminal Law Bill but argued without objections from the Howard League and other British-based organisations, it was unlikely it would have been defeated. The Howard League’s Annual Report 1936–1937 stated that its representations meant the ‘most objectionable’ clauses of the Palestinian Penal Code had been redrafted and ‘a wise effort made to relate the Code to the peculiar racial and other conditions existing in the territory’.14 The Howard League’s intervention garnered success in restricting the application of bodily punishment as judicial punishment in Palestine and, as such, was consistent with liberal colonial penality. The assumption that ‘peculiar racial and other conditions’ meant the suitability of different treatment of Palestinian Arabs and Jewish people from each other, as well as from white British people, reflected the long-standing division in colonial penality between practice in the colonies and the metropole. The account provided by Nixon about women and girls sheds light on one half of the dual penal regime in Palestine, as does the success of the Howard League’s campaign to revise the proposed penal code. However, the origins of the new penal code pointed towards the other half. The reason for advancing a mandatory death penalty for murder in the first place was because of the riots of 1929 – in other words, for reasons of shoring up colonial authority. This expansion of capital punishment was averted in terms of the criminal law, but the emergency penal regime enabled widespread repression, including executions. 4 THE NEW CRIMINAL CODE FOR PALESTINE Bentwich drew the attention of the Howard League’s Colonial Subcommittee to ‘certain reac- tionary features’ in the New Draft Penal Code and the Subcommittee resolved to send a resolution to the Colonial Office protesting against the capital and corporal punishment clauses.8 The Colo- nial Office referred the resolution to the High Commissioner for Palestine, who had legislative and executive authority.9 The Howard League and NCADP agreed to jointly make representations to MPs on the issue.10 The Howard League’s Colonial Subcommittee asked other organisations to contact the Colonial Office objecting to the proposed code; the Society of Friends Penal Com- mittee and the Women’s International League sent their own resolutions and the Association of Moral and Social Hygiene contacted a member in Palestine.11 The Colonial Subcommittee wrote to the Grand Mufti of Jerusalem, a senior Muslim cleric, with their concerns.12 ned by the applicable Creative Commons License THE HOWARD JOURNAL OF CRIME AND JUSTICE 157 and brutal counter-insurgency measures legal, including press censorship, curfews, collective fines, confiscation of property, demolition of houses, arrest and detention without warrant, and shooting of rioters by soldiers on sight. Collective punishment was not unique to the emergency regime but was permitted under ordinances introduced by Mandate authorities in 1922 (Hughes, 2010). In 1936, military courts could impose the death penalty for sabotage, arson, carrying arms or ammunition, firing on the police and throwing bombs with intent to cause death, serious injury or damage to property. Damaging railway lines and interfering with communications carried a life sentence, or the death penalty if these actions were deemed to endanger life. These emergency penal measures were racially coded in their effect, meaning hundreds of Palestinians were imprisoned and dozens executed (Ben-Natan, 2021). The introduction of emergency powers in Palestine was reported approvingly in the British press. The Manchester Guardian (1936) stated: ‘The authorities are finally indicating that there will be no surrender to intimidation or violence’, and The Times (1936) commented: ‘The severity of the new regulations is to be welcomed’ although made the qualification that regulations would only make an impression when enforced. This reporting is significant for its tone, but also because it reveals that discussion of the emergency powers was in the public domain and would have been known to the Howard League’s Colonial Subcommittee. The Rebellion resumed in 1937 after the Peel Commission (1937) recommended the partition of Palestine to create a separate Jewish state and continued until 1939. British counter-insurgency measures intensified and elements of spatial control such as the demolition of houses and buildings were used more widely. In addition to emergency penal measures, British soldiers and colonial police perpetrated unofficial brutality and torture, such as electric shocks, mock executions, beatings, waterboarding and indiscriminate killings (Hughes, 2009, 2010; Norris, 2008). By early 1938, Palestine was under de facto martial law, which enabled British troops ‘to operate with relative impunity’ (Hughes, 2010; Norris, 2008, p.28). The British government resisted imposing martial law officially, despite censure from the League of Nations Mandates Commission for not having done so, due to the perceived embarrassment of doing this in a ‘Category A’ mandate, one recognised as on the way to independence (Norris, 2008). 5 THE EMERGENCY REGIME IN PALESTINE The British imposed martial law when they conquered Palestine in 1917 and established military courts during the riots of 1929 (Ben-Natan, 2021; Bentwich, 1920). The Palestinian Arab Rebel- lion commenced in April 1936. This was a nationalist uprising against the Mandatory Power and against policies of open-ended immigration for Jewish people that aimed to establish a Jewish homeland. In the same year that the new penal code was introduced, the British also established Defence (Emergency) Regulations, emergency powers which were adapted from those previously deployed in Ireland and India. These regulations made the use of repressive THE HOWARD JOURNAL OF CRIME AND JUSTICE THE HOWARD JOURNAL OF CRIME AND JUSTICE 158 forces’ policy’ (p.108). Coercion through force ‘was everywhere the mainstay of British counter- insurgency policy’ (p.116). The significance of the counter-insurgency against the Arab Rebellion in Palestine 1936–1939 was that it established tactics subsequently used by the British during the era of decolonisation after the Second World War – again in Palestine, and in Malaya, Cyprus, Nyasaland, Aden and Kenya, with the Malaya and Kenya Emergencies representing the most brutal examples (Drohan, 2017; Norris, 2008). During the period of the Arab Rebellion in Palestine, the Howard League’s Colonial Subcom- mittee took no further action in relation to the death penalty, despite its expansion under the emergency regulations. Examples of death sentences and executions for carrying arms in Pales- tine were reported in The Times so were not difficult to discover (The Times, 1937a, 1937b). The Howard League’s Report for 1937–1938 records that the Colonial Subcommittee was approached by the National Council of Civil Liberties, who were receiving ‘frequent allegations of ill treatment in Palestinian prisons’. A meeting was held with the National Council for Civil Liberties and the Palestine Subcommittee of the Society of Friends. It was resolved that Norman Bentwich would investigate prison conditions during a visit to Palestine in 1938.15 Prison conditions in Palestine were discussed at the March 1938 meeting of the Colonial Subcommittee with concerns raised about unauthorised beatings of prisoners, and acknowledgement of the need to investigate con- centration camps and police stations as well as prisons.16 Bentwich’s visit to Palestine is recorded in the May 1938 minutes, although there is little detail about what he found.17 Prison conditions and the extension of rehabilitative measures such as probation and juvenile reformatories were the main focus of the Howard League’s international and colonial campaign- ing and work. What this focus demonstrates in relation to Palestine in 1938 is that the Howard League restricted itself to the ordinary penal regime and demarcated the emergency regime as beyond its purview. Collective punishment, whether by curfew, fine or demolition of homes, outrageously violated penal liberalism, as did the expanded use of capital punishment. Instead, it reflected the long-standing imperial logic that different, bluntly authoritarian measures were needed to assert power in the colonies (Wagner, 2016; Wiener, 2009). The dual penal regime sectioned off measures of colonial repression and brutality as unconnected to ordinary penality (which is not to deny that repression was an aspect of ordinary penality). However, as Ben- Natan (2021) argues, the emergency penal regime was not outside the penal realm but reflected the duality of penal power in colonial contexts. The difficulty of maintaining this separation of the ordinary and the emergency in terms of the penal realm is illustrated by the reference to concentration camps in the Howard League minutes. These were established in Palestine in 1936 to detain nationalist rebels (Moore, 2010). As such, they were part of the emergency regime. On 10 January 1939, the War Office issued a statement to refute allegations in the German Nazi controlled press ‘of atrocities by British troops in Palestine’ (Manchester Guardian, 1939a), which the Manchester Guardian published in full and The Times summarised. The statement explained in Palestine ‘the active rebel and the peaceful citizen are inextricably mixed’ and that ‘[v]irtually every village in the country has at one time or another harboured and supported the rebels and assisted in concealing their identity’. It adhered to the logic of colonial counter-insurgency that civilians’ connection to regime opponents justified collective violence (see Thomas, 2018). The statement denied that ‘wholesale demolition’ of Palestinians’ houses was taking place but acknowledged that demolition was ‘occasionally’ used as collective punishment, which was jus- tified as ‘fully recognised and understood by the Palestinian Arab’. This highlights another aspect of colonial counter-insurgency (and colonial penality more widely), the racial coding of violence (Thomas, 2018). The statement outlined three types of collective punishment in use: collective fines, collective demolition and curfews. The process for spatial control enacted via collective demolition was marking houses ‘either of suspected bad characters or of village notables who can, and should, control the village’ as a warning and demolishing them if there were further acts of terrorism (Manchester Guardian, 1939b). French (2011) argues that establishing physical control over the population was key to British military success in counter-insurgencies. The use of collective punishments was ‘not an incidental part of these operations, they were a deliberate and calculated part of the security THE HOWARD JOURNAL OF CRIME AND JUSTICE 159 Following the merger of NCADP with the Howard League in 1948, the League’s Capital Punishment Subcommittee was created. Members of the League provided written and oral evidence to the Royal Commission on Capital Punishment 1949–1953 (Seal, 2014). The only clear mention of the colonial death penalty in the Howard League archives for the post-war era concerned Kenya in 1951. The Colonial Office wrote to the League highlighting the ‘infliction of the death penalty for rape in Kenya’ and explaining that they had urged Kenya, North- ern Rhodesia and Nyasaland, the only three British territories where rape was a capital offence, to remove the death penalty for rape. The minutes of the Executive Committee sum up the discussion thus: Campaigning and work by the Howard League in relation to British colonies on particular issues was rare in comparison with the 1930s, especially when it came to the death penalty. The salience of capital punishment as an issue rose in Britain, with unsuccessful attempts at aboli- tion in 1948 and 1956. The Howard League waged an active domestic campaign against the death penalty during this period. Following the merger of NCADP with the Howard League in 1948, the League’s Capital Punishment Subcommittee was created. Members of the League provided written and oral evidence to the Royal Commission on Capital Punishment 1949–1953 (Seal, 2014). The only clear mention of the colonial death penalty in the Howard League archives for the post-war era concerned Kenya in 1951. The Colonial Office wrote to the League highlighting the ‘infliction of the death penalty for rape in Kenya’ and explaining that they had urged Kenya, North- ern Rhodesia and Nyasaland, the only three British territories where rape was a capital offence, to remove the death penalty for rape. The minutes of the Executive Committee sum up the discussion thus: Very few crimes were still punishable with death, and the Colonial Office was per- sistently trying to reduce them; but it was difficult for it to impose its will when the colonies had certain degrees of self-government and fuller self-government was the end in view. The committee felt that repressive punishments were the result of the insecurity felt by the small white population in the midst of a large coloured population, but without further knowledge no comment could be made.18 This summary excuses the Colonial Office’s apparent inaction as lack of authority. 6 LIBERAL COLONIAL PENALITY AFTER THE SECOND WORLD WAR The Howard League’s international and colonial campaigning paused during the Second World War. International work continued after the war via involvement in United Nations led meetings, conferences and initiatives related to penal issues and a new International Subcommittee was established in 1949. In 1947, Executive Committee members Margery Fry and Hermann Mannheim met with representatives from the Anti-Slavery and Aborigines’ Protection Society, the Colonial Bureau of the Fabian Society and the Royal African Society to explore the possibility of creating a new Colonial Subcommittee. Ultimately, these plans were shelved – in part because THE HOWARD JOURNAL OF CRIME AND JUSTICE 160 Executive Committee member Fry sat on the Treatment of Offenders Subcommittee of the Colonial Office’s Committee on Social Welfare (formerly the Standing Advisory Committee on Penal Administration). The original iteration of this subcommittee was set up by Howard League member Denis Pritt and Fry’s membership meant that the Howard League had a voice in relation to colonial matters, but – unlike its erstwhile Colonial Subcommittee – one that was not independent from government. p g In 1951, Fry published ‘Penal reform in the colonies’ in the Howard Journal, which elaborated a vision of liberal colonial penality. She characterised penal reform in the colonies as ‘the work of bringing colonial penal administration more into line with modern ideas’ (Fry, 1951, p.90). Fry commented that ‘communal responsibility, the duty of a tribe or village to make good the wrong- doing of one of its members, is not in accordance with our modern ideas of justice’ (p.91). She did not intend this reflection as a criticism; Fry was pioneering in her attempts to bring elements of restitution and restorative justice to the criminal justice system in Britain (Logan, 2017). However, this ‘modern’ notion of individual responsibility only applied to the ordinary penal regimes in British colonial states. Collective punishment as deployed in 1930s Palestine paid no regard to individual responsibility. Fry did not mention this contradiction but rather wrote approvingly of probation, ‘the necessary foundationstone of a rational treatment’, appearing in the colonies and of the spread of juvenile justice systems including children’s courts, approved schools and borstals (p.94). Fry concluded that much still needed to be done in terms of the modernisation of colonial penal systems, but a ‘new spirit’ of collaborative working had emerged (p.95). Exactly what Fry thought about colonialism is unknown. Her biographer notes: ‘there is no evidence that she addressed the wider issue of colonial power or the ethics of imperial domination’ but given Fry’s left-wing politics, she was unlikely to have enthusiastically embraced imperialism (Logan, 2017, p.130). Campaigning and work by the Howard League in relation to British colonies on particular issues was rare in comparison with the 1930s, especially when it came to the death penalty. The salience of capital punishment as an issue rose in Britain, with unsuccessful attempts at aboli- tion in 1948 and 1956. The Howard League waged an active domestic campaign against the death penalty during this period. 7 THE KENYA EMERGENCY Kenya in the 1950s makes an instructive example of this violent imperial collapse. The British waged a brutal and repressive counter-insurgency against the ‘Mau Mau’ rebellion 1952–1960, an uprising for self-government mainly fought by Gikuyu people. A state of emergency was declared in October 1952. The range of crimes eligible for the death penalty expanded under the emergency regulations and like in Palestine included firearms offences, as well as administration of oaths and consorting with terrorists (Anderson, 2005; Ben-Natan, 2021; Drohan, 2017). Over a thousand Kenyans were executed and summary execution was rumoured to have taken place (Anderson, 2005; Drohan, 2017). The British targeted individuals suspected of supporting the insurgency via mass arrests, deportation and detention without trial in forced labour camps. Confinement was used extensively, reaching 71,346 people in December 1954, 98% of whom were Gikuyu. As part of the dirty war, security forces engaged in beatings, torture and murder (Anderson, 2005). Other measures included collective punishments such as cordon and search operations, curfews, forcible population resettlement (villagisation), limitation of populations’ access to food and free fire zones (French, 2011). Thomas (2018) defines dirty war as ‘highly demonstrative acts of collec- tive violence that are designed to compel popular compliance, with other, more covert actions’ (p.505). The civilian population is the primary target. In late 1952, the Howard League was informed of a series of new ordinances in Kenya which included use of anonymous witnesses in trials, registering new organisations and printing presses with the authorities, blacklisting and banishment. Margery Fry and Violet Creech-Jones, Howard League members who sat on the Colonial Office’s Treatment of Offenders Subcommittee, raised the banishment law with the Colonial Office but accepted its opinion that it was a matter for the Kenyan Government and Parliament.19 Public attention was drawn to the Kenya Emergency in the mid-1950s. The Daily Mirror published a series of articles by Labour MP Barbara Castle, which highlighted the disappearance of people into prison camps and raised the deeply racialised use of these punishment measures by asking if it was possible for Black men in Kenya to get white justice (Gopal, 2019). that the use of such measures was related to race. The phrasing of a ‘small white population in the midst of a large coloured population’ suggested some sympathy with the ‘white popula- tion’, although the minutes do not provide further elaboration. The Executive Committee was not interested in taking this issue further, representing a shift from the 1930s when the repres- sive use of capital punishment in ordinary penal regimes was a matter for intervention. The early 1950s was the era of decolonisation during which there was frequent mass violence in the col- lapsing British and French Empires. Thomas & Thompson (2014) identify a humanitarian double standard, whereby disintegrating colonial powers such as Britain and France espoused a new lan- guage of global human rights while their Empires collapsed amid mass violence towards civilian populations. Members of the Executive Committee had an awareness of ‘repressive measures’ in colonial regimes and THE HOWARD JOURNAL OF CRIME AND JUSTICE 161 023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/hojo.12513 by Test, Wiley Online Library on [15/06/2023]. See the Terms and Conditio She highlighted practices of hard labour and solitary confinement for girls who sang ‘Mau Mau’ songs. Fletcher attributed the high number of executions of Black Africans and discrepancies in justice between white and Black people to ‘white supremacy’ (Bruce-Lockhart, 2015). In the House of Commons, Labour MP (and former Colonial Secretary), Howard League member and husband of Violet Creech Jones, Arthur Creech Jones, argued that confinement in the camps was essentially internment without trial and that lengthy and life sentences had been given to children. Fenner Brockway, Labour MP and former vice-president of the Howard League, stated that out of 1,015 people executed in Kenya over the previous four years, only 297 were executed for murder. More than 40,000 were detained without trial or held despite having been cleared by the emergency courts (Historic Hansard, 6 June 1956). In 1954, Brockway had founded the Movement for Colonial Freedom, which campaigned to end British colonialism. This group published Fletcher’s pamphlet (Bruce-Lockhart, 2015; Gopal, 2019). In response to the member query, Executive Committee member Cicely Craven suggested that Margery Fry and Violet Creech Jones, as the League’s representatives on the Standing Advisory Council at the Colonial Office, should ask whether conditions in Kenyan camps had been investigated.21 The Executive Committee agreed the allegations warranted an inquiry. Alan Lennox-Boyd, the Colonial Secretary, announced that a Parliamentary Delegation might be sent to Kenya and at the Executive Committee’s September 1956 meeting, Ulster Unionist MP Mont- gomery Hyde offered to join the delegation as representative of the Howard League.22 However, this delegation, which visited Kenya in 1957, did not include Hyde. These two examples – the new ordinances of 1952 and the member query about conditions in prison camps in 1956 – are the only instances of the Kenya Emergency appearing in the Howard League’s archives. The House of Commons ‘Hola Camp’ debate of June 1959 raised the mass beat- ings of 85 detainees in the Hola Detention Camp in March 1959, which killed eleven men. This camp was intended for ‘hardcore’ Mau Mau and coerced the men into hard labour (Anderson, 2005). The ‘Hola massacre’ was a scandal and generated outrage in Britain about late imperial- ism. The Kenyan authorities and the British government acknowledged that the massacre was a shocking tragedy (Toye, 2017). Neither the massacre nor the parliamentary debate is mentioned in the Howard League’s minutes. 023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/hojo.12513 by Test, Wiley Online Library on [15/06/2023]. See the Terms and Conditio The Malayan Emergency of 1948–1960 is not mentioned at all; counter-insurgency measures there involved mass executions, forcible population resettlement, collective punishment and massacres (Dixon, 2009). In her speech in the House of Commons that made the Hola massacre public, Barbara Castle asked how attitudes would have differed if it had happened in a British prison (Toye, 2017). This question was not posed or answered by the Howard League, whose archive is characterised mainly by silence about the violent and punitive actions taken under emergency regulations in the era of violent colonial collapse. From this silence, it appears that the organisation did not perceive emergency penal regimes to be within their sphere of interest. The Howard League’s Executive and Colonial Committees had in the 1930s included individuals such as Fenner Brockway and Denis Pritt who became high-profile anticolonial campaigners (Pritt, a QC, led the defence of Jomo Kenyatta when he was tried in 1953 for instigating the Mau Mau rebellion: Gopal (2019)). The imperative for the League to remain non-political and work closely with government, including with the Colonial Office, made an organisational anticolonial stance unlikely. 7 THE KENYA EMERGENCY In July 1956, a Howard League member asked the Executive Committee whether prison conditions in Kenya ‘fell within the scope of the League’.20 The issue of conditions in prison camps in Kenya had been debated in the House of Commons, following the publication of Eileen Fletcher’s whistleblowing pamphlet The truth about Kenya in March 1956, which caused a ‘political firestorm’ (cited in Bruce-Lockhart, 2015, p.816). Fletcher, a Quaker social worker with the Community Development and Welfare Department, designed the rehabilitation programme at the Kamiti Detention Camp for women and worked with juvenile girls in the camp as a ‘rehabilitation officer’. She made complaints to the Kenyan Government about conditions in the camp before writing the article. THE HOWARD JOURNAL OF CRIME AND JUSTICE 162 162 campaign against, or raise the issue of, punishment under emergency regimes in British colonies. In 1949, the Irish Prisoners Welfare Committee (IPWC) contacted the League about the sentences being served by Irish political prisoners and the conditions in Crumlin Gaol in Belfast. Cicely Craven sent a reply informing the IPWC that the Howard League was non-political, and stating that the IPWC needed to make representations to the authorities at a political level.23 Like the Howard League’s acceptance in 1952 of Colonial Office advice that repressive ordinances in Kenya were a matter for the Kenyan Government, this example sheds some light on how the Howard League drew boundaries around their areas of interest and intervention at this time. The organi- sation was less willing to intervene in relation to state violence in the era of decolonisation than it was in the 1930s, when it did highlight the perceived misuse or extension of the death penalty in ordinary penal regimes. From the perspective of 21st-century British criminology, it makes intuitive sense that the Howard League did not intervene in emergency penal regimes. Its influence did not stretch to matters of counter-insurgency and there would have been the risk of compromising its work in areas where it did hold sway. However, as Ben-Natan (2021) argues, accepting a separation between ordinary and emergency penal regimes obscures the penal element of emergency powers. This is not to argue that the Howard League could have prevented colo- nial atrocities in 1930s Palestine or 1950s Kenya, but to highlight that the development of 20th-century penal liberalism rested on silence about the violent, repressive tactics of illib- eral emergency penal regimes in the Empire and by implication construed those regimes as something other than penal. According to penal liberalism, penal power should be limited, ratio- nal, beneficial and fair. These aspirations clashed both with colonial and emergency penality, which operated on logics of different and worse treatment, which was often racially coded. Collective punishment, prison camps and mass execution illustrated this clash with penal liberalism. Criminological analyses of British penal modernism and penal liberalism, including in the mid-20th century, follow the same demarcation of colonial emergency penal regimes as beyond the scope of criminology and usually pay no attention to colonial penality at all. The legacy of penal liberalism at play in British criminology is reflected in the under analysis of both state- based violence and the interaction between normal and exceptional penal practice during war, occupation and counter-insurgency (Jamieson, 2016; Walklate & McGarry, 2015). The narrative has been purified of colonialism, which is an effect of colonialism (Agozino, 2003; Frenkel & Shenhav, 2006; King, 2017). Attention to emergency penal regimes provides a fuller picture of mid-20th-century British penality beyond its own shores and unsettles the narrative of a ‘kinder’ paradigm demolished by neo-liberalism.24 It upends the argument that penal modernism abjured bodily punishment – this discomfort applied in the metropole only (Brown 2002; Hogg & Brown, 2018). Integration of colonial penality into assessments of British penal modernism helps to push beyond a solely Eurocentric interpretation and exposes the ‘dark side’ of penal modernism. This integration can be understood as work towards a better understanding of how coloniality affects criminology (Dimou, 2021; Quijano, 2007). 8 CONCLUSION 8 The Howard League as an organisation did not outline its views on colonialism or colonial penal- ity. It was an avowedly non-political insider group and this probably accounts for why it did not THE HOWARD JOURNAL OF CRIME AND JUSTICE 163 E N D N O T E S 1Modern Records Centre, MSS 157/3/CAP/4/1–13, minutes of the meeting of the Colonial Subcommittee, 2 June 1930. All archival references are to the Modern Records Centre, unless otherwise stated (Howard League for Penal Reform Collection, 1788–2002; National Council for the Abolition of the Death Penalty Collection, 1923–1949). 2MSS 157/3/CAP/4/1–13, report on Howard League International Work 1929. 3MSS 16B/1/1, minutes of the meeting of the Colonial Subcommittee, 27 May 1930. 4MSS 16B/1/1, minutes of the meeting of the Colonial Subcommittee, 30 June 1930. 5MSS 16B/1/1, minutes of Colonial Subcommittee, 7 March 1932 and 18 April 1932. 6MSS 16B/1/6, Penal Reformer, current comment, October 1934. 7MSS 16B/1/6, Penal Reformer, current comment, January 1935. 8MSS.16B/1/2, minutes of the meeting of the Colonial Subcommittee, 13 November 1933. 9MSS 16B/1/2, minutes of the meeting of the Executive Committee, 19 January 1934. 10MSS 16B/ADP/1/5, minutes of NCADP Executive Committee, 17 January 1934. 11MSS 16B/HLP/1/2, minutes of the meeting of the Executive Committee, 16 February 1934. 12MSS 16B/1/2, minutes of the meeting of the Colonial Subcommittee, 27 November 1934. The National Archives/CO192/3. With many thanks to Anne Logan for sharing her notes with u d h f ff d h l 13The National Archives/CO192/3. With many thanks to Anne Logan for sharing her notes wit Advisory Committee on the Treatment of Offenders in the Colonies. y 14MSS 16B/4/1/1–16. 15MSS B/ / A l R 14MSS 16B/4/1/1–16. 15MSS 16B/1/5, Annual Report 1937–1938. 15MSS 16B/1/5, Annual Report 1937–1938. 16MSS 16B/1/3, minutes of the Colonial Subcommittee, 31 March 1938. 16MSS 16B/1/3, minutes of the Colonial Subcommittee, 31 March 1938. 17MSS 16B/1/3, minutes of the Colonial Subcommittee, 12 May 1938. 18MSS 16B/1/5, minutes of the Executive Committee, 20 April 1951. 19MSS 16B/1/6, minutes of the Executive Committee, 21 November 1952. 20MSS 16B/1/6, minutes of the Executive Committee, 20 July 1956. 21MSS 16B/1/6, minutes of the Executive Committee, 20 July 1956. 22MSS 16B/1/6, minutes of the Executive Committee, 14 September 1956. 23MSS 16B/1/5, minutes of the Executive Committee, 9 September 1949. 24With regard to Northern Ireland, failure to incorporate the emergency penal regime as part of British penality means failure to apply this analysis to the United Kingdom as a whole, nevermind further afield. 24With regard to Northern Ireland, failure to incorporate the emergency penal regime as part of British penality means failure to apply this analysis to the United Kingdom as a whole, nevermind further afield. 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Towards the connection between snow microphysics and melting layer: insights from multifrequency and dual-polarization radar observations during BAECC
Atmospheric chemistry and physics
2,020
cc-by
12,899
Haoran Li1, Jussi Tiira1, Annakaisa von Lerber2, and Dmitri Moisseev1,2 1Institute for Atmospheric and Earth System Research/Physics, Faculty of Science, University of Helsinki, Helsinki, Finland 2Finnish Meteorological Institute, Helsinki, Finland Correspondence: Haoran Li (haoran.li@helsinki.fi) Received: 9 January 2020 – Discussion started: 3 April 2020 Revised: 7 July 2020 – Accepted: 8 July 2020 – Published: 14 August 2020 Received: 9 January 2020 – Discussion started: 3 April 2020 Revised: 7 July 2020 – Accepted: 8 July 2020 – Published: 14 August 2020 Abstract. In stratiform rainfall, the melting layer (ML) is of- ten visible in radar observations as an enhanced reflectivity band, the so-called bright band. Despite the ongoing debate on the exact microphysical processes taking place in the ML and on how they translate into radar measurements, both model simulations and observations indicate that the radar- measured ML properties are influenced by snow microphys- ical processes that take place above it. There is still, how- ever, a lack of comprehensive observations to link the two. To advance our knowledge of precipitation formation in ice clouds and provide new insights into radar signatures of snow growth processes, we have investigated this link. This study is divided into two parts. Firstly, surface-based snowfall mea- surements are used to develop a new method for identify- ing rimed and unrimed snow from X- and Ka-band Doppler radar observations. Secondly, this classification is used in combination with multifrequency and dual-polarization radar observations collected during the Biogenic Aerosols – Ef- fects on Clouds and Climate (BAECC) experiment in 2014 to investigate the impact of precipitation intensity, aggre- gation, riming and dendritic growth on the ML properties. The results show that the radar-observed ML properties are highly related to the precipitation intensity. The previously reported bright band “sagging” is mainly connected to the increase in precipitation intensity. Ice particle riming plays a secondary role. In moderate to heavy rainfall, riming may cause additional bright band sagging, while in light precipi- tation the sagging is associated with unrimed snow. The cor- relation between ML properties and dual-polarization radar signatures in the snow region above appears to be arising through the connection of the radar signatures and ML prop- erties to the precipitation intensity. In addition to advancing our knowledge of the link between ML properties and snow processes, the presented analysis demonstrates how multifre- quency Doppler radar observations can be used to get a more detailed view of cloud processes and establish a link to pre- cipitation formation. 1 Introduction Stratiform precipitation is prevalent in middle to high lati- tudes. In such precipitation systems, ice particles nucleated at the cloud top descend and grow on their way down by going through various microphysical processes, e.g., vapor deposition, aggregation and/or riming (Lamb and Verlinde, 2011). In the case of rainfall, these ice particles transform into raindrops in the melting layer (ML). The melting of ice particles is capable of modulating the thermal structure of the ML through the exchange of latent heat with the envi- ronment (Stewart et al., 1984; Carlin and Ryzhkov, 2019) and, as a result, can change the dynamics of precipitation (e.g., Heymsfield, 1979; Szeto et al., 1988; Fabry and Za- wadzki, 1995). It has shown that ML properties are modified by the ambient environment such as relative humidity (RH; Willis and Heymsfield, 1989; Battaglia et al., 2003; Carlin and Ryzhkov, 2019), as well as microphysical processes tak- ing place in the ML (Heymsfield et al., 2015), and by snow microphysical processes occurring above, e.g., aggregation and riming (Stewart et al., 1984; Klaassen, 1988; Fabry and Atmos. Chem. Phys., 20, 9547–9562, 2020 https://doi.org/10.5194/acp-20-9547-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. Towards the connection between snow microphysics and melting layer: insights from multifrequency and dual-polarization radar observations during BAECC Haoran Li1, Jussi Tiira1, Annakaisa von Lerber2, and Dmitri Moisseev1,2 1Institute for Atmospheric and Earth System Research/Physics, Faculty of Science, University of Helsinki, Helsinki, Finland 2Finnish Meteorological Institute, Helsinki, Finland Atmos. Chem. Phys., 20, 9547–9562, 2020 https://doi.org/10.5194/acp-20-9547-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. Towards the connection between snow microphysics and melting layer: insights from multifrequency and dual-polarization radar observations during BAECC Haoran Li1, Jussi Tiira1, Annakaisa von Lerber2, and Dmitri Moisseev1,2 1Institute for Atmospheric and Earth System Research/Physics, Faculty of Science, University of Helsinki, Helsinki, Finland 2Finnish Meteorological Institute, Helsinki, Finland Haoran Li1, Jussi Tiira1, Annakaisa von Lerber2, and Dmitri Moisseev1,2 1Institute for Atmospheric and Earth System Research/Physics, Faculty of Science, University of Helsinki, Helsinki, Finland 2Finnish Meteorological Institute, Helsinki, Finland H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC Properties of the ML and its radar manifestation bright band are influenced by cloud dynamics and microphysics, which can be directly probed by aircraft-mounted in situ measurements (e.g., Stewart et al., 1984; Willis and Heymsfield, 1989; Heymsfield et al., 2015) despite the inability to conduct continuous long-term oper- ations with such setups. Remote sensing of the ML with radars dates back to the 1940s (Ryde, 1946). Atlas (1957) has found that the strength of the bright band is weakened when melting graupel particles are present, which was further con- firmed by Klaassen (1988) and Zawadzki et al. (2005). A comprehensive long-term analysis of the ML appearance in vertically pointing X-band radar and ultra high frequency (UHF) wind profiler observations has been performed by Fabry and Zawadzki (1995). They have compiled a record of the main ML features that were later used in modeling studies (e.g., Szyrmer and Zawadzki, 1999; Zawadzki et al., 2005; von Lerber et al., 2014). As the ML bridges snow and rain, the raindrop size distributions below the ML seem to be related to the bright band’s reflectivity values (Huggel et al., 1996; Sarma et al., 2016). As presented by Wolfensberger et al. (2016), the thickness of the ML depends on riming, particle fall velocities and the bright band intensity. Mean- while, the downward extension of the bright band, called the saggy bright band, may be linked to riming as suggested by previous studies (Trömel et al., 2014; Kumjian et al., 2016; Ryzhkov et al., 2016; Xie et al., 2016; Erlingis et al., 2018). Recently, Carlin and Ryzhkov (2019) have incorporated the cooling effects of melting snowflakes in the ML model and proposed that the saggy bright band may be explained by a combination of processes instead of a single factor. From the perspective of observation, there seems to be a lack of sta- tistical studies untangling the impacts of snow growth pro- cesses on the observed ML properties. y g p y The detailed properties of ice particles are complex as manifested by the extraordinary variety in their habit, size, mass and concentration (Korolev et al., 2000, 2003; Bai- ley and Hallett, 2009). This complexity is exacerbated by the diversity of ice growth processes that take place in ice clouds (Li et al., 2018; Oue et al., 2018; Barrett et al., 2019; Moisseev et al., 2015, 2017, 2019; Tiira and Moisseev, 2020). H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC 9548 Zawadzki, 1995; Zawadzki et al., 2005; von Lerber et al., 2014; Kumjian et al., 2016; Xie et al., 2016; Wolfensberger et al., 2016; Trömel et al., 2019). In addition, the microwave attenuation in the ML is sensitive to the parameterization of snow microphysics (von Lerber et al., 2014) and can be sig- nificant at millimeter wavelengths (Matrosov, 2008; Haynes et al., 2009; Li and Moisseev, 2019). ratios (DWRs) at Ka–W-bands and X–Ka-bands on riming was observed by Kneifel et al. (2015). Dias Neto et al. (2019) have presented the strong aggregation signatures close to the ML using multifrequency radar observations. This rapid ag- gregation could manifest itself as a dark band in W-band cloud radar observations, namely the dip in radar reflectiv- ity just above the ML top (Lhermitte, 1988; Sassen et al., 2005, 2007; Heymsfield et al., 2008). Such a reflectivity dip just above the ML may even be present in X-band radar measurements of light precipitation (Fabry and Zawadzki, 1995) but has not been well addressed. Mason et al. (2018) have incorporated the Doppler velocity and radar reflectiv- ity observations from vertically pointing Ka- and W-band radars into an optimal estimation scheme to infer the rim- ing fraction, among other parameters. In addition to multi- frequency radar observations, dual-polarization radar mea- surements show promise in improving our understanding of ice precipitation processes (e.g., Bechini et al., 2013; Gian- grande et al., 2016; Kumjian et al., 2016; Ryzhkov et al., 2016; Moisseev et al., 2015, 2017; Li et al., 2018; Oue et al., 2018; Vogel and Fabry, 2018; Moisseev et al., 2019; Tiira and Moisseev, 2020). Therefore, the utilization of collocated multifrequency and dual-polarization radar observations may pave the way for a better understanding of the connection be- tween dry and melting snow microphysics. , ; , ) To centimeter-wavelength weather radars, the ML appears as a band of the increased reflectivity, the so-called bright band, while to millimeter-wavelength radars, such an ap- pearance is less distinct (e.g., Lhermitte, 1988; Sassen et al., 2005; Kollias and Albrecht, 2005). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 2.3 NASA particle imaging package The PIP is an improved version of the Snowflake Video Im- ager (Newman et al., 2009), which uses a high frame rate camera operating at 380 frames per second to record the silhouettes of precipitation particles. The field of view of this camera is 48 mm×64 mm with a spatial resolution of 0.01 mm2. The focal plane of this camera is 1.3 m. Because the measurement volume is not enclosed, the wind-induced effects on the measurements are minimized (Newman et al., 2009). The data-processing software defines the size of each particle using the disk-equivalent diameter (Ddeq), which is the diameter of a disk with the same area of a particle shadow. Particle size distribution (PSD) and fall velocity are recorded as a function of Ddeq in the PIP software. Based on these PIP products, von Lerber et al. (2017) have derived particle mass and fall velocity as a function of the observed maximum par- ticle diameter (Dmax,ob), which is obtained by fitting an el- lipsoid model to each particle. Here and hereafter, D rep- resents Dmax,ob. The snowfall measurements started as part of the BAECC field campaign were continued, and data col- lected during the experiment and an additional three winters were used in this study. The collected data were processed using the method by von Lerber et al. (2017). H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC The paper is organized as follows. Section 2 introduces the instrumentation used in this study, followed by the illustra- tion of detecting the ML and separating unrimed and rimed snow in Sect. 3. A sanity check of the snow classification and the statistical results of multifrequency and dual-polarization radar observations are provided in Sect. 4. Conclusions are presented in Sect. 5. tivity can be significantly affected by the attenuation from the ML, rain and a wet radome (Li and Moisseev, 2019), the relative calibration was made at precipitation top where the Rayleigh assumption can be applied at Ka- and X-bands. During BAECC, a radiosonde was launched four times per day, out of which the temporally closest one was used as in- put to the millimeter-wavelength propagation model (Liebe, 1985) to correct for the gaseous attenuation at all radar fre- quencies. 2.2 Dual-polarization weather radar The BAECC field campaign was conducted at the Univer- sity of Helsinki’s Hyytiälä Station from February to Septem- ber 2014 (Petäjä et al., 2016). This experiment provides com- prehensive vertically pointing multifrequency radar rainfall observations, which are used in this study. A 2D video dis- drometer (2DVD) was used to measure rain rate and calibrate X-band radar reflectivity. The collocated observations were aided by the FMI C-band dual-polarization weather radar. In addition to the radar setup during BAECC, long-term snow observations were made by a National Aeronautics and Space Administration (NASA) Particle Imaging Pack- age (PIP; Newman et al., 2009; Tiira et al., 2016; von Lerber et al., 2017). The FMI C-band dual-polarization weather radar located in Ikaalinen, 64 km west from the Hyytiälä station, operates in the simultaneous transmission and receiving mode (Doviak et al., 2000). This radar performs RHI scans over the mea- surement site every 15 min. The range and azimuth resolu- tions are 500 m and 1◦, respectively. The dual-polarization measurements used in this study are Zdr, which was cali- brated during light rainfalls (Bringi and Chandrasekar, 2001; Li et al., 2018). For data analysis, the Python ARM Radar Toolkit (Helmus and Collis, 2016) was used. H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC Despite the recent attempts to resolve the ice micro- physics (e.g., Mason et al., 2018, 2019; Barrett et al., 2019), direct characterization of ice particles and their growth pro- cesses is still challenging. In some cases, ML properties could emphasize radar signatures of such processes (Za- wadzki et al., 2005; Kumjian et al., 2016; Li and Moisseev, 2020) and therefore provide additional information. How- ever, there is an ongoing debate on the link between snow growth processes, such as riming and aggregation, their radar signatures and ML properties (e.g., Kumjian et al., 2016; Carlin and Ryzhkov, 2019; Heymsfield et al., 2015). This study aims to advance our understanding of the link and re- solve at least some of the discussed topics. During the Bio- genic Aerosols – Effects on Clouds and Climate (BAECC) experiment (Petäjä et al., 2016), vertically pointing X-, Ka- and W-band cloud radars were deployed at the University of Helsinki research station in Hyytiälä, Finland. These observations were supplemented by range–height indica- tor (RHI) scans carried out by the Finnish Meteorological In- stitute (FMI) C-band dual-polarization radar, providing a set of unique synergistic observations ideally suited for studying the connection between the growth and melting processes of snowflakes. Over the last few years, multifrequency radar measure- ments of clouds and precipitation have become more eas- ily attainable, which has led to the proliferation of stud- ies demonstrating the advantages of using these observa- tions for the investigation of snow microphysical processes (e.g., Kneifel et al., 2011, 2015; Leinonen et al., 2012a; Leinonen et al., 2013, 2018; Tyynelä and Chandrasekar, 2014; Leinonen and Moisseev, 2015; Leinonen and Szyrmer, 2015; Grecu et al., 2018; Chase et al., 2018; Mason et al., 2018, 2019). The potential dependence of dual-wavelength https://doi.org/10.5194/acp-20-9547-2020 Atmos. Chem. Phys., 20, 9547–9562, 2020 H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC 9549 3.1 Detection of ML boundaries The height where melting starts ranges from the surface to several kilometers above, mainly depending on the temper- ature profiles. Thus, prior to addressing the general char- acteristics of ML, it is important to detect ML bound- aries. Fabry and Zawadzki (1995) have employed the gra- dient of reflectivity to determine the ML boundaries using single-polarization X-band radar measurements. The verti- cally pointing X- and Ka-band radars used in this study pro- vide dual-polarization observations, i.e., ρhv and LDR, re- spectively. These observations supply additional information to estimate the ML boundaries (Giangrande et al., 2008). However, care should be taken in how this information is used. Wolfensberger et al. (2016) have suggested that the use of ρhv could underestimate the ML top as the significant drop in ρhv may not happen until a significant amount of ice has al- ready melted. To mitigate this issue, we determined the upper boundary of ML by finding the local minimum of the X-band reflectivity gradient around the ρhv-detected ML top, which is similar to Wolfensberger et al. (2016). The validity of uti- lizing the radar reflectivity in determining the ML top is fur- ther confirmed in our recent study (Li and Moisseev, 2020). The ML bottom was determined in a similar way to derive the radar reflectivity at the melting bottom. Note that cases in which precipitation fall streaks are significantly slanted, as shown in Fabry and Zawadzki (1995), were excluded. Mason et al. (2018) have shown that the extent of rim- ing can be retrieved using radar-measured DWR (Matrosov, 1998; Hogan et al., 2000) and mean Doppler velocity (V ). If the radar reflectivity is expressed in decibels (dB), then the DWR can be written as DWR(λ1,λ2) = Zλ1 −Zλ2, (2) (2) where Zλ1 and Zλ2 are observed radar reflectivities at the wavelengths of λ1 and λ2, respectively. Zλ can be expressed as Zλ = 10log10   Dmax Z Dmin λ4 π5|Kλ|2 N(D)σb,λ (D,mob(D))dD  , (3 (3) where |Kλ|2 is the dielectric constant of liquid water and σb,λ(D,mob(D)) is the backscattering coefficient of snow particles at a given wavelength. In X-SACR, Ka- SACR and MWACR data files, |Kλ|2 is set to 0.93, 0.88 and 0.70, respectively. The values of σb,λ were taken from the single-scattering databases (Leinonen and Moisseev, 2015; Leinonen and Szyrmer, 2015; Tyynelä and von Ler- ber, 2019). 3.1 Detection of ML boundaries These three datasets were combined into a sin- gle lookup table of ice particle scattering properties de- fined as a function of maximum diameter and mass. For a given D and mob, the backscattering cross section was es- timated using linear interpolation in the log–log space. The mean Doppler velocity can be derived in the same way: H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC 9550 2.1 2DVD and vertically pointing radars The Atmospheric Radiation Measurement 2DVD (ARM 2DVD) used in this study is the new generation of the one described in Kruger and Krajewski (2002). It relies on two cameras and two light sources placed in orthog- onal directions and records image projections of raindrops as they fall cross the cameras’ field of view. The 2DVD is often used for recording the size distributions, fall velocities and shapes of raindrops. Based on this information, the rain rate and reflectivity at a given radar frequency can be derived. The X- and Ka-band scanning ARM cloud radar (X/Ka- SACR) and W-band ARM cloud radar (MWACR) have the range gate spacing of 25, 25 and 30 m, respectively (Kollias et al., 2014; Kneifel et al., 2015; Falconi et al., 2018). The original time resolution of 2 s was averaged to 10 s for these radars. The half-power beam widths of X-SACR, Ka-SACR and MWACR are 1.27, 0.33 and 0.38◦, respectively. X- and Ka-SACR are dual-polarization radar systems installed on the same pedestal, recording the co-polar (e.g., ρhv, Zdr) and cross-polar (e.g., cross-polar correlation coefficient and lin- ear depolarization ratio, LDR) measurements, respectively. MWACR had a small antenna pointing error of 0.5 to 1◦, which may lead to significant error in the vertical Doppler velocity but which does not affect reflectivity measurements. To mitigate the potential attenuation from wet radome and raindrops, the simulated X-band radar reflectivity from 2DVD data was used to match the measured X-band re- flectivity at 500 m where the near-field effect is minimized (Sekelsky, 2002; Falconi et al., 2018). As the Ka-band reflec- Atmos. Chem. Phys., 20, 9547–9562, 2020 https://doi.org/10.5194/acp-20-9547-2020 3 Methods mur(D) = 0.0053D2.05. This relation is similar to the one de- rived from aircraft measurements (Heymsfield et al., 2004). A further discussion on the definition of mur(D) is found in Moisseev et al. (2017) and Li et al. (2018). 4 Results To study how ML properties depend on the precipitation intensity, snowflake riming fraction and PSD, all rainfall cases observed during the BAECC experiment were ana- lyzed. Given the need for coinciding multifrequency ver- tically pointing radar measurements and the radar scans performed during the experiment, we have identified 4147 vertical profiles of observations in 24 stratiform rainfall events corresponding to about 11.5 h. Table 2 summaries the dates used in this study (quicklooks are available at https://doi.org/10.5281/zenodo.3979103). Due to the peri- odic changes in radar scans, the multifrequency radar mea- surements recorded in the vertically pointing mode were available only in some inconsecutive time periods for an event. During the analysis, the mean radar Doppler velocity was scaled to the air density at 1000 hPa and 0 ◦C, as pre- viously described. It should be noted that the RHI scans by the FMI C-band weather radar were performed every 15 min. Therefore, the profiles of specific differential phase and dif- ferential reflectivity are recorded much less frequently than the vertically pointing radar observations. The RHI obser- vations are nonetheless presented here in order to link the features observed in this study to the previous reports (Gi- angrande et al., 2016; Kumjian et al., 2016; Li et al., 2018; Vogel and Fabry, 2018). Figure 2 shows the flow chart of the data process in this study. N(D) = N0e−3D, (5) N(D) = N0e−3D, (5) where the intercept parameter N0 is canceled out while com- puting DWR and VX, so the radar variables depend on 3, which controls the average size of ice particles in N(D). We have varied 3−1 between 0 and 11 mm to mimic different snowfall conditions, which is similar to what was done in Leinonen and Szyrmer (2015). Table 1 summaries the fit- ted expressions of DWR(X, Ka) = aV b X for these three par- ticle types. Since snow microphysics and the corresponding radar measurements can significantly change with precipita- tion intensity (Moisseev et al., 2017), the computed values were separated into four subgroups according to precipita- tion rate (PR). Simulations of DWR(X, Ka)-VX for four groups of pre- cipitation rate are presented in Fig. 1. Most cases with FR ≤0.2 are centered around the curve of LH74 unrimed, whose velocity–diameter relation is similar to low-density snowflakes (Tiira et al., 2016). It seems that riming hap- pens more frequently in heavier precipitation. H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC 9551 in the order of 1 dB (Kneifel et al., 2015). Therefore, cases with DWR(X, Ka) < 1 dB were rejected when identifying un- rimed snow. the potential link between FR and simulated Doppler radar measurements at X- and Ka-bands was accessed with the uti- lization of in situ snowfall observations from BAECC to the winters of 2014–2018. the potential link between FR and simulated Doppler radar measurements at X- and Ka-bands was accessed with the uti- lization of in situ snowfall observations from BAECC to the winters of 2014–2018. The dependence of DWR(X, Ka) and VX on FR can be computed using Eqs. (1), (2) and (4). For comparison, the mob −D and v −D relations of aggregates of unrimed radiating assemblages, side planes, bullets, and columns (LH74 unrimed), aggregates of densely rimed radiating as- semblages of dendrites (LH74 rimed), and lump graupel (LH74 graupel) presented in Locatelli and Hobbs (1974) were used. To compute the DWR(X, Ka) and VX using re- lations from the literature, we assumed that N(D) can be pa- rameterized as 4 Results In contrast, far fewer unrimed cases are present in heavier precipitation (Fig. 1c and d). Heavily rimed snowflakes (FR > 0.5; red dots) are characterized by low DWR(X, Ka) and high VX, contrasting with the unrimed/lightly rimed cases (blue dots). Specifically, snowflakes with large sizes and low velocities usually are rather slightly rimed (FR ≤0.2). For the cases where FR exceeds 0.5, most DWR(X, Ka) values are be- low 3 dB, indicating that heavily rimed particles are usu- ally associated with small snowflakes. Inspired by this dis- tinct feature, we have fitted the DWR(X, Ka) = aV b X rela- tions for cases with FR ≤0.2 and 0.4 ≤FR ≤0.6 (shown in Table 1), which separate the observations into three types: unrimed, transitional and rimed snow. For the sake of com- parison, the power b for unrimed snow was adopted from the fit for LH74 unrimed. In this study, these fitted relations were employed for classifying unrimed and rimed snow. The pres- ence of supercooled liquid water does not significantly affect X-band reflectivity but may lead to appreciable attenuation at Ka-band which translates to enhanced DWR(X, Ka) after the relative calibration at precipitation top. For the liquid wa- ter path of 500 g m−2, the estimated Ka-band attenuation is 3.2 Diagnosing snowflake rime mass fraction The rime mass fraction (FR), defined as the ratio of accreted ice mass by riming to the total snowflake mass, has been used to quantify the riming extent in ice microphysical schemes (Morrison and Milbrandt, 2015) and in observational studies (e.g., Moisseev et al., 2017; Li et al., 2018). The rime mass fraction can be defined as Vλ = Dmax R Dmin v(D)N(D)σb,λ (D,mob(D))dD Dmax R Dmin N(D)σb,λ (D,mob(D))dD , (4) (4) (4) FR = 1 − Dmax R Dmin N(D)mur(D)dD Dmax R Dmin N(D)mob(D)dD , (1) Dmax R where v(D) is the fall velocity of snowflakes which was used to derive m(D) (von Lerber et al., 2017). To minimize the im- pact of varying air density (ρair), Vλ was adjusted to the air condition of 1000 hPa and 0 ◦C (air density ρair,0) with a fac- tor of ( ρair,0 ρair )0.54 (Heymsfield et al., 2007). ρair was derived from the temperature and relative humidity obtained from the temporally closest sounding. FR = 1 − R Dmin N(D)mur(D)dD Dmax R Dmin N(D)mob(D)dD , (1) (1) where Dmax and Dmin are maximum and minimum parti- cle sizes, respectively, mob(D) and mur(D) are masses of observed and unrimed snowflakes as a function of D, re- spectively, and N(D) is the PSD. In this study, FR was computed using ground-based observations of PSD and snowflake masses retrieved from PIP observations, as de- scribed in von Lerber et al. (2017). The masses of unrimed ice particles were derived assuming the following. Firstly, unrimed snowflakes were present in PIP observations. Sec- ondly, the ice particles belonging to the lightest 5 % are rep- resentative of unrimed snowflakes. Following these assump- tions, the mass of unrimed snowflakes can be expressed as Dias Neto et al. (2019) have shown that the size growth of snowflakes close to the ML is accelerated due to the en- hanced aggregation. Therefore, relatively large aggregates are prevalent snow types close to the ML and are better rep- resented by DWR(X, Ka) than DWR(Ka, W) (see the com- parison by Barrett et al., 2019). The use of a lower radar frequency (X- and Ka-bands) avoids estimating the non- neglectable W-band attenuation caused by ML, as well as su- percooled liquid water (Li and Moisseev, 2019). Therefore, Atmos. Chem. Phys., 20, 9547–9562, 2020 https://doi.org/10.5194/acp-20-9547-2020 H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC 9552 Figure 1. Scatter plot of DWR(X, Ka) versus VX (1000 hPa and 0 ◦C) colored with FR. Mass–size and velocity–size relations from Locatelli and Hobbs (1974) are adopted for reference (dashed lines). Observed mass–size and velocity–size relations are derived using the approach developed by von Lerber et al. (2017), and the particle backscattering coefficient σb,λ is adopted from Leinonen and Moisseev (2015) and Leinonen and Szyrmer (2015). The solid blue and red curves separate unrimed (light blue shading), transitional (no shading) and rimed snow (light red shading) in our classification scheme. Figure 1. Scatter plot of DWR(X, Ka) versus VX (1000 hPa and 0 ◦C) colored with FR. Mass–size and velocity–size relations from Locatelli and Hobbs (1974) are adopted for reference (dashed lines). Observed mass–size and velocity–size relations are derived using the approach developed by von Lerber et al. (2017), and the particle backscattering coefficient σb,λ is adopted from Leinonen and Moisseev (2015) and Leinonen and Szyrmer (2015). The solid blue and red curves separate unrimed (light blue shading), transitional (no shading) and rimed snow (light red shading) in our classification scheme. Table 1. Fitted parameters for DWR(X, Ka) = aV b X. Aggregates of unrimed radiating assemblages, side planes, bullets, and columns (LH74 unrimed), aggregates of densely rimed radiating assemblages of dendrites (LH74 rimed), and lump graupel (LH74 graupel) in Lo- catelli and Hobbs (1974) are shown for reference. The last column shows the root mean square error (RMSE) of fitting. The confidence interval is marked by “\” when the parameter is manually fixed. Fitted parameters a (95 % b (95 % RMSE confidence confidence (dB) interval) interval) LH74 Unrimed 2.6 (2.2 3) 7.3 (6.1 8.5) 1.9 Rimed 0.2 (0.09 0.31) 9.8 (8.1 11.5) 1.8 Graupel 0.35 (0.19 0.51) 2.5 (2.03 2.97) 0.8 PR ≤0.15 mm h−1 FR ∈[0 0.2] 1.3 (1.02 1.58) 7.3 (\) 2 FR ∈[0.4 0.6] 0.2 (0.14 0.26) 2.96 (2.26 3.66) 0.5 0.15 mm h−1 < PR ≤0.5 mm h−1 FR ∈[0 0.2] 0.75 (0.64 0.86) 7.3 (\) 2.4 FR ∈[0.4 0.6] 0.47 (0.37 0.57) 3.1 (2.7 3.5) 1.1 0.5 mm h−1 < PR ≤1 mm h−1 FR ∈[0 0.2] 0.69 (0.61 0.77) 7.3 (\) 2.2 FR ∈[0.4 0.6] 0.52 (0.4 0.64) 2.9 (2.3 3.5) 0.85 1 mm h−1 < PR ≤4 mm h−1 FR ∈[0 0.2] 0.6 (0.59 0.61) 7.3 (\) 2.3 FR ∈[0.4 0.6] 0.75 (0.59 0.91) 2.85 (2.16 3.54) 1.2 Atmos. Atmos. Chem. Phys., 20, 9547–9562, 2020 https://doi.org/10.5194/acp-20-9547-2020 4.1 Sanity check of the snow classification At the first step of our data analysis, the classification of unrimed and rimed snow using DWR(X, Ka)-VX observa- tions at the ML top, proposed in the previous section, was evaluated against previous studies. As shown in Fig. 3, both DWR(X, Ka) and VX tend to increase as the precipitation intensifies. Most cases of rimed snow fall in the region of DWR(X, Ka) < 4 dB and VX being higher than for unrimed snow. The VX of unrimed snow rarely exceeds 1.5 m s−1. Those outliers of rimed snow in Fig. 1a may be attributed to the local vertical air motions, which contaminate the mea- sured mean Doppler velocity. It should be noted that the snow observations in Fig. 1 are limited to PR ≤4 mm h−1; namely, the maximum radar reflectivity at the ML bottom (ZX,rain) is around 33 dBZ, as computed by using the localized Z–R re- lation (Leinonen et al., 2012b). The reflectivity enhancement in the ML, which is defined as the difference between the ZX maximum in the ML and the ZX at the melting bottom (ZX,rain), was also studied. Za- wadzki et al. (2005) have analyzed the UHF Doppler wind profiler observations in VUHF,snow/VUHF,rain reflectivity en- Atmos. Chem. Phys., 20, 9547–9562, 2020 https://doi.org/10.5194/acp-20-9547-2020 H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC indicating the reasonable snow classification employed in this study. https://doi.org/10.5194/acp-20-9547-2020 Atmos. Chem. Phys., 20, 9547–9562, 2020 Table 2. Summary of the studied events. Event Date Event Date 1 9 May 2014 13 10 August 2014 2 11 May 2014 14 12 August 2014 3 16 May 2014 15 13 August 2014 4 19 May 2014 16 14 August 2014 5 31 May 2014 17 18 August 2014 6 4 June 2014 18 19 August 2014 7 6 June 2014 19 20 August 2014 8 12 June 2014 20 24 August 2014 9 13 June 2014 21 25 August 2014 10 15 July 2014 22 26 August 2014 11 16 July 2014 23 27 August 2014 12 30 July 2014 24 9 September 2014 Table 2. Summary of the studied events. H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC Figure 2. Flow chart of the data process in this study. The snow classification part as discussed in Sect. 3.1 is in light blue. Variables used in snow classification are in green. Radar observations during BAECC are in black. The sanity check in the next section is represented by the dashed black diagram. Figure 2. Flow chart of the data process in this study. The snow classification part as discussed in Sect. 3.1 is in light blue. Variables used in snow classification are in green. Radar observations during BAECC are in black. The sanity check in the next section is represented by the dashed black diagram. Figure 2. Flow chart of the data process in this study. The snow classification part as discussed in Sect. 3.1 is in light blue. Variables used in snow classification are in green. Radar observations during BAECC are in black. The sanity check in the next section is represented by the dashed black diagram. Figure 3. Distribution of (a) DWR(X, Ka) and (b) VX above the ML as a function of ZX,rain. Note that no transitional snow type between unrimed and rimed is presented. Table 2. Summary of the studied events. Event Date Event Date 1 9 May 2014 13 10 August 2014 2 11 May 2014 14 12 August 2014 3 16 May 2014 15 13 August 2014 4 19 May 2014 16 14 August 2014 5 31 May 2014 17 18 August 2014 6 4 June 2014 18 19 August 2014 7 6 June 2014 19 20 August 2014 8 12 June 2014 20 24 August 2014 9 13 June 2014 21 25 August 2014 10 15 July 2014 22 26 August 2014 11 16 July 2014 23 27 August 2014 12 30 July 2014 24 9 September 2014 hancement space and found that the augmentation of rimed snowflake mass can increase VUHF,snow/VUHF,rain and de- crease reflectivity enhancement. As shown in Fig. 4, de- spite the scattered distribution of reflectivity enhancement, the majority of cases with high VX,snow/VX,rain is domi- nated by rimed snow, while most unrimed cases are below VX,snow/VX,rain = 0.25. Such dependence of VX,snow/VX,rain on riming is in line with the results in Zawadzki et al. (2005), Figure 3. Distribution of (a) DWR(X, Ka) and (b) VX above the ML as a function of ZX,rain. Note that no transitional snow type between unrimed and rimed is presented. H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC Chem. Phys., 20, 9547–9562, 2020 https://doi.org/10.5194/acp-20-9547-2020 https://doi.org/10.5194/acp-20-9547-2020 9553 Insights from multifrequency and dual-polarization radar observations during BAECC H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC Table 2. Summary of the studied events. Figure 3. Distribution of (a) DWR(X, Ka) and (b) VX above the ML as a function of ZX,rain. Note that no transitional snow type between unrimed and rimed is presented. indicating the reasonable snow classification employed in this study. hancement space and found that the augmentation of rimed snowflake mass can increase VUHF,snow/VUHF,rain and de- crease reflectivity enhancement. As shown in Fig. 4, de- spite the scattered distribution of reflectivity enhancement, the majority of cases with high VX,snow/VX,rain is domi- nated by rimed snow, while most unrimed cases are below VX,snow/VX,rain = 0.25. Such dependence of VX,snow/VX,rain on riming is in line with the results in Zawadzki et al. (2005), hancement space and found that the augmentation of rimed snowflake mass can increase VUHF,snow/VUHF,rain and de- crease reflectivity enhancement. As shown in Fig. 4, de- spite the scattered distribution of reflectivity enhancement, the majority of cases with high VX,snow/VX,rain is domi- nated by rimed snow, while most unrimed cases are below VX,snow/VX,rain = 0.25. Such dependence of VX,snow/VX,rain on riming is in line with the results in Zawadzki et al. (2005), Atmos. Chem. Phys., 20, 9547–9562, 2020 Atmos. Chem. Phys., 20, 9547–9562, 2020 https://doi.org/10.5194/acp-20-9547-2020 H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC 9554 Figure 4. Scatter plot of VX,snow/VX,rain versus reflectivity en- hancement in the ML. The reflectivity enhancement is defined as the difference between the reflectivity peak in the ML and the re- flectivity in rain just below the ML. by riming (Kumjian et al., 2016). In our observations, both ρhv dip and reflectivity peak descend with the increase in PR. Therefore, it appears that precipitation intensity is an impor- tant factor affecting the formation of the saggy bright band. This finding is in line with a recent simulation study (Carlin and Ryzhkov, 2019), which proposes that the saggy bright band can also be attributed to other factors, such as the ag- gregation process, the increased precipitation intensity and the sudden decrease in RH. For unrimed snow, the response of ρhv to the melting is obviously later than X-band reflec- tivity, which indicates that the utilization of ρhv for detecting the ML top should be applied with caution. p pp The reflectivity peak is smaller for rimed snow than un- rimed for a given PR provided that the Rayleigh scattering is not violated. 4.2.1 X-band reflectivity, ρhv and DWR(X, Ka) Figure 5 shows the profiles of radar reflectivity and ρhv mea- sured by X-SACR and grouped by PR. Note that to gener- alize the observations, the vertical axis is shifted such that the ML top is the reference height of 0 m, and each reflec- tivity profile was normalized by offsetting the difference be- tween ZX,rain and the median value of ZX,rain in the corre- sponding PR group. The same procedure was made to ρhv and the following measurements. For most cases, the rela- tive humidity (RH) around the ML top is above 95 % with no dependence on PR. Thus, the effect of dry air infiltration, e.g., decreasing reflectivity and ML thickness and descend- ing dual-polarization measurements (Carlin and Ryzhkov, 2019), should be minimized. Considering the general aspects of Fig. 5, it is clear that the ML thickness and reflectivity peak increase with PR, which is in line with previous re- sults (Fabry and Zawadzki, 1995; Wolfensberger et al., 2016; Trömel et al., 2019). g ( ) Interestingly, the DWR(X, Ka) profile below the ML is higher for the rimed cases and progressively converges to- wards the unrimed profile as PR increases. For light pre- cipitation, the rain drops are small enough to be Rayleigh scatterers at Ka-band; thus the difference of DWR(X, Ka) in rain between unrimed and rimed cases is rooted in the differences in attenuation. If the supercooled liquid water attenuation of rimed cases is more significant, the corre- sponding DWR(X, Ka) in rain would be smaller than the unrimed cases. However, the reverse is observed. von Ler- ber et al. (2014) have shown that the melting layer atten- uation of rimed snowflakes is smaller than unrimed ones, which could possibly explain the larger DWR(X, Ka) of rimed cases in rain. With the increase in precipitation inten- sity, the DWR(X, Ka) of rimed cases in rain decreases to- wards the unrimed profile. This can be attributed to several factors, such as the enhanced liquid attenuation above the melting layer and the non-Rayleigh scattering of large rain- drops at Ka-band. We hesitate to determine the role of non- Rayleigh scattering in rain since raindrops characterized by The ρhv and radar reflectivity have been used in identifying the bright band sagging (Kumjian et al., 2016; Ryzhkov et al., 2016; Xie et al., 2016). Table 2. Summary of the studied events. When PR is greater than 1 mm h−1, the re- flectivity peaks of rimed and unrimed snow are closer, which can be explained by the non-Rayleigh scattering of very large aggregates at X-band, as discussed by Fabry and Zawadzki (1995). Another notable finding is that the ZX at the ML top for rimed snow is smaller than unrimed, which indicates that rimed snowflakes may have smaller sizes for a given PR. This is further confirmed in the DWR(X, Ka) profiles as shown in Fig. 6. From the aggregation region to the ML top, the DWR(X, Ka) of rimed snow is significantly smaller than un- rimed snow. In particular, very weak DWR(X, Ka) for rimed snow could be identified just above the ML. This indicates that the aggregation process, the dominating factor of grow- ing snow size close to the ML (Fabry and Zawadzki, 1995), can be heavily suppressed for rimed snow. Heymsfield et al. (2015) have reported the enhanced maximum particle size below the 0 ◦C isotherm using in situ measurements and at- tributed it to the continuing aggregation in the ML. Such con- tinuing aggregation in conjunction with the changing scatter- ing properties (the water coating) may be responsible for the continuing increase in DWR(X, Ka) in the ML. Figure 4. Scatter plot of VX,snow/VX,rain versus reflectivity en- hancement in the ML. The reflectivity enhancement is defined as the difference between the reflectivity peak in the ML and the re- flectivity in rain just below the ML. 4.2 Vertical profiles of multifrequency radar measurements in the ML To obtain a general idea of how the ML is modulated by rim- ing and aggregation, statistics of vertically pointing radar ob- servations were made. As the ML properties are modulated by precipitation intensity (Fabry and Zawadzki, 1995; Carlin and Ryzhkov, 2019), the observations were grouped by PR. For ease of comparison, the vertical axis is shifted such that the reference height is the ML top. H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC 9555 Figure 5. Normalized X-band radar profiles grouped by PR. tunrimed and trimed indicate the total observing time in each group for unrimed and rimed cases, respectively. The median values of X-band reflectivity at the ML bottom for unrimed (ZX,unrimed) and rimed (ZX,rimed) cases are marked just below the ML bottom with the standard values in brackets. The median and standard deviations (in parentheses) of relative humidity (RH) at the ML top for unrimed (RHunrimed) and rimed (RHrimed) cases in each group are presented near the ML top. The median and standard deviations (in parentheses) of PR for unrimed (PRunrimed) and rimed (PRrimed) cases in each group are presented in the lower part. Shaded regions represent the standard derivation. Figure 5. Normalized X-band radar profiles grouped by PR. tunrimed and trimed indicate the total observing time in each group for unrimed and rimed cases, respectively. The median values of X-band reflectivity at the ML bottom for unrimed (ZX unrimed) and rimed (ZX rimed Figure 5. Normalized X-band radar profiles grouped by PR. tunrimed and trimed indicate the total observing time in each group for unrimed and rimed cases, respectively. The median values of X-band reflectivity at the ML bottom for unrimed (ZX,unrimed) and rimed (ZX,rimed) cases are marked just below the ML bottom with the standard values in brackets. The median and standard deviations (in parentheses) of relative humidity (RH) at the ML top for unrimed (RHunrimed) and rimed (RHrimed) cases in each group are presented near the ML top. The median and standard deviations (in parentheses) of PR for unrimed (PRunrimed) and rimed (PRrimed) cases in each group are presented in the lower part. Shaded regions represent the standard derivation. Figure 6. Same as Fig. 5 but for DWR(X, Ka). Note that the radar calibration is made by matching the X- and Ka-band radar reflectiv- ities at precipitation top. Figure 6. Same as Fig. 5 but for DWR(X, Ka). Note that the radar different non-Rayleigh scattering size regions can lead to op- posite effects on DWR(X, Ka), as shown by Li and Moisseev (2019). 4.2.1 X-band reflectivity, ρhv and DWR(X, Ka) When PR is greater than 1 mm h−1, the level of ρhv minimum of rimed snow seems to be lower than the unrimed; however, the opposite holds when PR is less than or equal to 1 mm h−1, which seems controversial to the expectation that the bright band sagging is mainly caused Atmos. Chem. Phys., 20, 9547–9562, 2020 https://doi.org/10.5194/acp-20-9547-2020 4.2.3 W-band reflectivity W-band reflectivity can be heavily affected by a wet radome, rain, ML, supercooled liquid water and gaseous attenuation (Kneifel et al., 2015; Li and Moisseev, 2019). Such attenu- ation coupled with precipitation microphysical processes, as well as the change of particle scattering regimes, can modu- late the W-band reflectivity profiles. As shown in Fig. 9, the decrease in W-band reflectivity with height is mainly caused by rain attenuation. This effect is enhanced as PR increases, which has been adopted to retrieve PR (Matrosov, 2007). From dry to melting snow, there is a jump in W-band reflec- tivity, and the extent of such a jump seems dependent on PR. The bright band signature is partially visible when PR is less than or equal to 0.15 mm h−1 but is absent as the precipita- tion intensifies. This is expected, given the increased non- Rayleigh scattering at W-band for large snowflakes (Sassen et al., 2005). When PR is less than or equal to 0.15 mm h−1, the dark band is present for both unrimed and rimed snow, while the reflectivity dip near the ML top for unrimed snow is stronger than rimed. Below 1 mm h−1, the dark band is present for unrimed snow, in contrast with its absence for rimed snow when PR is greater than 0.15 mm h−1, which may indicate that the dark band is more frequently observed for the scenario of unrimed snow. Figure 7. Same as Fig. 5 but for LDR observed by Ka-SACR. As shown in Fig. 8, the Ka-band reflectivity enhancement in the ML decreases as the precipitation intensifies. This is similar to the observations presented by Fabry and Za- wadzki (1995) who found that the reflectivity peak in the ML observed by an X-band radar is less pronounced than that measured by an UHF radar when the reflectivity in rain ex- ceeds 25 dBZ. With the increase in precipitation intensity, the size of snowflakes generally grows. Therefore, there are less hydrometeors satisfying the Rayleigh criteria, and the non- Rayleigh scattering becomes more significant. As a result, the reflectivity peak in the ML is not as pronounced as in the scenario of Rayleigh scattering. In addition, the ML atten- uation increases as PR intensifies (Li and Moisseev, 2019), which further impedes the increase in reflectivity in the ML. H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC 9556 Figure 7. Same as Fig. 5 but for LDR observed by Ka-SACR. above the ML top, as shown in Sassen et al. (2005, 2007) and Heymsfield et al. (2008). above the ML top, as shown in Sassen et al. (2005, 2007) and Heymsfield et al. (2008). 4.2.3 W-band reflectivity This also explains the lower reflectivity enhancement in the ML for rimed snow when PR is greater than 0.5 mm h−1. As shown in Fig. 6, rimed snowflakes are usually smaller; thus the non-Rayleigh effect and ML attenuation (von Ler- ber et al., 2014) are not as significant as the larger unrimed ice particles. Sassen et al. (2005) have proposed that the dark band observed by W-band radars is due to the combination of Rayleigh and non-Rayleigh scattering effects modulated by the PSD. Heymsfield et al. (2008) have pinpointed that such a reflectivity dip is linked to the aggregation process, which consumes small ice while growing large snowflakes whose backscattering cross sections at W-band are much smaller than the scenario of Rayleigh scattering. This statement is ev- idenced in our statistical results since the dark band feature is more significant for unrimed snow and is more distinct at W- band than at Ka-band. Furthermore, the obscured dark band for rimed snow may indicate that the aggregation of rimed snow can be weaker than unrimed snow. When PR is less than or equal to 0.15 mm h−1, a weak re- flectivity dip, the dark band, appears at the top of the ML for unrimed snow, which may also be observed by centimeter- wavelength radars (Fabry and Zawadzki, 1995). In the lit- erature, the dark band has different definitions. For ground- based radars, Kollias and Albrecht (2005) referred the dip in radar reflectivity below the ML top as dark band. The dark band which is present just above the ML top, as observed by the spaceborne W-band radar, can be caused by the strong signal attenuation from large snow aggregates, as discussed in Sassen et al. (2007). Meanwhile, the change of PSD during the aggregation process can also contribute to this reflectiv- ity dip, which is named dark band by Sassen et al. (2005) and dim band by Heymsfield et al. (2008). In this study, the dark band is identified as the decrease in radar reflectivity just 4.2.2 Ka-band LDR and reflectivity LDR usually increases in the ML as melting increases the di- electric constant of nonspherical ice particles. Figure 7 shows the profiles of LDR (Ka-SACR) and ρhv (X-SACR). Both LDR peak and ρhv dip in rimed snow are lower than un- rimed snow when PR is greater than 1 mm h−1, while the re- verse is observed for lighter precipitation. Despite the rather good agreement between LDR and ρhv observations, it ap- pears that LDR systematically reveals a lower ML bottom than ρhv, indicating that LDR can be suitable in discriminat- ing between rain and melting snow (Illingworth and Thomp- son, 2011; Dias Neto et al., 2019). The smaller LDR peak for rimed snow is correlated with the smaller X-band reflec- tivity enhancement as shown in Fig. 5, which is consistent with Illingworth and Thompson (2011) and Sandford et al. (2017). Figure 6. Same as Fig. 5 but for DWR(X, Ka). Note that the radar calibration is made by matching the X- and Ka-band radar reflectiv- ities at precipitation top. https://doi.org/10.5194/acp-20-9547-2020 Atmos. Chem. Phys., 20, 9547–9562, 2020 H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC 9557 Figure 8. Same as Fig. 5 but for Ka-band radar. Note that the calibration is made by matching the Ka-band reflectivity with X-band at precipitation top, while the attenuation in the profile is not accounted for. To be in line with Fig. 5, ρhv observed by X-SACR is presented instead of the LDR measured by Ka-SACR. Figure 8. Same as Fig. 5 but for Ka-band radar. Note that the calibration is made by matching the Ka-band reflectivity with X-band at precipitation top, while the attenuation in the profile is not accounted for. To be in line with Fig. 5, ρhv observed by X-SACR is presented instead of the LDR measured by Ka-SACR. Figure 9. Same as Fig. 5 but for W-band. Note that the reflectivity profiles at W-band are shifted by matching the radar reflectivity at the ML bottom (ZW,rain), while the value of ZW,rain is not shown due to the unknown W-band attenuation. gation happens in light precipitation. Li et al. (2018) have shown that Zdr is a function of snow shape, canting angle distribution and density, and it generally decreases with the increase in radar reflectivity. It would be interesting to study the riming impact on Zdr profiles (Vogel and Fabry, 2018); unfortunately, we were not able to perform such a compar- ison due to the very limited number of RHI profiles during the studied events. It should be noted that the beam width of FMI C-band radar is 1◦, resulting in a vertical projection of around 1.1 km over the Hyytiälä station. This explains why the height at which Zdr starts increasing is approximately 500 m higher than the ML top determined by X-SACR. High Kdp values were observed when PR exceeds 1 mm h−1, while no detectable Kdp signal can be found when PR is less than or equal to 1 mm h−1. This is in line with the previous finding that the enhanced Kdp is indicative of in- tense precipitation (Bechini et al., 2013). When PR is greater than 1 mm h−1, the enhanced Kdp starts at around 3000 m above the ML with the expected temperature of around −20 ◦C, which is related to the dendritic growth region (Be- chini et al., 2013; Moisseev et al., 2015). Atmos. Chem. Phys., 20, 9547–9562, 2020 4.3 Weather radar measurements Recent studies have demonstrated the potential of polarimet- ric measurements in revealing cloud microphysics and im- proving precipitation forecasts (Tiira and Moisseev, 2020; Trömel et al., 2019). Given the importance of precipitation intensity to the ML, it is necessary to address how the dual- polarization observations are dependent on PR. Therefore, we have analyzed the statistical profiles of Zdr and Kdp ob- served by the RHI scan of the FMI C-band dual-polarization radar. The vertical axis of weather radar RHI observations was shifted to the same level as we did for vertically pointing radars. https://doi.org/10.5194/acp-20-9547-2020 Atmos. Chem. Phys., 20, 9547–9562, 2020 H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC 9558 Figure 10. Normalized Zdr and Kdp profiles observed by FMI C- band radar with RHI scanning. Number of RHI profiles is presented at the bottom. The ML top (as retrieved from X-SACR observa- tions) is used as the reference height in the vertical axis. b. X-band radar reflectivity peak is smaller for rimed snow than unrimed for a given precipitation inten- sity if the non-Rayleigh scattering effect is not sig- nificant. b. X-band radar reflectivity peak is smaller for rimed snow than unrimed for a given precipitation inten- sity if the non-Rayleigh scattering effect is not sig- nificant. c. If the non-Rayleigh scattering effect is distinct, e.g., at Ka- or W-band, the reflectivity peak can be larger for rimed snow. d. The reflectivity dip at the melting layer top (dark band) is obscured for rimed snow, while it is pro- nounced for unrimed snow. This suggests that the aggregation process may be suppressed by riming. 3. The decrease in Zdr towards the melting layer is pro- nounced in heavy precipitation but is insignificant in light precipitation. A well-calibrated triple-frequency radar setup has been shown potential in studying the microphysics of snowfall. However, such measurements may not be well suited to rain- fall due to the highly uncertain W-band attenuation caused by the melting layer, as well as the supercooled water. The approach presented explores the possibility of adding the Doppler velocity to distinguish between unrimed and rimed conditions and is less affected by the attenuation from su- percooled water. Such instrumentation as the X/Ka-SACR mounted on the same platform takes much less effort in pointing alignment. Its application may also be expanded to space-borne radars. For example, instead of launching triple-frequency radars, implementing the Doppler capability with sufficient sensitivity on either of the radars on a dual- wavelength platform may be served as an option. Figure 10. Normalized Zdr and Kdp profiles observed by FMI C- band radar with RHI scanning. Number of RHI profiles is presented at the bottom. The ML top (as retrieved from X-SACR observa- tions) is used as the reference height in the vertical axis. H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC Overall, these ob- servations indicate that the dependence of ML properties on the dual-polarization signatures above may mainly be due to the correlation of these signatures with precipitation inten- sity. Figure 9. Same as Fig. 5 but for W-band. Note that the reflectivity profiles at W-band are shifted by matching the radar reflectivity at the ML bottom (ZW,rain), while the value of ZW,rain is not shown due to the unknown W-band attenuation. Significant dependence of Zdr and Kdp on PR can be found in Fig. 10. In cases when PR is greater than 0.15 mm h−1, Zdr decreases significantly to around 0 dB just above the ML. This is mainly due to the aggregation process, which leads to increased particle size and decreased density. In contrast, Zdr does not change just above the ML when PR is less than or equal to 0.15 mm h−1, indicating that very weak aggre- Atmos. Chem. Phys., 20, 9547–9562, 2020 https://doi.org/10.5194/acp-20-9547-2020 H. Li et al.: Insights from multifrequency and dual-polarization radar observations during BAECC 5 Conclusions In this work, the connection between the precipitation melt- ing layer and snow microphysics was studied using ver- tically pointing multifrequency Doppler radar and C-band dual-polarization weather radar observations. Using surface- based snowfall measurements collected over five winters at the University of Helsinki measurement station and supple- mented by the single particle scattering datasets of aggre- gated snowflakes and rimed ice particles, a connection be- tween rime mass fraction and radar observations at X- and Ka-bands was established and used in classifying unrimed and rimed snow. The sanity checks show that the results of this classification are consistent with the previous study using single-frequency radar observations (Zawadzki et al., 2005). Statistics of vertically pointing multifrequency dual- polarization radars and RHI scans of C-band polarimetric weather radar show the following. A coordinated radar setup as employed during BAECC fa- cilitates the synergy of multiple radar frequencies and polari- metric observations at various scan modes. Due to the peri- odical changes of radar scanning modes during BAECC, the total stratiform rainfall cases are limited to ∼11.5 h. More of such observations can be utilized to evaluate and consolidate the presented conclusions. If such coordinated measurements with high time resolutions can be obtained in the future, our understanding of snow microphysical processes may be fur- ther advanced. 1. The radar-observed melting layer properties show a de- tectable connection to the precipitation intensity. The in- crease in precipitation intensity can lead to the saggy bright band, i.e., the descending of reflectivity peak and ρhv dip. Data availability. Quicklooks of radar observations used in this study are available at https://doi.org/10.5281/zenodo.3979103 (Li, 2020). The FMI radar data are available from the Atmospheric Ra- diation Measurement (ARM) Climate Research Facility (https: //iop.archive.arm.gov/arm-iop/2014/tmp/baecc/moisseev-radar_ cband/?uid=LIH2&st=5f32748d&home=arm-archive, last access: 11 August 2020) (von Lerber, 2020). 2. Riming can affect melting layer properties in the fol- lowing ways. The ARM data used in this study are available from Atmospheric Radiation Measurement (ARM) Climate Research Facility (ARM Climate Research Facility, 2006, 2010, 2011). The ARM data used in this study are available from Atmospheric Radiation Measurement (ARM) Climate Research Facility (ARM Climate Research Facility, 2006, 2010, 2011). a. In moderate to heavy rainfall, riming may cause ad- ditional bright band sagging. However, the oppo- site effect is observed in light precipitation, namely, such sagging is associated with unrimed snow. PIP data are available from https://doi.org/10.5281/zenodo.3977959 (Moisseev, 2020). Competing interests. The authors declare that they have no conflict of interest. Competing interests. The authors declare that they have no conflict of interest. ARM Climate Research Facility: Ka-Band Scanning ARM Cloud Radar (KASACRVPT). 2014-01-15 to 2014-09-13, ARM Mo- bile Facility (TMP) U. of Helsinki Research Station (SMEAR II), Hyytiala, Finland; AMF2 (M1), Compiled by: Isom, B., Bharad- waj, N., Lindenmaier, I., Nelson, D., Hardin, J., and Matthews, A., Atmospheric Radiation Measurement (ARM) Climate Re- search Facility Data Archive, Oak Ridge, Tennessee, USA, https://doi.org/10.5439/1046201, 2010. Acknowledgements. We would like to thank the personnel of Hyytiälä Station for their support in field observations. We espe- cially thank Matti Leskinen for his help in data analysis. The re- search of Haoran Li, Jussi Tiira and Dmitri Moisseev was sup- ported by the Academy of Finland’s Centers of Excellence program (grant 307331) and ERA-PLANET’s transnational project iCUPE (grant agreement 689443), funded under the EU Horizon 2020 framework program. Annakaisa von Lerber was funded by the Academy of Finland (postdoc grant 333901). Haoran Li was also funded by the China Scholarship Council. The instrumentation used in this study was supported by NASA’s Global Precipitation Mea- surement Mission ground validation program and by the US De- partment of Energy’s Office of Science ARM program. ARM Climate Research Facility: X-Band Scanning ARM Cloud Radar (XSACRVPT). 2014-01-15 to 2014-09-13, ARM Mobile Facility (TMP) U. of Helsinki Research Station (SMEAR II), Hyytiala, Finland; AMF2 (M1), Compiled by: Isom, B., Bharad- waj, N., Lindenmaier, I., Nelson, D., Hardin, J., and Matthews, A., Atmospheric Radiation Measurement (ARM) Climate Re- search Facility Data Archive, Oak Ridge, Tennessee, USA, https://doi.org/10.5439/1150303, 2011. Atlas, D.: Drop size and radar structure of a precipitation streamer, J. Meteorol., 14, 261–271, 1957. Financial support. This research has been supported by the Academy of Finland (grant no. 307331), the European Commission (grant no. ERA-PLANET (689443)) and the China Scholarship Council (grant no. 201603170181). Bailey, M. P. and Hallett, J.: A comprehensive habit diagram for atmospheric ice crystals: confirmation from the laboratory, AIRS II, and other field studies, J. Atmos. Sci., 66, 2888–2899, 2009. 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DEVELOPMENT OF SCIENCE PROCESS SKILLS STUDENTS WITH PROJECT BASED LEARNING MODEL- BASED TRAINING IN LEARNING PHYSICS
Jurnal Pendidikan Fisika
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DEVELOPMENT OF SCIENCE PROCESS SKILLS STUDENTS WITH PROJECT BASED LEARNING MODEL- BASED TRAINING IN LEARNING PHYSICS Ratna Malawati1, Sahyar2 1Student Alumni of Physics Education Study Programs Postgraduate School UNIMED 2Physics Education Study Programs Postgraduate School UNIMED PENINGKATAN KETERAMPILAN PROSES SAINS MAHASISWA DENGAN MODEL PROJECT BASED LEARNING BERBASIS PELATIHAN DALAM PEMBELAJARAN FISIKA Ratna Malawati1, Sahyar2 1Alumni Mahasiswa Program Studi Pendidikan Fisika Program Pascasarjana UNIMED 2Program Studi Pendidikan Fisika Program Pascasarjana UNIMED R. Malawati dan Sahyar: Peningkatan Keterampilan Proses Sains Mahasiswa Dengan Model Project Based Learning Berbasis Pelatihan Dalam Pembelajaran Fisika R. Malawati dan Sahyar: Peningkatan Keterampilan Proses Sains Mahasiswa Dengan Model Project Based Learning Berbasis Pelatihan Dalam Pembelajaran Fisika Jurnal Pendidikan Fisika p-ISSN2252-732X e-ISSN 2301-7651 email:sahyarpasca@gmail.com Abstrak. Penelitian ini bertujuan untuk meningkatkan Keterampilan Proses Sains fisika Mahasiswa pada aspek kognitif dan psikomotor dengan menggunakan model pembelajaran Project Based Learning berbasis pelatihan. Objek penelitian ini adalah model pembelajaran Project Based Learning yang digunakan dalam proses pembelajaran Fisika Komputasi. Metode penelitian yang digunakan adalah penelitian tindakan kelas melalui dua siklus pembelajaran, setiap siklus terdiri dari tahapan perencanaan, pelaksanaan, observasi dan refleksi. Pada siklus I diberikan penekanan perlakuan dengan adanya pelatihan pada fase pertama hingga ketiga dalam model Project Based Learning, sedangkan pada siklus II diberikan tambahan perlakuan dengan menekankan diskusi bersifat kolaborasi dalam mencapai hasil produk terbaik untuk setiap kelompok. Hasil analisis data menunjukkan ada peningkatan Kemampuan berpikir Mahasiswa pada ranah kognitif dan Keterampilan Proses Sains pada ranah psikomotor. Katakunci: Keterampilan Proses Sains, Project Based Learning, Pelatihan. PENDAHULUAN dengan metode sains, sehingga memudahkan internalisasi nilai-nilai dan semangat Mahasiswa. Ravitz, dkk, (2004) mengatakan bahwa model PjBL menggunakan kolaborasi dan penelitian untuk melakukan evaluasi. Bell (2010) menyatakan PjBL sebagai inovasi dalam pendekatan pembelajaran oleh pendidik dengan beberapa strategi penting untuk sukses di abad dua puluh satu. Pengembangan kurikulum yang dilakukan saat ini merupakan proses perbaikan yang dilakukan pemerintah dalam meningkatkan kualitas dan kuantitas pendidikan. Pengembangan itu dilakukan untuk menciptakan generasi yang lebih kompeten dan berkarakter dalam melaksanakan kegiatan sesuai dengan bidangnya. Pembentukan generasi difokuskan pada pihak-pihak yang secara langsung bergerak di bidang pendidikan. Hal ini bertujuan menghasilkan Sumber Daya Manusia yang kompeten di bidangnya dan berkarakter yang dapat menunjang aktifitas yang dilakukan. Penilaian karakter saat ini lebih ditekankan dikarenakan kemampuan dan keahlian dinilai kurang cukup tanpa ditunjang dengan karakter yang baik (good skill dan good attitude). Hal ini dikarenakan adanya keterkaitan antara prestasi kerja dengan hubungan yang terjalin dalam lingkungan kerja yang dapat mempengaruhi iklim dari lingkungan kerja tersebut. p g p Namun menurut Roessingh dan Chambers (2011) PjBL memiliki karakteristik pada professionalitas Pendidik, fasilitas pendukung, kemampuan menjelaskan, kompetensi, disposisi yang diperlukan untuk membuat transisi yang sukses. Di sisi lain, Barron dan Hammond (2007) menyatakan PjBL sebagai model untuk mengeksplorasi Mahasiswa belajar untuk masalah dunia nyata, bersaing, dan berkolaborasi dalam kelompok. Dalam sebuah studi Mahanal, dkk, (2012) PjBL terbukti efektif dalam meningkatkan sikap, memberdayakan sikap terhadap lingkungan, interaksi dalam kelompok, dan Keterampilan Proses Sains. g g j Faktor-faktor tersebut dibutuhkan sebagai indikator penilaian dalam pemilihan model pembelajaran yang digunakan khususnya dalam pembelajaran Fisika. Dengan kriteria tersebut digunakan model pembelajaran Project Based Learning (PjBL). Model pembelajaran ini digunakan juga berdasarkan pertimbangan dari beberapa penelitian. Menurut Hong, dkk, (2010) Model Pembelajaran Project Based Learning adalah pendekatan yang signifikan dalam meningkatkan potensi mengubah cara pengajaran dan pembelajaran pasif untuk memungkinkan siswa dengan alat dan dukungan media untuk meningkatkan Keterampilan Proses Sains. Hal ini ditegaskan Holubova (2008) yang menyatakan PjBL memiliki kelebihan dalam jenis mengajar pada kegiatan mahasiswa dan kesempatan untuk memecahkan masalah multidisiplin. Berdasarkan penelitian Mihardi (2013) mengenai efek model pembelajaran Project Based Learning di Unimed menyatakan bahwa model pembelajaran PjBL merupakan model yang baik untuk mengarahkan Mahasiswa pada proses berpikir dalam pencapaian hasil pembelajaran. Meskipun demikian diharapkan dalam penggunaan model pembelajaran tersebut diarahkan tidak pada sub materi dari suatu mata kuliah untuk tingkat Perguruan Tinggi. Hal ini dikarenakan penggunaan waktu pada Perguruan Tinggi sangat singkat untuk sub materi. Jurnal Pendidikan Fisika p-ISSN2252-732X e-ISSN 2301-7651 email: sahyarpasca@gmail.com Abstract This study aims to improve the physics Science Process Skills Students on cognitive and psychomotor aspects by using model- based Project Based Learning training. The object of this study is the Project Based Learning learning model used in the learning process of Computational Physics. The method used is classroom action research through two learning cycles, each cycle consisting of the stages of planning, implementation, observation and reflection. In the first cycle of treatment with their emphasis given training in the first phase up to Vol.5 No.1 Juni 2016 Vol.5 No.1 Juni 2016 Juni 2016 http://jurnal.unimed.ac.id/2012/index.php/jpf http://jurnal.unimed.ac.id/2012/index.php/jpf 58 R. Malawati dan Sahyar: Peningkatan Keterampilan Proses Sains Mahasiswa Dengan Model Project Based Learning Berbasis Pelatihan Dalam Pembelajaran Fisika third in the model Project Based Learning, while the second cycle is given additional treatment with emphasis discussion is collaboration in achieving the best results for each group of products. The results of data analysis showed increased ability to think Students on cognitive and Science Process Skills in the psychomotor. third in the model Project Based Learning, while the second cycle is given additional treatment with emphasis discussion is collaboration in achieving the best results for each group of products. The results of data analysis showed increased ability to think Students on cognitive and Science Process Skills in the psychomotor. Keywords: Science Process Skills, Project Based Learning, Training http://jurnal.unimed.ac.id/2012/index.php/jpf Jurnal Pendidikan Fisika p-ISSN2252-732X e-ISSN 2301-7651 Kedua, kriteria sentralitas berarti bahwa proyek-proyek di mana siswa belajar hal-hal yang berada di luar kurikulum. Project Based Learning atau Pembelajaran berbasis proyek (PjBL) merupakan pengembangan dalam mengajar sebagai pendekatan pembelajaran diperkenalkan oleh John Dewey. Namun, dalam perkembangannya PjBL mulai digunakan sebagai metode belajar menggambar dan menunjukkan kreativitas siswa. Pembelajaran Berbasis Proyek telah didefinisikan dalam banyak cara. Dalam definisi yang diberikan, PjBL telah disebut sebagai "model", "pendekatan" atau "teknik", atau sebagai "pembelajaran" atau "mengajar". Berikut adalah beberapa pandangan di PjBL dalam belajar. Menurut Baker, dkk (2011) bahwa Model Pembelajaran Berbasis Proyek melibatkan para siswa dalam pembelajaran yang relevan yang berdampak positif masyarakat dan ekosistem lokal mereka. Hal ini ditegaskan Cakmakci dan Tasar (2010) yang menjelaskan bahwa dalam proyek berdasarkan perspektif pembelajaran, pembelajaran ditangani dengan reorganisasi struktur kognitif peserta didik. Belajar permanen dan efisien adalah target dalam proyek pembelajaran berbasis dengan partisipasi aktif siswa. Dalam konteks ini, pelaksana proyek memiliki tanggung jawab penting seperti penyusunan rencana proyek, penentuan sumber dan alat-alat, suplementasi terus menerus dari proyek dengan perubahan inovatif melalui observasi, dan pengendalian kegiatan mahasiswa dengan transfer pengetahuan. Hal yang sama pasti dijelaskan Laffey, dkk (1998) yang menjelaskan bahwa proyek ini berhubungan dengan dunia nyata pelajar, memerlukan penyelidikan kolaboratif dan produksi dari serangkaian artefak proyek, peserta didik mampu memperoleh keterampilan proses seperti perencanaan, pelaksanaan dan pemantauan proyek serta isi pengetahuan. Menurut Bell (2010) Pembelajaran Berbasis Proyek merupakan pendekatan inovatif untuk pembelajaran yang mengajarkan banyak strategi penting untuk sukses di abad kedua puluh satu. Menurut Klein, dkk, (2009) pembelajaran berbasis proyek adalah strategi pembelajaran memberdayakan peserta didik untuk mengejar pengetahuan konten sendiri dan menunjukkan pemahaman baru mereka melalui berbagai mode presentasi. Hal yang senada dijelaskan Han dan Bhattacharya (2001) bahwa Pembelajaran Berbasis Proyek adalah strategi pengajaran dan pembelajaran yang melibatkan peserta didik dalam kegiatan yang kompleks. Sementara, Hadgraft (2012) menjelaskan Pembelajaran Berbasis Proyek (PjBL) yang berpusat pada pembelajaran yang berasal dari proyek rekayasa nyata. Pembelajaran lebih penting daripada solusi proyek. Hadgraft (2012) mengatakan PjBL sebagai metode dalam pembelajaran. Hal yang senada didefinisikan Doppelt (2003) yang didefinisikan bahwa pembelajaran berbasis proyek (PjBL) adalah metode terkenal untuk menyampaikan kompetensi berpikir dan menciptakan lingkungan belajar yang fleksibel. Sebuah murid memajukan rendah mencapai merupakan tantangan on- going untuk sistem pendidikan. Blumenfeld, dkk (1991) menggambarkan dua komponen PjBL: masalah yang harus diselesaikan (atau tugas yang harus diselesaikan), dan produk nyata sebagai hasil dari proyek. Jurnal Pendidikan Fisika p-ISSN2252-732X e-ISSN 2301-7651 R. Malawati dan Sahyar: Peningkatan Keterampilan Proses Sains Mahasiswa Dengan Model Project Based Learning Berbasis Pelatihan Dalam Pembelajaran Fisika ternyata proses pembelajaran dengan model pembelajaran Project Based Learning yang dilaksanakan masih ada kekurangan dalam waktu dan pengembangan materi sebagaimana semestinya. Kegiatan belajar di laboratorium sebagai sarana yang dapat meningkatkan minat dan motivasi belajar, mengembangkan keterampilan kerja ilmiah, membantu memahami konsep, pengembangan kemampuan kognitif, meningkatkan kemampuan berpikir kreatif dan inovatif, dan menumbuhkan sikap ilmiah masih jarang dilakukan, sehingga kemampuan berpikir siswa kurang berkembang. ternyata proses pembelajaran dengan model pembelajaran Project Based Learning yang dilaksanakan masih ada kekurangan dalam waktu dan pengembangan materi sebagaimana semestinya. Kegiatan belajar di laboratorium sebagai sarana yang dapat meningkatkan minat dan motivasi belajar, mengembangkan keterampilan kerja ilmiah, membantu memahami konsep, pengembangan kemampuan kognitif, meningkatkan kemampuan berpikir kreatif dan inovatif, dan menumbuhkan sikap ilmiah masih jarang dilakukan, sehingga kemampuan berpikir siswa kurang berkembang. Heo, et al, (2010) mendefinisikan pembelajaran berbasis proyek (PjBL) adalah model penting untuk mewujudkan perspektif sosial-budaya belajar di lingkungan pendidikan. Menurut Thomas (2000) Pembelajaran Berbasis Proyek adalah model yang menyelenggarakan pembelajaran sekitar proyek, dengan didasarkan pada pertanyaan menantang atau masalah. Dari beberapa penjelasan ini dapat dilihat dengan jelas bahwa model PjBL dirancang sebagai model pembelajaran yang melibatkan siswa dalam investigasi masalah menarik yang berujung pada produk otentik. Proyek-proyek yang membuat peluang kelas belajar yang lebih kuat dapat bervariasi dalam materi pelajaran dan cakupan, dan dapat disampaikan di berbagai tingkatan kelas. Peneliti memilih model pembelajaran Project Based Learning adalah karena model ini merupakan suatu model pembelajaran yang memiliki kelebihan (1) Membuat situasi anak menjadi lebih aktif, bersemangat, bermutu dan berdaya guna. (2) Siswa dapat menguasai bahan pelajaran lebih mendalam dan melatih berfikir ilmiah dalam menghadapi penyelesaian masalah secara kreatif. (3) Dapat menumbuhkan sikap objektif, percaya diri, kesungguhan, keberanian, dan rasa tanggung jawab. Sehingga, tujuan penelitian ini untuk mengetahui bagaimanakah peningkatan Keterampilan Proses Sains Mahasiswa (kognitif, psikomotorik, dan afektif) dengan menggunakan model pembelajaran Project Based Learning berbasis pelatihan. Menurut Thomas (2000) bahwa proyek-proyek PjBL adalah pusat, tidak perifer dengan kurikulum. Kriteria ini memiliki dua akibat wajar. Pertama, menurut fitur ini didefinisikan sebagai proyek kurikulum. Dalam PjBL, proyek ini adalah pusat strategi mengajar, siswa menemukan dan mempelajari konsep disiplin melalui proyek. Ada kasus dimana pekerjaan proyek berikut instruksi tradisional sedemikian rupa bahwa proyek berfungsi untuk memberikan ilustrasi, contoh, praktik tambahan, atau aplikasi praktis untuk materi pelajaran yang diajarkan pada awalnya dengan cara lain. http://jurnal.unimed.ac.id/2012/index.php/jpf PENDAHULUAN Dengan demikian penelitian ini diarahkan untuk meneliti Keterampilan Proses Sains Mahasiswa melalui proses pembelajaran pada mata kuliah Fisika Komputasi. Pencapaian Keterampilan Proses Sains Mahasiswa pada ranah kognitif sudah sangat baik. Akan tetapi, untuk tingkat berpikir secara kognitif masih perlu ditingkatkan untuk menghasilkan lulusan yang kreatif. Pada ranah psikomotor pencapaian kompetensi Mahasiswa masih dinilai kurang karena saat praktikum hanya 30% dari Mahasiswa yang mampu melakukan percobaan dan memahami apa yang sedang dilakukan. Berdasarkan pada pencapaian Keterampilan Proses Sains siswa pada ketiga ranah di atas dapat disimpulkan bahwa terdapat permasalahan dalam proses pembelajaran Fisika di Universitas Negeri Medan. Hasil refleksi tentang proses pembelajaran Fisika yang telah dilakukan disimpulkan bahwa penilaian belum sesuai dengan standar penilaian pada ranah afektif dan psikomotorik khususnya. Merefleksi kegiatan pembelajaran yang telah dilakukan Mihardi (2013) Selain itu, PjBL dapat dilakukan di lingkungan luar sekolah, melatih Mahasiswa dalam bekerja sama, menggunakan berbagai peralatan, teknologi, dan bahan. Hal ini dikonfirmasi ChanLin (2008) yang menyatakan bahwa penting untuk melakukan implementasi PjBL dengan mengintegrasikan teknologi dalam pembelajaran sebagai pengalaman eksplorasi diri. Dalam kasus ini, Doppelt (2003) menunjukkan bahwa teknologi bisa digunakan sebagai cara meningkatkan motivasi Mahasiswa. Kteily dan Hawa (2010) juga menjelaskan bahwa penerapan Mahasiswa PjBL melalui proses panjang dari penyelidikan dalam menghadapi masalah, pertanyaan yang kompleks, dan tantangan. Hal ini dilakukan karena menurut Nurohman (2008) PjBL memiliki tahap pembelajaran yang konsisten Vol.5 No.1 Juni 2016 Vol.5 No.1 Juni 2016 Vol.5 No.1 Juni 2016 http://jurnal.unimed.ac.id/2012/index.php/jpf http://jurnal.unimed.ac.id/2012/index.php/jpf 59 Jurnal Pendidikan Fisika p-ISSN2252-732X e-ISSN 2301-7651 Jurnal Pendidikan Fisika p-ISSN2252-732X e-ISSN 2301-7651 METODE PENELITIAN Jenis penelitian yang dilakukan merupakan penelitian tindakan kelas. Penelitian tindakan kelas merupakan suatu pencermatan terhadap kegiatan belajar berupa sebuah tindakan, yang sengaja dimunculkan dan terjadi dalam sebuah kelas secara bersama (Arikunto, dkk, 2011). Untuk peningkatan Keterampilan Proses Sains Fisika Umum Mahasiswa dinyatakan dalam persentase rerata skor gain yang dinormalisasi (N-gain). N-gain dihitung dengan persamaan: p p Model pembelajaran Project Based Learning berbasis pelatihan memiliki karakteristik pada proses pembelajaran fase I hingga fase III yang menekankan pelatihan kepada Mahasiswa sebelum memulai diskusi. Pelatihan yang diberikan dengan mengarahkan Mahasiswa untuk mengikuti instruksi program yang dibuat sebagai latihan awal sebelum merancang program yang sesuai dengan mater yang diberikan. Pelatihan ini juga bermanfaat sebagai pengenalan awal Mahasiswa dalam membuat rancangan coding baru. Dalam pelatihan yang dilakukan tidak terlepas dari bimbingan Dosen sebagai pengajar dan instruktur yang membimbing dalam pembelajaran. Pada tahap ini Dosen dapat menjadi pemimpin dalam melatih dan membimbing pembelajaran sehingga Mahasiswa dapat fokus dalam mengikuti langkah-langkah kerja yang diberikan sebagai latihan kerja. Dalam hal ini, kepemimpinan yang diberikan melalui kurikulum, perlakuan, integerasi, dan pengaturan kerja dan kinerja untuk mengoptimalkan hasil produk yang disajikan Mahasiswa sebagai hasil dalam pembelajaran. % g = pre maks pre post S S S S   x 100% %g adalah gain yang dinormalisasi, Smaks adalah skor maksimum (ideal) dari tes awal dan tes akhir, Spost adalah skor tes akhir, sedangkan Spre adalah skor tes awal. Tinggi rendahnya N-gain dapat diklasifikasikan sebagai berikut: , g Spre w Tinggi rendahnya N-gain dapat diklasifikasikan sebagai berikut: (1) jika %g > 40%, maka N-gain yang dihasilkan dalam kategori tinggi; g gg (2) jika 21% ≤%g ≤ 40%, maka N-gain yang dihasilkan dalam kategori sedang; (3) jika %g < 21%, maka N-gain yang dihasilkan dalam kategori rendah g p j Menurut Padilla (1990) keterampilan proses sains dapat dibagi menjadi Basic Science Process Skill dan Integrated Science Process Skill. Pada Basic Science Process Skill memiliki indikator sebagai berikut: Observing, Inferring, Measuring, Communicating, Classifying, dan Predicting. Indikator yang dimiliki Basic Science Process Skill merupakan kemampuan dasar yang harus dimiliki seorang saintis. Kemampuan ini dapat ditunjukkan pada kegiatan eksperimen. Integrated Science Skill merupakan tahap peningkatan dalam kemampuan proses sains. Hal ini ditunjukkan dengan indikator yang dimiliki pada penilaian secara Integrated berbeda dengan Basic. Pada Integrated indikator yang dimiliki adalah: Controlling variables, Defining, operationally, Formulating hypotheses, Interpreting data, Experimenting, dan Formulating models. Dengan indikator ini Siswa lebih dapat berkembang menjadi saintis yang lebih kompeten. METODE PENELITIAN Hal ini ditunjukkan oleh penelitian Espinosa, dkk (2013) yang menyatakan bahwa dengan pembelajaran yang diarahkan ke Integrated Science Process Skill Siswa lebih dapat terarah dan menghemat waktu pembelajaran dalam pemahaman materi sehingga hasil yang diperoleh lebih optimal dalam kegiatan investigasi yang dilakukan. Jurnal Pendidikan Fisika p-ISSN2252-732X e-ISSN 2301-7651 Sedangkan menurut Capraro dan Slough (2009) mengatakan bahwa PjBL untuk tujuan disini adalah penggunaan sebuah proyek yang sering menyebabkan munculnya berbagai hasil pembelajaran. Hal lain dijelaskan Thomas (2000) bahwa ada sejumlah cara dari desain PjBL diadaptasi dengan variabel karakteristik Mahasiswa. Ada sejumlah variabel karakteristik siswa yang mungkin Vol.5 No.1 Juni 2016 Juni 2016 Vol.5 No.1 http://jurnal.unimed.ac.id/2012/index.php/jpf 60 Jurnal Pendidikan Fisika p-ISSN2252-732X e-ISSN 2301-7651 Jurnal Pendidikan Fisika p-ISSN2252-732X e-ISSN 2301-7651 R. Malawati dan Sahyar: Peningkatan Keterampilan Proses Sains Mahasiswa Dengan Model Project Based Learning Berbasis Pelatihan Dalam Pembelajaran Fisika diselidiki dalam konteks Pembelajaran Berbasis Proyek di lima perilaku berpikir kritis (sintesis, peramalan, produksi, evaluasi, dan refleksi) dan lima perilaku partisipasi sosial (bekerja sama, memulai, mengelola, kesadaran antar kelompok, dan antar kelompok memulai). SIMPULAN Berdasarkan hasil analisis data penelitian dapat disimpulkan: (1) Ada peningkatan Keterampilan Proses Sains Mahasiswa pada ranah kognitif dengan menggunakan model pembelajaran Project Based Learning berbasis pelatihan. (2) Ada peningkatan Keterampilan Proses Sains Mahasiswa pada ranah psikomotor dengan menggunakan model pembelajaran Project Based Learning berbasis pelatihan. Espinosa, A. A., Monterola, S. L. C., dan Punzalan, A. E. Career-Oriented Performance Tasks in Chemistry: Effects on Students’ Integrated Science Process Skills. Cypriot Journal of Educational Sciences Volume 8, Issue 2 (2013) 211-226. http://www.awer-center.org/cjes/ Jurnal Pendidikan Fisika p-ISSN2252-732X e-ISSN 2301-7651 Perbandingan Tingkat Ketuntasan Psikomotorik ( = Tuntas; = Tidak Tuntas) ChanLin, L. J. 2008. Technology Integration Applied to Project-Based Learning In Science. Department of Library & Information Science, Fu-Jen Catholic University, Hsin-Chuang, Taiwan. Innovations in Education And Teaching International. Vol. 45, no. 1, February 2008, 55– 65. Email: lins1005@mails.fju.edu.tw. ISSN 1470-3297 © 2008 Taylor & Francis. Http://www.informaworld.com. Gambar 2. Perbandingan Tingkat Ketuntasan Psikomotorik ( = Tuntas; = Tidak Tuntas) Doppelt, Y. 2003. Implementation and Assessment of Project-Based Learning In A Flexible Environment. International Journal of Technology and Design Education 13, 255–272, 2003. Science & Technology Youth Center, Technion, Israel Institute of Technology, Israel. E-mail: yaron@noar.technion.ac.il ©2003 Kluwer Academic Publishers. Printed In The Netherlands. Jurnal Pendidikan Fisika p-ISSN2252-732X e-ISSN 2301-7651 Jurnal Pendidikan Fisika p-ISSN2252-732X e-ISSN 2301-7651 R. Malawati dan Sahyar: Peningkatan Keterampilan Proses Sains Mahasiswa Dengan Model Project Based Learning Berbasis Pelatihan Dalam Pembelajaran Fisika kelompok pada siklus I. Dengan adanya kolaborasi Mahaiswa dapat terlatih untuk memodifikasi dan memperbaiki kekurangan yang ada. Selain itu, Mahasiswa melatih untuk dapat menerima saran dan mempertimbangkannya. Dengan demikian, Mahasiswa merasa tidak ada persaingan antar kelompok selama pembelajaran melainkan kerjasama antar kelompok yang berbeda tanpa harus menilai buruk hasil kelompok lain. Hal ini sesuai dengan penelitian Ravitz, dkk (2004) yang menjelaskan bahwa PjBL digunakan sebagai model pembelajaran yang dapat mengarahkan kelompok untuk berkolaborasi dan berbagi informasi penting yang dibutuhkan dalam pembelajaran dari segala sumber belajar. Hal ini juga dipertegas oleh Bell (2010) yang menjelaskan bahwa dalam pembelajaran dan aktifitas sangat dibutuhkan kolaborasi dalam penelitian proyek yang sedang dicapai. Baker, E., Trygg, B., Otto, P., Tudor, M. & Ferguson, L. 2011. Project-Based Learning Model Relevant Learning For The 21st Century. Pacific Education Institute, www.pacificeducationinstitute.org. Barron, B. & Hammond, L. D. 2007. Teaching For Meaningful Learning: A Review Of Research On Inquiry-Based And Cooperative Learning Implementing Project-Based Learning Districtwide. Stanford University. Adapted From Edutopia Article, “River Journeys And Life Without Bathing: Immersive Education,” By Laura Scholes (May 15, 2007). Bell, S. 2010. Project-Based Learning For The 21st Century: Skills For The Future. The Clearing House, 83: 39–43, 2010. Copyright © Taylor & Francis Group, Llc. Gambar 1. Perbandingan Tingkat Ketuntasan Kognitif ( = Tuntas; = Tidak Tuntas) Gambar 2. Perbandingan Tingkat Ketuntasan Psikomotorik ( = Tuntas; = Tidak Tuntas) Blumenfeld, P. C., Soloway, E., Marx, R. W., Krajcik, J. S., Guzdial, M. & Palincsar, A. 1991. Motivating Project-Based Learning: Sustaining The Doing, Supporting The Learning. Educational Psychologist, 26(3&4), 369-398. Gambar 1. Perbandingan Tingkat Ketuntasan Kognitif ( = Tuntas; = Tidak Tuntas) Gambar 1. Perbandingan Tingkat Ketuntasan Kognitif ( = Tuntas; = Tidak Tuntas) Cakmakci, G. & Tasar, M. F. 2010. Contemporary Science Education Research: Learning And Assessment A Collection of Papers Presented At ESERA 2009 Conference. ISBN 9786053640332 © Copyright ESERA, 2010. Capraro, R. M. & Slough, S. W. 2009. Project-Based Learning An Integrated Science, Technology, Engineering, And Mathematics (STEM) Approach. Published by: Sense Publishers, Rotterdam, The Netherlands. Http://www.sensepublishers.com. All Rights Reserved © 2009 Sense Publishers. Gambar 1. Perbandingan Tingkat Ketuntasan Kognitif ( = Tuntas; = Tidak Tuntas) Gambar 2. Perbandingan Tingkat Ketuntasan Psikomotorik ( = Tuntas; = Tidak Tuntas) Gambar 2. HASIL DAN PEMBAHASAN Berdasarkan hasil postes kemampuan kognitif Mahasiswa pada siklus I dan siklus II yang meningkat. Peningkatan nilai rata-rata dan ketuntasan belajar ranah kognitif dapat dilihat pada Gambar 1. Kenaikan peningkatan kognitif yang drastis ini disebabkan adanya kegiatan pembelajaran yang dapat mengarahkan Mahasiswa menjadi aktif baik dalam diskusi maupun dalam berkolaborasi kepada kelompok lain. Dengan berkolaborasi Mahasiswa mendapat pengetahuan baru dan ide baru yang muncul dalam memperbaiki produk yang dihasilkan. Hal ini dapat memicu terjadinya pengembangan produk yang dihasilkan dalam pembelajaran sehingga tidak hanya menjadikan Mahasiswa lebih kreatif tetapi juga dapat melatih inovasi mereka. Hal yang sama ditunjukkan dalam penelitian Mihardi (2013) dalam hasil peningkatan kemampuan berpikir yang dimiliki Mahasiswa dalam mengikuti pembelajaran dengan Model PjBL. Hal ini juga dipertegas oleh penelitian ChanLin (2008) dan Bell (2010) yang menjelaskan mengenai keberhasilan PjBL dalam membentuk pengetahuan pada proses pembelajaran. Dari data analisis lembar hasil observasi psikomotor pada siklus I dan siklus II pada tingkat ketuntasan belajar ranah psikomotor dapat dilihat pada Gambar 2 berikut. Kenaikan yang drastis juga ditunjukkan dari penilaian psikomotorik yang difokuskan pada kemampuan sains dalam pemrograman komputer Mahasiswa yang menunjukkan tingkat ketidaktuntasan turun lebih dari 50%. Hal ini dikarenakan meningkatnya aktifitas Mahasiswa saat berkolaborasi pada siklus II dibandingkan dengan hanya melakukan presentasi antar Selain itu, dengan Integrated Science Process Skill Siswa dapat lebih berkolaborasi dengan team lain dan dapat menjadi pembelajaran baru yang lebih kompleks sehingga dapat menerapkan dalam kehidupan sebagai relevansi sains. Hal ini ditegaskan oleh Mei, dkk (2007) yang menyatakan dengan Science Process Skill siswa lebih dapat terarah kepada kegiatan kolaborasi dan dapat membantu mereka untuk bebas menentukan investigasi serta penulisan laporan dalam meningkatkan refleksi pada pembelajaran serta kedalaman materi yang dipelajari. Vol.5 No.1 Juni 2016 Juni 2016 Vol.5 No.1 http://jurnal.unimed.ac.id/2012/index.php/jpf 61 Jurnal Pendidikan Fisika p-ISSN2252-732X e-ISSN 2301-7651 Jurnal Pendidikan Fisika p-ISSN2252-732X e-ISSN 2301-7651 R. Malawati dan Sahyar: Peningkatan Keterampilan Proses Sains Mahasiswa Dengan Model Project Based Learning Berbasis Pelatihan Dalam Pembelajaran Fisika Through Science Alive! Programme. Proceedings of the Redesigning Pedagogy: Culture, Knowledge and Understanding Conference, Singapore, May 2007. Hadgraft, R. 2012. Project handbook 2012: Project based learning. Accessed in http://www.google.com/project+based+learnin g.pdf. Downloaded in PDF Files at http://pbworks.com. Mihardi, S. 2013. Effect of Project Based Learning Model With KWL (Know-Want-Learn) Worksheet on Creative Thinking in Solved Physics Problems. Thesis in State University of Medan (Unimed). Indonesia, Medan: Universitas Negeri Medan. Han, S. & Bhattacharya, K. 2001. Constructionism, Learning By Design, And Project-Based Learning. In M. Orey (ed.), Emerging Perspectives On Learning, Teaching, And Technology. Ebook Learning, Teaching & Technology, Michael Orey, Editor. Available Accessed In Website: http://www.coe.uga.edu/epltt/learningbydesign. htm Nurohman, S. 2008. Pendekatan Project Based Learning Sebagai Upaya Internalisasi Scientific Method Bagi Mahasiswa Calon Guru Fisika. Yogyakarta: Universitas Negeri Yogyakarta. Heo, H., Lim, K. Y. & Kim, Y. 2010. Exploratory Study on The Patterns of Online Interaction And Knowledge Co-Construction In Project-Based Learning. Computers & Education Journal. Homepage: www.elsevier.com/locate/compedu Padilla, M. J. 1990. The Science Process Skills. Research Matters - to the Science Teacher No. 9004, March 1 (1990). http://www.educ.sfu.ca/narstsite/publications/re search/skill.htm Ravitz, J., Mergendoller, J., Markham, T., Thorsen, C., Rice, K., Snelson, C. & Reberry, S. 2004. Online Professional Development For Project Based Learning: Pathways To Systematic Improvement. Running Head: Online Professional Development For Pbl Paper Presented At Meetings Of The Association For Educational Communications And Technology. October 21, 2004. Chicago, il. Buck Institute For Education And Boise State University. Holubova, R. 2008. Effective Teaching Methods—Project- Based Learning In Physics. Faculty of Science, Palacky University Olomouc, Svobody 2677146, Czech Republic. Dec. 2008, volume 5, no.12 (serial no.49). Us-China Education Review, ISSN 1548-6613, USA. Hong, L., Yam, S. & Rossini, P. 2010. Implementing A Project-Based Learning Approach In An Introductory Property Course. 16th Pacific Rim Real Estate Society Conference Wellington, New Zealand, January 2010. University of South Australia. Roessingh, H. & Chambers, W. 2011. Project-Based Learning and Pedagogy In Teacher Preparation: Staking Out The Theoretical Mid-Ground. International Journal Of Teaching And Learning In Higher Education, 2011, Volume 23, Number 1, 60-71, ISSN 1812-9129. 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Promoting Science Process Skills and The Relevance of Science http://jurnal.unimed.ac.id/2012/index.php/jpf Vol.5 No.1 Juni 2016 Vol.5 No.1 Juni 2016 63
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The Planar Ising Model and Total Positivity
Journal of statistical physics
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J Stat Phys (2017) 166:72–89 DOI 10.1007/s10955-016-1690-x J Stat Phys (2017) 166:72–89 DOI 10.1007/s10955-016-1690-x The Planar Ising Model and Total Positivity Marcin Lis1 Received: 30 June 2016 / Accepted: 2 December 2016 / Published online: 29 December 2016 © The Author(s) 2016. This article is published with open access at Springerlink.com Abstract A matrix is called totally positive (resp. totally nonnegative) if all its minors are positive (resp. nonnegative). Consider the Ising model with free boundary conditions and no external field on a planar graph G. Let a1, . . . , ak, bk, . . . , b1 be vertices placed in a counterclockwise order on the outer face of G. We show that the k × k matrix of the two- point spin correlation functions Mi, j = ⟨σai σb j ⟩ is totally nonnegative. Moreover, det M > 0 if and only if there exist k pairwise vertex- disjoint paths that connect ai with bi. We also compute the scaling limit at criticality of the probability that there are k parallel and disjoint connections between ai and bi in the double random current model. Our results are based on a new distributional relation between double random currents and random alternating flows of Talaska [37]. is totally nonnegative. Moreover, det M > 0 if and only if there exist k pairwise vertex- disjoint paths that connect ai with bi. We also compute the scaling limit at criticality of the probability that there are k parallel and disjoint connections between ai and bi in the double random current model. Our results are based on a new distributional relation between double random currents and random alternating flows of Talaska [37]. Keywords Ising model · Total positivity · Random currents · Alternating flows Mathematics Subject Classification 82B20 · 60C05 · 05C50 Mathematics Subject Classification 82B20 · 60C05 · 05C50 B Marcin Lis m.lis@statslab.cam.ac.uk 1 Statistical Laboratory, Centre for Mathematical Sciences, Cambridge University, Wilberforce Road, Cambridge CB3 0WB, UK B Marcin Lis m.lis@statslab.cam.ac.uk 1 Statistical Laboratory, Centre for Mathematical Sciences, Cambridge University, Wilberforce Roa Cambridge CB3 0WB, UK 1 Introduction The Ising model was introduced by Lenz with the intention to describe the behaviour of ferromagnets, and was first solved in dimension 1 by Ising [19]. Peierls later showed that the model does undergo a phase transition in dimensions 2 or more [32], and it has been since the subject of extensive study both in the physics and mathematics literature. Notable results in the planar case include the exact solution obtained by Onsager [31] and Yang [38], and the 123 The Planar Ising Model and Total Positivity 73 recent breakthrough of Smirnov et al. showing conformal invariance in the critical scaling limit [7,8,17,18,36]. recent breakthrough of Smirnov et al. showing conformal invariance in the critical scaling limit [7,8,17,18,36]. A seemingly unrelated notion is that of totally positive matrices characterized by having all their minors positive. They appear in various areas of mathematics and physics including oscillations in mechanical systems [12,13] (which was the original motivation to study them), stochastic processes and statistical mechanics [9,11,22], quantum groups [28–30], and alge- braic geometry [33,34]. Some of their fundamental properties include the simplicity and positivity of the spectrum obtained by Gantmacher and Krein [12], and the variation dimin- ishing property discovered by Shoenberg [35] which says that the number of sign changes in a vector does not increase after multiplying by a totally positive matrix. In this article we identify total positivity in the planar Ising model. Our results on the Ising boundary two-point correlation functions are analogous to the results of Fomin [11] on the walk matrices of random walks on planar graphs. Indeed, the walk matrix can be interpreted as the two-point correlation function matrix of another very well known ferromagnetic spin system—the discrete Gaussian free field. Furthermore, Fomin provides an interpretation of the determinants of walk matrices in terms of probabilities of non-intersection events involving loop-erased walks of Lawler [25]. In our setting the relevant events concern the double random current model. The random current model is derived from the power series expansion of the Ising model partition function. It was introduced by Griffiths, Hurst and Sherman [15], and was later used by Aizenman et al. [1–3] to obtain a detailed description of the behaviour of the Ising model on Zd. The model has recently received revived attention from the mathematical community. 2 Main Results Let G = (V, E) be a finite connected planar graph. For positive coupling constants J : E → (0, ∞), we consider the Ising model on G with free boundary conditions and no external field, i.e., a probability measure on the space of spin configurations {−1, 1}V given by PIsg(σ) = 1 ZIsg  {u,v}∈E exp(J{u,v}σuσv), σ ∈{−1, 1}V , where where ZIsg =  σ∈{−1,1}V  {u,v}∈E exp(J{u,v}σuσv) is the partition function. Since the coupling constants are positive, the model is ferromagnetic, i.e., it assigns larger probability to configurations where more pairs of adjacent spins assume the same value. For u, v ∈V , the two-point spin correlation function is defined to be the expectation is the partition function. Since the coupling constants are positive, the model is ferromagnetic, i.e., it assigns larger probability to configurations where more pairs of adjacent spins assume the same value. For u, v ∈V , the two-point spin correlation function is defined to be the expectation ⟨σuσv⟩=  σ∈{−1,1}V σuσvPIsg(σ). A classical argument says that in a ferromagnetic spin system, all two-point functions are positive (see Lemma 5.1). A classical argument says that in a ferromagnetic spin system, all two-point functions are positive (see Lemma 5.1). Let W = {w1, w2, . . . , wn} be the set of vertices on the outer face of G listed counterclock- wise, and let W◦∪W• = W be a partition of W into two sets (one of them possibly empty). We will refer to W as the boundary vertices and for A ⊆W, we will write A◦= A ∩W◦ and A• = A ∩W•. For two natural numbers i, j, we define Ii, j to be the set of numbers strictly between i and j, e.g. I1,1 = ∅, and I1,3 = I3,1 = {2}. The following result yields positivity of determinants of matrices whose entries are, up to a sign, the boundary two-point spin correlation functions. Theorem 2.1 Let A = {wl1, wl2, . . . , wlk} ⊆W with l1 < l2 < · · · < lk. Consider the k ×n matrix N A i, j = (−1)s(i, j)⟨σwli σw j ⟩, where s(i, j) = |Ili, j ∩{l1, . . . ,lk}| + 1{w j∈A◦, j<li} + 1{w j∈A•, j>li}. 1 Introduction The interpretation of the determinants of the two-point functions in terms of random current probabilities is achieved by a new distributional relation between double random currents and random alternating flows. Postnikov [33]. The interpretation of the determinants of the two-point functions in terms of random current probabilities is achieved by a new distributional relation between double random currents and random alternating flows. This article is organized as follows: The next section introduces basic notation and presents the main results. Section 3 gives a distributional identity for double random currents, and Sect. 4 provides a closely related identity for random alternating flows. The relationship between these two, together with the total positivity of alternating flows, is the basis for our main results, whose proofs are given in Sect. 5. 1 Introduction The most notable example is the result of Aizenman, Duminil-Copin and Sidoravicius [4] who studied percolation properties of double random currents to show continuity of magneti- zation for a wide range of Ising models on Zd (including the d = 3 case). Their argument was later generalized by Björnberg to the setting of quantum Ising models [5]. Also, a new dis- tributional relation between random currents, Bernoulli percolation and the FK-Ising model was discovered by Lupu and Werner [27]. One of the main tools used to study the random current model is the switching lemma [15], and our result may be thought of as its planar generalization (see Example 2.2). For a recent overview of the applications of the random current model, see [10]. Since the work of Groeneveld, Boel and Kasteleyn [16], the boundary Ising correlation functions have been known to satisfy exact Pfaffian relations. We provide an alternative proof of this fact using the expansion of the Pfaffian as a sum of determinants (see Lemma 5.6). Our results are hence a refinement of those in [16] in that we relate each of these determinants to an explicit event in the double random current model. A special property of the (dis-)connection events appearing in our results is that even though they are non-local, i.e., they depend on a macroscopic (in terms of the size of the graph) number of edge variables, their probabilities are simple functions of the boundary measurements which are defined as expectations of local variables. This in particular implies that these probabilities do not depend on the structure of the graph as long as the boundary measurements are preserved. This phenomenon also exists in uniform groves and double dimers as was discovered by Kenyon and Wilson [23,24]. Moreover, the authors proved that the probability of seeing any type of partition of the boundary vertices induced by cluster connectivities in these models is a homogenous multi-linear polynomial in the boundary measurements. We arrive at the total positivity of the Ising boundary two-point functions by representing them in terms of alternating flows of Talaska which satisfy total positivity [37]. They, in turn, appear in the combinatorial study of the totally nonnegative Grassmannian initiated by 123 74 M. Lis M. Lis Postnikov [33]. 2 Main Results Then, for any B ⊂W with |B| = k, Then, for any B ⊂W with |B| = k, det N A,B ≥0, 123 75 The Planar Ising Model and Total Positivity where N A,B is the k×k submatrix of N A with columns indexed by B. Moreover, det N A,B > 0 if and only if there exist k pairwise edge-disjoint (possibly empty) directed paths in G such that each of them starts in A and ends in B, and if two paths meet at a vertex, then their edges alternate in orientation around it (edge oriented towards or away from the vertex). Here, we assume that an empty path joining v ∈A◦∩B◦(resp. v ∈A• ∩B•) with itself is represented by a counterclockwise loop (resp. clockwise loop) attached to v within the outer face. where N A,B is the k×k submatrix of N A with columns indexed by B. Moreover, det N A,B > 0 if and only if there exist k pairwise edge-disjoint (possibly empty) directed paths in G such that each of them starts in A and ends in B, and if two paths meet at a vertex, then their edges alternate in orientation around it (edge oriented towards or away from the vertex). Here, we assume that an empty path joining v ∈A◦∩B◦(resp. v ∈A• ∩B•) with itself is represented by a counterclockwise loop (resp. clockwise loop) attached to v within the outer face. We note that the choice of W◦and W• determines both the sign factors appearing in the definition of N A, and the interpretation of the associated determinants in terms of alternating flows (see Lemma 5.5). This is inherited from the results of [37] through the construction of Sect. 4 (see Fig. 2). Example 2.1 For any distinct a, b, c ∈W, we have 1 + ⟨σaσb⟩> ⟨σaσc⟩+ ⟨σbσc⟩, and 1 + ⟨σaσb⟩2 > ⟨σaσc⟩2 + ⟨σbσc⟩2. Indeed, assume that a, b, c are ordered counterclockwise, a, b ∈W◦, c ∈W•, and take A, B = {a, b, c}. We have Indeed, assume that a, b, c are ordered counterclockwise, a, b ∈W◦, c ∈W•, and take A, B = {a, b, c}. We have N A,B = ⎡ ⎣ 1 ⟨σaσb⟩ ⟨σaσc⟩ −⟨σaσb⟩1 −⟨σbσc⟩ ⟨σaσc⟩ −⟨σbσc⟩1 ⎤ ⎦, and by Theorem 2.1, det N A,B = 1 + ⟨σaσb⟩2 −⟨σaσc⟩2 −⟨σbσc⟩2 > 0, which gives the second inequality. 2 Main Results We define xing(π) to be the number of crossings between the line segments. It is easy to see that when A, B are as in Corollary 2.2, then xing(π) is the number of inversions of π treated as a permutation of the index set {1, . . . , k}. Hence, by the definition of the determinant, det M A,B =  π:A→B (−1)xing(π)  {a,b}∈π ⟨σaσb⟩. (2.1) (2.1) Moreover, the choice of signs in the definition of the matrix N A yields an analogous expansion of the determinant for general choices of A and B: Moreover, the choice of signs in the definition of the matrix N A yields an analogous expansion of the determinant for general choices of A and B: det N A,B =  π:A→B (−1)xing(π)  {a,b}∈π ⟨σaσb⟩. (2.2) (2.2) (2.2) A verification of (2.2) can be found in [33, Proposition 5.2] or [37, Proposition 2.12]. A verification of (2.2) can be found in [33, Proposition 5.2] or [37, Proposition 2.12]. Our second main result provides an interpretation of these determinants in terms of random currents. A current on G is a function n : E →{0, 1, 2, . . . }. By ∂n we denote the set of sources of n, i.e., vertices v such that  u: {u,v}∈E n{u,v} is odd. For A ⊆V , we define A = {n | ∂n = A}. The weight of a current n is defined by w(n) =  e∈E (Je)ne ne! , (2.3) (2.3) and the random current probability measure with boundary conditions A is given by and the random current probability measure with boundary conditions A is given by PA curr(n) = w(n) Z Acurr , n ∈A, PA curr(n) = w(n) Z Acurr , n ∈A, where Z A curr =  n∈A w(n) is the partition function. where Z A curr =  n∈A w(n) is the partition function. where Z A curr =  n∈A w(n) is the partition function. A Note that if n1 ∈A and n2 ∈∅, then n1 + n2 ∈A. The double random current probability measure PA d-curr with boundary conditions A is defined to be the measure of the sum of two independent random currents with A and ∅boundary conditions respectively: PA d-curr(n) = PA curr ⊗P∅ curr({(n1, n2) ∈A × ∅| n1 + n2 = n}), n ∈A. 2 Main Results The inequality is strict since we can take empty paths connecting the vertices with themselves. To obtain the first inequality, we use the second one together with the Griffiths inequality ⟨σaσb⟩≥⟨σaσc⟩⟨σcσb⟩[14]. which gives the second inequality. The inequality is strict since we can take empty paths connecting the vertices with themselves. To obtain the first inequality, we use the second one together with the Griffiths inequality ⟨σaσb⟩≥⟨σaσc⟩⟨σcσb⟩[14]. In the special case when A and B form disjoint contiguous sets of boundary vertices, we obtain the following total positivity property of the boundary two-point spin correlation functions with no additional signs. Corollary 2.2 (Total positivity) Let A = {a1, a2, . . . , ak}, B = {b1, b2, . . . bk} be contigu- ous sets of boundary vertices, i.e., such that a1, . . . , ak, bk, . . . b1 is a counterclockwise order on A ∪B. Then, the k × k matrix M A,B i, j = ⟨σai σb j ⟩ M A,B i, j = ⟨σai σb j ⟩ is totally nonnegative. Moreover, M A,B is totally positive if and only if for any A′ ⊂A and B′ ⊂B such that |A′| = |B′| = l, there exist l pairwise vertex-disjoint paths that connect A′ and B′. Remark 1 One can prove that if A and B are as above, then det M A,B = det N A,B by showing that the additional signs in N A,B make the same sign contribution to the determinant as the reverse order on columns in M A,B. This is evident in the following expansion of these determinants. Let A, B be any subsets of W of equal cardinality, and let π : A →B be a bijection. One can interpret π as a pairing of the disjoint union A ⊔B, i.e., a partition of A ⊔B into pairs {a, π(a)}. One can then think of a diagrammatic representation of π where points representing A and B are placed in the corresponding order on the boundary of a disk, and straight line segments connect the points according to π. Here, for each v ∈A◦∩B◦ 123 76 M. Lis (resp. each v ∈A• ∩B•), two copies of v are placed on the circle, and the one corresponding to a point in A comes immediately after (resp. before) the one corresponding to a point in B in the counterclockwise order. 2 Main Results For u, v ∈V and a current n, we will write u n↔v if u and v are connected in G by a path of edges with non-zero values of n, and we will write u n↮v otherwise. Let A and B be contiguous sets of boundary vertices as in Corollary 2.2. We define the event of having parallel and disjoint connections between A and B (see Fig. 1) by PA,B = {n ∈A∪B | ai n↔bi for all i, ai n↮b j for all i ̸= j}. PA,B = {n ∈A∪B | ai n↔bi for all i, ai n↮b j for all i ̸= j}. As a consequence of the proof of Theorem 2.1 and Corollary 2.2, we can compute the probability of PA,B under the double random current measure: Theorem 2.3 Let A and B be contiguous sets of boundary vertices as in Corollary 2.2, and let v1, v2, . . . , v2k be a counterclockwise order on A ∪B. Consider the 2k ×2k skew-symmetric matrix satisfying K A∪B i, j = ⟨σvi σv j ⟩ for i < j. 123 77 The Planar Ising Model and Total Positivity Fig. 1 The solid edges represent non-zero values of a current n ∈PA,B with A = {a1, a2, a3} and B = {b1, b2, b3}. The black edges represent odd values and the grey edges represent even values a1 a2 a3 b3 b2 b1 Then, PA∪B d-curr(PA,B) = det M A,B Pf K A∪B , where Pf denotes the Pfaffian of a skew-symmetric matrix. Fig. 1 The solid edges represent non-zero values of a current n ∈PA,B with A = {a1, a2, a3} and B = {b1, b2, b3}. The black edges represent odd values and the grey edges represent even values Fig. 1 The solid edges represent non-zero values of a current n ∈PA,B with A = {a1, a2, a3} and B = {b1, b2, b3}. The black edges represent odd values and the grey edges represent even values b2 b1 Then, where Pf denotes the Pfaffian of a skew-symmetric matrix. where Pf denotes the Pfaffian of a skew-symmetric matrix. Example 2.2 We consider the simplest non-trivial case of Theorem 2.3 when k = 2. To this end, let S = {a, b, c, d} ⊆W be ordered counterclockwise, and let X = {n ∈S | a n↔b n↔c n↔d}. 2 Main Results Note that by planarity of G, and the fact that each connected component of n contains an even number of vertices in ∂n, the complement of X in S is P{a,b},{c,d} ∪ P{a,d},{b,c}. We have Pf K S = ⟨σaσb⟩⟨σcσd⟩+ ⟨σaσd⟩⟨σbσc⟩−⟨σaσc⟩⟨σbσd⟩, and hence by Theorem 2.3, PS d-curr(P{a,b},{c,d}) = ⟨σaσd⟩⟨σbσc⟩−⟨σaσc⟩⟨σbσd⟩ Pf K S , PS d-curr(P{a,d},{b,c}) = ⟨σaσb⟩⟨σcσd⟩−⟨σaσc⟩⟨σbσd⟩ Pf K S , PS d-curr(X) = ⟨σaσc⟩⟨σbσd⟩ Pf K S . We note that these formulas follow from the switching lemma of Griffiths, Hurst and Sher- man [15] (see e.g. [4] for a modern treatment of the lemma). The statement of Theorem 2.3 for k > 2 does not however seem to be a direct consequence of this lemma. Remark 2 For future reference, recall that the Pfaffian of the skew-symmetric matrix K A∪B is the square root of its determinant, and it is a well known fact that it can be written as Pf K A∪B =  π: pairing of A∪B (−1)xing(π)  {u,v}∈π ⟨σuσv⟩, (2.4 Pf K A∪B =  π: pairing of A∪B (−1)xing(π)  {u,v}∈π ⟨σuσv⟩, (2.4) (2.4) where the sum is over all pairings of A ∪B, and where xing is defined as in (2.2). where the sum is over all pairings of A ∪B, and where xing is defined as in (2.2). Our last main result concerns the scaling limit at criticality of the parallel connection probability in double random currents. The proof relies on the computation of Hongler [17] of the scaling limit of the boundary two-point functions themselves. Analogous and more general results were obtained by Kenyon and Wilson [24] for the double dimer model, multichordal loop erased walk, and grove Peano curves. Theorem 2.4 Let D be a bounded finitely-connected domain in the complex plane with a piecewise C1 boundary. Let ∂sD be the straight part of the boundary, i.e., the part composed 123 123 M. Lis 78 of intervals parallel either to the real or imaginary axis. Let A = {a1, a2, . . . , ak} ⊂∂sD and B = {b1, b2, . . . bk} ⊂∂sD be such that a1, . . . , ak, bk, . . . , b1 is a counterclockwise ordering of A ∪B around the outer boundary of D. For ϵ > 0, let Dϵ be the maximal connected component of the rescaled square nearest-neighbor lattice ϵZ2 contained in D. 2 Main Results Consider the double random current measure PAϵ∪Bϵ d-curr on Dϵ with homogeneous critical coupling constants Je = 1 2 log( √ 2 + 1), where Aϵ and Bϵ are sets of vertices on the outer face of Dϵ approximating A and B. Then, the following limit exists pA,B(D) := lim ϵ→0 PAϵ∪Bϵ d-curr (PAϵ,Bϵ). Moreover, if H is the upper half-plane and ϕ : D →H is a conformal equivalence between D and H, then pA,B(D) = det ˜M A,B Pf ˜K A∪B , where ˜M A,B is a k × k matrix, and ˜K A∪B is a 2k × 2k skew-symmetric matrix satisfying ˜M A,B i, j = 1 |ϕ(ai) −ϕ(b j)|, and ˜K A∪B i, j = 1 |ϕ(vi) −ϕ(v j)| for i < j, where v1, v2, . . . , v2k is a counterclockwise order on A ∪B. 3 Random Currents In this section G = (V, E) is a finite (not necessarily planar) graph. Note that the definitions of the Ising model and the random current model generalize verbatim to arbitrary finite graphs. g p Let A ⊆V be a set of even cardinality (possibly empty). With a current n ∈A, we associate a pair of sets of edges ω(n) = (ω1(n), ω2(n)), where ω1(n) = {e ∈E | ne is odd}, and ω2(n) = {e ∈E | ne is even, ne ̸= 0}. Let EA be the collection of sets of edges for which A is the set of vertices with odd degree in the induced subgraph. One can see that A := {ω(n) | n ∈A} = {(ω1, ω2) | ω1 ∈EA, ω2 ⊆E \ ω1}. A := {ω(n) | n ∈A} = {(ω1, ω2) | ω1 ∈EA, ω2 ⊆E \ ω1}. We will often identify ω ∈ A with the set ω1 ∪ω2 ⊆E. Note that for u, v ∈V , u n↔v if and only if u and v belong to the same connected component of ω(n). A Let xe = tanh Je and ye = (cosh Je)−1. The next result describes the measure P A curr on A induced from the random current measure. Lemma 3.1 The probability of ω ∈ A induced from the random current measure is given by P A curr(ω) = 1 ¯Z Acurr  e∈ω1 xe  e∈ω2 (1 −ye)  e∈E\ω ye, where ¯Z A curr = Z A curr e∈E ye. where ¯Z A curr = Z A curr e∈E ye. 123 79 The Planar Ising Model and Total Positivity P A curr(ω) =  n∈A: ω(n)=ω PA curr(n) = 1 Z Acurr  n∈A: ω(n)=ω  e∈E (Je)ne ne! = 1 Z Acurr  e∈ω1 ∞  k=1 (Je)2k−1 (2k −1)!  e∈ω2 ∞  k=1 (Je)2k (2k)! = 1 Z Acurr  e∈ω1 sinh Je  e∈ω2 (cosh Je −1) = 1 ¯Z Acurr  e∈ω1 xe  e∈ω2 (1 −ye)  e∈E\ω ye. ⊓⊔ ⊓⊔ ⊓⊔ For ω ⊆E, let E∅(ω) = E∅∩{ω′ | ω′ ⊆ω}. The main result of this section gives a formula for the probability measure P A d-curr on A induced from the double random current measure. 3 Random Currents Theorem 3.2 The probability of ω ∈ A induced from the double random current measure is given by is given by P A d-curr(ω) = 1 ¯Z A d-curr |E∅(ω)|  e∈ω1 xe  e∈ω2 x2 e  e∈E\ω (1 −x2 e ), where ¯Z A d-curr = Z A curr Z∅ curr e∈E(1 −x2 e ). where ¯Z A d-curr = Z A curr Z∅ curr e∈E(1 −x2 e ). Proof Let n1 ∈A, n2 ∈∅, n = n1 + n2, and ω1 = ω(n1), ω2 = ω(n2), ω = ω(n). Note that e ∈ω1 if and only if e ∈ω1 1△ω2 1. Therefore, by Lemma 3.1, each e ∈ω1 carries a multiplicative weight of xe[(1 −ye) + ye] = xe in the double random current measure. Similarly, e ∈ω2 if and only if e ∈ω1 1 ∩ω2 1 or e ∈(ω1 2 ∪ω2 2) \ (ω1 1 ∪ω2 1). By Lemma 3.1, the multiplicative contribution of e ∈ω2 is hence x2 e = (1 −ye + ye)2 −y2 e in both cases. The contribution of an edge e ∈E \ ω is then 1 −x2 e . It is now enough to show that there are exactly |E∅(ω)| choices of ω1 1 and ω2 1 such that ω1 1△ω2 1 = ω1 and ω1 1 ∩ω2 1 ⊂ω2. Indeed, this is equivalent to freely choosing ω2 1 ∈E∅(ω) and setting ω1 1 = (ω1 \ ω2 1) ∪(ω2 1 ∩ω2) ∈EA. ⊓⊔ ⊓⊔ We note that one can express |E∅(ω)| in terms of the number of vertices, edges, and connected components of ω (see Lemma 4.2). 4 Random Alternating Flows In this section we assume that G is planar, and we explain how the double random current measure is related to a measure on alternating flows. To this end, we define a directed modification ⃗G = (V ∪W + ∪W −, ⃗E) of G as follows. Each edge e ∈E is replaced by three directed edges ⃗e ∈⃗E: one middle edge ⃗em, and two side edges ⃗es1, ⃗es2 (see Fig. 2). M. Lis 80 wi wi−1 wi+1 w w G 1 wi wi−1 wi+1 w+ i+1 w− i+1 w− i w+ i w+ i−1 w− i−1 G wi wi−1 wi+1 wi wi−1 wi+1 w+ i+1 w− i+1 w− i w+ i w+ i−1 w− i−1 G G Fig. 2 A choice of orientations of edges in a directed modification ⃗G of G. For each boundary vertex w, a source w+ and a sink w−are added in an order depending on whether w ∈W◦or w ∈W• G Fig. 2 A choice of orientations of edges in a directed modification ⃗G of G. For each boundary vertex w, a source w+ and a sink w−are added in an order depending on whether w ∈W◦or w ∈W• The side edges lie on opposite sides of ⃗em and have the opposite orientation to ⃗em. The orientation of each middle edge is chosen arbitrarily, and the edge weights are given by The side edges lie on opposite sides of ⃗em and have the opposite orientation to ⃗em. The orientation of each middle edge is chosen arbitrarily, and the edge weights are given by x⃗em = sinh 2Je 2 = xe 1 −x2e , x⃗es1 = x⃗es2 = tanh Je 2 = xe 2 . (4.1) (4.1) Moreover, for each w ∈W, two vertices w+ ∈W + and w−∈W −, and two directed edges (w, w−), (w+, w) ∈⃗E are placed in the external face of G so that w−becomes a sink and w+ becomes a source. The weights of these edges are set to 1. The vertices are placed in such a way that w−is comes immediately before (resp. after) w+ if w ∈W◦(resp. if w ∈W•) in the counterclockwise order around the external face of G (see Fig. 2). ⃗ ⃗ A flow on ⃗G is a set of edges F ⊆⃗E such that each vertex has the same number of ingoing and outgoing edges in F. 4 Random Alternating Flows An alternating flow is a flow such that for each v ∈V , the edges of the flow alternate in orientation around v (edge oriented towards v or away from v). For U ⊆W, we will write U + ⊆W + and U −⊆W −for the the corresponding sets of sources and sinks in ⃗G. For two (possibly intersecting) sets A, B ⊂W of equal cardinality, let FA,B = {alternating flow F on ⃗G | V (F) ∩W + = A+, V (F) ∩W −= B−}, FA,B = {alternating flow F on ⃗G | V (F) ∩W + = A+, V (F) ∩W −= B−}, where V (F) is the set of vertices incident on at least one edge of F. Following Talaska [37], we define the weight of an alternating flow F ∈FA,B by w(F) = 2|A|+|F|−|V (F)|  ⃗e∈F x⃗e, (4.2) (4.2) and we consider the probability measure on alternating flows with boundary conditions A, B given by and we consider the probability measure on alternating flows with boundary conditions A, B given by PA,B a-flow(F) = w(F) Z A,B a-flow , F ∈FA,B, where Z A,B a-flow =  F∈FA,B w(F) is the partition function. Note that in [37] general oriented planar graphs are considered, and the connection with double random currents described in this section is realized by our particular choice of ⃗G and its edge weights. where Z A,B a-flow =  F∈FA,B w(F) is the partition function. Note that in [37] general oriented planar graphs are considered, and the connection with double random currents described in this section is realized by our particular choice of ⃗G and its edge weights. There are four different types of local (interior) edge configurations in an alternating flow F on ⃗G according to the total amount of flow at the endpoints (the number of incoming edges minus the number of outgoing edges) and the orientation of the edges (see Fig. 3). Note that it is not possible that F contains ⃗eR1 and ⃗eR2 but not ⃗eB since then the alternating 123 The Planar Ising Model and Total Positivity The Planar Ising Model and Total Positivity 81 Fig. 3 Four types of local edge configurations in alternating flows on ⃗G (1a) (2a) (2b) (1b) condition is violated. 4 Random Alternating Flows Also note that there are three different configurations of type (1a) which are indistinguishable from the point of view of alternating flows—they can be interchanged without forcing any other changes in the flow. (1a) (2a) (2b) (1b) Fig. 3 Four types of local edge configurations in alternating flows on ⃗G (1b) (1a) condition is violated. Also note that there are three different configurations of type (1a) which are indistinguishable from the point of view of alternating flows—they can be interchanged without forcing any other changes in the flow. condition is violated. Also note that there are three different configurations of type (1a) which are indistinguishable from the point of view of alternating flows—they can be interchanged without forcing any other changes in the flow. With each F ∈FA,B, as in Sect. 3, we associate a pair of sets ω(F) = (ω1(F), ω2(F)), where ω1(F) is the set of edges e ∈E such that the local configuration of F at e is of type (1a) or (1b), and where ω2(F) is the set of edges e ∈E with the local configuration of type (2a) or (2b). We denote by A,B the image of FA,B under this map. Note that ω1(F) ∈EA△B since, by the flow condition, one can split ω1(F) into a sourceless part in E∅and a collection of |A| edge-disjoint directed paths starting at A and ending at B. In particular, every v ∈A ∩B is the starting and ending point of exactly one such path, and hence, the degree of ω1(F) at v is even. This means that A,B ⊆ A△B, and hence the map above induces a probability measure P A,B a flow on A△B, which is supported on A B. The main result of this section casts this measure into a form related to that of the induced double random current measure from Theorem 3.2. Theorem 4.1 Let A, B ⊂W be such that |A| = |B|, and let ⃗G be one of the directed modifications of G described above. Let ω ∈ A,B, and let k′(ω) be the number of connected components of ω that contain a vertex in A ∪B. 4 Random Alternating Flows Then, the probability of ω induced from the random alternating flow measure is given by P A,B a−flow(ω) = 1 ¯Z A,B a-flow 2|A|−k′(ω)|E∅(ω)|  e∈ω1 xe  e∈ω2 x2 e  e∈E\ω (1 −x2 e ), where ¯Z A,B a-flow = Z A,B a-flow e∈E(1 −x2 e ). In particular, P A,B a-flow = P B,A a-flow. P A,B a−flow(ω) = 1 ¯Z A,B a-flow 2|A|−k′(ω)|E∅(ω)|  e∈ω1 xe  e∈ω2 x2 e  e∈E\ω (1 −x2 e ), Before proving the theorem, we need to recall a standard result about the cardinality of E∅(ω). We give a proof for completeness. For ω ⊆E, we denote by k(ω) the number of connected components of ω. Lemma 4.2 Let ω ⊆E be such that E∅(ω) is nonempty. Then, Lemma 4.2 Let ω ⊆E be such that E∅(ω) is nonempty. Then, |E∅(ω)| = 2|ω|−|V (ω)|+k(ω). Proof Consider a maximal spanning forest T ⊆ω of ω, and note that |ω\T| = |ω|−|V (ω)|+ k(ω). It is hence enough to construct a bijection between E∅(ω) and the set of subsets of ω\T. To this end, to each e ∈ω \ T we assign the unique cycle Ce ⊆(ω \ T ) ∪{e}. Note that Ce ∈E∅(ω). The bijection is given by assigning to each ω′ ∈E∅(ω) the set ω′ ∩(ω \ T ), and its inverse by assigning to each {e1, . . . , el} ⊆ω \ T the set Ce1△· · · △Cel ∈E∅(ω). ⊓⊔ 123 82 M. Lis M. Lis Proof of Theorem 4.1 Let ¯ω be a modification of ω seen as a subset of E where each edge in ω2 is replaced by two parallel edges. By an alternating orientation of ¯ω we mean an assignment of orientations to the edges of ¯ω such that for each v ∈V , the edges alternate in orientation around v. Let F be such that ω(F) = ω, and let ˜F be its equivalence class under identifying the three local edge configurations of type (1a). Note that such equivalence classes are in one-to-one correspondence with alternating orientations of ¯ω. We claim that the number of such alternating orientations is 2k(ω)−k′(ω). (4.3) 2k(ω)−k′(ω). (4.3) Indeed, consider a connected component κ of ¯ω that does not contain a vertex in A ∪B. We claim that κ has exactly 2 different alternating orientations. 4 Random Alternating Flows To see this, consider the outer boundary ∂outκ of κ, i.e., the set of edges in κ incident on the unbounded face of κ. Because of the alternating condition, the choice of the orientation for one edge determines the orientation of all other edges in ∂outκ. Moreover, there are no conflicts of orientations since each vertex of κ has even degree, and hence ∂outκ can be deformed to a cycle by splitting vertices incident on more than 2 edges from ∂outκ. Then, the orientations of all edges agree with the clockwise or anticlockwise order on the cycle. After orienting ∂outκ, one has to consider all connected components of κ \ ∂outκ, and repeat the reasoning with the only difference being that there is no more freedom of choice of the orientation (if ∂outκ was oriented clockwise (resp. counterclockwise), then all outer boundaries of the connected components of κ \∂outκ have to be oriented counterclockwise (resp. clockwise)). One repeats this procedure until all edges of κ are oriented. On the other hand, there is no freedom of orientation for components containing a vertex in A ∪B since each such component has to be oriented from a vertex in A to a vertex in B. Therefore (4.3) holds true. Indeed, consider a connected component κ of ¯ω that does not contain a vertex in A ∪B. We claim that κ has exactly 2 different alternating orientations. To see this, consider the outer boundary ∂outκ of κ, i.e., the set of edges in κ incident on the unbounded face of κ. Because of the alternating condition, the choice of the orientation for one edge determines the orientation of all other edges in ∂outκ. Moreover, there are no conflicts of orientations since each vertex of κ has even degree, and hence ∂outκ can be deformed to a cycle by splitting vertices incident on more than 2 edges from ∂outκ. Then, the orientations of all edges agree with the clockwise or anticlockwise order on the cycle. After orienting ∂outκ, one has to consider all connected components of κ \ ∂outκ, and repeat the reasoning with the only difference being that there is no more freedom of choice of the orientation (if ∂outκ was oriented clockwise (resp. counterclockwise), then all outer boundaries of the connected components of κ \∂outκ have to be oriented counterclockwise (resp. clockwise)). 4 Random Alternating Flows Combining all the previous observations, and using the definition of PA,B a-flow, we can write 123 83 The Planar Ising Model and Total Positivity P A,B a-flow(ω) =  F: ω(F)=ω PA,B a-flow(F) = 1 Z A,B a-flow  F: ω(F)=ω w(F) = 1 Z A,B a-flow  ˜F: ω( ˜F)=ω w( ˜F) = 1 Z A,B a-flow  ˜F: ω( ˜F)=ω 2|A|+|ω|−|V (ω)|  e∈ω1 xe 1 −x2e  e∈ω2 x2 e 1 −x2e = 1 ¯Z A,B a-flow  ˜F: ω( ˜F)=ω 2|A|+|ω|−|V (ω)|  e∈ω1 xe  e∈ω2 x2 e  e∈E\ω (1 −x2 e ) = 1 ¯Z A,B a-flow 2k(ω)−k′(ω)2|A|+|ω|−|V (ω)|  e∈ω1 xe  e∈ω2 x2 e  e∈E\ω (1 −x2 e ) = 1 ¯Z A,B a-flow 2|A|−k′(ω)|E∅(ω)|  e∈ω1 xe  e∈ω2 x2 e  e∈E\ω (1 −x2 e ), P A,B a-flow(ω) =  F: ω(F)=ω PA,B a-flow(F) = 1 Z A,B a-flow  F: ω(F)=ω w(F) = 1 Z A,B a-flow  ˜F: ω( ˜F)=ω w( ˜F) = 1 Z A,B a-flow  ˜F: ω( ˜F)=ω 2|A|+|ω|−|V (ω)|  e∈ω1 xe 1 −x2e  e∈ω2 x2 e 1 −x2e = 1 ¯Z A,B a-flow  ˜F: ω( ˜F)=ω 2|A|+|ω|−|V (ω)|  e∈ω1 xe  e∈ω2 x2 e  e∈E\ω (1 −x2 e ) = 1 ¯Z A,B a-flow 2k(ω)−k′(ω)2|A|+|ω|−|V (ω)|  e∈ω1 xe  e∈ω2 x2 e  e∈E\ω (1 −x2 e ) = 1 ¯Z A,B a-flow 2|A|−k′(ω)|E∅(ω)|  e∈ω1 xe  e∈ω2 x2 e  e∈E\ω (1 −x2 e ), PA,B a-flow(F) where the second to last equality follows from (4.3), and the last one from Lemma 4.2. ⊓⊔ where the second to last equality follows from (4.3), and the last one from Lemma 4.2. ⊓⊔ In two special cases the measures induced from double random currents and alternating flows are the same. In two special cases the measures induced from double random currents and alternating flows are the same. Corollary 4.3 We have that P ∅ d-curr = P ∅,∅ a-flow and P {a,b} d-curr = P {a},{b} a-flow for any a, b∈W, a ̸= b. Proof By Lemma 5.4, we have that ∅,∅= ∅and {a},{b} = {a,b}, and the statement follows from Theorems 3.2 and 4.1. ⊓⊔ 4 Random Alternating Flows One repeats this procedure until all edges of κ are oriented. On the other hand, there is no freedom of orientation for components containing a vertex in A ∪B since each such component has to be oriented from a vertex in A to a vertex in B. Therefore (4.3) holds true. We now turn to the total weight w( ˜F) of each equivalence class ˜F, which we define to be the sum of weights of all flows in ˜F. By (4.1), the total multiplicative contribution to w( ˜F) of configurations of type (1a) is 23 xe 2 2 xe 1 −x2e + 2xe = 2xe 1 −x2e , where we took the factor 2# edges and did not take the factor 2|A|−# vertices from (4.2) into account. This contribution is therefore equal to the contribution of a configuration of type (1b). The contributions of configurations of type (2a) and (2b) also agree and are equal to where we took the factor 2# edges and did not take the factor 2|A|−# vertices from (4.2) into account. This contribution is therefore equal to the contribution of a configuration of type (1b). The contributions of configurations of type (2a) and (2b) also agree and are equal to 22 xe 2 xe 1 −x2e = 2x2 e 1 −x2e . Hence, by (4.2), we can write Hence, by (4.2), we can write w( ˜F) = 2|A|−|V (ω)|  e∈ω1 2xe 1 −x2e  e∈ω2 2x2 e 1 −x2e = 2|A|+|ω|−|V (ω)|  e∈ω1 xe 1 −x2e  e∈ω2 x2 e 1 −x2e . Note that the map ω is constant on each equivalence class ˜F, and hence, with a slight abuse of notation, we can write ω( ˜F) for the value of ω evaluated at any representative of ˜F. 5 Proofs of Main Results We need to state a few necessary lemmas. The first one goes back to the work of Griffiths, Hurst and Sherman [15], and we give a proof for completeness. We will write Za,b = Z{a,b}, and we define Za,b curr = Z∅ curr for a = b. Lemma 5.1 For a, b ∈V , we have ⟨σaσb⟩= Za,b curr Z∅curr . Proof If a = b, then the equality is trivial. Otherwise, for a vertex v and a current n, we define nv =  u: {u,v}∈E n{u,v}, and we have ⟨σaσb⟩=  σ∈{−1,1}V σaσb {u,v}∈E exp(J{u,v}σuσv)  σ∈{−1,1}V {u,v}∈E exp(J{u,v}σuσv) ⟨σaσb⟩=  σ∈{−1,1}V σaσb {u,v}∈E exp(J{u,v}σuσv)  σ∈{−1,1}V {u,v}∈E exp(J{u,v}σuσv) =  σ∈{−1,1}V σaσb {u,v}∈E ∞  k=0 (J{u,v})kσ k u σ k v /k!  σ∈{−1,1}V {u,v}∈E ∞  k=0 (J{u,v})kσ ku σ kv /k! =  σ∈{−1,1}V σaσb {u,v}∈E ∞  k=0 (J{u,v})kσ k u σ k v /k!  σ∈{−1,1}V {u,v}∈E ∞  k=0 (J{u,v})kσ ku σ kv /k! 123 M. Lis 84 =  σ∈{−1,1}V σaσb  n∈ w(n) v∈V σ nv v  σ∈{−1,1}V  n∈ w(n) v∈V σ nv v =  σ∈{−1,1}V  n∈ w(n) v∈V σ nv+1{v∈{a,b}} v  σ∈{−1,1}V  n∈ w(n) v∈V σ nv v = 2|V |  n∈{a,b} w(n) 2|V |  n∈∅ w(n) = Za,b curr Z∅curr . The second to last equality holds true since the only currents that survive the summation over the symmetric set {−1, 1}|V | are the ones for which the exponent of σv is even at every vertex. ⊓⊔ The second to last equality holds true since the only currents that survive the summation over the symmetric set {−1, 1}|V | are the ones for which the exponent of σv is even at every vertex. ⊓⊔ The next lemma expresses the two-point spin correlation function as a ratio of partition functions of alternating flows. Recall that we defined A,B ⊆ A△B to be the image of FA,B under the map ω from Sect. 4. Lemma 5.2 For a, b ∈W, we have Lemma 5.2 For a, b ∈W, we have Lemma 5.2 For a, b ∈W, we have ⟨σaσb⟩= Za,b a-flow Z∅ a-flow . ⟨σaσb⟩= Za,b a-flow Z∅ a-flow . Proof Note that for each F ∈F{a},{b}, the graph ω(F) contains exactly k′ = 1 connected component that connects a and b. The following is the reverse of the lemma above. Lemma 5.4 Let ω ∈ A△B be such that for each connected component κ of ω, the vertices in A+ ∪B−that are adjacent to V (κ) alternate between the sources in A+ and the sinks in B−as one goes around the external face. Then, there exists F ∈FA,B such that ω(F) = ω. Proof Fix a connected component κ of ω. Similarly to the proof of Theorem 4.1, we consider a graph ¯κ where each edge of κ ∩ω2 is replaced by two parallel edges, and moreover, the edges connecting each vertex in V (κ) ∩W to the corresponding vertices in A+ ∪B−are added. It is now enough to show that there exists an alternating orientation of ¯κ where each vertex in V (¯κ) ∩A+ becomes a source and each vertex in V (¯κ) ∩B−becomes a sink. By the assumption on κ, we can add (in one of two possible ways) |V (¯κ) ∩(A+ ∪B−)|/2 vertex-disjoint edges connecting in pairs consecutive vertices in V (¯κ) ∩A+ and V (¯κ) ∩B−. We call the resulting graph ¯κ′. It is now enough to construct an alternating orientation of ¯κ′ such that each of the additional edges is directed from a vertex in V (¯κ) ∩B−to a vertex in V (¯κ) ∩A+, and then restrict the orientation to ¯κ. To this end, note that each vertex of ¯κ′ has even degree. Therefore, as in the proof Theorem 4.1, the outer boundary of ¯κ′ can be deformed to a cycle, and can be directed clockwise or counterclockwise so that the additional edges are directed properly. We can then orient the rest of ¯κ′ consistently as in Theorem 4.1, and the lemma is proved. ⊓⊔ The next lemma is an adaptation of a result of [37]. Lemma 5.5 [37, Corollary 4.3] Let N A,B be as in Theorem 2.1. We have det N A,B = Z A,B a-flow Z∅ a-flow . Proof The proof is a matter of translation of the results of [37] to our setting. To this end, consider the boundary measurement matrix M from Definition 2.4 of [37] defined for ⃗G with weights as in Sect. 4. By Corollary 5.3 of [37], the entry corresponding to source a+ and sink b−is equal to Za,b a-flow/Z∅ a-flow, and hence, by Lemma 5.2, to ⟨σaσb⟩. Therefore, the boundary measurement matrix for ⃗G is the matrix of Ising boundary spin correlation functions. 5 Proofs of Main Results Moreover, by Lemma 5.4 we have that {a},{b} = {a}△{b}, and ∅,∅= ∅. Hence, by comparing the formulas in Theorem 3.2 and 4.1, and using Lemma 5.1, we get Za,b a-flow Z∅ a-flow = 2|{a}|2−k′ Za,b curr Z∅ curr Z∅curr Z∅curr = Za,b curr Z∅curr = ⟨σaσb⟩. ⊓⊔ The next lemma describes an alternating property of sources and sinks in a connected component of an alternating flow. The next lemma describes an alternating property of sources and sinks in a connected component of an alternating flow. Lemma 5.3 Let F ∈FA,B, and let κ ⊂F be one of its connected components. Then, the sources and sinks of κ interlace, i.e., as one goes around the external face, the vertices in V (κ) ∩(A+ ∪B−) alternate between the sources in A+ and the sinks in B−. Proof Note that κ is an alternating flow itself. Take a source vertex v ∈V (κ) ∩A+, and traverse the edges of the external face of κ in a counterclockwise order. Since at each vertex in V that you visit, you take the rightmost possible turn, and since κ is alternating, all the directed edges are aligned with the direction of traversal until you encounter the consecutive vertex in v′ ∈V (κ) ∩(A+ ∪B−). Since v′ is either a sink or a source, and since there exists an edge directed towards v′, it must be a sink. An analogous argument can be made when starting at v′ but this time the edges are directed against the direction of traversal. Hence, the next vertex encountered in V (κ) ∩(A+ ∪B−) is again a source, and the lemma is proved. ⊓⊔ 123 123 85 The Planar Ising Model and Total Positivity The following is the reverse of the lemma above. We are now ready to prove our first two main results. The following is the reverse of the lemma above. Indeed, in a flow F ∈FA,B there cannot be a connected component that contains more than 2 vertices in A ∪B since then the alternating condition from Lemma 5.3 would be violated. On the other hand, if there exists k vertex disjoint paths connecting A and B, then by Lemma 5.4, there exists a flow in FA,B which maps to these paths under ω. ⊓⊔ Furthermore, by Lemma 5.3 and 5.4, Z A,B a-flow > 0 if and only if there are k vertex disjoint paths connecting A and B. Indeed, in a flow F ∈FA,B there cannot be a connected component that contains more than 2 vertices in A ∪B since then the alternating condition from Lemma 5.3 would be violated. On the other hand, if there exists k vertex disjoint paths connecting A and B, then by Lemma 5.4, there exists a flow in FA,B which maps to these paths under ω. ⊓⊔ Before proving the rest of our main results, we will need a classical Pfaffian formula of Groeneveld, Boel and Kasteleyn [16]. We present here a different proof involving the connection between double random currents and alternating flows. For yet another proof involving the dimer model, see the recent treatment of the combinatorics of the planar Ising model [6]. Lemma 5.6 [16, Theorem A] Let K A∪B be as in Theorem 2.3. We have Lemma 5.6 [16, Theorem A] Let K A∪B be as in Theorem 2.3. We have Pf K A∪B = Z A∪B curr Z∅curr . Proof By (2.2), for any A′ ⊂A ∪B with |A′| = |A| and B′ = (A ∪B) \ A′, we have det N A′,B′ =  π: bijection A′→B′ (−1)xing(π)  {a,b}∈π ⟨σaσb⟩ Recall that a bijection between A′ and B′ can be thought of as a pairing of A ∪B, i.e., a partition of A ∪B into pairs {a, π(a)}, a ∈A′. Note that each pairing π of A ∪B appears in a sum as above for exactly 2|A| choices of A′. Indeed, for each of the |A| pairs in π, we can choose one vertex that will belong to A′. Hence, by (2.4) and by Lemma 5.5, we have Pf K A∪B = 2−|A|  A′,B′ det N A′,B′ = 2−|A| Z∅ a-flow  A′,B′ Z A′,B′ a-flow. (5.1) (5.1) Recall that A′,B′ ⊂ A∪B is the image of FA′,B′ under the map ω from Sect. The following is the reverse of the lemma above. By Corollary 4.3of [37], it is hence enough to check that the signs in N A agree with those in the matrix in Corollary 4.3 of [37] where only the columns corresponding to sinks are considered. In [37], the sign of the entry corresponding to source a+ and sink b−is negative if the number of sources strictly between a+ and b−in a fixed clockwise order is odd, and it is positive otherwise. By our choice, for every w ∈W◦(resp. w ∈W•), the sink w− immediately precedes (resp. succeeds) the source w+ in the counterclockwise order. One can easily check that as a result, s(i, j) from the definition of N A,B counts the number of sources strictly between the source w+ li ∈A+ and the sink w− j ∈B−. Therefore, the signs in the definition of N A agree with those in [37] (up to changing a clockwise to a counterclockwise order). Hence, the result follows from Corollary 4.3 of [37]. ⊓⊔ ⊓⊔ Remark 3 The proof of Corollary 4.3 of [37] uses the signed walk interpretation of the boundary measurement matrix of Postnikov [33]. One can compare this with the Kac–Ward representation of the planar Ising model [20], where the Ising boundary two-point functions can be expressed as partition functions of signed non-backtracking walks on the undirected graph G (see e.g. [21], or [26] for a concise proof of the Kac–Ward formula). 123 123 123 M. Lis 86 M. Lis Proof of Theorem 2.1 It is a direct consequence of Lemma 5.5. The interpretation of empty paths as counterclockwise or clockwise loops follows from the way the sources and sinks are placed around the external face (see Sect. 4). ⊓⊔ ⊓⊔ Proof of Corollary 2.2 Note that every square submatrix of M A,B satisfies the assumptions of Corollary 2.2. Hence, it is enough to prove that det M A,B ≥0, and det M A,B > 0 if and only if there are k vertex disjoint paths connecting A and B. To this end, note that by (2.1), (2.2) and Lemma 5.5, det M A,B = det N A,B = Z A,B a-flow Z∅ a-flow ≥0. Furthermore, by Lemma 5.3 and 5.4, Z A,B a-flow > 0 if and only if there are k vertex disjoint paths connecting A and B. The following is the reverse of the lemma above. Comparing the formulas in Theorem 3.2 and 4.1, we have PA∪B d-curr(PA,B) = P A∪B d-curr(P A,B) =  ω∈P A,B P A∪B d-curr(ω) = Z A,B a-flow Z A∪B curr Z∅curr  ω∈P A,B 2k′(ω)−|A|P A,B a-flow(ω) = Z A,B a-flow Z A∪B curr Z∅curr  ω∈P A,B P A,B a-flow(ω) = Z A,B a-flow Z A∪B curr Z∅curr , ( = Z A,B a-flow Z A∪B curr Z∅curr  ω∈P A,B 2k′(ω)−|A|P A,B a-flow(ω) = Z A,B a-flow Z A∪B curr Z∅curr  ω∈P A,B P A,B a-flow(ω) (5.2) where the second to last equality holds true since, by the definition of PA,B, k′(ω) = |A| for every ω ∈P A,B. Moreover, since P A,B a-flow is supported on A,B, to justify the last equality we have to show that P A,B = A,B. To this end, note that by Lemma 5.3, for each F ∈FA,B, we have that ω(F) ∈P A,B, where ω denotes the map from Sect. 4. Indeed, for contiguous sets A and B, there is only one way of distributing the sources A+ and sinks B−between the connected components of an alternating flow in such a way that they alternate around the external face for each component. This means that each connected component of F connects a single point in A to a single point in B. On the other hand, by Lemma 5.4, for each ω ∈P A,B, there exists F ∈FA,B such that ω(F) = ω. Therefore, P A,B = A,B and (5.2) holds true. Furthermore, using Lemma 5.5 and 5.6, we get where the second to last equality holds true since, by the definition of PA,B, k′(ω) = |A| for every ω ∈P A,B. Moreover, since P A,B a-flow is supported on A,B, to justify the last equality we have to show that P A,B = A,B. To this end, note that by Lemma 5.3, for each F ∈FA,B, we have that ω(F) ∈P A,B, where ω denotes the map from Sect. 4. Indeed, for contiguous sets A and B, there is only one way of distributing the sources A+ and sinks B−between the connected components of an alternating flow in such a way that they alternate around the external face for each component. This means that each connected component of F connects a single point in A to a single point in B. The following is the reverse of the lemma above. 4. We claim that each ω ∈ A∪B belongs to A′,B′ for exactly 2k′(ω) choices of A′, where k′(ω) is the number of connected components of ω containing a vertex in A ∪B. Indeed, by Lemma 5.3 and 5.4, for each connected component κ of ω that intersects A ∪B, there are exactly two ways of distributing the vertices in V (κ) ∩(A ∪B) between A′ and B′ (choosing one vertex to be connected to a source or a sink fixes the choices for all other vertices in V (κ)∩(A ∪B) 123 The Planar Ising Model and Total Positivity 87 by the alternating property). Hence, by combining Theorem 3.2 and 4.1, we have (5.1) = 1 Z∅ a-flow  A′,B′  ω∈ A′,B′ 2−k′(ω)|E∅(ω)|  e∈ω1 xe 1 −x2e  e∈ω2 x2 e 1 −x2e = 1 Z∅ a-flow  ω∈ A∪B 2k′(ω)−k′(ω)|E∅(ω)|  e∈ω1 xe 1 −x2e  e∈ω2 x2 e 1 −x2e = Z A∪B curr Z∅ curr Z∅ a-flow = Z A∪B curr Z∅curr , (5.1) = 1 Z∅ a-flow  A′,B′  ω∈ A′,B′ 2−k′(ω)|E∅(ω)|  e∈ω1 xe 1 −x2e  e∈ω2 x2 e 1 −x2e = 1 Z∅ a-flow  ω∈ A∪B 2k′(ω)−k′(ω)|E∅(ω)|  e∈ω1 xe 1 −x2e  e∈ω2 x2 e 1 −x2e = Z A∪B curr Z∅ curr Z∅ a-flow = Z A∪B curr Z∅curr , where in the last equality we used Corollary 4.3 to get Z∅ a-flow = (Z∅ curr)2. ⊓⊔ where in the last equality we used Corollary 4.3 to get Z∅ a-flow = (Z∅ curr)2. ⊓⊔ We are now in a position to prove the rest of our main results. We are now in a position to prove the rest of our main results. Proof of Theorem 2.3 Recall that A and B are placed around the outer face in such a way that allverticesof A comebeforeeveryvertexof B inacounterclockwiseorder.Let P A,B ⊆ A∪B be the image of the event PA,B under the map from Sect. 3. The following is the reverse of the lemma above. On the other hand, by Lemma 5.4, for each ω ∈P A,B, there exists F ∈FA,B such that ω(F) = ω. Therefore, P A,B = A,B and (5.2) holds true. Furthermore, using Lemma 5.5 and 5.6, we get det M A,B Pf K A∪B = Z A,B a-flowZ∅ curr Z∅ a-flowZ A∪B curr = Z A,B a-flow Z A∪B curr Z∅curr , where we used Corollary 4.3 to obtain that Z∅ a-flow = (Z∅ curr)2. Together with (5.2), this finishes the proof. ⊓⊔ where we used Corollary 4.3 to obtain that Z∅ a-flow = (Z∅ curr)2. Together with (5.2), this finishes the proof. ⊓⊔ 123 123 88 M. Lis Proof of Theorem 2.4 Hongler in his PhD thesis [17] showed that in the setting of Theo- rem 2.4, for any a ∈A and b ∈B, the following limit exists Proof of Theorem 2.4 Hongler in his PhD thesis [17] showed that in the setting of Theo- rem 2.4, for any a ∈A and b ∈B, the following limit exists fa,b(D) := lim ϵ→0 ϵ−1⟨σaϵσbϵ⟩, (5.3) (5.3) where aϵ and bϵ are vertices on the outer face of Dϵ approximating a and b. Moreover, the limit is conformally covariant, i.e., for a domain D′ and a conformal equivalence ϕ : D →D′, we have where aϵ and bϵ are vertices on the outer face of Dϵ approximating a and b. Moreover, the limit is conformally covariant, i.e., for a domain D′ and a conformal equivalence ϕ : D →D′, we have fa,b(D) = fϕ(a),ϕ(b)(D′)|ϕ′(a)ϕ′(b)| 1 2 . (5.4) (5.4) Furthermore, for real numbers a, b, we have fa,b(H) = 2( √ 2 + 1) π|a −b| . 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Convergence and almost sure T-stability for a random iterative sequence generated by a generalized random operator
Journal of inequalities and applications
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© 2015 Okeke and Abbas; licensee Springer. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduc- tion in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. R ES EA RCH Open Access Abstract The aim of this paper is to introduce the concept of generalized φ-weakly contraction random operators and then to prove the convergence and almost sure T-stability of Mann and Ishikawa-type random iterative schemes. We also prove that a random fixed point of such operators is Bochner integrable. Our results generalize, extend and improve various results in the existing literature including the results in Berinde (Bul. ¸Stiin¸t. - Univ. Baia Mare, Ser. B Fasc. Mat.-Inform. 18(1):7-14, 2002), Olatinwo (J. Adv. Math. Stud. 1(1):5-14, 2008), Rhoades (Trans. Am. Math. Soc. 196:161-176, 1974; Indian J. Pure Appl. Math. 21(1):1-9, 1990; Indian J. Pure Appl. Math. 24(11):691-703, 1993) and Zhang et al. (Appl. Math. Mech. 32(6):805-810, 2011). MSC: 47H09; 47H10; 49M05; 54H25 Keywords: Ishikawa-type random iterative scheme; Mann-type random iterative scheme; almost sure T-stability; separable Banach spaces; Bochner integrability; generalized φ-weakly contractive random operator Godwin Amechi Okeke1* and Mujahid Abbas2,3 Godwin Amechi Okeke1* and Mujahid Abbas2,3 *Correspondence: gaokeke1@yahoo.co.uk 1Department of Mathematics, College of Science and Technology, Covenant University, Canaanland, KM 10 Idiroko Road, P.M.B. 1023, Ota, Ogun State, Nigeria Full list of author information is available at the end of the article *Correspondence: gaokeke1@yahoo.co.uk 1Department of Mathematics, College of Science and Technology, Covenant University, Canaanland, KM 10 Idiroko Road, P.M.B. 1023, Ota, Ogun State, Nigeria Full list of author information is available at the end of the article Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 DOI 10.1186/s13660-015-0666-8 Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 DOI 10.1186/s13660-015-0666-8 Convergence and almost sure T-stability for a random iterative sequence generated by a generalized random operator Convergence and almost sure T-stability for a random iterative sequence generated by a generalized random operator Godwin Amechi Okeke1* and Mujahid Abbas2,3 1 Introduction Real world problems are embedded with uncertainties and ambiguities. To deal with prob- abilistic models, probabilistic functional analysis has emerged as one of the momentous mathematical disciplines and attracted the attention of several mathematicians over the years in view of its applications in diverse areas from pure mathematics to applied sciences. Random nonlinear analysis, an important branch of probabilistic functional analysis, deals with the solution of various classes of random operator equations and related problems. Of course, the development of random methods has revolutionized financial markets. Ran- dom fixed point theorems are stochastic generalizations of classical or deterministic fixed point theorems and are required for the theory of random equations, random matrices, random partial differential equations and various classes of random operators arising in physical systems (see [, ]). Random fixed point theory was initiated in s by Prague school of probabilists. Spacek [] and Hans [] established a stochastic analogue of the Ba- nach fixed point theorem in a separable complete metric space. Itoh [] in generalized and extended Spacek and Han’s theorem to a multivalued contraction random operator. The survey article by Bharucha-Reid [] in , where he studied sufficient conditions for a stochastic analogue of Schauder’s fixed point theorem for random operators, gave Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Page 2 of 11 Page 2 of 11 wings to random fixed point theory. Now this area has become full fledged research area, and many interesting techniques to obtain the solution of nonlinear random system have appeared in the literature (see [–, , –]). Papageorgiou [] established an existence of random fixed point of measurable closed and nonclosed valued multifunctions satisfying general continuity conditions and hence improved the results in [, ] and []. Xu [] extended the results of Itoh to a nonself- random operator T, where T satisfies weakly inward or the Leray-Schauder condition. Shahzad and Latif [] proved a general random fixed point theorem for continuous ran- dom operators. As applications, they derived a number of random fixed points theorems for various classes of -set and -ball contractive random operators. Arunchai and Plub- tieng [] obtained some random fixed point results for the sum of a weakly-strongly con- tinuous random operator and a nonexpansive random operator in Banach spaces. 1 Introduction Mann [] introduced an iterative scheme and employed it to approximate the solution of a fixed point problem defined by a nonexpansive mapping where the Picard iterative scheme fails to converge. Later, in , Ishikawa [] introduced an iterative scheme to obtain the convergence of a Lipschitzian pseudocontractive operator when a Mann itera- tive scheme is not applicable. The study of convergence of different random iterative processes constructed for var- ious random operators is a recent development (see [–] and references mentioned therein). Recently, Zhang et al. [] studied the almost sure T-stability and convergence of Ishikawa-type and Mann-type random algorithms for certain φ-weakly contractive- type random operators in the setup of a separable Banach space. They also established the Bochner integrability of a random fixed point for such random operators. In this paper, we introduce the notion of generalized φ-weakly contractive random op- erator and obtain the convergence and almost sure T-stability of Ishikawa-type random iterative scheme and Mann-type random iterative scheme for such operators. Our results extend, unify and generalize the comparable results in [–] and []. Our results improves and generalizes the deterministic fixed points results of Berinde [], Olatinwo [], Rhoades [–] in stochastic verse. Moreover, it extends and im- proves the results of Zhang et al. []. 2 Preliminaries Let (,,μ) be a complete probability measure space and (E,B(E)) be a measurable space, where E is a separable Banach space, B(E) is Borel sigma algebra of E, (,) is a mea- surable space (-sigma algebra) and μ is a probability measure on , that is, a measure with total measure one. A mapping ξ :  →E is called (a) E-valued random variable if ξ is (,B(E))-measurable, (b) strongly μ-measurable if there exists a sequence {ξn} of μ- simple functions converging to ξ μ-almost everywhere. Due to the separability of a Ba- nach space E, the sum of two E-valued random variables is an E-valued random variable. A mapping T : ×E →E is called a random operator if for each fixed e in E, the mapping T(·,e) :  →E is measurable. The following definitions and results will be needed in the sequel. Definition .[] Let (,ξ,μ) be a complete probability measure space. A random vari- able ξ :  →X is Bochner integrable if for each ω ∈, Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Page 3 of 11   ξ(ω) dμ(ω) < ∞,   ξ(ω) dμ(ω) < ∞, (.) (.) where ∥ξ(ω)∥is a nonnegative real-valued random variable. where ∥ξ(ω)∥is a nonnegative real-valued random variable. The Bochner integral is a natural generalization of the familiar Lebesgue integral to the vector-valued setting. Proposition .[] A random variable ξ is Bochner integrable if and only if there exists a sequence of random variables {ξn}∞ n=converging strongly to ξ almost surely such that lim n→∞   ξn(ω) – ξ(ω) dμ(ω) = . lim n→∞   ξn(ω) – ξ(ω) dμ(ω) = . (.) (.) Definition .[] Let (,ξ,μ) be a complete probability measure space, E be a nonempty subset of a separable Banach space X, and T :  × E →E be a random op- erator. Define F(T) = {ξ ∗:  →E such that T(ω,ξ ∗(ω)) = ξ ∗(ω) for each ω ∈} (the random fixed point set of T). Definition .[] Let (,,μ) be a complete probability measure space and E be a nonempty subset of a separable Banach space X. 2 Preliminaries (.) (.) The random Mann-type iterative scheme is a sequence of functions {ξn} defined by he random Mann-type iterative scheme is a sequence of functions {ξn} defined by The random Mann-type iterative scheme is a sequence of functions {ξn} defined by ξ(ω) ∈E, ξ (ω) = (– a )ξ (ω) + a T(ω ξ (ω)) (.) ξ(ω) ∈E, ξn+(ω) = (– an)ξn(ω) + anT(ω,ξn(ω)), (.) (.) ξ( ) , ξn+(ω) = (– an)ξn(ω) + anT(ω,ξn(ω)), (.) ξn+(ω) = (– an)ξn(ω) + anT(ω,ξn(ω)), where ≤an,cn ≤and ξ:  →E is an arbitrary measurable mapping. where ≤an,cn ≤and ξ:  →E is an arbitrary measurable mapping. For any given random variable ξ:  →E, define a random iterative scheme with the help of functions {ξn}∞ n=as follows: ξn+(ω) = f T;ξn(ω)  , n = ,,,..., (.) ξn+(ω) = f T;ξn(ω)  , n = ,,,..., (.) where f is some function measurable in the second variable. Definition .[] Let ξ ∗be a random fixed point of a random operator T and Bochner integrable with respect to {ξn}∞ n=. Let {ζn}∞ n=be an arbitrary sequence of random variables. Set ϵn(ω) = ζn+(ω) – f T;ζn(ω) , (.) (.) and assume that ∥ϵn(ω)∥∈L((ξ,μ)) (n = ,,...). The iterative scheme (.) is almost surely T-stable (or the iterative scheme (.) is almost surely stable with respect to T) if and only if and assume that ∥ϵn(ω)∥∈L((ξ,μ)) (n = ,,...). The iterative scheme (.) is almost surely T-stable (or the iterative scheme (.) is almost surely stable with respect to T) if and only if lim n→∞   ϵn(ω) dμ(ω) =  (.) (.) implies that ξ ∗(ω) is Bochner integrable with respect to {ζn(ω)}∞ n=. implies that ξ ∗(ω) is Bochner integrable with respect to {ζn(ω)}∞ n=. The following lemma will be needed in the sequel. Lemma .[] Let {γn} and {λn} be two sequences of nonnegative real numbers, {σn} be a sequence of positive numbers satisfying the conditions: ∞ n=σn = ∞and limn→∞ γn σn = . If λn+≤λn – σnφ(λn) + γn hold for each n ≥, where φ : R+ →R+ is a continuous and strictly increasing function with φ() = , then {λn} converges to as n →∞. 2 Preliminaries A random operator T :  × E →E is called a φ-weakly contractive-type random operator if there exists a continuous and non- decreasing function φ : R+ →R+ with φ(t) > for each t ∈(,∞) and φ() = such that for each x,ς ∈E, ω ∈, we have   T(ω,x) – T(ω,ς) dμ(ω) ≤   ∥x – ς∥dμ(ω) – φ   ∥x – ς∥dμ(ω)  . (.) (.) Motivated by the above results, we hereby introduce the following contractive condition. Motivated by the above results, we hereby introduce the following contractive condition. Definition .Let (,ξ,μ) be a complete probability measure space and E be a nonempty subset of a separable Banach space X. A random operator T :  × E →E is of generalized φ-weakly contractive-type if there exists L(ω) ≥and a continuous and nondecreasing function φ : R+ →R+ with φ(t) > for each t ∈(,∞) and φ() = such that for each x,ς ∈E, ω ∈,   T(ω,x) – T(ω,ς) dμ(ω) ≤eL(ω)∥x–ς∥   ∥x – ς∥dμ(ω) – φ   ∥x – ς∥dμ(ω)  . (.)   ≤eL(ω)∥x–ς∥   ∥x – ς∥dμ(ω) – φ   ∥x – ς∥dμ(ω)  . (.) (.) If L(ω) = for each ω ∈ in (.), then it reduces to condition (.). The study of nonlinear operators have attracted the attention of several mathematicians (see, e.g. [–]). Several interesting fixed points results have emerged as a result of such study. Let T :  × E →E be a random operator, where E is a nonempty convex subset of a separable Banach space X. Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Page 4 of 11 Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Page 4 of 11 Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 The random Ishikawa-type iterative scheme is a sequence of functions {ξn} and {ηn} defined by ⎧ ⎪⎨ ⎪⎩ ξ(ω) ∈E, ξn+(ω) = (– an)ξn(ω) + anT(ω,ηn(ω)), ηn(ω) = (– cn)ξn(ω) + cnT(ω,ξn(ω)). 3 Main results We start with the following result. Theorem .Let (E,∥· ∥) be a separable Banach space, T :  × E →E be a generalized φ-weakly contractive-type random operator with F(T) ̸= ∅, and {ξn} be a random itera- tive sequence as defined in (.) where {an} and {cn} are real sequences in (,) such that ∞ n=ancn = ∞. Then the random fixed point ξ ∗of T is Bochner integrable. Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Page 5 of 11 Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Page 5 of 11 Page 5 of 11 Proof It suffices to show that limn→∞   ∥ξn(ω) – ξ ∗(ω)∥dμ(ω) = . By (.) and (.), we have Proof It suffices to show that limn→∞   ∥ξn(ω) – ξ ∗(ω)∥dμ(ω) = . By (.) and (.), we have   ξn+(ω) – ξ ∗(ω) dμ(ω) ≤(– an)   ξn(ω) – ξ ∗(ω) dμ(ω) + an   T ω,ηn(ω) – ξ ∗(ω) dμ(ω) ≤(– an)   ξn(ω) – ξ ∗(ω) dμ(ω) + an  eL(ω)∥ηn(ω)–ξ∗(ω)∥   ηn(ω) – ξ ∗(ω) dμ(ω) – φ   ηn(ω) – ξ ∗(ω) dμ(ω)  ≤(– an)   ξn(ω) – ξ ∗(ω) dμ(ω) ∗  ≤(– an)   ξn(ω) – ξ ∗(ω) dμ(ω) + aneL(ω)∥ηn(ω)–ξ∗(ω)∥   ηn(ω) – ξ ∗(ω) dμ(ω). (.) (.) we compute the following estimate: Now we compute the following estimate:   ηn(ω) – ξ ∗(ω) dμ(ω) ≤(– cn)   ξn(ω) – ξ ∗(ω) dμ(ω) + cn   T ω,ξn(ω)  – ξ ∗(ω) dμ(ω) ≤(– cn)   ξn(ω) – ξ ∗(ω) dμ(ω) + cn  eL(ω)∥ξn(ω)–ξ∗(ω)∥   ξn(ω) – ξ ∗(ω) dμ(ω) – φ   ξn(ω) – ξ ∗(ω) dμ(ω)  ≤(– cn)   ξn(ω) – ξ ∗(ω) dμ(ω) + cneL(ω)∥ξn(ω)–ξ∗(ω)∥   ξn(ω) – ξ ∗(ω) dμ(ω) ≤(– cn)   ξn(ω) – ξ ∗(ω) dμ(ω) + cneL(ω)∥ξn(ω)–ξ∗(ω)∥   ξn(ω) – ξ ∗(ω) dμ(ω) – cneL(ω)∥ξn(ω)–ξ∗(ω)∥φ   ξn(ω) – ξ ∗(ω) dμ(ω)  . 3 Main results (.) (.) Using (.) in (.), we obtain Using (.) in (.), we obtain Using (.) in (.), we obtain   ξn+(ω) – ξ ∗(ω) dμ(ω) ≤(– an)   ξn(ω) – ξ ∗(ω) dμ(ω) + aneL(ω)∥ξn(ω)–ξ∗(ω)∥  (– cn) ×   ξn(ω) – ξ ∗(ω) dμ(ω) + cne(L(ω)∥ξn(ω)–ξ∗(ω)∥)   ξn(ω) – ξ ∗(ω) dμ(ω) – cne(L(ω)∥ξn(ω)–ξ∗(ω)∥)φ   ξn(ω) – ξ ∗(ω) dμ(ω)    ξn+(ω) – ξ ∗(ω) dμ(ω) = (– an)   ξn(ω) – ξ ∗(ω) dμ(ω) + aneL(ω)∥ξn(ω)–ξ∗(ω)∥(– cn) = (– an)   ξn(ω) – ξ ∗(ω) dμ(ω) + aneL(ω)∥ξn(ω)–ξ∗(ω)∥(– cn) lities and Applications ( 2015) 2015:146 Page 6 of 11 Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Page 6 of 11 Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Page 6 of 11 Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Page 6 of 11 ×   ξn(ω) – ξ ∗(ω) dμ(ω) + ancne(L(ω)∥ξn(ω)–ξ∗(ω)∥)   ξn(ω) – ξ ∗(ω) dμ(ω) – ancne(L(ω)∥ξn(ω)–ξ∗(ω)∥)φ   ξn(ω) – ξ ∗(ω) dμ(ω)     L( )∥ξ ( ) ξ∗( )∥ – ancne(L(ω)∥ξn(ω)–ξ∗(ω)∥)φ   ξn(ω) – ξ ∗(ω) dμ(ω)  ≤   ξn(ω) – ξ ∗(ω) dμ(ω) + aneL(ω)∥ξn(ω)–ξ∗(ω)∥   ×   ξn(ω) – ξ ∗(ω) dμ(ω) + ancneL(ω)∥ξn(ω)–ξ∗(ω)∥   ξn(ω) – ξ ∗(ω) dμ(ω) – ancneL(ω)∥ξn(ω)–ξ∗(ω)∥φ   ξn(ω) – ξ ∗(ω) dμ(ω)  = + aneL(ω)∥ξn(ω)–ξ∗(ω)∥+ ancneL(ω)∥ξn(ω)–ξ∗(ω)∥ ×   ξn(ω) – ξ ∗(ω) dμ(ω) – ancneL(ω)∥ξn(ω)–ξ∗(ω)∥ × φ   ξn(ω) – ξ ∗(ω) dμ(ω)  . (.) ×   ξn(ω) – ξ ∗(ω) dμ(ω) + ancneL(ω)∥ξn(ω)–ξ∗(ω)∥   ξn(ω) – ξ ∗(ω) dμ(ω) ×   ξn(ω) – ξ ∗(ω) dμ(ω) + ancneL(ω)∥ξn(ω)–ξ∗(ω)∥   ξn(ω) – ξ ∗(ω) dμ(ω) – ancneL(ω)∥ξn(ω)–ξ∗(ω)∥φ   ξn(ω) – ξ ∗(ω) dμ(ω)  = + aneL(ω)∥ξn(ω)–ξ∗(ω)∥+ ancneL(ω)∥ξn(ω)–ξ∗(ω)∥ ×  ξn(ω) – ξ ∗(ω) dμ(ω) – ancneL(ω)∥ξn(ω)–ξ∗(ω)∥ – ancneL(ω)∥ξn(ω)–ξ∗(ω)∥φ   ξn(ω) – ξ ∗(ω) dμ(ω)  = + aneL(ω)∥ξn(ω)–ξ∗(ω)∥+ ancneL(ω)∥ξn(ω)–ξ∗(ω)∥ (.) Set Set λn = + ancneL(ω)∥ξn(ω)–ξ∗(ω)∥  ξn(ω) – ξ ∗(ω) dμ(ω), σn = ancneL(ω)∥ξn(ω)–ξ∗(ω)∥ and γn = aneL(ω)∥ξn(ω)–ξ∗(ω)∥   ξn(ω) – ξ ∗(ω) dμ(ω) in Lemma ., it follows that conditions of Lemma .are satisfied. Hence in Lemma ., it follows that conditions of Lemma .are satisfied. Hence (.) (.) Using (.), we have Using (.), we have   T ω,kn(ω)  – ξ ∗(ω) dμ(ω) ≤eL(ω)∥kn(ω)–ξ∗(ω)∥   kn(ω) – ξ ∗(ω) dμ(ω) – φ   kn(ω) – ξ ∗(ω) dμ(ω)  ≤eL(ω)∥kn(ω)–ξ∗(ω)∥   kn(ω) – ξ ∗(ω) dμ(ω) ≤eL(ω)∥kn(ω)–ξ∗(ω)∥  (– cn)   ςn(ω) – ξ ∗(ω) dμ(ω) + cn   T ω,ςn(ω)  – ξ ∗(ω) dμ(ω)  ≤eL(ω)∥kn(ω)–ξ∗(ω)∥(– cn)   ςn(ω) – ξ ∗(ω) dμ(ω) + cneL(ω)∥kn(ω)–ξ∗(ω)∥  eL(ω)∥ςn(ω)–ξ∗(ω)∥   ςn(ω) – ξ ∗(ω) dμ(ω) – φ   ςn(ω) – ξ ∗(ω) dμ(ω)  ≤eL(ω)∥kn(ω)–ξ∗(ω)∥   ςn(ω) – ξ ∗(ω) dμ(ω) + cneL(ω)∥kn(ω)–ξ∗(ω)∥   ςn(ω) – ξ ∗(ω) dμ(ω) – cneL(ω)∥kn(ω)–ξ∗(ω)∥ × φ   ςn(ω) – ξ ∗(ω) dμ(ω)  . (.)   T ω,kn(ω)  – ξ ∗(ω) dμ(ω) ≤eL(ω)∥kn(ω)–ξ∗(ω)∥   kn(ω) – ξ ∗(ω) dμ(ω) – φ   kn(ω) – ξ ∗(ω) dμ(ω)  ≤eL(ω)∥kn(ω)–ξ∗(ω)∥   kn(ω) – ξ ∗(ω) dμ(ω) ≤eL(ω)∥kn(ω)–ξ∗(ω)∥  (– cn)   ςn(ω) – ξ ∗(ω) dμ(ω) + cn   T ω,ςn(ω)  – ξ ∗(ω) dμ(ω)  ≤eL(ω)∥kn(ω)–ξ∗(ω)∥(– cn)   ςn(ω) – ξ ∗(ω) dμ(ω) + cneL(ω)∥kn(ω)–ξ∗(ω)∥  eL(ω)∥ςn(ω)–ξ∗(ω)∥   ςn(ω) – ξ ∗(ω) dμ(ω) – φ   ςn(ω) – ξ ∗(ω) dμ(ω)  ≤eL(ω)∥kn(ω)–ξ∗(ω)∥   ςn(ω) – ξ ∗(ω) dμ(ω) + cneL(ω)∥kn(ω)–ξ∗(ω)∥   ςn(ω) – ξ ∗(ω) dμ(ω) – cneL(ω)∥kn(ω)–ξ∗(ω)∥ × φ   ςn(ω) – ξ ∗(ω) dμ(ω)  . (.)   T ω,kn(ω)  – ξ ∗(ω) dμ(ω)   ≤eL(ω)∥kn(ω)–ξ∗(ω)∥   kn(ω) – ξ ∗(ω) dμ(ω) – φ   kn(ω) – ξ ∗(ω) dμ(ω)  ≤eL(ω)∥kn(ω)–ξ∗(ω)∥   kn(ω) – ξ ∗(ω) dμ(ω) ≤eL(ω)∥kn(ω)–ξ∗(ω)∥  (– cn)   ςn(ω) – ξ ∗(ω) dμ(ω) + cn   T ω,ςn(ω)  – ξ ∗(ω) dμ(ω)  ≤eL(ω)∥kn(ω)–ξ∗(ω)∥(– cn)   ςn(ω) – ξ ∗(ω) dμ(ω) ∗  ∗  × φ   ςn(ω) – ξ ∗(ω) dμ(ω)  . in Lemma ., it follows that conditions of Lemma .are satisfied. Hence in Lemma ., it follows that conditions of Lemma .are satisfied. Hence lim n→∞   ξn(ω) – ξ ∗(ω) dμ(ω) = . lim n→∞   ξn(ω) – ξ ∗(ω) dμ(ω) = . (.) □ (.) □ Remark .Theorem .improves and generalizes the results of Akewe and Okeke [], Akewe et al. [], Berinde [, ], Olatinwo [], Zhang et al. [] and Rhoades [–]. Remark .Theorem .improves and generalizes the results of Akewe and Okeke [], Akewe et al. [], Berinde [, ], Olatinwo [], Zhang et al. [] and Rhoades [–]. Now we obtain the following theorem as a special case of Theorem .. Theorem .Let (E,∥· ∥) be a separable Banach space, T :  × E →E be a generalized φ-weakly contractive-type random operator with F(T) ̸= ∅, and {ξn} be a random iterative sequence as defined in (.) where {an} is a real sequence in (,) such that ∞ n=an = ∞. Then the random fixed point ξ ∗of T is Bochner integrable. Theorem .Let (E,∥· ∥) be a separable Banach space, T :  × E →E be a generalized φ-weakly contractive-type random operator with F(T) ̸= ∅, and {ξn} be a random iterative sequence as defined in (.) converging strongly to the random fixed ξ ∗of T almost surely, where {an} and {cn} are real sequences in (,) such that < a ≤an and < c ≤cn (n ≥). Then {ξn}∞ n=is almost surely T-stable. Proof Let {ςn}∞ n=be any sequence of random variables and ϵn(ω)  = ςn+(ω) – (– an)ςn(ω) – anT ω,kn(ω) , n = ,,..., (.) (.) Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Page 7 of 11 where kn(ω) = (– cn)ςn(ω) + cnT(ω,ςn(ω)) and limn→∞   ∥ϵn(ω)∥dμ(ω) = . Next, we show that ξ ∗(ω) is Bochner integrable with respect to the sequence {ςn(ω)}∞ n=. From (.), we obtain   ςn+(ω) – ξ ∗(ω) dμ(ω) ≤   ςn+(ω) – (– an)ςn(ω) – anT ω,kn(ω) dμ(ω) + (– an)   ςn(ω) – ξ ∗(ω) dμ(ω) + an   T ω,kn(ω)  – ξ ∗(ω) dμ(ω) ≤   ϵn(ω) dμ(ω) +   ςn(ω) – ξ ∗(ω) dμ(ω) + an   T ω,kn(ω)  – ξ ∗(ω) dμ(ω). (. + an   T ω,kn(ω)  – ξ ∗(ω) dμ(ω). in Lemma ., it follows that conditions of Lemma .are satisfied. Hence (.) (.) By (.) in (.), we obtain that By (.) in (.), we obtain that   ςn+(ω) – ξ ∗(ω) dμ(ω) ≤   ϵn(ω) dμ(ω) +   ςn(ω) – ξ ∗(ω) dμ(ω) + aneL(ω)∥kn(ω)–ξ∗(ω)∥   ςn(ω) – ξ ∗(ω) dμ(ω)   ςn+(ω) – ξ ∗(ω) dμ(ω) ≤   ϵn(ω) dμ(ω) +   ςn(ω) – ξ ∗(ω) dμ(ω) + aneL(ω)∥kn(ω)–ξ∗(ω)∥   ςn(ω) – ξ ∗(ω) dμ(ω) lities and Applications ( 2015) 2015:146 Page 8 of 11 Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Page 8 of 11 Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Page 8 of 11 Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Page 8 of 11 Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Page 8 of 11 Page 8 of 11 + ancneL(ω)∥kn(ω)–ξ∗(ω)∥   ςn(ω) – ξ ∗(ω) dμ(ω) – ancneL(ω)∥kn(ω)–ξ∗(ω)∥φ   ςn(ω) – ξ ∗(ω) dμ(ω)  =   ϵn(ω) dμ(ω) + + aneL(ω)∥kn(ω)–ξ∗(ω)∥ + ancneL(ω)∥kn(ω)–ξ∗(ω)∥  ςn(ω) – ξ ∗(ω) dμ(ω) – ancneL(ω)∥kn(ω)–ξ∗(ω)∥ × φ   ςn(ω) – ξ ∗(ω) dμ(ω)  . (.) + ancneL(ω)∥kn(ω)–ξ∗(ω)∥   ςn(ω) – ξ ∗(ω) dμ(ω) – ancneL(ω)∥kn(ω)–ξ∗(ω)∥φ   ςn(ω) – ξ ∗(ω) dμ(ω)  =   ϵn(ω) dμ(ω) + + aneL(ω)∥kn(ω)–ξ∗(ω)∥ + ancneL(ω)∥kn(ω)–ξ∗(ω)∥  ςn(ω) – ξ ∗(ω) dμ(ω) – ancneL(ω)∥kn(ω)–ξ∗(ω)∥ × φ   ςn(ω) – ξ ∗(ω) dμ(ω)  . (.) (.) Using the conditions that limn→∞   ∥ϵn(ω)∥dμ(ω) = , < a ≤an and < c ≤cn (n ≥), we have Using the conditions that limn→∞   ∥ϵn(ω)∥dμ(ω) = , < a ≤an and < c ≤cn (n ≥), we have lim n→∞   ∥ϵn(ω)∥dμ(ω) ancn ≤lim n→∞   ∥ϵn(ω)∥dμ(ω) ac = . (.) (.) Take λn = (+ aneL(ω)∥kn(ω)–ξ∗(ω)∥+ ancneL(ω)∥kn(ω)–ξ∗(ω)∥)   ∥ςn(ω) – ξ ∗(ω)∥dμ(ω), σn = ancneL(ω)∥kn(ω)–ξ∗(ω)∥, γn =   ∥ϵn(ω)∥dμ(ω). Note that all the conditions in Lemma . are satisfied. Therefore, we obtain Take λn = (+ aneL(ω)∥kn(ω)–ξ∗(ω)∥+ ancneL(ω)∥kn(ω)–ξ∗(ω)∥)   ∥ςn(ω) – ξ ∗(ω)∥dμ(ω), σn = ancneL(ω)∥kn(ω)–ξ∗(ω)∥, γn =   ∥ϵn(ω)∥dμ(ω). Note that all the conditions in Lemma . are satisfied. Therefore, we obtain lim n→∞   ςn(ω) – ξ ∗(ω) dμ(ω) = . in Lemma ., it follows that conditions of Lemma .are satisfied. Hence (.) (.) Conversely, if ξ ∗(ω) is Bochner integrable with respect to the sequence {ςn(ω)}∞ n=, we have Conversely, if ξ ∗(ω) is Bochner integrable with respect to the sequence {ςn(ω)}∞ n=, we have   ϵn(ω) dμ(ω) =   ςn+(ω) – (– an)ςn(ω) – anT ω,kn(ω) dμ(ω) ≤   ςn+(ω) – ξ ∗(ω) dμ(ω) + (– an)   ξ ∗(ω) – ςn(ω) dμ(ω) + an   ξ ∗(ω) – T ω,kn(ω) dμ(ω). (.) + an   ξ ∗(ω) – T ω,kn(ω) dμ(ω). (.) (.) Using (.), we have   ξ ∗(ω) – T ω,kn(ω) dμ(ω)   ξ ∗(ω) – T ω,kn(ω) dμ(ω) =   T ω,ξ ∗(ω)  – T ω,kn(ω) dμ(ω) ≤eL(ω)∥ξ∗(ω)–kn(ω)∥   ξ ∗(ω) – kn(ω) dμ(ω) – φ   ξ ∗(ω) – kn(ω) dμ(ω)  ≤eL(ω)∥ξ∗(ω)–kn(ω)∥   ξ ∗(ω) – kn(ω) dμ(ω) ≤eL(ω)∥ξ∗(ω)–kn(ω)∥  (– cn)   ξ ∗(ω) – ςn(ω) dμ(ω) =   T ω,ξ ∗(ω)  – T ω,kn(ω) dμ(ω) Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Page 9 of 11 + cn   T ω,ξ ∗(ω)  – T ω,ςn(ω) dμ(ω)  ≤eL(ω)∥ξ∗(ω)–kn(ω)∥  (– cn)   ξ ∗(ω) – ςn(ω) dμ(ω) + cn  eL(ω)∥ξ∗(ω)–ςn(ω)∥   ξ ∗(ω) – ςn(ω) dμ(ω) – φ   ξ ∗(ω) – ςn(ω) dμ(ω)  ≤(– cn)eL(ω)∥ξ∗(ω)–kn(ω)∥   ξ ∗(ω) – ςn(ω) dμ(ω) + cneL(ω)∥ξ∗(ω)–kn(ω)∥   ξ ∗(ω) – ςn(ω) dμ(ω) – cneL(ω)∥ξ∗(ω)–kn(ω)∥φ   ξ ∗(ω) – ςn(ω) dμ(ω)  ≤ eL(ω)∥ξ∗(ω)–kn(ω)∥+ cneL(ω)∥ξ∗(ω)–kn(ω)∥ ×   ξ ∗(ω) – ςn(ω) dμ(ω) – cneL(ω)∥ξ∗(ω)–kn(ω)∥ × φ   ξ ∗(ω) – ςn(ω) dμ(ω)  . (.) Using (.) in (.), we have Using (.) in (.), we have Using (.) in (.), we have   ϵn(ω) dμ(ω) ≤   ςn+(ω) – ξ ∗(ω) dμ(ω) +   ξ ∗(ω) – ςn(ω) dμ(ω) + an eL(ω)∥ξ∗(ω)–kn(ω)∥+ cneL(ω)∥ξ∗(ω)–kn(ω)∥ ×   ξ ∗(ω) – ςn(ω) dμ(ω) – ancneL(ω)∥ξ∗(ω)–kn(ω)∥ × φ   ξ ∗(ω) – ςn(ω) dμ(ω)  =   ςn+(ω) – ξ ∗(ω) dμ(ω) + + aneL(ω)∥ξ∗(ω)–kn(ω)∥ + ancneL(ω)∥ξ∗(ω)–kn(ω)∥  ξ ∗(ω) – ςn(ω) dμ(ω) – ancneL(ω)∥ξ∗(ω)–kn(ω)∥φ   ξ ∗(ω) – ςn(ω) dμ(ω)  . We now state the following theorem as a special case of Theorem .. References 1. Joshi, MC, Bose, RK: Some Topics in Nonlinear Functional Analysis. Wiley Eastern, New Delhi (1985) p y y 2. Zhang, SS: Fixed Point Theory and Applications. Chongqing Publishing Press, Chongqing (1984) (i g y g g g 3. Spacek, A: Zufallige gleichungen. Czechoslov. Math. J. 5, 462-466 (1955) 4. Hans, O: Random operator equations. In: Proceedings of the Fourth Berkeley Symposium on Mathematical Statistics 4. Hans, O: Random operator equations. In: Proceedings of the Fourth Berkeley Symposium on Mathematical Stati and Probability Vol II, Part I, pp 185-202 University of California Press, California (1961) 4. Hans, O: Random operator equations. In: Proceedings of the Fourth Berkeley Symposium on M and Probability. Vol. II, Part I, pp. 185-202. University of California Press, California (1961) 4. Hans, O: Random operator equations. In: Proceedings of the Fourth Berkeley Symposium on Mathema and Probability. Vol. II, Part I, pp. 185-202. University of California Press, California (1961) y y 5. Itoh, S: Random fixed point theorems with an application to random differential equations in Banach spaces. J. Math. Anal. Appl. 67(2), 261-273 (1979) 6. Bharucha-Reid, AT: Fixed point theorems in probabilistic analysis. Bull. Am. Math. Soc. 82, 641-657 (1976) 6. Bharucha-Reid, AT: Fixed point theorems in probabilistic analysis. Bull. Am. Math. Soc. 82, 641-657 (1976) 7. Arunchai, A, Plubtieng, S: Random fixed point of Krasnoselskii type for the sum of two operators. Fixed Point Theory Appl. 2013, Article ID 142 (2013) 8. Beg, I, Abbas, M: Equivalence and stability of random fixe Article ID 23297 (2006). doi:10.1155/JAMSA/2006/23297 8. Beg, I, Abbas, M: Equivalence and stability of random fixed point iterative procedures. J. Appl. Math. Stoch. Anal. 2006 Article ID 23297 (2006) doi:10 1155/JAMSA/2006/23297 8. Beg, I, Abbas, M: Equivalence and stability of random fixe Article ID 23297 (2006). doi:10.1155/JAMSA/2006/23297 9. Beg, I, Abbas, M: Random fixed point theorems for Caristi type random operators. J. Appl. Math. Comput. 25(1-2), 425-434 (2007) ( ) 10. Beg, I, Abbas, M, Azam, A: Periodic fixed points of random operators. Ann. Math. Inform. 37, 39-49 (2010) 11. Chang, SS, Cho, YJ, Kim, JK, Zhou, HY: Random Ishikawa iterative sequence with applications. Stoch. Anal. Appl. 23, 69-77 (2005) ( ) 12. Moore, C, Nnanwa, CP, Ugwu, BC: Approximation of common random fixed points of finite families of N-uniformly 12. Author details 1 Author details 1Department of Mathematics, College of Science and Technology, Covenant University, Canaanland, KM 10 Idiroko Road, P.M.B. 1023, Ota, Ogun State, Nigeria. 2Department of Mathematics and Applied Mathematics, University of Pretoria, Pretoria, 0002, South Africa. 3Department of Mathematics, LUMS, Lahore, Pakistan. Authors’ contributions Authors’ contributions Both authors contributed equally to this work Both authors read and approved the final manuscript Authors’ contributions Both authors contributed equally to this work. Both authors read and approved the final manuscript. Both authors contributed equally to this work. Both authors read and approved the final manuscript. in Lemma ., it follows that conditions of Lemma .are satisfied. Hence (.   ϵn(ω) dμ(ω) ≤   ςn+(ω) – ξ ∗(ω) dμ(ω) +   ξ ∗(ω) – ςn(ω) dμ(ω) + an eL(ω)∥ξ∗(ω)–kn(ω)∥+ cneL(ω)∥ξ∗(ω)–kn(ω)∥ ×   ξ ∗(ω) – ςn(ω) dμ(ω) – ancneL(ω)∥ξ∗(ω)–kn(ω)∥ × φ   ξ ∗(ω) – ςn(ω) dμ(ω)   ∗ ×   ξ ∗(ω) – ςn(ω) dμ(ω) – ancneL(ω)∥ξ∗(ω)–kn(ω)∥ × φ   ξ ∗(ω) – ςn(ω) dμ(ω)  =   ςn+(ω) – ξ ∗(ω) dμ(ω) + + aneL(ω)∥ξ∗(ω)–kn(ω)∥ =   ςn+(ω) – ξ ∗(ω) dμ(ω) + + aneL(ω)∥ξ∗(ω)–kn(ω)∥ + ancneL(ω)∥ξ∗(ω)–kn(ω)∥  ξ ∗(ω) – ςn(ω) dμ(ω) – ancneL(ω)∥ξ∗(ω)–kn(ω)∥φ   ξ ∗(ω) – ςn(ω) dμ(ω)  . (.) Hence lim n→∞   ϵn(ω) dμ(ω) = . (.) □ lim n→∞   ϵn(ω) dμ(ω) = . (.) Remark .Theorem .generalizes and improves the results of Zhang et al. [], Berinde [, ], Olatinwo [], Rhoades [–] and several others in the literature. Remark .Theorem .generalizes and improves the results of Zhang et al. [], Berinde [, ], Olatinwo [], Rhoades [–] and several others in the literature. We now state the following theorem as a special case of Theorem .. We now state the following theorem as a special case of Theorem .. Page 10 of 11 Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 Theorem .Let (E,∥· ∥) be a separable Banach space, T :  × E →E be a generalized φ-weakly contractive-type random operator with F(T) ̸= ∅, and {ξn} be a random iterative sequence as defined in (.) converging strongly to the random fixed ξ ∗of T almost surely, where {an} is a real sequence in (,) such that < a ≤an (n ≥). Then {ξn}∞ n=is almost surely T-stable. 4 Example p Example .Consider the following nonlinear stochastic integral equation: p Example .Consider the following nonlinear stochastic integral equation: (ξ;ω) =  ∞  (– t)e|ξ(t;ω)–ς(t;ω)| (+ |ξ(s;ω)|) ds ≤e|ξ(t;ω)–ς(t;ω)|  ∞   (+ |ξ(s;ω)|) ds – t  ∞   (+ |ξ(s;ω)|) ds  . (.) From (.), we see that (.) is satisfied with φ(t) = t. (ξ;ω) =  ∞  (– t)e|ξ(t;ω)–ς(t;ω)| (+ |ξ(s;ω)|) ds ≤e|ξ(t;ω)–ς(t;ω)|  ∞   (+ |ξ(s;ω)|) ds – t  ∞   (+ |ξ(s;ω)|) ds  . (.) (.) From (.), we see that (.) is satisfied with φ(t) = t. From (.), we see that (.) is satisfied with φ(t) = t. From (.), we see that (.) is satisfied with φ(t) = t. Competing interests Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. Acknowledgements c o edge e ts The authors wish to thank the anonymous referees for their comments. The first author is grateful to the Covenant University Centre for Research and Development (CUCERD) for supporting his research. Received: 6 November 2014 Accepted: 15 April 2015 Received: 6 November 2014 Accepted: 15 April 2015 Okeke and Abbas Journal of Inequalities and Applications ( 2015) 2015:146 References Moore, C, Nnanwa, CP, Ugwu, BC: Approximation of common random fixed points of finite families of N-uniformly Li-Lipschitzian asymptotically hemicontractive random maps in Banach spaces. Banach J. Math. Anal. 3(2), 77-85 (2009) Li-Lipschitzian asymptotically hemicontractive random maps in Banach spaces. Banach J. Math. Anal. 3(2), 77-85 (2009) 13. Papageorgiou, NS: Random fixed point theorems for measurable multifunctions in Banach spaces. Proc. Am. Math. Soc. 97(3), 507-514 (1986) 14. Shahzad, N, Latif, S: Random fixed points for several classes of 1-ball-contractive and 1-set-contractive random maps. J. Math. Anal. Appl. 237, 83-92 (1999) J. Math. Anal. Appl. 237, 83-92 (1999) 15. Xu, HK: Some random fixed point theorems for condensing and nonexpansive operators. Proc. Am. Math. 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A generalisable integrated natural capital methodology for targeting investment in coastal defence
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Journal of Environmental Economics and Policy ISSN: 2160-6544 (Print) 2160-6552 (Online) Journal homepage: https://www.tandfonline.com/loi/teep20 A generalisable integrated natural capital methodology for targeting investment in coastal defence Katrina J. Davis, Amy Binner, Andrew Bell, Brett Day, Timothy Poate, Siân Rees, Greg Smith, Kerrie Wilson & Ian Bateman To cite this article: Katrina J. Davis, Amy Binner, Andrew Bell, Brett Day, Timothy Poate, Siân Rees, Greg Smith, Kerrie Wilson & Ian Bateman (2019) A generalisable integrated natural capital methodology for targeting investment in coastal defence, Journal of Environmental Economics and Policy, 8:4, 429-446, DOI: 10.1080/21606544.2018.1537197 To link to this article: https://doi.org/10.1080/21606544.2018.1537197 To link to this article: https://doi.org/10.1080/21606544.2018.1537197 © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 31 Oct 2018. Submit your article to this journal Article views: 1541 View related articles View Crossmark data Citing articles: 1 View citing articles p g © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 31 Oct 2018. Submit your article to this journal Article views: 1541 View related articles View Crossmark data Citing articles: 1 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=teep20 JOURNAL OF ENVIRONMENTAL ECONOMICS AND POLICY 2019, VOL. 8, NO. 4, 429–446 https://doi.org/10.1080/21606544.2018.1537197 A generalisable integrated natural capital methodology for targeting investment in coastal defence Katrina J. Davis a, Amy Binner a, Andrew Bell b, Brett Daya, Timothy Poate c, Siân Rees d, Greg Smith a, Kerrie Wilson e and Ian Bateman a Katrina J. Davis a, Amy Binner a, Andrew Bell b, Brett Daya, Timothy Poate c, Siân Rees d, Greg Smith a, Kerrie Wilson e and Ian Bateman a aLand, Environment, Economics and Policy Institute, The University of Exeter Business School, Exeter, UK; bCentre for Rural Policy Research, The University of Exeter, Exeter, UK; cSchool of Biological and Marine Sciences, University of Plymouth, Plymouth, UK; dMarine Institute, School of Biological and Marine Sciences, University of Plymouth, Plymouth, UK; eSchool of Biological Sciences and ARC Centre of Excellence for Environmental Decisions, The University of Queensland, St Lucia, Australia University of Queensland, St Lucia, Australia ABSTRACT Coastal ecosystems, such as saltmarsh, produce a range of ecosystem services that underpin human well-being. In the UK, and globally, saltmarsh extent and quality is declining due to coastal squeeze, deteriorating water quality, and agricultural activities. Here, we develop a general framework to evaluate changes in coastal defence. Using this framework, we identify priority areas for saltmarsh re-alignment: re- creation of saltmarsh in areas that have been saltmarsh in the past – but that have been claimed for a variety of land uses, particularly agriculture. We base our re-alignment prioritisation on the ecosystem services provided by saltmarsh in the North Devon Biosphere Reserve: specifically carbon sequestration and recreational benefits, and the economic values of those services. We compare potential economic benefits with the economic costs of creating new saltmarsh areas – specifically lost agricultural output, property damages and direct re- alignment costs. We identify a number of priority areas for managed re- alignment that generate high recreational values in areas where properties would not be damaged. These findings provide a necessary and timely analysis for the managers of the North Devon Biosphere Reserve. Furthermore, we outline a comprehensive methodology to plan future management of coastal zones. © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ARTICLE HISTORY Received 19 July 2018 Accepted 7 October 2018 KEYWORDS Coastal planning; ecosystem services; managed re- alignment; natural capital; opportunity costs; saltmarsh CONTACT Katrina J. Davis k.davis2@exeter.ac.uk Land, Environment, Economics and Policy Institute, Th Exeter Business School, Exeter, UK © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CONTACT Katrina J. Davis k.davis2@exeter.ac.uk Land, Environment, Economics and Policy Institute, The University of Exeter Business School, Exeter, UK 1. Introduction Marine and coastal ecosystems provide a number of essential functions, such as primary production and climate regulation, which underpin life on Earth (MEA 2005). These essential functions deliver flows of ecosystem services that support human well-being, including food, flood protection and opportunities for recreation (Roberts et al. 2001; Rees et al. 2010; Arkema et al. 2013; Potts et al. 2014; Rees et al. 2014; Arkema et al. 2015). In recognition of the crucial interdependencies between natural and human systems, targets to sustainably manage marine and coastal ecosystems are embedded in international (CBD 1992, 2010; OSPAR Convention 2002; UN 2014) and national pol- icy (Ostle et al. 2009; H.M. Government 2011, 2018). In the UK, managed re-alignment is a policy to recreate saltmarsh, intertidal grasslands, in areas where they have occurred historically, for example, K. J. DAVIS ET AL. 430 in areas converted to agriculture or other land uses (Luisetti et al. 2014). Saltmarsh produces a range of ecosystem services including carbon sequestration (Beaumont et al. 2014), recreational benefits (Barbier et al. 2011) and fisheries support services. The relevant policy question is: where should re-alignment occur, to maximise the benefits that new saltmarsh provides to society, relative to the costs of removing land from its current use? In the North Devon Biosphere Reserve (Figure 1), a programme of managed re-alignment is cur- rently being undertaken. Understanding where managed re-alignment should be prioritised is a pressing question for the Biosphere managers. In this research, we worked closely with the Biosphere managers to identify areas where the ecosystem services generated by new saltmarsh areas in the Bio- sphere would generate the greatest economic benefits, relative to the economic costs. We develop a general framework to guide the assessment of projects that involve changes in coastal defence activi- ties. As part of this framework, we describe a methodology outlining the biophysical and socio-econ- omic analyses that are necessary to conduct a complete economic assessment of potential changes in coastal defence. The application of this framework to the assessment of changes in saltmarsh extent, managed re- alignment, has focused on identifying priority areas for re-alignment. This approach contrasts with previous exercises that have asked a more general question – whether managed re-alignment can provide economic benefits. For example, Turner et al. (2007) and Luisetti et al. 1. Introduction (2011) took a spatially explicit approach to the assessment of the potential costs and benefits specific to managed re-align- ment. In particular, Luisetti et al. (2011) aimed to provide decision support by quantifying the costs and benefits of existing re-alignment areas. However, little work has been undertaken to identify pri- ority areas for future managed re-alignment. A key innovation in our prioritisation approach has been to incorporate temporally discrete carbon sequestration rates by saltmarsh, and the lost carbon sequestration values of previous land use. Previous studies (e.g. Turner et al. 2007) have assumed a single value for carbon sequestration by saltmarsh and a single carbon price, this approach ignores differences in sequestration rates following the establishment of saltmarsh. We follow best-practice described by Bateman et al. (2014) to consider changes in marginal abatement costs over time. Finally, we conduct an initial assessment of property damage caused by managed re-alignment. Our approach is to identify the full range of biophysical and socio-economic components that should be analysed for a complete assessment of changes in coastal flood defence, e.g. managed Figure 1. North Devon Biosphere Reserve (left) includes all the catchment areas draining to the north Devon coast and extends to 12 nautical miles beyond Lundy island. The different designations of the reserve: core, buffer and transition areas are indicated. South West England (right) with location of North Devon Biosphere outlined. Figure 1. North Devon Biosphere Reserve (left) includes all the catchment areas draining to the north Devon coast and extends to 12 nautical miles beyond Lundy island. The different designations of the reserve: core, buffer and transition areas are indicated. South West England (right) with location of North Devon Biosphere outlined. JOURNAL OF ENVIRONMENTAL ECONOMICS AND POLICY 431 re-alignment. Our analysis focuses on a subset of these components for which data is available – we also identify important areas for future research to overcome existing data limitations. Specifically, we evaluate the following costs and benefits associated with managed re-alignment of saltmarsh in the North Devon Biosphere: opportunity costs to agricultural production, property damages, direct re-alignment costs, carbon sequestration benefits and recreational benefits. We use data on the tidal flood frame in the North Devon Biosphere (Figure 1) to identify potential managed re-alignment areas. The opportunity costs of lost future agricultural production, capitalised in land value, are based on the agricultural land classifications (ALC, MAFF 1988) and land-sale price data (DEFRA 2006). 2. North Devon Biosphere Reserve 2. North Devon Biosphere Reserve The North Devon Biosphere Reserve (Figure 1) is one of 669 reserves worldwide designated by UNESCO’s Man and the Biosphere Programme. In total, the terrestrial extent is 233,495 ha, and the marine extent is 291,583 ha. The Biosphere contains a number of Local Nature Reserves, Sites of Special Scientific Interest and Special Areas of Conservation and the majority of the coast is desig- nated as an Area of Outstanding Natural Beauty. Collectively, these designations make up the differ- ent zones of the reserve: core, buffer and transition zones (see Figure 1). The historical extent of saltmarsh areas in the biosphere is estimated at 968.8 ha, while the current extent is 230.7 ha. 1. Introduction We use ORVAL (Day and Smith 2018b) to estimate recreational benefits, land use data from Bateman et al. (2013), and the CoolFarm Tool (Hillier et al. 2011) to estimate carbon sequestration benefits. Candidate re-alignment sites are prioritised by assessing the annualised costs and benefits of conversion to saltmarsh, adjusted to 2016 prices. j p Using an integrated natural capital methodology, we identify priority areas for saltmarsh re-align- ment. We identify four sites within the North Devon Biosphere Reserve that are prioritised for man- aged re-alignment under three assumptions about how property damages could be treated: ignoring property damages, excluding sites with properties from the analysis, and including an initial assess- ment of property damages into our assessment. Incorporating property damages changes our prior- itisation, and reduces the annualised net present value of new re-alignment areas by 17%. In what follows we describe the current extent of saltmarsh areas in North Devon and the selection process we followed to identify potential re-alignment sites. We describe the costs of managed re-alignment estimated for each site, the ecosystem services provided by saltmarsh, and the economic values of these services. We finish by identifying priority areas for managed re-alignment in the North Devon Biosphere, and exploring the sensitivity of results to different assumptions regarding the treatment of property damages. 3. Methods We present a conceptual framework defining key areas of consideration in evaluating potential coastal defence projects (Figure 2). While applicable to the assessment of any coastal defence project, in the present analysis we focus on priority areas for saltmarsh managed-re-alignment. We further provide a complete description of how a sites’ geomorphology and tidal dynamics could be assessed to understand whether a site would be suitable for managed re-alignment – although this assessment is beyond the current scope of this research. In our analysis, we address a component of the evalu- ation problem described in Figure 2; putting to one side the framework steps that assess climate con- ditions, changes in socio-economic drivers and the estuary regime. All analyses are conducted in R (R Core Team 2018) using applied spatial data analysis methods from packages “rgeos”, “sp”, “ras- ter” and “rgdal” (Pebesma and Bivand 2005; Bivand, Pebesma, and Gomez-Rubio 2013; Brunsdon and Chen 2014; Hijmans 2017; Bivand and Rundel 2018; Bivand, Keitt, and Rowlingson 2018). Our approach is based on work conducted by Turner et al. (2007) and Luisetti et al. (2011) – and focuses on the costs and potential ecosystem service benefits that could be generated by returning 432 K. J. DAVIS ET AL. 432 Figure 2. Conceptual framework to evaluate potential coastal defence projects, including managed re-alignment of saltmarsh. . Conceptual framework to evaluate potential coastal defence projects, including managed re-alignment of saltma areas in North Devon to saltmarsh. Each of these costs and benefits is discussed in more detail in the following sections. areas in North Devon to saltmarsh. Each of these costs and benefits is discussed in more detail in the following sections. 3.1. Geomorphology and tidal hydrodynamics A complete understanding of whether a site would be suitable for managed re-alignment can be achieved by evaluating its geomorphology and tidal hydrodynamics. The elevation and Figure 3. Indicative UK intertidal mudflat and saltmarsh profile. Adapted from Foster et al. (2013). Tides: HAT: highest astronomical tides; MHWS: mean high water springs; MHW: mean high water; MHWN: mean high water neaps. Figure 3. Indicative UK intertidal mudflat and saltmarsh profile. Adapted from Foster et al. (2013). Tides: HAT: highest astronomical tides; MHWS: mean high water springs; MHW: mean high water; MHWN: mean high water neaps. JOURNAL OF ENVIRONMENTAL ECONOMICS AND POLICY 433 geomorphology of a site is a crucial factor in helping establish a healthy saltmarsh. A network of creeks across the site is fundamental in providing sediment transport pathways into the saltmarsh, facilitating sediment deposition and saltmarsh aggradation. The network of channels also helps regu- late tidal flows by increasing frictional drag, more conducive to depositional environments. Salt- marsh habitats are found high in the tidal frame (Figure 3) and consequently, their colonisation is closely linked to the tidal inundation of a site. The tidal prism (i.e. volume of tidal water exchange passing a given point in an estuary) at a location determines the frequency and duration of inundation. This, in turn, impacts sedimentation, salinity, soil redox potential and propagule delivery to the site (Mossman, Davy, and Grant 2012; Mossman et al. 2012; Spencer and Harvey 2012). While consensus on the optimum inundation regime is lacking, Table 1 provides a summary of the habitat types most likely to colonise a site based on elevation within the tidal range. Depending on the site, the method of habitat creation can be tailored to maximise the optimum geomorphological and hydrodynamic conditions required. A Regulated Tidal Exchange allows control over inundation rates, to ensure – through careful man- agement – tidal flows across the site are suitable. This technique requires close monitoring as biofoul- ing and mechanical faults can result in poor inundation rates, limiting scheme success (Masselink et al. 2017). Where coastal defence is not a factor for the site, barrier breaches can be undertaken, providing a more natural channel to match local tide levels. Further, full bank retreats that remove the entire structure are a less controlled but more naturalistic approach. 3.1. Geomorphology and tidal hydrodynamics Consequently, the physical parameters of a selected area, in conjunction with the tidal hydrodynamics and the proximity of neighbouring marsh, will all need to be carefully considered for a successful re-alignment site. 3.2. Identifying candidate managed re-alignment sites We identified candidate areas for managed re-alignment from data provided by the Environment Agency (n.d.). Using ordnance survey mapping products and Light Detection and Ranging (LIDAR) land surface information, these data identify historic saltmarsh areas that have been sub- sequently claimed for other land use. In particular, ‘landclaim’ area is identified as any location below the highest astronomical tide that is adjacent to the estuary and sitting behind an artificial flood defence. Examination of the landclaim area in the North Devon Biosphere Reserve identified 57 can- didate sites for managed re-alignment that ranged from 0.3 ha to 339 ha in size, with an average of 15.32 ha. 3.3. Economic costs of managed re-alignment sites A full assessment of managed re-alignment must count all service flows (market and non-market) coming from current land use as a cost. The change in ecosystem services delivered by existing land uses – relative to potential saltmarsh areas – must be captured to appropriately assess whether there will be a net gain in the economic value of ecosystem service provision under re-alignment. Here we focus on lost agricultural output, property damages, and the direct costs of re-alignment. Table 1. Summary of optimum hydrodynamic conditions for intertidal habitat generation. Habitat type Site gradient Annual inundation p/yr Tidal range Mudflats 1–3% 450–600 Between MHWN and MHWS, <2.1 m ODN Saltmarsh (salinity > 10) 1–3% Pioneer marsh = 300–450 Lower marsh = 30–300 Upper marsh = <30 Transitional marsh ∼MHWN ∼MHW ∼MHWS MHWS – HAT Note: MHWN: mean high water neaps; MHWS: mean high water springs; HAT: highest astronomical tides; ODN: Ordnance Datum Newlyn, a mean sea level datum in the UK. Source: Environment Agency (2003), Nottage and Robertson (2005). able 1. Summary of optimum hydrodynamic conditions for intertidal habitat generation. abitat t pe Site gradient Ann al in ndation p/ r Ti K. J. DAVIS ET AL. 434 In all cases, there is an upfront cost associated with re-alignment. In the context of our analysis, we consider these costs as occurring in year 1, but annualise these values for comparison with the benefits of re-alignment. Our calculation of annualised net present value is derived from the equiv- alent annual annuity formula: In all cases, there is an upfront cost associated with re-alignment. In the context of our analysis, we consider these costs as occurring in year 1, but annualise these values for comparison with the benefits of re-alignment. Our calculation of annualised net present value is derived from the equiv- alent annual annuity formula: (NPV × r) 1 −(1 + r)−n (1) (1) where r is the discount rate, and n is the number of periods. With an infinite time horizon, as is the case in our analysis where we assume a stream of recreational benefits that continue indefinitely, then this equation collapses to: where r is the discount rate, and n is the number of periods. 3.3. Economic costs of managed re-alignment sites With an infinite time horizon, as is the case in our analysis where we assume a stream of recreational benefits that continue indefinitely, then this equation collapses to: x = NPV × r (2) (2) x = NPV × r (2) x = NPV × r where x is the annualised net present value. 3.3.1. Opportunity cost to agricultural production 3.3.1. Opportunity cost to agricultural production 3.3.1. Opportunity cost to agricultural production 3.3.2. Implications for flood risk and property losses A major consideration in projects assessing changes in coastal defences are potential changes in the risk of flooding to which properties are exposed. To fully understand the economic costs (or benefits) of this change, we would ideally have a high-resolution digital terrain model with property location data – interacting with a flood inundation model to calculate the probabilities of flooding. We would then apply those probabilities to published data on flood damage costings (Penning-Rowsell et al. 2013). In the absence of such information, we take a simplified approach in this case study, which examines the direct property loss that arises when sites are flooded to re-create saltmarsh. Understanding how changes to coastal defence changes flood risk (either increased or decreased) in properties neighbouring candidate re-alignment sites is identified as an important area for further research. In our approach, we draw on high-resolution data on property locations (Ordnance Survey 2017b) and potential saltmarsh extent. By interpreting this information, we can make assumptions about the economic impacts of managed re-alignment on properties. This implies that there can be no simple, single answer to the prioritisation of saltmarsh re-creation, rather we show that there are different ways of assessing changes to coastal defence that yield different results. We examine three scenarios with different treatment of direct property damage. Scenario 1: Ignoring property damage – equivalent to assuming that these damages will be zero. This is an extreme approach where we ignore property impacts. We note that, despite the outcomes of this assessment, where managed re-alignment would flood private properties, it is unlikely that these sites would be realistic candidates for future re-alignment. Scenario 2: Excluding all candidate managed re-alignment sites with properties within their bounds. This gives us a new prioritisation – a second extreme where any property impacts are con- sidered unacceptable. Scenario 3: Incorporating property losses. In this scenario, we take a simplified approach to esti- mating property losses incurred if sites with properties were flooded (converted to saltmarsh). First, for every site, we identify the number of properties within the site. Then, we take an average property value (H.M. Land Registry 2018) for each sites’ postcode(s) (Ordnance Survey 2017a) and set the damage costs for each site equal to the number of properties in the site multiplied by the average property value for the postcode(s). 3.3.1. Opportunity cost to agricultural production We assume that, with a well-functioning land market, the selling price at which a landowner would be willing to trade productive agricultural land will be the net present value of the flow of future profits from that land. As such, land prices provide a guide to the value of the agricultural output emanating from that land. To calculate the land prices for each of our managed re-alignment sites, we used spatially explicit data on ALC grades (MAFF 1988) and sale price data specific to those grades (DEFRA 2006) (Figure 4)1. The ALC framework classifies land according to the extent to which its physical or chemical characteristics impose long-term limitations on agricultural use (MAFF 1988). The principal physical factors influencing agricultural production are climate, site and soil. These factors, together with their interactions, form the basis for classifying land into one of five ranked grades: from Grade 1 land being of excellent quality down to Grade 5 land of very poor quality (MAFF 1988). Figure 4. Spatial distribution of ranked agricultural land classifications (MAFF 1988) in the North Devon Biosphere Reserve. Grade 1 land is classified as excellent quality while Grade 5 land is classified as very poor quality. Overlap with candidate saltmarsh re-align- ment areas is indicated. Figure 4. Spatial distribution of ranked agricultural land classifications (MAFF 1988) in the North Devon Biosphere Reserve. Grade 1 land is classified as excellent quality while Grade 5 land is classified as very poor quality. Overlap with candidate saltmarsh re-align- ment areas is indicated. JOURNAL OF ENVIRONMENTAL ECONOMICS AND POLICY 435 We identified the spatial extent of each ALC grade in each of our 57 sites. Following Turner et al. (2007), we calculated opportunity costs by identifying the proportion of each site in each ALC grade, then multiplying this proportion by the sale price specific to the land grade and summing all areas. All values were converted to 2016 prices using the GDP deflators published by H.M. treasury (H.M. Treasury 2018). We then calculated the annualised stream of costs for comparison with other econ- omic costs (e.g. expected property damages and direct costs) and benefits (e.g. recreational and car- bon sequestration) as per Equation (2). We use a private discount rate, set at 2.5% in line with the 2016 Bank of England interest rate (Bank of England 2018). 3.3.2. Implications for flood risk and property losses Once we convert this figure to an annualised stream (see Equation 1), we obtain a cost value of property losses if sites were converted to saltmarsh that we can compare with other economic costs and benefits. This allows us to perform another prioritisation exercise with an initial estimate of the economic costs of property losses due to managed re-alignment. Note that a further approach, beyond the scope of the current exercise, could consider stress related to flooding (Tapsell et al. 2002) – as experienced by property owners. This approach is rel- evant in cases where (a) managed retreat increases the probability of flooding or (b) managed retreat requires the compulsory purchase of properties that are then abandoned or cleared to allow flooding (Penning-Rowsell et al. 2013). This approach would move the prioritisation back towards Scenario 2. 3.4.1. Recreational benefits To estimate spatially explicit recreational values, we utilise ORVal (the Outdoor Recreational Valua- tion tool) (Day and Smith 2018b). ORVal estimates visitation to existing or newly created green spaces across the whole of England and Wales and derives monetary estimates of the value house- holds attach to the recreational opportunities provided by those green spaces. ORVal has recently been incorporated into the UK Treasury’s Green Book – the government’s guidance for project appraisal and evaluation (H.M. Treasury 2018) and features in the government’s 25-Year Environ- ment Plan (H.M. Government 2018). The recreation demand model that underpins ORVal is a Random Utility Model (RUM) using a cross-nested multinomial logit specification estimated on data drawn from the Monitor of Engage- ment with the Natural Environment (MENE) survey (Natural England 2017). The ORVal recreation demand model allows for three different dimensions of choice: (i) whether to take an outdoor recrea- tion trip on a particular day, (ii) whether to walk or drive to a recreation site when taking a trip and (iii) which particular site to visit (for full details of the ORVal modelling, see Day and Smith 2017, 2018a). The fundamental assumption of the ORVal model is that the choices observed in the MENE data are welfare-maximising. So, when an individual is observed to have taken a trip to enjoy green- space, it is assumed that the welfare of taking a trip at that time exceeds the welfare of doing some- thing entirely different. Likewise, when an individual is observed to have chosen a visit to one particular recreational site, it is assumed that the welfare derived from that visit exceeds the welfare that would be enjoyed from visiting an alternative site. Ultimately, ORVal makes probabilistic predictions about how likely it is that people with particu- lar characteristics in particular locations visit a particular greenspace given the characteristics of the greenspaces available and the cost of travelling to them. For estimating the recreation value of new sites, the model adds that new site to each individual’s set of potential choices and calculates how much welfare each gains from that additional possible trip location. The total welfare value of that new site is calculated by summing up those welfare gains for each adult across England and Wales over the course of a year. 3.3.3. Direct costs Published estimates of the direct costs of saltmarsh re-alignment vary greatly across different regions and re-alignment projects. For example, Economics for the Environment Consultancy Ltd, eftec K. J. DAVIS ET AL. K. J. DAVIS ET AL. 436 (2015b) refers to costs as high as £50,000 per hectare for ‘intertidal habitat creation’ in the UK. Fol- lowing published guidelines by Hudson et al. (2015), we assume a direct cost for re-alignment ‘with- out major new defence construction’ of £15,000 per ha. This estimate was consistent with the experience of the Biosphere managers regarding previous re-alignment projects. 3.4. Economic benefits of managed re-alignment sites In this analysis, we focus on two economic benefits of managed re-alignment: recreational and car- bon sequestration benefits. We calculate an annual stream of recreational benefits that are assumed to continue indefinitely. These benefits are annualised following Equation (2). For carbon, we calcu- late an annual stream of benefits across a 20-year period. This benefit stream is then annualised fol- lowing Equation (2). 3.4.2. Carbon sequestration 3.4.2. Carbon sequestration We compared annual carbon sequestration rates for each potential managed re-alignment site under current land use versus saltmarsh. To estimate the annual carbon sequestration rates of existing land use we first identified existing land use from a data set (Bateman et al. 2013) describing the percen- tage of area at a resolution of 2 km grid squares (400 ha) attributed to the following land use cat- egories: temporary grassland, permanent grassland, rough grazing, root crops, cereals and ‘other’. Carbon emissions from these different land use categories were then estimated using the ‘CoolFarm Tool’ (Hillier et al. 2011). The CoolFarm Tool incorporates data on soil types and climate to estimate carbon emissions under different land uses. We calculated the annual carbon emissions in each site under current land use. Where sites were located outside of the 2 km grid, we assumed that the emis- sions would match emission from the ‘nearest neighbour’ grid cell. We further calculated the carbon stock in each site under existing land use based on previous UK estimates (Ostle et al. 2009). We assumed that this entire stock of carbon would be released upon conversion to saltmarsh – a con- servative assessment. The carbon sequestration benefits of new saltmarsh areas for sites less than 15 years old (4 tCO2 yr−1) and established sites (2 tCO2 yr−1) were estimated using the method followed by (Economics for the Environment Consultancy Ltd, eftec 2017). We based our valuation of carbon sequestration benefits on work by Bateman et al. (2014) and calculated the costs of carbon emis- sions using an estimate of marginal abatement costs (untraded). In addition, we estimated the time it would take saltmarsh to reach ‘equilibrium’, e.g. to have stabilised carbon and no longer be sequestering this from the atmosphere, at 20 years and this became the project time horizon. It is worth noting that where landward migration is not prevented by the presence of sea walls, saltmarsh could continue to accrete as sea level rises. This would imply that saltmarsh could con- tinue to secrete carbon indefinitely. Our approach, therefore, represents a lower-bound estimate of the carbon sequestration benefits of managed re-alignment. We discounted all benefits and costs across this time horizon to calculate the net present value. We then annualised net present value as per Equation (2). 3.4.1. Recreational benefits y The online ORVal tool (version 2.0) available at http://leep.exeter.ac.uk/orval (accessed on 12 May 2018) was used to calculate the value that might be realised if each of the 57 potential re-align- ment sites was opened up to recreation. The details of the re-alignment sites were inputted into the ORVal tool, the centroid was used as the location and the sites were defined as ‘path’ features with the length of the path approximated based on the size of the site and the potential length of new high tide boundary. Finally, the sites were assigned land covers of 50% saltmarsh and 50% agriculture with an estuary water margin equal to the path length. The ORVal tool allows the travel cost calculations to be either ‘crude’ (straight line distances), ‘good’ (road networks) or ‘exact’ (road and path net- works). In this analysis, the ‘exact’ method was used to allow for accurate costs to be calculated for both walking and driving recreation visits. All recreational values are outputted from ORVal in 2016 prices. JOURNAL OF ENVIRONMENTAL ECONOMICS AND POLICY 437 3.4.2. Carbon sequestration 4. Results We identify priority sites for saltmarsh managed re-alignment in the North Devon Biosphere Reserve, based on an assessment of lost agricultural output, potential property damages, direct re- alignment costs, changes in carbon sequestration benefits and the generation of recreational benefits. In Figure 5, we present priority sites for re-alignment under three scenarios regarding willingness to accept property damages: (1) ignoring property damages; (2) excluding potential sites where prop- erties were located; and (3) accounting for a basic assessment of property damages. The top site prioritised for re-alignment in Scenario 1 is site 41 with an annualised net present value of £185,217. In Scenarios 2 and 3, the optimal site for managed re-alignment is site 49, with an annual- ised net present value of £152,408. It is worth noting that potential annualised property damages in site 41 – the site with the highest annualised net present value when property damages are ignored – is £382,666: implying that the annualised cost of ignoring property damages when prioritising man- aged re-alignment would be £230,259. In Scenarios 1 and 2, recreational values are a primary driver of the prioritisation (Figure 6). In Scenario 1, prioritisation is also given to sites with low opportunity costs to agriculture. In Scenario 3, prioritisation is highly influenced by recreational values (Figure 6), but also by property damage costs. The distribution of annualised costs (Table 2) indicates that a few sites with large costs reduce the mean, such that the absolute value of the mean is an order of magnitude greater than the median, and approximately double the value of the third quartile. This skew is particularly apparent for property damage costs, where mean property damages are −£225 k, but the median and third quartile values are −£33 k and −£101 k, respectively. This skew is also present in the annualised benefits but to a lesser degree. Here means and medians are of the same magnitude. Across all costs and benefits, 438 K. J. DAVIS ET AL. of sites for managed re-alignment of saltmarsh across three scenarios vary damages, (2) excluding sites with properties from the analysis, and (3) inc oritisation is based on an assessment of candidate sites’ costs: opportun osts (Scenario 3), and benefits: recreational and carbon sequestration. The rcled in red and annualised net present value reported. Figure 5. 4. Results Prioritisation of sites for managed re-alignment of saltmarsh across three scenarios varying in their treatment of property damage: (1) ignoring damages, (2) excluding sites with properties from the analysis, and (3) incorporating a basic assessment of property damages. Prioritisation is based on an assessment of candidate sites’ costs: opportunity costs to agriculture, property damages and direct costs (Scenario 3), and benefits: recreational and carbon sequestration. The site with the highest annualised net present value is circled in red and annualised net present value reported. Figure 5. Prioritisation of sites for managed re-alignment of saltmarsh across three scenarios varying in their treatment of property damage: (1) ignoring damages, (2) excluding sites with properties from the analysis, and (3) incorporating a basic assessment of property damages. Prioritisation is based on an assessment of candidate sites’ costs: opportunity costs to agriculture, property damages and direct costs (Scenario 3), and benefits: recreational and carbon sequestration. The site with the highest annualised net present value is circled in red and annualised net present value reported. JOURNAL OF ENVIRONMENTAL ECONOMICS AND POLICY 439 Figure 6. Annualised recreational benefits (£2016) from saltmarsh in candidate managed re-alignment sites in the North Devon Biosphere Reserve. Figure 6. Annualised recreational benefits (£2016) from saltmarsh in candidate managed re-alignment sites in the North Devon Biosphere Reserve. Table 2. Summary of annualised costs and benefits generated by the creation of new saltmarsh areas in the North Devon Biosphere, and annualised net present value across all property damage scenarios. when assessed at the mean, the value of property damages dominate. However, closer inspection reveals that this result arises from a small number of sites with high property damages. Considering the overall distributions, it is notable that in absolute terms when assessed at the median, it is rec- reational benefits that deliver the highest values. The mean annualised net present value for Scenarios 1 and 2 is positive, but becomes negative under Scenario 3. This effect is once again due to a small number of sites with large property damage values that skew the mean downwards, as reflected in the positive median value for Scenario 3. Annualised costs (£) Annualised benefits (£) Annualised net present value (£) Opportunity costs to agriculture Property damages Direct Carbon sequestration Recreational Scenario 1 Scenario 2 Scenario 3 Min. 0 –5,513 –150 13 10,933 –124,283 15,112 –2,069,841 1st Qu. 4. Results –46 –11,025 –384 45 60,393 54,483 48,296 39,158 Median –138 –33,075 –909 130 77,553 71,819 64,214 63,020 3rd Qu. –752 –101,320 –4,015 534 120,672 117,592 87,761 84,517 Max. –52,396 –2,232,207 –152,419 11,165 186,610 185,217 152,408 152,408 Mean –1,847 –225,030 –6,896 773 89,045 81,075 71,942 –1,831 when assessed at the mean, the value of property damages dominate. However, closer inspection reveals that this result arises from a small number of sites with high property damages. Considering the overall distributions, it is notable that in absolute terms when assessed at the median, it is rec- reational benefits that deliver the highest values. The mean annualised net present value for Scenarios 1 and 2 is positive, but becomes negative under Scenario 3. This effect is once again due to a small number of sites with large property damage values that skew the mean downwards, as reflected in the positive median value for Scenario 3. when assessed at the mean, the value of property damages dominate. However, closer inspection reveals that this result arises from a small number of sites with high property damages. Considering the overall distributions, it is notable that in absolute terms when assessed at the median, it is rec- reational benefits that deliver the highest values. The mean annualised net present value for Scenarios 1 and 2 is positive, but becomes negative under Scenario 3. This effect is once again due to a small number of sites with large property damage values that skew the mean downwards, as reflected in the positive median value for Scenario 3. Across the three scenarios, there is some agreement regarding the top 10 sites that should be prioritised for re-alignment (Figure 7). Four sites are consistently prioritised across all scenarios: sites 26, 34, 47, and 49. The annualised net present value flows from these sites are all within the top quartile across the three scenarios. There are no properties in any of these sites. Conversion of site 49 to saltmarsh would not impose any opportunity costs on agricultural production, however there are small opportunity costs to agriculture (within the second quartile of annualised costs, see Table 2) in sites 26, 34 and 47. Not surprisingly, Scenarios 2 and 3 have a high degree of overlap: seven sites are in the top 10 for both scenarios. 440 K. J. DAVIS ET AL. Figure 7. 5. Discussion We identify priority sites for managed re-alignment of saltmarsh in the North Devon Biosphere Reserve. The study was developed in close consultation with the managers of the North Devon Bio- sphere Reserve and was designed to provide decision support for the prioritisation of new saltmarsh areas. Saltmarsh is rapidly degrading and decreasing in the UK and globally (Barbier et al. 2011), making managed re-alignment an environmental policy priority. At the same time, public funding for environmental programmes is limited. Therefore, new saltmarsh sites should be located in areas where they will provide the greatest benefits, relative to the costs. Here, we have focused on benefit (and cost) flows that arise as ecosystem services. Sites that were high priorities for re-alignment were sites with high recreational values, as well as low opportunity costs to agriculture (Scenario 1), and low property damage costs (Scenario 3). In general, our findings suggest that targeted re-alignment in the North Devon Biosphere can result in positive net present values. This result is in line with the analysis of natural capital invest- ment opportunities in the UK undertaken by Economics for the Environment Consultancy Ltd, eftec (2015a), which found that the net present value of investment in saltmarsh regeneration across the UK was £730 million (2014 prices) over the next 50 years. In our case, we find a positive change in the net present value generated by the following ecosystem services: carbon sequestration and rec- reational benefits, relative to re-alignment costs: including lost agricultural production, property damages and the direct costs of re-alignment. Only one site would generate negative annualised net present values if converted to saltmarsh under the assumptions of Scenario 1, and seven sites (∼12% of total sites) would generate negative values under Scenario 3. There was substantial hetero- geneity in the annualised net present value of sites when converted to saltmarsh: across Scenarios 1 and 3 this value differed by several orders of magnitude. This suggests that prioritising managed re- alignment will offer substantial gains for planners, and result in a more efficient use of resources. Irrespective of the scenario, we identify four sites that are high priorities for managed re-align- ment. The annualised net present value generated by re-alignment at these sites is within the top quartile across all scenarios and there are no properties located in any of these sites. 4. Results Sites in the North Devon Biosphere Reserve that rank among the top 10 sites prioritised for saltmarsh managed re-align- ment across all (red) or two (amber and green) scenarios of property damage: (1) ignoring damages, (2) excluding sites with prop- erties from the analysis, and (3) incorporating a basic assessment of property damages. Figure 7. Sites in the North Devon Biosphere Reserve that rank among the top 10 sites prioritised for saltmarsh managed re-align- ment across all (red) or two (amber and green) scenarios of property damage: (1) ignoring damages, (2) excluding sites with prop- erties from the analysis, and (3) incorporating a basic assessment of property damages. Figure 8. Annualised net present value relative to site ranking. Note that in Scenarios 1 and 3, sites are ranked 1–57. In Scenario 2, there are 36 sites, ranked here from 21 to 57 for comparison with other scenarios. Property damage scenarios are: (1) ignoring property damages, (2) excluding sites with properties from the analysis, and (3) incorporating a basic assessment of property damages. Sites with negative annualised net present value have been excluded for display purposes. Figure 8. Annualised net present value relative to site ranking. Note that in Scenarios 1 and 3, sites are ranked 1–57. In Scenario 2, there are 36 sites, ranked here from 21 to 57 for comparison with other scenarios. Property damage scenarios are: (1) ignoring property damages, (2) excluding sites with properties from the analysis, and (3) incorporating a basic assessment of property damages. Sites with negative annualised net present value have been excluded for display purposes. Figure 8. Annualised net present value relative to site ranking. Note that in Scenarios 1 and 3, sites are ranked 1–57. In Scenario 2, there are 36 sites, ranked here from 21 to 57 for comparison with other scenarios. Property damage scenarios are: (1) ignoring property damages, (2) excluding sites with properties from the analysis, and (3) incorporating a basic assessment of property damages. Sites with negative annualised net present value have been excluded for display purposes. JOURNAL OF ENVIRONMENTAL ECONOMICS AND POLICY 441 Further analysis of the sites prioritised for re-alignment in Scenario 1 shows that there is a steep improvement in annualised net present value among the highest ranked sites (Figure 8). 4. Results In Scenarios 2 and 3, there is a clear difference in annualised net present value separating the top one (and two in Scenario 3’s case) site and the next ranked sites. This indicates that if there are limited resources for managed re-alignment, substantial gains can be made from prioritisation. We can also analyse sites with the greatest annualised net present values per m2 (Figure 9). This analysis provides us with a heat map of priority areas for re-alignment – independent of the sites’ size. Across all scenarios, the top site prioritised for re-alignment is different when evaluated from a site (Figure 5) versus m2 (Figure 9) perspective. Similar to the site-based analysis, small areas con- tinue to be prioritised for re-alignment. It should be noted that areas where partial re-alignment of a site was being considered, planners would also need to consider the sites’ geomorphology, tidal hydrodynamics (see Section 3.1), and whether additional ‘hard’ infrastructure would be required. 5. Discussion These qualities may make re-alignment in these sites more popular with local communities. Three of the four high- priority sites are located in agricultural areas. If these sites were converted to saltmarsh, some of the principal ecosystem service benefits: namely recreational and carbon benefits, would be widespread, with knock-on health and well-being effects. However, the costs of lost agricultural production would be incurred by a comparatively small number of landowners. This implies that one section of society would disproportionately incur the costs of new saltmarsh areas relative to the benefits. In this case, an equitable decision-making approach would need to be considered, which balanced economic trade-offs with a consideration for the bearers of the cost burden, for example property owners. 442 K. J. DAVIS ET AL. ites for managed re-alignment of saltmarsh across three scenarios var ages, (2) excluding sites with properties from the analysis, and (3) in sation is based on an assessment of candidate sites’ costs: opportu (Scenario 3), and benefits: recreational and carbon sequestration, ue per m2 is circled in red and annualised net present value reporte Figure 9. Prioritisation of sites for managed re-alignment of saltmarsh across three scenarios varying in their treatment of property damage: (1) ignoring damages, (2) excluding sites with properties from the analysis, and (3) incorporating a basic assessment of property damages. Prioritisation is based on an assessment of candidate sites’ costs: opportunity costs to agriculture, property damages and direct costs (Scenario 3), and benefits: recreational and carbon sequestration, per m2. The site with the highest annualised net present value per m2 is circled in red and annualised net present value reported. Figure 9. Prioritisation of sites for managed re-alignment of saltmarsh across three scenarios varying in their treatment of property damage: (1) ignoring damages, (2) excluding sites with properties from the analysis, and (3) incorporating a basic assessment of property damages. Prioritisation is based on an assessment of candidate sites’ costs: opportunity costs to agriculture, property damages and direct costs (Scenario 3), and benefits: recreational and carbon sequestration, per m2. The site with the highest annualised net present value per m2 is circled in red and annualised net present value reported. Figure 9. Prioritisation of sites for managed re-alignment of saltmarsh across three scenarios varying in their treatment of property damage: (1) ignoring damages, (2) excluding sites with properties from the analysis, and (3) incorporating a basic assessment of property damages. 5. Discussion Prioritisation is based on an assessment of candidate sites’ costs: opportunity costs to agriculture, property damages and direct costs (Scenario 3), and benefits: recreational and carbon sequestration, per m2. The site with the highest annualised net present value per m2 is circled in red and annualised net present value reported. JOURNAL OF ENVIRONMENTAL ECONOMICS AND POLICY 443 This study is confined to the prioritisation of sites by purely economic assessment. However, we emphasise that re-alignment should also be determined by the geomorphology and tidal dynamics of the estuary. Re-alignment in the wrong place can lead to erosion of important areas elsewhere in the system, resulting in no net gain or even loss of upper intertidal habitats such as saltmarsh. Future research should include a geomorphological model as part of the decision support tool. Planners must also be aware that the land where re-alignment is planned may currently be providing valuable freshwater flood storage that will need to be replaced to sustain existing flood defence for commu- nities around the estuary. Other potentially significant non-monetised benefits (or costs) of re-align- ment should also be considered. For example, impacts on biodiversity, the productivity of fisheries, and water quality. Re-alignment may also have important implications for landscape-scale processes like habitat connectivity, for example, saltmarsh is thought to be a key habitat for migratory birds (Iwamura et al. 2013; Murray et al. 2014). An important area for future research is to identify how the condition of saltmarsh (e.g. JNCC 2010) will impact provision of ecosystem services generated by saltmarsh. According to the 2000 Natura assessment (JNCC 2010), 57% of saltmarsh in the UK is in unfavourable condition, with 43% in favour- able condition. Natural England, as the UK statutory conservation advisor to Government, has a duty to report on the condition of saltmarsh features within conservation designations every six years. Con- dition is divided into favourable or unfavourable based on an assessment of habitat extent; physical structure (creeks and pans); vegetation structure (zonation and sward structure), vegetation compo- sition (characteristic species, indicators of a negative trend) and; other negative indicators. The identifi- cation of quality indicators (notable species or important, distinctive species) is not mandatory within this process. Further evidence is required as to how the condition of the saltmarsh interacts with the provision of ecosystem services e.g. carbon sequestration. Note 1. Over long timescales (e.g. >50 years) it is expected that climate change will result in the flooding of some coastal areas. Arguably, this should be incorporated in a social cost benefit analysis of managed re-alignment, for example, by reducing the future value of land. However, when undertaking policy action in the present, the responsible agency has to purchase land at current market prices. Impacts such as the effects of climate change on coastal land will be reflected in market prices only to the extent that those prices incorporate future changes. 1. Over long timescales (e.g. >50 years) it is expected that climate change will result in the flooding of some coastal areas. Arguably, this should be incorporated in a social cost benefit analysis of managed re-alignment, for example, by reducing the future value of land. However, when undertaking policy action in the present, the responsible agency has to purchase land at current market prices. Impacts such as the effects of climate change on coastal land will be reflected in market prices only to the extent that those prices incorporate future changes. 5. Discussion In addition, saltmarsh are particularly sensi- tive to pressures linked to sea level rise, storm events and human use (including agriculture). Assess- ments of how condition supports the resilience of saltmarsh and the levels of ecosystem services flows will serve to improve our understanding of how and where to prioritise managed re-alignment. g g g In conclusion, we outline a comprehensive methodology for identifying priority areas for mana- ged re-alignment of coastal defences based upon consequent costs and benefits. We illustrate this methodology through an application to the North Devon Biosphere Reserve. This, in turn, allows us to demonstrate the flexibility of the approach when faced with a multi-functional environment, such as saltmarsh. Results show that our framework could be used to prioritise managed re-align- ment projects and predict the impact on ecosystem service provision of different scenarios of change: including climate change, agricultural policy (e.g. under Brexit) and water quality scenarios. As research is increasingly identifying the importance of saltmarsh for flood defence and the provision of other ecosystem services, our methodology provides the necessary basis for future management of coastal zones. Developing a decision support tool capable of incorporating the flexibility of this methodology would be particularly timely given ongoing and rapid policy change both in response to Brexit and in line with the UK Government’s recent commitments to a 25-year plan to improve the environment (H.M. Government 2018). ORCID Katrina J. Davis http://orcid.org/0000-0002-6982-9381 Amy Binner http://orcid.org/0000-0003-3235-0585 Andrew Bell http://orcid.org/0000-0002-6274-0157 Timothy Poate http://orcid.org/0000-0002-4285-3066 Siân Rees http://orcid.org/0000-0001-9606-783X Greg Smith http://orcid.org/0000-0001-9278-1593 Kerrie Wilson http://orcid.org/0000-0002-0092-935X Ian Bateman http://orcid.org/0000-0002-2791-6137 Underlying research materials The underlying research materials for this article are available upon request from the corresponding author. Acknowledgements This research contains public sector information licensed under the Open Government Licence v3.0 and OS data ©Crown copyright and database right 2017. This research formed part of the Valuing Nature Programme (valuing- K. J. DAVIS ET AL. K. J. DAVIS ET AL. 444 nature.net) which is funded by the Natural Environment Research Council, the Economic and Social Research Coun- cil, the Biotechnology and Biological Sciences Research Council, the Arts and Humanities Research Council and the Department for Environment, Food and Rural Affairs. nature.net) which is funded by the Natural Environment Research Council, the Economic and Social Research Coun- cil, the Biotechnology and Biological Sciences Research Council, the Arts and Humanities Research Council and the Department for Environment, Food and Rural Affairs. Disclosure statement No potential conflict of interest was reported by the authors. Funding This work was supported by the Natural Environment Research Council under Grants NE/P011217/1 and NE/ N013573/1, and the Australian Research Council Centre of Excellence for Environmental Decisions under Grant CE11001000104. References g pp y Economics for the Environment Consultancy Ltd, eftec. 2017. 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Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach
International Journal of Islamic Economics and Finance
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p y y y 2 Institute of Islamic Banking and Finance, International Islamic University Malaysia, Malaysia Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Mohamed Asmy Bin Mohd Thas Thaker1*, Salina Kassim2, Md Fouad Bin Amin3, Marhanum Che Mohd Salleh4, Nadhrah Othman5, Siti Nadhirah Kassim6 *) Corresponding email: asmy@iium.edu.my Article History Received: May 20th, 2021 Revised: June 17th, 2021 Accepted: November 29th, 2021 1 Associate Professor, Department of Economics, International Islamic University Malaysia, Malaysia g 3 Department of Economics, King Saud University, Saudi Arabia Abstract The access to capital is very crucial for ensuring the financial sustainability of microfinance clients. It is also equally important to determine the demand for microfinance services among the clients. This study aims to identify the factors affecting the demand for Islamic microfinance (IsMF) services among the women micro- entrepreneurs in Malaysia. This study has collected a total of 250 samples from the field survey on women micro-entrepreneurs who are also the clients of Amanah Ikhtiar Malaysia (AIM). In addition, Partial Least Squares (PLS) method used to identify the potential factors (4As) i.e., affordability, accessibility, adequacy and awareness affecting the demand for IsMF. The results show that only “accessibility” has significant and positive relation with the demand for IsMF. Besides, the measurements items of accessibility such as distance of IsMF institution, collateral requirement, guarantor requirement, application procedure and process, repayment method, service efficiency, advise and consultation, and number of IsMF centers are the key factors affecting the demand for IsMF services in Malaysia. This paper provides some insights for the policy makers of Islamic microfinance and recommends that IsMF providers should take accessibility factor into greater consideration for the economic upliftment of women in microenterprises in Malaysia. Keywords: Islamic microfinance; Women; Amanah Ikthiar Malaysia; 4As JEL Classification: C83; D02; G21; G23 Type of paper: Research Paper Keywords: Islamic microfinance; Women; Amanah Ikthiar Malaysia; 4As JEL Classification: C83; D02; G21; G23 Type of paper: Research Paper DOI: https://doi.org/10.18196/ijief.v5i1.11776 https://journal.umy.ac.id/index.php/ijief/article/view/11776 ttps://doi.org/10.18196/ijief.v5i1.11776 https://journal.umy.ac.id/index.php/ijief/article/view/11776 https://doi.org/10.18196/ijief.v5i1.11776 https://journal.umy.ac.id/index.php/ijief/article/view/11776 Citation: g y y y 6 Institute of Islamic Banking and Finance, International Islamic University Malaysia, Malaysia p y y y 5 Institute of Islamic Banking and Finance, International Islamic University Malaysia, Malaysia 6 Institute of Islamic Banking and Finance International Islamic University Malaysia Malaysia p , y y , y 5 Institute of Islamic Banking and Finance, International Islamic University Malaysia, Malaysia Thaker, M. A. M., Kassim, S., Amin, M. F. B., Salleh, M. C. M., Othman, N., & Kassim, S. N. (2022) Modeling the demand for Islamic microfinance services: An application of PLS-SEM approach. International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106. DOI: https://doi.org/10.18196/ijief.v5i1.11776. International Journal of Islamic Economics and Finance (IJIEF) Vol. 5 No. 1, January 2022, pages 89-106 5 Institute of Islamic Banking and Finance, International Islamic University Malaysia, Malaysia 6 Institute of Islamic Banking and Finance, International Islamic University Malaysia, Malaysia g , y y , y 3 Department of Economics, King Saud University, Saudi Arabia Citation: Thaker, M. A. M., Kassim, S., Amin, M. F. B., Salleh, M. C. M., Othman, N., & Kassim, S. N. (2022) Modeling the demand for Islamic microfinance services: An application of PLS-SEM approach. International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106. DOI: https://doi.org/10.18196/ijief.v5i1.11776. Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach 1.1. Background Over the last few decades, the most widely discussed topic across the globe is on the concept of poverty alleviation. There are numerous approaches and tools supported by the government and non-government organizations in least developed and developing countries for poverty alleviation and economic well-being of the underprivileged group who are excluded from the conventional financial services. With the unique idea of poverty alleviation, Dr. M. Yunus won the noble prize in 2006 to support the poor through micro- credit facility. Later on, the concept of micro-credit has expanded to microfinance covering a wide range of financial services such saving and insurance. The main goal of establishing microfinance institutions (MFIs) is to enhance the accessibility of microfinance products and services to the poor who become the entrepreneurs. The MFIs help the clients to strengthen their entrepreneurships, awareness and skills. Since poverty alleviation is one of the biggest global concerns and an essential requirement for sustainable development, several efforts have been undertaken to address this issue with the help of education, medical care, economic development via microfinance services. As a factor of production, capital is considered an indispensable condition for economic growth. Microfinance services play a pivotal role and appear as an effective tool of improving the economic conditions of the poor and low-income group (Khandker, 2003; Gertler et al., 2003, and Park & Ren, 2001). In Malaysia, the number of microenterprises has increased significantly in various sectors including, food, craft, textile, and agriculture, which are mainly contributed by the microfinance services. Indeed, microenterprises has become one of the alternatives sources to generate income, but micro- entrepreneurs require small start-up capital, few workers, and good management system. More specifically, the number of women involved in microenterprises have increased remarkably across developing and least developed countries and Malaysia is not an exception where many women become economically self-reliant, promising micro-entrepreneurs and out- performing their male counterpart. In Malaysia, the number of microenterprises has increased significantly in various sectors including, food, craft, textile, and agriculture, which are mainly contributed by the microfinance services. Indeed, microenterprises has become one of the alternatives sources to generate income, but micro- entrepreneurs require small start-up capital, few workers, and good management system. 1.1. Background More specifically, the number of women involved in microenterprises have increased remarkably across developing and least developed countries and Malaysia is not an exception where many women become economically self-reliant, promising micro-entrepreneurs and out- performing their male counterpart. Malaysian government established Amanah Ikhtiar Malaysia (AIM) in the 1980s with the aim to assist the poor by improving their living standards. The AIM initiated various economic empowerment programs to support microenterprises. Inspired by the success story of Grameen Bank in Bangladesh, AIM started with a pioneer project in 1986 with 373 clients (known as “Sahabat” in Bahasa Malaysia or “good friend” in English). In due International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│90 Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach course of time, Malaysian government has extended direct financial support which helped AIM to turned into an effective Islamic Microfinance Institution (IsMFI). The government used to allocate special fund for AIM in every budget, for instance, it has received a fund equivalent to RM2.7 billion in 2018. The main reason of allocating special funds to Microfinance Institutions (MFIs) is because of their outstanding contribution to the national economy. Over the last few decades, AIM has experienced many reforms in terms of loan diversifications which has changed from subsidies to commercial financing. As of February 2015, AIM has sanctioned loans a total of RM 12.2 billion among 356,458 members from 135 different branches whereas this loan was only RM891,488 distributed among 3,220 members from 27 branches in 1990. It is also estimated that the total market share of AIM will reach to 50% in 2018 from 40 percent in 2013 (Amanah Ikhtiar Malaysia, n.d.). 1.2. Objectives Since the capital availability is a pre-condition for ensuring financial sustainability of microfinance clients, it is important to identify the factors affecting the demand for MFIs among the clients. This study aims to identify the key factors affecting the demand for Islamic microfinance (IsMF) among the women micro-entrepreneurs in Malaysia by surveying a total of 250 respondents from AIM, the largest IsMFI in Malaysia. The demand factors are categorized into four areas (4As), namely affordability, accessibility, adequacy and awareness. This study intended to identify the most significant factors from 4As. The remaining part of this paper is organized as follow: next section introduces background theory of microfinance with the finding of previous studies followed by methodology and data, analysis and the last section contain conclusions and recommendations. 2.1. Background Theory Microfinance theory is based on the concept of changing the economic life- cycle of the poor who are excluded from formal financial services. The theory is tested by targeting the clients who are either absolute or hard-core poor. The gross monthly household income is measured based on the poverty line income (PLI) which is estimated based on the price of basic necessities. Households below the PLI are considered as “absolute poor” category, whereas households with income lower than half of the PLI are categorised as International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│91 Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach “hardcore poor” category, where both categories are selected of credit assistance (Al-Mamun et al., 2011). “hardcore poor” category, where both categories are selected of credit assistance (Al-Mamun et al., 2011). According to the Microfinance theory, poor clients become the members of a microfinance institution and obtain loan for income generating activities, inculcate saving behavior for the repayment of earlier loans and receive the subsequent loan for the extension of their small businesses to microenterprises. The theory describes life changing cycle of the poor who start the journey with small informal loan and end up as successful micro- entrepreneurs with the financial knowledge, business management and technical skills. This theory is proven as an effective tool to economically empower the poor in all most every part of the world. The concept of Islamic Microfinance is developed by blending of microfinance theory and Islamic principles. Based on this, AIM was established in 1987 to enhance the economic status of the poor in Malaysia through micro-credit and later on extended to microfinance scheme. As of June 2017, AIM has extended its outreach to 23 states in Malaysia with a total of 136 branches and serving a total of 45,382 clients known as ‘Sahabat’ (Amanah Ikhtiar Malaysia, n.d.). The core of microfinance theory is based on group lending without any collateral. The group responsibility acts as the social collateral which is proven successful in terms of loan repayment history. Likewise, AIM employs the group-based lending mechanism by offering collateral-free loans based on qard-hasan concept with a 10% service charge. Other features of microfinance are the mandatory requirements of attending skills upgrading training and weekly meeting programs. International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│92 2.1. Background Theory In this aspect, various benevolent financing schemes are offered: the basic financing scheme is i-Mesra scheme (up to RM 10,000) that aims to finance economic projects expected to generate income to the clients; the i-Srikandi scheme (up to RM 20,000) that is eligible for individuals with sustainable and successful projects; and the i-Wibawa scheme that is specifically designed for individuals taking i-Mesra or i-Srikandi scheme and providing soft loans to those who are in need of additional capital for the expansion of business to grab seasonal opportunities. AIM also offer non- economic financing schemes such as the i-Bistari scheme (education loan), i- Sejahtera scheme (housing/multipurpose loan), i-Penyayang scheme (recovery loan for the defaulter) and the i-Emas scheme (loan for old people whose age are 75 years and above) (Mason et al., 2015). In addition to the conventional microfinancing mechanism, AIM sets the objectives in such way that help enhancing the economic condition of the target groups. In this aspect, various benevolent financing schemes are offered: the basic financing scheme is i-Mesra scheme (up to RM 10,000) that aims to finance economic projects expected to generate income to the clients; the i-Srikandi scheme (up to RM 20,000) that is eligible for individuals with sustainable and successful projects; and the i-Wibawa scheme that is specifically designed for individuals taking i-Mesra or i-Srikandi scheme and providing soft loans to those who are in need of additional capital for the expansion of business to grab seasonal opportunities. AIM also offer non- economic financing schemes such as the i-Bistari scheme (education loan), i- Sejahtera scheme (housing/multipurpose loan), i-Penyayang scheme (recovery loan for the defaulter) and the i-Emas scheme (loan for old people whose age are 75 years and above) (Mason et al., 2015). Apart from the financing mechanism, AIM also gives stresses on the importance of acquiring their clients with appropriate entrepreneurship skill and financial knowledge to ensure that clients can manage their businesses efficiently. Some of the programs offered include business development, human capital development and training program. Additionally, AIM established the Charity and Welfare Fund for Sahabat (Tabung Kebajikan dan Kesejahteraan Sahabat) in 2006 to reduce their financial burden and mental distress during the emergency situations like chronic illnesses and hospitalisation, funeral ceremony, natural disasters and other calamities. 2.1. Background Theory Fresh applicants are required to take part a one-week training program so that they can gather information on the rules and regulations, IsMF schemes, and repayment schedules before receiving the loan as a Sahabat. Upon passing the certain evaluation criterion, groups of five Sahabats are formed and the groups are then assigned to particular center consisting of two to twelve groups per center (Amanah Ikhtiar Malaysia, n.d.). The responsible officers used to conduct review sessions during their weekly meeting on the loan performances as well as participation of Sahabats in other social services. of the Sahabats. The amount of loan can be varied depending on the microfinance schemes but loan repayment is scheduled on weekly basis. A key to success of microfinance theory lies on the group-based lending mechanism to mitigate the risk of loan default. AIM’s rule state that all the Sahabats are required to be in a group of five members which is intended to mitigate default risk. This works well as the group members would be vouching for one another throughout the loan disbursement process although it is considered as individual loans. In case of loan default within the group International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│92 Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach members, the pooled funds are utilized to assist the defaulting members to repay their weekly loan instalment. Mason et al. (2015) advocate that the feeling of this peer pressure and accountability among the Sahabats turn into a mechanism of collective responsibility as reflected in the highest loan repayment rate (99.6%). In addition to the conventional microfinancing mechanism, AIM sets the objectives in such way that help enhancing the economic condition of the target groups. International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│93 2.2. Previous Studies The main purpose of this section is to review literatures that focus on the factors affecting the demand for microfinance product and services. Even though many studies are conducted on this area, limited studies are available that focuses on Islamic microfinance. Despite the fact, we attempt to review the existing literatures as the basis of theoretical underpinning of our structural model which to be empirically tested on the clients of AIM. Over last two decades, the number of poor people receiving microcredit has increased substantially across the world. Many researchers have illustrated the key deriving factors of demands for microfinance and innovative strategies to improve its outreach to the Bottom of Pyramid (BOP). The BOP based on the model of Four A’s introduced by C.K Prahalad and applied in the International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│93 Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach area of international marketing (Kamande and Jarhult, 2013). These Four A’s are Accessibility, Awareness, Affordability, and Availability (Prahalad, 2010). Sometimes the term Accessibility is replaced by Acceptability (Anderson and Billou, 2007). In our study, we have taken these 4As into account to identify different dimensions of demands factor of Islamic microfinance. We have considered the factor Adequacy instead of Availability. According to Selos and Mair (2007), the 4As model is receiving rather lackluster recognition due to inadequate researches to validate its relevance, hence resulting in the lack of literature covering its application to cater for the BOP segment. London & Hart (2011) highlight the importance of the 4As model to ensure the success of people in BOP segment. However, a proper theoretical framework on implementing the model has yet to be established. The first concept of 4As model is ‘Accessibility’ which is achieved, according to Prahalad (2012), when consumers are located in remote and suburban areas with the accessibility of any products and services. In contrast to this, some areas have fragile infrastructures such as poor road conditions where transport of goods and services are extremely difficult particularly for trucks, trailers and lorries. Anderson and Billou (2007) view that most of the rural markets have the characteristics of poor infrastructures causing a disruption of normal supply chain and distribution channel. 2.2. Previous Studies The same study also added that the opportunity of various markets i.e., capital, product and labor markets are mostly unavailable in the rural markets. To overcome this situation, it is suggested that the producers and local organizations can work on partnership basis within the BOP markets. It is also recommended to set up a close collaboration with local institutions, non-governmental organizations as well as local entrepreneurs so that all the parties involved in this chain can become a part of effective networking system in the BOP segment (Shah, 2012; Soete, 2010; Seelos & Mair, 2007). The second factor of 4As model is ‘Awareness’ that measures the extent of customers’ level of awareness on particular products or services. Chikweche and Fletcher (2012) assert that creating awareness in the BOP segment pose a real challenge for the organizations because their target groups are usually selected from the people in low-income bracket and majority of them have neither access to mass media such as radio, television, internet nor to print media like newspapers. It is important to create ‘Awareness’ among the BOP consumers and producers. This helps consumers to make the best use of information and grab the optimal benefits from the products and services. This also supports producers to evaluate the market demands of their products. Pralahad (2012) emphasizes on the awareness creation program within BOP segment with the organizational support that designed for specific target group with diverse needs. International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│94 Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach The third factor of 4As model is ‘Affordability’ which is one of the most challenging factors for the BOP segment due to low-income status of consumers. More specifically, the organization must take into consideration of their products and services with their cash flows which they receive as incomes on a daily basis. Thus, the pricing strategy is identified as a key challenging factor in BOP segment (Anderson & Billou (2007). Chikweche & Fletcher (2012) claim that the researchers attempting to implement a well- developed market-price mechanism under any BOP segment have a very limited outcome. 2.2. Previous Studies The last factor of 4As model is ‘Availability’ which is measured by the time scale when the customers are able to own and utilize the products or services. It is observed that the BOP consumers would only purchase products when they have enough disposable income, and the product is readily available in the market. According to Prahalad (2011), an uninterrupted supply chain is key to gain trust and loyalty of customer at BOP segment. In light of above literature review, we observed that most of the studies focus on the 4As model in determining the demand for products and services other than microfinance. In this study, we attempt to fill the gaps in existing literatures by analyzing the 4As model in determining the demand factors of Islamic Microfinance services in Malaysia. A few factors have been taken into consideration as potential variables in building a conceptual framework for the research model. As shown in Figure 1, the dependent variable for this conceptual framework is the demand for Islamic Microfinance (IsMF) and the independent variables that are identified as the factors affecting the demand for IsMF are accessibility, awareness, adequacy and affordability. These four variables described the products and services offered by AIM under their IsMF schemes. Figure 1. Research Framework Source: Adopted & Adapted from Prahalad (2010) Accessibility Awareness Adequacy Affordability Demand for Microfinancing Demand for Microfinancing Affordability Figure 1. Research Framework Source: Adopted & Adapted from Prahalad (2010) Source: Adopted & Adapted from Prahalad (2010) Source: Adopted & Adapted from Prahalad (2010) International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│95 International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│95 Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach 2.3. Hypothesis In line with the objective of this study, which is to identify the factors determining the demand for IsMF among women micro-entrepreneurs in Malaysia, the following hypotheses are constructed based on the 4A model suggested by Prahalad (2009): In line with the objective of this study, which is to identify the factors determining the demand for IsMF among women micro-entrepreneurs in Malaysia, the following hypotheses are constructed based on the 4A model suggested by Prahalad (2009): H1 : Accessibility has positive relationship in affecting the demand for IsMF H2 : Awareness has positive relationship in affecting the demand for IsMF H3 : Adequacy has positive relationship in affecting the demand for IsMF H4 : Affordability has positive relationship in affecting the demand for IsMF H1 : Accessibility has positive relationship in affecting the demand for IsMF H2 : Awareness has positive relationship in affecting the demand for IsMF H3 : Adequacy has positive relationship in affecting the demand for IsMF H4 : Affordability has positive relationship in affecting the demand for IsMF H1 : Accessibility has positive relationship in affecting the demand for IsMF H2 : Awareness has positive relationship in affecting the demand for IsMF H3 : Adequacy has positive relationship in affecting the demand for IsMF H4 : Affordability has positive relationship in affecting the demand for IsMF 3.1. Data Collection Procedure and Sample In this study, we have collected data directly from the field survey. We distributed structured questionnaires to the clients of Amanah Ikhtiar Malaysia (AIM), Hulu Selangor branch. Purposive sampling has used to gather the data from the client of AIM. The total of 402 questionnaires were distributed and only a total of 250 used for data analysis. It happened due to missing or incompleteness of data during the process of data cleaning and recoding. International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│96 3.2.1 Research Instruments This paper computed the demographic question using a nominal scale as suggested by Haque and Raihan (2004) and Amin et al. (2007). This nominal scale provides a range of values for obtaining the age of respondents. The choice of the answers or items under each construct was based on a five-point Likert type scale, ranking as 1 (strongly disagree), 2 (disagree), 3 (neutral), 4 (agree), 5 (strongly agree), which is suggested by Bhatti (2007) and Amin et al. (2007). Using a Likert type scale, the respondents choose a point on the scale reflecting his or her position towards the statement. Extant literatures have been used to extract the constructs and the items. All the constructs and items are adopted and adapted to serve the purpose of this study. All major scale items are adapted from Riquelme & Rios (2010) where each construct consists (several items) i.e., adequacy (five items), affordability (six items), awareness (ten 10 items), accessibility (nine items), and demand for Islamic microfinance (eight items). 3.2. Model Development The data are analyzed by using SPSS Statistics version 25.0 and SmartPLS version 3.2.7. This study adopted Partial Least Square (PLS) method because its’ its ability to measure causal relationships among all the latent constructs simultaneously and ability to focus on structural model for dealing with measurement errors (Farooq, 2016; Hair et al., 2017). Since this study is explanatory in nature, the application of PLS model show the best fit indices for the current study (Hair et al., 2017). As suggested by Hair et al. (2017), measurement models are examined separately before evaluating the structural model. Before conducting measurement models, we much ensure that there is no common method bias which indicates a certain context where data for dependent and independent variables are gathered from the same respondents using the same instruments. For this purpose, the current study employs Harman (1976) one factor test. In conducting the one-factor test, the researchers observed the guidelines and approaches suggested by Podsakoff et al. (2003). All the items of measurement scale are entered into a principal component analysis with varimax rotation, so that any signs of single factor can be identified from factor analysis. Based on the finding, it showed that the International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│96 Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach first factor accounted for 40.42% of variance. This figure meets the threshold level which is less than 50 percent suggested by Podsakoff et al. (2003). 4.1. Descriptive Statistics We have collected the data from AIM clients to determine the factors affecting the demand for IsMF services. As shown in Table 1, most of the respondents were female (51.6%) and followed by male (48.4%). Almost many of them were fall under the age category of 40 and below (85.2%) with the dominance of married micro-entrepreneurs (59.2%), while most of them (84%) have an average of more than 3 family members. It is also important to know the educational qualification of respondents because it plays a key role in making the economic decision. Thereby, majority of the respondents (81.6%) have diploma and degree level educational qualification. This indicates that AIM’s clients are capable of enhancing their financial literacy by attending various training programs. This factor is also very much related to their average incomes. Table 1 also demonstrates the respondents’ average monthly income. The respondents were asked about their monthly average income over the last one year and a quarter of (24.4%) and one-fifth of them (19.6%) have monthly average income less than RM 1000 and more than RM 5000, respectively. Apart from these two income levels, less the half of the respondents (43%) International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│97 Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach have achieved monthly average income with a range between RM 2000-RM 5000. This higher income pattern of the respondents strongly suggests the effectiveness of Islamic microfinance program offered by AIM. Table 1. Descriptive Statistics of the Respondents Demographic Factors Category Frequency Percent Gender Male 121 48.4 Female 129 51.6 Age 20-30 148 59.2 31-40 65 26.0 41-50 31 12.4 more than 50 6 2.4 Marital Status Single 148 59.2 Married 102 40.8 Education No Education 1 .4 Secondary (SPM) 29 11.6 Diploma 36 14.4 Degree 168 67.2 Postgraduate (Master/PhD) 16 6.4 Family Size 1-3 40 16.0 More than 3 210 84.0 Monthly Income Less than RM1000 61 24.4 RM1001-RM2000 32 12.8 RM2001-RM3000 35 14.0 RM3001-RM4000 40 16.0 RM4001-RM5000 33 13.2 More Than RM5000 49 19.6 Table 1. Descriptive Statistics of the Respondents Table 1. Descriptive Statistics of the Respondents h Table 1. Descriptive Statistics of the Respondents International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│98 4.2. Measurement Model The convergent validity is tested at the initial stage. During the test of convergent validity, indicator or items loadings, average variance extracted (AVE) and composite reliability (CR) are taken into the consideration. Based on the results presented in Table 2, items’ loading exceeded 0.6 for items, which meet the recommended value suggested by Hair et al., (2009). The threshold value of AVE is at least 0.50 as recommended by Hair et al., (2009) and we obtain the values of AVE within a range of 0.547 and 0.706 and CR value ranged from 0.854 to 0.936 which meet the recommended value of 0.7. Table 2 shows the results of measurement model with the values of individual item loading, AVE and CR. After conducting the test of convergent validity, the next stage is to test the discriminant validity. Because of certain limitations of conducting test for discriminant validity under the Fornell-Larcker (1981) criterion, Henseler et International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│98 Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach al., (2015) have suggested an alternative approach to evaluate discriminant validity by using heterotrait-monotrait ratio of correlations. Henseler et al. (2015) also demonstrate the superior performance of this method with the support of Monte Carlo simulation study. We have adopted this approach to test the discriminant validity as shown in Table 3. If the HTMT value is greater than HTMT0.85 value of 0.85 (Kline 2011), or HTMT0.90 value of 0.90 (Gold et al., 2001), then there is a problem of discriminant validity. In our obtained result, all the values passed the criterion of HTMT0.90 (Gold et al., 2001) and the HTMT0.85 (Kline, 2011) as shown in Table 3 which indicates that discriminant validity has been ascertained. Based on these results, it indicates that measurement model has adequate convergent validity and discriminant validity. y Table 2. International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│99 International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│99 International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│99 4.3. Structural Model Ramayah et al. (2016) have suggested using R2 to observe the goodness of the structural model. According to Hair et al. (2011), coefficient of determination and the level of significance of the path coefficients (beta values) can be captured by R2. The R2 for the current research is 0.42, suggesting that 42% of the variance of demand for Islamic Microfinance can be explained by accessibility, adequacy, affordability, and awareness. Subsequently, in order to assess the statistical significance of path coefficients, the current study has calculated the path coefficients of the structural model and performed bootstrap analysis (re-sampling = 500). The results presented in table 3 reveal that accessibility (b = 0.37, p < 0.01), affordability (b = 0.188, p < 0.01) and awareness (b = 0.24, p < 0.01) have a positive and significant relationship with the demand for Islamic Microfinance. Thus, the hypotheses of H1, H3 and H4 are supported. In addition, the results of structural model also indicated that adequacy factor is found to be insignificant. Thus, H2 is not supported. Table 4. Results of Structural Model Hypothesis R/ship Std. Beta Std. error t-value Decision H1 ACC -> DD 0.37 0.094 3.944 Supported H2 ADEQ -> DD -0.078 0.113 0.697 Not supported H3 AFFORD -> DD 0.188 0.097 1.932 Supported H4 AWARE -> DD 0.24 0.115 2.085 Supported 4 3 I t P f M A l i 4.2. Measurement Model Results of Measurement Model Construct Items Loadings AVE CR Affordability CD1 0.888 0.682 0.928 CD2 0.888 CD3 0.823 CD4 0.815 CD5 0.782 CD6 0.749 Awareness CB1 0.800 0.619 0.936 CB2 0.815 CB3 0.768 CB4 0.769 CB5 0.805 CB6 0.816 CB7 0.763 CB8 0.778 CB9 0.764 Accessibility CA2 0.622 0.547 0.905 CA3 0.734 CA4 0.832 CA5 0.739 CA6 0.674 CA7 0.751 CA8 0.787 CA9 0.755 Adequacy CC1 0.766 0.706 0.923 CC2 0.807 CC3 0.86 CC4 0.874 CC5 0.888 Demand B1 0.638 0.596 0.854 B6 0.834 B7 0.821 B8 0.779 Note: B3, B4, B2, B5, CA1 and CB10 were deleted due to low loadings. Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Table 3. HTMT Criterion Accessibility Adequacy Affordability Awareness Accessibility Adequacy 0.693 Affordability 0.661 0.804 Awareness 0.762 0.845 0.783 Demand 0.695 0.551 0.591 0.662 4.3. Structural Model Table 3. HTMT Criterion International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│100 International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│101 4.3. Importance-Performance Map Analysis Moreover, the cost of borrowing becomes higher if we consider the opportunity cost of access to IsMF. Sometimes, the borrowers need to attend various meeting and maintain other loan procedures which require them to visit the branches frequently and in most cases they have to spend time, money and give up some other works, which are considered as the opportunity cost. This higher borrowing cost demotivate them to apply for IsMF. Moreover, the repayment method is important for the borrowers. This is vital because not all the borrowers have same type of businesses and they have different cash-flows. Thus, the identical or standard loan repayment schedule for all the borrowers regardless of their business types cannot be justified. Other aspects that affect the demand for IsMF are the product services including the efficiency of staffs, rules, regulations and procedure of loan approval. Sometimes, IsMF institution like AIM become very rigid on their loan approval procedures. The borrowers who are in urgent need for IsMF must follow the same procedures as regular borrowers, and this whole cumbersome loan approval procedures in some cases demotivate borrowers to stick to the IsMF. It is also important to increase the efficiency of IsMF product and services. This efficiency leads to retain and attract new members which is cost effective for the institution and also beneficial for the borrowers. 4.3. Importance-Performance Map Analysis 4.3. Importance-Performance Map Analysis In order the confirm the obtained results, we have conducted Importance- Performance Map Analysis (IPMA) as suggested by Ringle and Sarstedt (2016). The main advantage of IPMA is that it can identify predecessors which have a relatively low performance but high importance for the target constructs. IPMA becomes a very useful analytical tool in PLS-SEM. IPMA graphically extends the standard path coefficient estimates in more practical way (Ringle & Sarstedt, 2016). nternational Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│100 International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│100 Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Figure 2. Importance-Performance Map Analysis Figure 2. Importance-Performance Map Analysis This paper applied Importance-Performance Map Analysis (IPMA) in order to evaluate the most influencing factors as represented by four predecessors (accessibility, adequacy, affordability and awareness) affecting the demand for Islamic Microfinance (Figure 1). The IPMA’s results is presented in table 4 which reveals that the construct Adequacy has higher value in performance index but not under importance index in predicting the demand for Islamic Microfinance. This paper found the higher weights of three other constructs i.e., accessibility, affordability and awareness whereas affordability factor remains in the highest performance domain followed by awareness and affordability. Table 5. Importance-Performance Map Analysis Statistics Table 5. Importance-Performance Map Analysis Statistics Construct Importance (Total Effect) Performance (Index Values) Accessibility 0.461 79.896 Adequacy -0.093 79.461 Affordability 0.237 82.377 Awareness 0.316 81.042 In this study, it shows that certain factors are important that can ensure the higher demand for Islamic Microfinance. The key items under the accessibility factors that have greater influence on Islamic Microfinance products and services are: i) the distances of AIM’s branches which is related to higher borrowing cost, ii) the loan size is relatively small in amount that the borrowers’ have to incur a higher cost to access the loan, iii) collateral requirements, guarantor requirement, and repayment method which are found to be highly important, iv) efficiency of Islamic Microfinance service provides like AIM provided by the microfinance providers, v) continuous advise and consultation, and vi) easy and simple loan application procedures and process. International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│102 4.3. Importance-Performance Map Analysis nternational Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│101 Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach The loan size is important for the clients regardless of their status as fresh or old members. Most of the members are either involved in micro-enterprises or planning to start new ventures which requires adequate amount of capital. That is why the members of AIM consider this item as one of the top priority items affecting their demand for Islamic Microfinance (IsMF). Moreover, the cost of borrowing becomes higher if we consider the opportunity cost of access to IsMF. Sometimes, the borrowers need to attend various meeting and maintain other loan procedures which require them to visit the branches frequently and in most cases they have to spend time, money and give up some other works, which are considered as the opportunity cost. This higher borrowing cost demotivate them to apply for IsMF. Moreover, the repayment method is important for the borrowers. This is vital because not all the borrowers have same type of businesses and they have different cash-flows. Thus, the identical or standard loan repayment schedule for all the borrowers regardless of their business types cannot be justified. Other aspects that affect the demand for IsMF are the product services including the efficiency of staffs, rules, regulations and procedure of loan approval. Sometimes, IsMF institution like AIM become very rigid on their loan approval procedures. The borrowers who are in urgent need for IsMF must follow the same procedures as regular borrowers, and this whole cumbersome loan approval procedures in some cases demotivate borrowers to stick to the IsMF. It is also important to increase the efficiency of IsMF product and services. This efficiency leads to retain and attract new members which is cost effective for the institution and also beneficial for the borrowers. The loan size is important for the clients regardless of their status as fresh or old members. Most of the members are either involved in micro-enterprises or planning to start new ventures which requires adequate amount of capital. That is why the members of AIM consider this item as one of the top priority items affecting their demand for Islamic Microfinance (IsMF). 5.2. Recommendation In view of above findings, this paper recommends certain aspects of products and services of AIM. The IsMF institutions like AIM need to focus on accessibility factors to ensure that the microfinance product is appealing to the clients. The sufficient number of IMF services centres should be established along with the reduction of the cost of IsMF products and services. Similarly, IsMF providers should also take into account of its product’s features including collateral requirements, guarantor requirement, and repayment method which are highly valued by the clients in determining the demand for IsMF. the IsMF providers need to ensure that these requirements are not set at highest stringent level. We also suggest that IsMF providers may adopt innovative risk mitigations mechanism by refining the group lending and weekly repayment methods. This study also suggest that MFIs should continue to strive in providing better services to ensure customers retention as well as attraction of new clients for their services. It is expected that adopting the above recommendations can enhance the participation of women in microenterprises and uplift them toward higher economic living standards in Malaysia. 5.1. Conclusion The aim of this study is to identify factors determining the demand for Islamic microfinance (IsMF) among women micro-entrepreneurs in Malaysia. This study has conducted a survey with the clients of Amanah Ikhtiar Malaysia in Selangor. This study adopts the Partial Least Squares (PLS) method in detecting the potential demand factors of IsMF, namely affordability, accessibility, adequacy and awareness. The results show that only “accessibility” factor has significant and positive relationship with the demand for IsMF. More specifically, a few measurements items under accessibility such as, distance to microfinance institution, collateral requirements, guarantor requirement, application procedure and process, repayment method, efficiency of services by microfinance providers, continuous advice and consultation, and number of microfinance centers which are significantly affecting the demand for IsMF in Malaysia. It shows that for the clients of International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│102 Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach microfinance, easier accessible and convenience accessible would encourage them to use continuously the services that offered by microfinance institutions. At the same time, IsMF institutions can increase their marketing know-how by implementing effective accessibility strategies that generate services availability to the clients. Based on the analysis of Importance-Performance Map Analysis (IPMA), it shows that IsMF institutions should not focus much on adequacy as it will possibly reduce the profit of the institution. The three other constructs which are more important are accessibility, affordability and awareness. Affordability is already high in performance but awareness is slightly lower, so the microfinance institutions should focus on enhancing affordability matter. In terms of accessibility, it is important for IsMF institution to simplify the application and approval process to obtain IsMF. Acknowledgement This paper originates from of a research project approved in 2015 funded by the Ministry of Higher Education (MOHE) Malaysia under the Fundamental Research Grant Scheme (Project ID: FRGS15-230-0471). The authors would like to thank MOHE Malaysia for generously funding this research project. nternational Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│103 Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│104 References Amanah Ikhtiar Malaysia. (n.d.). Retrieved from http://www.aim.gov.my Amanah Ikhtiar Malaysia. (n.d.). Retrieved from http://www.aim.gov.my Al Mamun, A. (2011). Impact of Amanah Ikhtiar Malaysia’s schemes on hardcore poor households in Peninsular Malaysia. (Doctoral Thesis). Faculty of Management, Multimedia University, Malaysia. Amin, H., Baba, R. and Muhammad, M. Z. (2007). An analysis of mobile banking acceptance by Malaysian customers. Sunway Academic Journal, 4, 1-12. Anderson, J. and Billou, N. (2007). Serving the world's poor: innovation at the base of the Economic pyramid, Journal of Business Strategy, 28 (2), 14-21. Bhatti, T. (2007). Exploring factors influencing the adoption of mobile commerce. Journal of Internet Banking & Commerce, 12(3), 1–14. Chikweche, T. and Fletcher R. (2012). Revisiting the marketing mix at the bottom of Pyramid (BOP): From theoretical considerations to practical implications, Journal of Consumer Marketing, 29(7),507-520. Fornell, C., and Larcker, D. F. (1981). Evaluating structural equation models with unobservable variables and measurement error. J. Mark. Res. 18, 39–50. Gertler, P., Levine, D.I., Moreti, E. (2003). Do microfinance programs help families iinsure consumption against illness? (Working Paper series C03-129) Center for International Development Economics Research (CIDER), University of California, Berkeley. Gold, A. H., Malhotra, A., & Segars, A. H. (2001). Knowledge management: An organizational capabilities perspective. Journal of Management Information Systems, 18(1), 185–214. Hair, J. F., Black, W. C., Babin, B. J., & Anderson, R. E. (2009). Multivariate data analysis. Upper Saddle River, NJ: Prentice Hall. Hair, J. F., Ringle, C. M., & Sarstedt, M. (2011). PLS-SEM: Indeed a silver bullet. Journal of Marketing Theory and Practice, 19(2), 139–151. Harman, H.H. (1976), Modern Factor Analysis, Chicago: University of Chicago Press. Henseler, J., Ringle, C.M. and Sarstedt, M. (2015). A new criterion for assessing discriminant validity in variance-based structural equation modeling. Journal of the Academy of Marketing Science, 43(1), 115–135. Kamande, S., & Jarhult, W. (2013). Reaching the mass market of the base of the pyramid-using the five A’s concept. (Master Thesis). Linnaeus University, Sweden. International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│104 Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Khandker, S. (2003). Micro finance and poverty: Evidence using panel data from Bangladesh. (Research Paper series 2945), World Bank Policy. World Bank. Washington. Kline, R. B. (2011). Principles and practice of structural equation modeling. New York: Guilford Press. London, T. and Hart, S. (2011). International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│105 International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│106 References Next-generation business strategies for the base of the pyramid: New approaches for building mutual value, in a. upper saddle river (Eds.), Financial Times Press. Mason, D. J., Jones, D. A., Roy, C., Sullivan, C. G., & Wood, L. J. (2015). Commonalities of nurse-designed models of health care. 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New Jersey (Eds.), Prentice Hall. Ramayah, T., Cheah, J., Chuah, F., Ting, H., & Memon, M. A. (2016). Partial least squares structural equation modeling (PLS-SEM) using SmartPLS 3.0: An updated and practical guide to statistical analysis. Singapore: Pearson. Ringle, C. M. and Sarstedt, M. (2016). gain more insight from your pls-sem results: The importance-performance map analysis. Industrial Management & Data Systems, 116(9), 1865-1886. Riquelme, H. E. and Rios, R. E. (2010). The moderating effect of gender in the adoption of mobile banking. The International Journal of Bank Marketing, 28, 328-341. International Journal of Islamic Economics and Finance (IJIEF), 5(1), 89-106│105 Thaker, Kassim, Amin, Salleh, Othman, & Kassim│ Modeling the Demand for Islamic Microfinance Services: An Application of PLS-SEM Approach Seelos, C. and Mair, J. (2007). 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Factors influencing implementation of a patient decision aid in a developing country: an exploratory study
Implementation science
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* Correspondence: leeyk@um.edu.my 1Department of Primary Care Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Full list of author information is available at the end of the article Factors influencing implementation of a patient decision aid in a developing country: an exploratory study Wen Ting Tong1, Yew Kong Lee1* , Chirk Jenn Ng1 and Ping Yein Lee2 Wen Ting Tong1, Yew Kong Lee1* , Chirk Jenn Ng1 and Ping Yein Lee2 © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tong et al. Implementation Science (2017) 12:40 DOI 10.1186/s13012-017-0569-9 Tong et al. Implementation Science (2017) 12:40 DOI 10.1186/s13012-017-0569-9 Open Access Abstract Background: Most studies on barriers and facilitators to implementation of patient decision aids (PDAs) are conducted in the west; hence, the findings may not be transferable to developing countries. This study aims to use a locally developed insulin PDA as an exemplar to explore the barriers and facilitators to implementing PDAs in Malaysia, an upper middle-income country in Asia. Methods: Qualitative methodology was adopted. Nine in-depth interviews (IDIs) and three focus group discussions (FGDs) were conducted with policymakers (n = 6), medical officers (n = 13), diabetes educators (n = 5) and a nurse, who were involved in insulin initiation management at an academic primary care clinic. The interviews were conducted with the aid of a semi-structured interview guide based on the Theoretical Domains Framework. The interviews were audio-recorded, transcribed verbatim and analyzed using a thematic approach. Results: Five themes emerged, and they were lack of shared decision-making (SDM) culture, role boundary, lack of continuity of care, impact on consultation time and reminder network. Healthcare providers’ (HCPs) paternalistic attitude, patients’ passivity and patient trust in physicians rendered SDM challenging which affected the implementation of the PDA. Clear role boundaries between the doctors and nurses made collaborative implementation of the PDA challenging, as nurses may not view the use of insulin PDA to be part of their job scope. The lack of continuity of care might cause difficulties for doctors to follow up on insulin PDA use with their patient. While time was the most commonly cited barrier for PDA implementation, use of the PDA might reduce consultation time. A reminder network was suggested to address the issue of forgetfulness as well as to trigger interest in using the PDA. The suggested reminders were peer reminders (i.e. HCPs reminding one another to use the PDA) and system reminders (e.g. incorporating electronic medical record prompts, displaying posters/notices, making the insulin PDA available and visible in the consultation rooms). Conclusions: When implementing PDAs, it is crucial to consider the healthcare culture and system, particularly in developing countries such as Malaysia where concepts of SDM and PDAs are still novel. Keywords: Implementation, Insulin patient decision aid, Developing countries, Insulin initiation, Type 2 diabetes mellitus, Decision support, Barriers, Facilitators, Factors © The Author(s). Study setting and participants Data collection was carried out at an academic primary care clinic at the University of Malaya Medical Center, an urban government teaching hospital. In Malaysia, the majority of diabetes patients are managed in the public sector where healthcare services are subsidized by the government. The primary care clinic is an outpatient clinic which accepts patients from all over Malaysia, and therefore, it has a high patient load with patients from various backgrounds. On average, there are 25 doctors in the clinic, two diabetes educators (DE) and 26 regis- tered nurses (RN). Generally, patients in the clinic do not get to decide which doctor to see; hence, they may not consult the same doctor during the follow up. As most of the studies on barriers and facilitators to im- plementation of PDAs are conducted in the west [6–8], the findings may not be transferable to Asian countries, most of which are developing countries, and issues such as socio-cultural barriers (language barrier and physician paternalism) and lack of resources (infrastructure or tech- nology development) may be more significant compared to developed countries. Nevertheless, studies have shown that patients in Asia want to be involved in SDM [9, 10] and there are an increasing number of PDAs being devel- oped in the region [11, 12]. However, no Asian studies have reported the implementation of these PDAs. g p There are three groups of healthcare professionals in- volved in insulin initiation in the clinic: doctors, DEs and RNs. In the clinic, only doctors (i.e. family medicine specialists) and medical officers were allowed to pre- scribe insulin to patients. There is a diabetes education centre located in the clinic where three DEs, who are nurses, provide patient education on diabetes, blood glu- cose monitoring and insulin initiation and injection techniques after receiving referrals from doctors. To qualify as a DE, RNs need to complete an advanced diploma in diabetes education. In the course, the RNs are trained on diabetes education, treatment and nursing of diabetes patients (including on insulin initiation). However, training on SDM and PDA is not part of the curriculum as the insulin PDA has not been formally in- troduced or implemented in the healthcare system in Malaysia. In contrast to DEs, RNs’ tasks are to assist doctors in the consultation rooms. Of 26 individuals Malaysia is a developing upper middle-income country in Southeast Asia [13]. Study design There are many factors that influence the implementa- tion of SDM and PDAs. Studies have highlighted barriers such as time constraints, healthcare professionals’ attitude, perceived legitimacy of the PDA, lack of applicability due to patient characteristics, clinic capacity, processes of care and the healthcare environment [6–8]. Among the facilita- tors were provider’s motivation, provision of training and skills development for providers, identification of a clinical champion, introduction of a system to identify eligible patients to use PDAs ahead of clinical consultations, posi- tive impact on the clinical process and patient outcomes [7, 8]. However, the relative influence of these factors varies across different countries. This study adopted a qualitative methodology to explore the factors influencing implementation of the insulin PDA. In-depth interviews (IDIs) and focus group discussions (FGDs) were conducted. Background attempt to advance SDM, several Malaysian PDAs have been developed for patients who are making a decision on insulin use [12], early breast cancer [16] and early prostate cancer treatment [17]. These PDAs are available in the Malay, Chinese, Tamil and English language to facilitate decision-making as they will be able to read and under- stand the PDA in their preferred language. Furthermore, these PDAs explored cultural beliefs of the major eth- nicities in Malaysia. Nevertheless, formal implementa- tion of these PDAs has not been conducted. Therefore, this study aims to use a locally developed insulin PDA (http://dmit.um.edu.my/?modul=DMIT_PDA) [12, 18] as an exemplar to explore the barriers and facilitators to implementing PDAs in a developing country. Shared decision-making (SDM) is part of patient-centred care whereby patients and clinicians decide on a treat- ment together. This is particularly relevant when the decision is preference-sensitive [1]. One way to promote SDM is to use a patient decision aid (PDA), which pro- vides information about the decision, available treatment options, benefits and risks of each option and ways to clarify patient values [2]. In a Cochrane Review of 115 randomized controlled trials, PDAs have been proven to be effective in involving patients in SDM and improving their decision quality [2]. However, the adoption and im- plementation of PDAs in clinical practice remains poor [3–5]. One study has found that only about 10% of eligible primary care patients actually received PDAs despite clinic-wide PDA adoption [4]. Abstract 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tong et al. Implementation Science (2017) 12:40 Page 2 of 12 Page 2 of 12 Table 1 Summary of the study interview guide Preamble: We would like to implement the insulin PDA in the clinic. We would like to hear your honest opinion on what are the barriers and facilitators to implementing the insulin PDA in your healthcare organization so that it is effective and sustainable to be used. This study used purposive sampling to recruit partici- pants who were healthcare policymakers (HPMs) and healthcare providers (HCPs) working in the medical centre [19]. The inclusion criteria for HPM were those who were responsible for or involved in making deci- sions on whether a particular health intervention should be implemented in the hospital, while for HCPs were those who were involved in advising patients about insu- lin initiation. The HPMs recruited included the director- level hospital manager, Endocrine and Primary Care Medicine policymakers and hospital matrons. They set standards of care and implement programmes to im- prove diabetes care in the hospital. While they perform managerial tasks, they are also practicing HCPs. HCPs in- cluded the doctors, DEs and RNs. In order to achieve maximum variation, we recruited participants from differ- ent socio-demographic backgrounds (ethnicity, gender) and included those with and without experience in using the PDA. The PDA was previously pilot-tested with a small group of HCPs in the clinic to test its acceptability, and a few of them had continued using it after the pilot- testing phase had ended. HPMs were not involved in the pilot study. Insulin PDA • What do you think about the insulin PDA? (Probe: feelings: afraid, hopeful) (emotions) Would you use this PDA? (intention, beliefs about consequences, optimism) • What do you think about the insulin PDA? (Probe: feelings: afraid, hopeful) (emotions) Would you use this PDA? (intention, beliefs about consequences, optimism) • Do you think that HCPs’ will want to use the insulin PDA? Why? (intentions) • Do you think that insulin PDA will affect your/HCP role? How? (social professional role and identity) • Do you think that you/HCPs would remember to use the PDA if it is implemented? Why? Why not? (memory, attention and decision processess) p • Do you think that you/HCPs will be confident to use the insulin PDA? Why? (beliefs about capabilities) (beliefs about capabilities) • How doctors and nurses or other HCPs such as dieticians or pharmacists play a role in implementing the insulin PDA? Table 1 Summary of the study interview guide (social professional role and identity) • Do you think that you/HCPs have the knowledge and skills to use the insulin PDA? What are the knowledge and skills needed? (knowledge and skills) • Do you see any benefit or harm in implementing the PDA in the current healthcare system? (beliefs about consequences) • Who do you think can influence you/HCPs whether to use or not to use the insulin PDA? How? (social influences) • What can you/HCPs do to use the insulin PDA in their consultation? (behavioural regulation) • If you will implement the insulin PDA, what are the goals you/patients want to achieve? (goals) Study setting and participants It has a population of 28.3 million, comprising three main ethnic groups namely Malay (67.4%), Chinese (24.6%) and Indian (7.3%) [14]. The im- plementation of SDM in Malaysia is hampered by several factors including the multi-cultural and language diversity of the population and the lack of patient involvement in healthcare [15]. In Malaysia, although the national lan- guage is the Malay language, not all Chinese or Indian patients are fluent in this language. Furthermore, each ethnic group has their own cultural norms and beliefs when it comes to health. Thus, doctors and patients may be mismatched linguistically as well as culturally. As an Tong et al. Implementation Science (2017) 12:40 Page 3 of 12 who were approached to participate in the study, one staff nurse refused due to time constraints. Study instrument The interviews were conducted with the aid of a semi- structured interview guide (Table 1), which was devel- oped based on the Theoretical Domains Framework (TDF) [20] and a literature review on barriers and facil- itators to implementing PDAs [7]. The TDF is an over- arching framework of 14 theoretical domains synthesized from behaviour change constructs found in 33 behaviour change theories (Table 2). The TDF was selected as the theoretical basis for this study because implementation success is largely dependent on the behaviour change of the users involved on whether to adopt or reject the im- plementation innovation. The TDF is comprehensive as it not only looks into the impact of rational and cognitive process of an individual but also looks into emotional factors [21] as well as external, organizational factors such as work- ing environment and resources that may influence them on whether or not to adopt a new behaviour or innovation such as the practice of SDM and use of PDA. Furthermore, the TDF has been used in many clinical behaviour change im- plementation research projects [22], which is why it was felt to be appropriate for this study to understand the factors in- fluencing implementation of insulin PDA. Inner context – the clinic • Does your institution have the resources to implement the insulin PDA? What are the resources available and what are needed? • How do you think your organization’s working culture (general beliefs, values, assumptions that people embrace, receptivity of a new intervention) will affect the implementation of the PDA? (environmental context and resources) Outer context the healthcare system • Are you confident/positive/optimistic that the current healthcare system is capable to successfully implement the insulin PDA despite any difficulty? Yes/No, why? (beliefs about capabilities; optimism) • What can be done by the higher-level authority to ensure the success of the implementation of the PDA? (reinforcements) • In your opinion, how can we make the implementation of the insulin PDA sustainable? participate in the study were given information about the insulin PDA, its objective and content, the concept of SDM and the various modalities (booklet, tablet, web- site) available. A video, which demonstrates how the in- sulin PDA can be used during a consultation, was shown to the participants to give them an idea on how the insu- lin PDA can be used in the clinic. The participants were also encouraged to think about other ways of imple- menting the insulin PDA in their practice. The interviews were conducted between December 2015 and March 2016 by the researchers (WTT, YKL, CJN) who asked the participants ‘open’ and ‘probing’ questions to explore the factors influencing implementa- tion of the insulin PDA in their practice. All the interviews Data collection process HPMs and HCPs who fulfilled the study inclusion criteria were invited to participate in the study. Prior to the interviews, eligible participants who consented to Page 4 of 12 Page 4 of 12 Tong et al. Implementation Science (2017) 12:40 Table 2 TDF [20] Domain Definition (American Psychological Associations’ Dictionary of Psychology) 1 Knowledge An awareness of the existence of something 2 Skills An ability or proficiency acquired through practice 3 Beliefs about capabilities Acceptance of the truth, reality or validity about an ability, talent or facility that a person can put to constructive use 4 Optimism The confidence that things will happen for the best or that desired goals will be attained 5 Beliefs about consequences Acceptance of the truth, reality, or validity about outcomes of a behaviour in a given situation 6 Reinforcement Increasing the probability of a response by arranging a dependent relationship, or contingency, between the response and a given stimulus 7 Intentions A conscious decision to perform a behaviour or a resolve to act in a certain way 8 Goals Mental representations of outcomes or end states that an individual wants to achieve 9 Memory, attention and decision processes The ability to retain information, focus selectively on aspects of the environment and choose between two or more alternatives 10 Emotion A complex reaction pattern, involving experiential, behavioural and physiological elements, by which the individual attempts to deal with a personally significant matter or event 11 Social/professional role and identity A coherent set of behaviours and displayed personal qualities of an individual in a social or work setting 12 Environmental context and resources Any circumstance of a person’s situation or environment that discourages or encourages the development of skills and abilities, independence, social competence, and adaptive behaviour 13 Social influences Those interpersonal processes that can cause individuals to change their thoughts, feelings, or behaviours 14 Behavioural regulation Anything aimed at managing or changing objectively observed or measured actions participants as they are based at this university. WTT is a PhD student, YKL is a lecturer, while both CJN and PYL are clinical lecturers who specialize in family medicine. All researchers are experienced in conducting qualitative re- search. As WTT did not know any of the participants prior to the interviews, WTT conducted most of the inter- views (8/12) as YKL and CJN refrained from interviewing participants whom they knew. PYL did not conduct any interview. Ethics approval Ethics approval This study received ethics approval from the University of Malaya Medical Centre Medical Ethics Committee (reference: MECID.NO: 20158-1600). Data collection process The interviews were audio-recorded, and field notes were written to document the content of and reflec- tions on the interviews. The data collection ceased when data saturation was achieved; that is when the barriers or facilitators that emerged from the data became repetitive and there were no more new findings. Data analysis ll h All the interviews were transcribed verbatim and checked for accuracy before being imported into Nvivo software for data analysis. The researchers (WTT, YKL, CJN, PYL) familiarized themselves with the first three transcripts and coded the transcripts independently using a thematic approach. The transcripts were read and coded line by line. A code is a short text which represents the meaning of the text segment. Codes that have similar meaning were grouped together to form a category, and later the cat- egories were compared and merged into bigger themes [23]. Researchers met to discuss the categories and themes which emerged from their individual analysis. Any dis- crepancies in the categories and themes were resolved through consensus before finalizing the coding frame- work, which was used to analyze the remaining transcripts by one of the researcher (WTT). Any new codes and categories that emerged were added to the list of themes and categories upon consultation with the research team. The written field notes and interview reflections were tri- angulated with the results to ensure that the findings were correctly interpreted and no information were missed out and to supplement the interview findings. By compar- ing data from multiple sources, we hope to enhance the credibility of the findings [24]. Emerging themes Five themes emerged from the interviews, and they were lack of SDM culture, role boundary, lack of continuity of care (COC), impact on consultation time and reminder network. Theme 1: Lack of SDM culture The participants highlighted that there is a lack of SDM culture in the current practice as HCPs’ paternalistic attitudes still prevailed whereby doctors would make treatment decision for patients and did not like patients to ask too many questions. In addition, patients tended to play a passive role in decision- Theme 1: Lack of SDM culture The participants highlighted that there is a lack of SDM culture in the current practice as HCPs’ paternalistic attitudes still prevailed whereby doctors would make treatment decision for patients and did not like patients to ask too many questions. In addition, patients tended to play a passive role in decision- Because we are busy in the clinic, sometimes we don’t like people to ask us a lot of questions. Socio-demographic and practice profile of participants Socio-demographic and practice profile of participants Nine IDIs and three FGDs were conducted with 25 indi- viduals who participated in the study: the hospital pol- icymakers (n = 6), medical officers (n = 13), DEs (n = 5) and a RN. Two of the FGDs were conduced with med- ical officers (n = 7; n = 6). Another FGD with the nursing policymakers was conducted with smaller number of were face-to-face and were conducted in the clinic consultation rooms during rest times in between clin- ical consultations, or at the office of the participants. The interviews lasted from 50 to 90 min. This study was conducted in the university hospital setting, and three of the researchers (WTT, YKL, CJN) were known to the Tong et al. Implementation Science (2017) 12:40 Page 5 of 12 making and trusted the physicians to make the decision for them. This rendered the lack of need for SDM and use of the insulin PDA. participants (n = 3) as they were the only three senior nursing staff at management level who were felt to be able to provide feedback on the implementation of the insulin PDA. There were six participants who have had experiences in using the insulin PDA (Table 3). I think in our Asian context, we still have this idea of doctor telling you what to do. I think this shared decision approach is a concept that developed countries have but I don’t know whether our culture has reached this stage or not.//Our practitioners don’t give patients chance to decide on their own. We like to tell the patient what to do. So they don’t see the need for this book. The patients still have the mindset of ‘You tell me what to do. It’s not my decision, it is your decision, I just follow you’.//I don’t know whether our patient is up to that yet for the shared decision part. – IDI 1_DE 1_have not used insulin PDA Theme 3: Lack of COC Theme 3: Lack of COC Some medical officers have continued the insulin PDA use after the pilot study because they felt that the PDA helped to reduce consultation time and address language barrier when they provided information to patients who spoke a different language. However, the use of PDA was later discontinued due to lack of COC. Furthermore, as there was no proper record that the insulin PDA had been given to patients, the medical officer might not be aware that the patient had already been introduced to the insulin PDA by another doctor. This sentiment is shared by both medical officers who have and have not used the PDA. But we don’t have so much opportunity to initiate (insulin PDA use) because instructions are passed down from the top,‘ok you do this’. They (doctors) are the first liner. IDI 1_DE 1_have not used insulin PDA However, assigning RNs to use the PDA with patients would not be accepted if it fell outside the scope of RNs’ current duties. For example, when asked if RNs had a role in using the insulin PDA with patients, participants voiced that RNs would not perceive the use of insulin PDA to be part of their job scope. RNs confined their duties only to pre-determined tasks and would not be interested to per- form additional tasks. They perceived using the insulin PDA as an added workload. Basically, COC is not there. Because doctors don’t get to see the same patients. We might give this PDA to a patient this visit. And next visit, you may not be the one who sees the patient. – FDG 1_Medical officer 4_have not used insulin PDA The book was in the diabetic education room. So I did refer patient there so that the nurse can explain but I’m not sure what the nurse explained to the patient. FGD 2_Medical officer 13_have used PDA But I think because they [RNs] don’t see it as their role to advise patients about their disease. It is more of getting the patient in, sorted out, weight, height. So, I think to get them to start [using insulin PDA] it might be a bit harder than to get the medical officers to use it. – IDI 7_Primary care policymaker_have not used insulin PDA Furthermore, as the setting was a teaching hospital, trainee doctors leave after their training is completed. Emerging themes FGD 3_Hospital matron 1_have not used insulin PDA Table 3 Characteristics of participants Interview Role Ethnicity Age Sex Experience with insulin PDA IDI 1_ Diabetes educator 1 Healthcare provider Chinese 56 Female No IDI 2_ Diabetes educator 2 Healthcare provider Malay 57 Female Yes IDI 3_ Diabetes educator 3 Healthcare provider Malay 53 Female No IDI 4_ Diabetes educator 4 Healthcare provider Malay 35 Female No IDI 5_ Diabetes educator 5 Healthcare provider Malay 57 Female No IDI 6_ Registered nurse Healthcare provider Indian 55 Female No IDI 7_ Primary care policymaker Healthcare policymaker Malay 48 Female No IDI 8_ Endocrine policymaker Healthcare policymaker Chinese 37 Male No IDI 11_ Director-level hospital manager Healthcare policymaker Malay 43 Male No FGD 1_ Medical officer 1 Healthcare provider Malay 35 Female No FGD 1_ Medical officer 2 Healthcare provider Chinese 32 Female No FGD 1_ Medical officer 3 Healthcare provider Indian 37 Female Yes FGD 1_ Medical officer 4 Healthcare provider Indian 35 Male No FGD 1_ Medical officer 5 Healthcare provider Malay 32 Male No FGD 1_ Medical officer 6 Healthcare provider Malay 32 Male No FGD 1_ Medical officer 7 Healthcare provider Indian 39 Male Yes FGD 2_ Medical officer 8 Healthcare provider Malay 31 Female Yes FGD 2_ Medical officer 9 Healthcare provider Malay 33 Female No FGD 2_ Medical officer 10 Healthcare provider Chinese 33 Female Yes FGD 2_ Medical officer 11 Healthcare provider Chinese 35 Female No FGD 2_ Medical officer 12 Healthcare provider Malay 36 Female No FGD 2_ Medical officer 13 Healthcare provider Malay 36 Female Yes FGD 3_ Hospital matron 1 Healthcare policymaker Malay 56 Female No FGD 3_ Hospital matron 2 Healthcare policymaker Malay 56 Female No FGD 3_ Hospital matron 3 Healthcare policymaker Chinese 57 Female No Table 3 Characteristics of participants Page 6 of 12 Page 6 of 12 Tong et al. Implementation Science (2017) 12:40 Tong et al. Implementation Science (2017) 12:40 Theme 2: Role boundary As for DEs, some felt that their role in the PDA implementation was just ‘taking orders from doctors’ on when to use the PDA with patients, while some felt that they could inform patients on when to initiate insulin. Theme 2: Role boundary There was a clear role boundary between the doctors and the nurses which made collaborative implementation of the PDAs challenging. Emerging themes All participants, including medical officers, felt that the doctor should be the key person to introduce and use the insulin PDA with patients because the decision to initiate insulin treatment lies within the doctor after reviewing patient’s glycaemic control and health profile. Furthermore, only doctors have the authority to prescribe insulin for patients. Doctor will say to the patient they have no time [to use PDA] ‘You go and see the DE’. The decision is made by doctor. I just follow order. IDI 2_DE 2_have used insulin PDA Actually we also have a clinic of our own and patients come to us we will monitor them. So when we see that they are losing control then we can tell them that ‘I think you need insulin already’. So we also have a little part to play. IDI 1_DE 1_have not used insulin PDA I think the initiation [to use insulin PDA] has to come from the doctor because we are the ones who will know whether it is appropriate or not to talk about insulin initiation. I think only doctors would understand circumstances such as if a patient has cataract and will not be able to inject herself or have no social support. Rather than at the pharmacy end ‘Oh, your Hba1c is very bad. You should be on insulin, here, take this book’ I think that is not right. FGD 2_Medical officer 8_have used insulin PDA Theme 3: Lack of COC Therefore, this posed another issue for implementation as doctors who were trained on how to use the insulin PDA may leave and new doctors may not be familiar with the PDA. Nurses, we only want to do what is nursing. We don’t want to do beyond that. ‘I’m a nurse … why should I do this, this is nothing concerning me, this is doctors’ job, not my job’.//‘Oh another booklet. Another thing to talk to the patient now’. Anything else is added work. – IDI 6_RN_have not used insulin PDA We have to train them (on PDA use), so we have to give them the exposure of what it is like, what patients are like, but you know it takes several consultations to fully get that. They can deploy the book, but then they may not be around next month to Page 7 of 12 Tong et al. Implementation Science (2017) 12:40 even follow up. – IDI 8_Endocrine policymaker_have not used insulin PDA even follow up. – IDI 8_Endocrine policymaker_have not used insulin PDA of the PDA should be made available in the computer’s desktop which can serve as a reminder for the doctor to use the insulin PDA. Theme 4: Impact on consultation time Time constraints were the most commonly raised implementation barrier of the PDA among the participants. Due to high patient load, there were concerns about taking up extra time to go through the insulin PDA with patients during consultations. So I suppose what you can do is… if their HbA1c is above certain level, then have the EMR goes alert, ‘PDA, PDA!’. So either I can give the book or I can alert them to go to the website. – IDI 7_Primary care policymaker_have not used insulin PDA Every day when we see patients, we have to use the computer. So maybe it should be in the desktop in PDF so that when we see patients, we just open up the PDF. FD4 1_Medical officer 4_have not used insulin PDA Time is a problem because there are many patients. Whether we have enough time to go through (the insulin PDA) with the patient. – FGD 1_Medical officer 4_have not used insulin PDA In contrast, some participants felt that the insulin PDA might help to reduce consultation time. Theme 3: Lack of COC However, this is only if patients used the insulin PDA prior to the clinical consultation such as while waiting for the doctor or at home. It was felt that patients who have read the PDA were better prepared to discuss their concerns with doctors during clinical consultation and this might help to save time. Another type of reminder is to put up posters or notices about the insulin PDA, as well as making the insulin PDAs available and visible in the consultation room. This will help HCPs remember to use the insulin PDA as well as creating awareness about the insulin PDA among patients. Like putting posters in the clinic so that patients or their carers can see. Then patient can remind doctors about the book. – FGD 2_Medical officer 13_have used insulin PDA This saved time for the doctor. ‘Mr, you read first, what you don’t understand please come back to me’. So he would have read the whole thing. What they didn’t understand they highlight to you and you address. So your time management is shortened. – IDI 6_RN_ have not used insulin PDA But I think if we alert them more they will use it. So you sort of like, make it available. So I suppose if it is available in the room, and they are reminded all the time. I think it is keep being reminded that there is such a thing. – IDI 7_Primary care policymaker_have not used insulin PDA Theme 5: Reminder network The PDA was not integrated into the care pathway, and hence, the HCPs might forget to use it with patients. The participants wanted reminders and suggested different approaches including peer reminder, incorporating the PDA into the clinic electronic medical record (EMR), and displaying posters or PDAs in the clinic to remind both HCPs and patients to use the PDA. Discussion In Switzerland, however, hierarchical structures and asym- metric physician-patient relationship were reported to be barriers for SDM implementation despite doctors recognizing its importance [31]. This shows similarity to the hierarchical social pattern in Asian culture whereby doctors are placed at a higher societal stratum [33], and patients may therefore tend to consign their healthcare decisions to HCPs. Furthermore, doctors may not wish to offer a choice to patients as it is considered a good practice to initiate insulin in patients with T2DM who are unable to achieve glycaemic control despite taking maximal oral medications. Patients in the public health facilities may feel that they may not have control over their health decision as they only pay a nominal fee (i.e. ‘I get what I pay for’). In addition, they do not have much freedom to choose the HCP they want to see. However, Asian studies have shown that many patients preferred an autonomous (active and shared) role in decision-making [9, 10]. Thus, to facilitate the use of PDAs, the concept of SDM needs to be promoted among HCPs and patients in developing countries. Zhang et al. [34] highlighted that one way to increase patient involvement in making treatment decisions is to increase healthcare professionals’ knowledge about this concept. HCPs also need to be trained on respecting pa- tients’ autonomy and how to engage patients in making decisions about their health care. The Patient Decision Aids Research Group has created continuous education such as online tutorials, interactive workshops, perform- ance feedback and structured protocols in providing de- cision support [18]. Efforts are also needed to empower patient in being more involved in their healthcare, and one way to do that is to conduct public health cam- paigns [15]. In developed countries such as Australia and the UK, the ‘AskShareKnow’ [35, 36] and the ‘Ask three questions’ [37, 38] campaigns are conducted to en- courage patients to ask their HCPs the three questions (‘What are my options?’, ‘What are the benefits and harms?’ and ‘How likely are these going to happen?’) to increase patient involvement in healthcare. Discussion While it has been shown to improve patient-doctor communication in SDM in Australia [35], it is unsure if such campaign would be effective on countries with strong hierarchical social pattern like in developing countries such as M l i A th t t i th t h b d Another factor influencing implementation that was raised was role boundary of HCPs in the clinic setting. Role boundary may act both as a facilitator or barrier for the implementation of the insulin PDA. Being clear of one’s job responsibilities helped participants decide if they were the right person to use the insulin PDA. For example, all participants felt that doctors should intro- duce and use the insulin PDA, given their authority to prescribe insulin and familiarity with the patient’s health profile. Relying solely on doctors would hinder an inter- professional team approach to using the PDA. Involve- ment of other HCPs besides physicians may help to disperse the work needed in providing decision support. For example, compared to primary care doctors who has to see high number of patients with various conditions, DEs would have more time to provide patient counsel- ling as they only see diabetes patients. Many western studies have also shown HCPs other than doctors, such as nurses, social workers, psychologists and allied health professionals, who play a significant role in ensuring PDAs are implemented successfully by identifying eli- gible patients, contacting patients about the PDA and providing decision coaching [39–41]. Participants of this current study also suggested that nurses could remind doctors to use the PDA. To increase DEs’ and nurses’ involvement in insulin PDA implementation, they can be trained in decision coaching skills, which have been found to be effective in guiding patients to make an in- formed decision [42]. SDM and decision coaching training can be included in the advanced diploma in diabetes edu- cation and even basic nursing programmes. In addition, doctors also need to be trained to work with the nurses as a team to implement PDA in the clinic. A recent study showed that very few SDM training programmes adopt an interprofessional approach [43], which could help to utilize the strengths of each team members to create a more coordinated and efficient PDA implementation process. Time constraints are a universal barrier in implement- ing SDM as well as PDAs [6, 44]. Discussion This study provides insights into factors influencing implementation of PDAs from a developing country, a setting which is scarcely reported. The factors revealed in our study are not too different from those commonly reported in the western countries such as lack of SDM culture [25–27], time constraints [6, 25] and reminders. Less commonly reported factors which emerged from our study were role boundary and COC. This study highlighted a few important domains from the TDF that could influence SDM implementation in developing countries. For example, under the ‘environmental con- text and resources domain’, we found that there was low awareness and receptivity to SDM among the HCPs, which then hindered the implementation of PDA in the Malaysian primary care setting. Doctors, among doctors [to remind one another]. Sometimes the attendants are also assisting the doctors. They may not know how to use it, but they will know there is such a tool.//Nurses who are working in the clinic with the doctors, they can remind the doctor ‘Hey doctor you can use the book you know’. – IDI 1_DE 1_have not used insulin PDA It was proposed that the EMR could be programmed to identify patients suitable for using the PDA and to notify the doctors during the consultation. In addition, the electronic copy The participants of this study were doubtful if the concept of SDM is culturally acceptable in Malaysia due to HCP paternalism and patient’s submissiveness Tong et al. Implementation Science (2017) 12:40 Page 8 of 12 Page 8 of 12 to promote SDM in clinical practice, however, at the macro level in Malaysia are the following: (1) incorpor- ation of SDM in clinical practice guidelines, (2) advocating the use of patient decision aids or other decision support tools in patient care, (3) inclusion of patient involvement in decision-making as a quality indicator and (4) payment/ reimbursement for practices which implement SDM or use decision aids [15]. towards doctors. This resistance to SDM would hinder the adoption of SDM and subsequently lead to non-use of the insulin PDA. There is a distinct contrast in SDM implementation between developed countries (such as the USA, UK, Germany, Netherlands, Australia, Italy and Spain) where SDM and the use of PDAs have already been implemented at policy and legislation levels [28–31] and developing countries in Asia see slower progress in adopting SDM and PDAs [15, 32]. Discussion More studies are needed to look at the effectiveness of having promo- tional materials such as posters or notices on HCPs in remembering to use as well as on patients to initiate discussions with HCPs on PDAs. Ultimately, HCPs and patients need to be made aware that long-term patient engagement in self-management for chronic conditions like diabetes is crucial in improving diabetes control and outcome. This would then obviate the need for reminders. The use of PDA should be integrated as part of the training on longitudinality of care whereby HCPs and patients should be made aware that insulin is an option as diabetes progresses and that PDAs are available to h l h k h d achieved more quickly over time [49]. While the first consultation using the PDA may take a longer time, in- sulin decision-making over subsequent consultations may be shorter, and this reduces delay in decision- making. The participants of this study also felt that pre- visit usage of the insulin PDA might help to save time. Brackett et al. [50] have tested a pre-visit model and found that in addition to time saving, physicians also re- ported that discussions during a consultation were more focused and helped to address patient values and prefer- ences, which may otherwise be overlooked. It also facili- tated decision-making as patients arrived at the clinic appointment more prepared to discuss and make a decision [50]. One strategy used to allay fears over in- creased time was by asking a mentor or peer expert to demonstrate how the insulin PDA can be incorpo- rated into a standard clinical consultation [51, 52] without taking more time and to emphasize that the delivery of PDA only becomes easier and possibly faster with training and practice. Another important aspect of the implementation of the insulin PDA is the need to follow up patients (ideally by the same HCP) to ensure that there is continuity in the delivery of the insulin PDA. The use of PDAs is not a one-off event as SDM is a continuous interactive process between HCPs and patients; hence, COC is cru- cial in ensuring effective delivery of the insulin PDA. Discussion Two studies reported longer consultation time with PDA use compared to usual care [45, 46]. However, some studies indicate otherwise and one strategy that can be used to address the time issue is to highlight to HCPs that the use of PDAs does not always require more time compared to usual care [2, 47, 48] and that informed decision may be Tong et al. Implementation Science (2017) 12:40 Page 9 of 12 Page 9 of 12 of our study highlighted a reminder network to address the issue of forgetfulness as well as to trigger interest in using the insulin PDA. The use of EMR and IT system has been raised as an effective reminder platform in the implementation of SDM and PDA; for example, they can be used to identify eligible patients for PDAs [44] before their clinic visit, to prescribe PDAs as well as to cue for PDA use [60]. While HCPs have reported they would be more likely to use the PDAs if they were reminded through EMR [4], some felt that an electronic, inter- active decision aid linked to a computerized reminder system may not necessarily be better compared to trad- itional paper resources because of technical issues. The integration of PDA reminders into the EMR will require technology support, which can be a significant barrier in developing countries that are still using paper-based sys- tems [61]. If technology cannot be adopted to facilitate implementation, peer support would be another useful form of reminder, which was also raised by participants in our study. For peer support to be effective, the work- ing culture needs to be one that holds the belief that the use of PDA is the preferred practice style. Efforts are needed to create awareness on the benefits of PDAs so that HCPs will be willing to use them. Physician cham- pion plays an important role in creating awareness about the availability and the importance of PDAs, and encour- age the staff to use them; this has been reported as one of the key factors in the successful implementation of PDAs [62]. Another reminder that was raised was having posters and notices to promote the insulin PDA. Pro- motional brochures placed at exam rooms have been implemented to increase patients’ interest in decision aids [4, 26]. However, while it prompted discussions about PDAs, it did not significantly lead to physicians prescribing PDA to patients [26]. Discussion The lack of COC in the use of PDAs may cause difficul- ties for patients to raise or discuss issues pertaining to the PDAs that were brought up in the previous consult- ation [27]; it also prevents rapport-building between HCPs and patients that could facilitate informed decision-making. In Asia, COC is a challenge in health- care delivery due to high patient load, lack of manpower, time constraints [53, 54], lack of family physicians and uncoordinated referral mechanisms [55]. COC is also difficult particularly in an academic healthcare setting, where there is a high turnover of staff after the medical trainees have completed their training [53, 56, 57]. To address this barrier, systematic documentation of the use of PDA and its discussions with patients can help to facilitate communication between different doctors who follow up the same patients. Furthermore, new HCPs need to be educated on decision support and PDA use. Such training can be introduced during the orientation programme for new HCPs, having a resource person that provide more information about the PDA in the im- plementation setting as well as having on-going training and support to familiarize HCPs with the concept of SDM and the use of PDAs. Ethics approval and consent to participate This study received ethics approval from the University of Malaya Medical Centre Medical Ethics Committee (reference: MECID.NO: 20158-1600). Consent to participate was obtained from all the participants in a written consent form. Consent to participate was obtained from all the participants in a written consent form. Strengths and limitations of study ‘Forgetting to use’ has been reported as a barrier in the implementation of PDAs [58, 59] even after provision of training and continuing medical education credits to encourage use among HCPs [58]. Participants This study highlights implementation issues that are per- tinent to developing countries where literature on barriers and facilitators to implementation of PDA is scanty. This Page 10 of 12 Tong et al. Implementation Science (2017) 12:40 study explored views from a diverse group of key stake- holders from policymaker, physician, nurse and DE, and this helps to elicit factors influencing implementation from various professional levels. interview; PDA: Patient decision aid; RN: Registered nurse; SDM: Shared decision-making; TDF: Theoretical domains framework Acknowledgements The authors would like to thank the University of Malaya Research Grant (UMRG) – RP041C-15HTM for funding this study. References 1. Coulter A, Collins A. Making shared decision-making a reality: no decision about me, without me. London: King's Fund; 2011. 2. Stacey D, Legare F, Col NF, Bennett CL, Barry MJ, Eden KB, et al. Decision aids for people facing health treatment or screening decisions. Cochrane Database Syst Rev. 2014; 28(1):Cd001431. 3. Hill L, Mueller MR, Roussos S, Hovell M, Fontanesi J, Hill J, et al. Opportunities for the use of decision aids in primary care. Fam Med. 2009; 41(5):350–5. 4. Lin GA, Halley M, Rendle KAS, Tietbohl C, May SG, Trujillo L, et al. An effort to spread decision aids in five California primary care practices yielded low distribution, highlighting hurdles. Health Aff. 2013;32(2):311–20. 5. Holmes-Rovner M, Valade D, Orlowski C, Draus C, Nabozny-Valerio B, Keiser S. Implementing shared decision-making in routine practice: barriers and opportunities. Health Expect. 2000;3(3):182–91. 6. Shultz CG, Jimbo M. Decision aid use in primary care: an overview and theory-based framework. Fam Med. 2015;47(9):679–92. 7. Elwyn G, Scholl I, Tietbohl C, Mann M, Edwards AGK, Clay C, et al. “Many miles to go…”: a systematic review of the implementation of patient decision support interventions into routine clinical practice. BMC Med Inform Decis Mak. 2013;13 Suppl 2:S14. 1. Coulter A, Collins A. Making shared decision-making a reality: no decision about me, without me. London: King's Fund; 2011. 1. Coulter A, Collins A. Making shared decision-making a reality: no decision about me, without me. London: King's Fund; 2011. Competing interests Th h d l h Competing interests The authors declare that they have no competing interests. Acknowledgements Acknowledgements The authors would like to thank the University of Malaya Research Grant (UMRG) – RP041C-15HTM for funding this study. p There are a few limitations in this study. First, there were participants who had not been exposed to or had no experience in using the insulin PDA; they may not be able to grasp the concept of the insulin PDA, which in- volves the process of shared decision-making between HCP and patients. Instead, they may have thought that the insulin PDA is just another educational material. There is a possibility that some issues pertaining to the implementation processes for PDA may have been omit- ted. The researchers tried to reduce this limitation by explaining to the participants the purpose of the insulin PDA, concept of SDM, its content, as well as showing a video of a mock consultation using the PDA before con- ducting the interviews. Second, the data collection was only conducted in an academic primary care clinic and did not include other healthcare settings such as public community health clinics or private practices. Hence, the findings of this study may not be transferable to other settings. Third, it is noteworthy that there was a preponderance of female participants in this study, as more female than male candidates are interested to pur- sue family medicine as a specialty in Malaysia. However, the researchers felt that the participants’ sex may not have significant impact on the study findings given that the topic of this study is one that is not gender-sensitive. Fourth, only HPMs’ and HCPs’ views were gathered in this study. Patient’s perspectives should also be taken into account, which are currently being explored in the next phase of this study. Lastly, there is a possibility that participants may have provided positive responses or so- cially desirable answers, as they did not want to criticize their own practice; researchers tried to offset this bias by reassuring the participants that confidentiality would be kept and that their participation in this study would not affect their work or career. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details 1 1Department of Primary Care Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. 2Department of Family Medicine, Faculty of Medicine and Health Sciences, University Putra Malaysia, Serdang, Malaysia. Received: 5 November 2016 Accepted: 13 March 2017 Received: 5 November 2016 Accepted: 13 March 2017 COC: Continuity of care; DE: Diabetes educator; FGD: Focus group discussion; HCP: Healthcare provider; HPM: Healthcare policymaker; IDI: In-depth Consent for publication f Participants were informed that any identifiable information obtained would be removed so that they cannot be identified when reporting the data. No individual data were reported in this study but reported as pooled findings. Written informed consent was obtained from the participants. Authors’ contributions All authors (WTT, YKL, CJN, PYL) of this study conceived and designed the study, analyzed the data and contributed to the discussion and wrote and edited the manuscript. WTT, YKL and NCJ conducted the interviews. WTT wrote the first draft of the manuscript. All authors read and approved the final manuscript. Conclusions 2. Stacey D, Legare F, Col NF, Bennett CL, Barry MJ, Eden KB, et al. Decision aids for people facing health treatment or screening decisions. Cochrane Database Syst Rev. 2014; 28(1):Cd001431. When implementing PDAs, it is crucial to consider the healthcare culture and system, particularly in developing countries such as Malaysia. This study highlights that to facilitate the implementation and effective use of PDA, there is a need to change physician’s paternalistic attitude as well as to promote patient empowerment so that the concept of SDM can be embraced. The use of an interpro- fessional approach and reminders should also be consid- ered when designing strategies for PDAs implementation. y 3. Hill L, Mueller MR, Roussos S, Hovell M, Fontanesi J, Hill J, et a O t iti f th f d i i id i i F 3. Hill L, Mueller MR, Roussos S, Hovell M, Fontanesi J, Hill J, et al. Opportunities for the use of decision aids in primary care. Fam Med. 2009; 41(5):350–5. 4. Lin GA, Halley M, Rendle KAS, Tietbohl C, May SG, Trujillo L, et al. An effort to spread decision aids in five California primary care practices yielded low distribution, highlighting hurdles. Health Aff. 2013;32(2):311–20. 5. Holmes-Rovner M, Valade D, Orlowski C, Draus C, Nabozny-Valerio B, Keiser S. Implementing shared decision-making in routine practice: barriers and opportunities. Health Expect. 2000;3(3):182–91. 6. Shultz CG, Jimbo M. Decision aid use in primary care: an overview and theory-based framework. Fam Med. 2015;47(9):679–92. 7. Elwyn G, Scholl I, Tietbohl C, Mann M, Edwards AGK, Clay C, et al. “Many miles to go…”: a systematic review of the implementation of patient decision support interventions into routine clinical practice. BMC Med Inform Decis Mak. 2013;13 Suppl 2:S14. Funding g This research study was funded by the University of Malaya Research Grant (UMRG) – RP041C-15HTM. g This research study was funded by the University of Malaya Research Grant (UMRG) – RP041C-15HTM. 1. Coulter A, Collins A. Making shared decision-making a reality: no decision about me, without me. London: King's Fund; 2011. 2. Stacey D, Legare F, Col NF, Bennett CL, Barry MJ, Eden KB, et al. Decision aids for people facing health treatment or screening decisions. Cochrane Database Syst Rev. 2014; 28(1):Cd001431. 3. Hill L, Mueller MR, Roussos S, Hovell M, Fontanesi J, Hill J, et al. Opportunities for the use of decision aids in primary care. Fam Med. 2009; 41(5):350–5. 4. Lin GA, Halley M, Rendle KAS, Tietbohl C, May SG, Trujillo L, et al. An effort to spread decision aids in five California primary care practices yielded low distribution, highlighting hurdles. Health Aff. 2013;32(2):311–20. 5. Holmes-Rovner M, Valade D, Orlowski C, Draus C, Nabozny-Valerio B, Keiser S. Implementing shared decision-making in routine practice: barriers and opportunities. Health Expect. 2000;3(3):182–91. 6. Shultz CG, Jimbo M. Decision aid use in primary care: an overview and theory-based framework. Fam Med. 2015;47(9):679–92. 7. Elwyn G, Scholl I, Tietbohl C, Mann M, Edwards AGK, Clay C, et al. “Many miles to go…”: a systematic review of the implementation of patient decision support interventions into routine clinical practice. BMC Med Inform Decis Mak. 2013;13 Suppl 2:S14. 4. Lin GA, Halley M, Rendle KAS, Tietbohl C, May SG, Trujillo L, et al. An effort to spread decision aids in five California primary care practices yielded low distribution, highlighting hurdles. Health Aff. 2013;32(2):311–20. 5. Holmes-Rovner M, Valade D, Orlowski C, Draus C, Nabozny-Valerio B, Keiser S. Implementing shared decision-making in routine practice: barriers and opportunities. Health Expect. 2000;3(3):182–91. 6. Shultz CG, Jimbo M. 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Implementation Science (2017) 12:40 Abbreviations COC C • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step:
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imilar names and terms in dermatology; an appraisal
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Dermatology Eponyms Dermatology Eponyms DOI: 10.7241/ourd.20124.86 Corresponding author: Dr. Khalid Al Aboud The first is the pigmentation of the nail fold in association with melanonychia as a sign of melanoma. Hutchinson’s sign is a clinical sign which may refer to two different things [6,7]. The first is the pigmentation of the nail fold in association with melanonychia as a sign of melanoma. The second thing, is skin lesion on the tip of the nose as a sign of ophthalmic herpes zoster. This occurs because the nasociliary branch of the trigeminal nerve innervates both the cornea and the tip of the nose. This sign is named after Sir Jonathan Hutchinson (1828 –1913), who was an English surgeon, ophthalmologist, dermatologist, venereologist and pathologist [7]. Errors involving look-alike names are common when the names are handwritten and errors with sound-alike names are common when the names are spoken. The problem involves both brand names and generic drug names. However, brand (proprietary) names are the most common to be confused. The second thing, is skin lesion on the tip of the nose as a sign of ophthalmic herpes zoster. This occurs because the nasociliary branch of the trigeminal nerve innervates both the cornea and the tip of the nose. This sign is named after Sir Jonathan Hutchinson (1828 –1913), who was an English surgeon, ophthalmologist, dermatologist, venereologist and pathologist [7]. Examples of the numerous drug names that have been confused because they look and/or sound similar include Celebrex (celecoxib), Cerebyx (fosphenytoin), and Celexa (citalopram) [3]. In another example, the antihistamine Zyrtec syrup (cetirizine) has been confused with the histamine H2- receptor antagonist Zantac syrup (ranitidine) for pediatric patients. The most common type of names which may cause confusion with other names is the eponyms. An eponym is a name that comes from a person’s name [8,9]. Possibly for non-dermatologist, one may think that „Sweet’’ in „Sweet’s syndrome’’, is „a taste of sugar’’. But, this syndrome was named for Dr Robert Douglas Sweet, who first described it 1964 [10]. Factors such as poor handwriting and clinical similarity may exacerbate the problem. Several Measures to decrease medication errors due to confusing drug nomenclature are suggested, in order to maximize patient safety [1-4]. 366 © Our Dermatol Online 4.2012 Corresponding author: Dr. Khalid Al Aboud amoa65@hotmail.com Our Dermatol Online. 2012; 3(4): 366-367 Our Dermatol Online. 2012; 3(4): 366-367 Date of submission: 26.06.2012 / acceptance: 21.07.2012 Our Dermatol Online. 2012; 3(4): 366-367 Ahmad Al Aboud, Khalid Al Aboud: Similar names and terms in dermatology; an appraisal. Our Dermatol Online. 2012; 3( device. It is recommended that drug names be confirmed by spelling the name, providing both the brand name and the generic name, or providing the indication for use. It is also recommended that the person receiving the order repeat it to the person transmitting the order. Storing similarly named drugs separately and using auxiliary labels to differentiate the products in medication storage areas, was also suggested [1-4]. In medicine, one can find easily an abbreviation which stand for few different things as well as many similar words.i The similarities in the words in medical field include the names of the drugs and the names of the diseases. The names might be similar in „written communications’’ which is known as ”look alike’’ or in „verbal communications’’ which is known as „sound alike’’. Overall, this problem can be alleviated through actions by regulatory agencies, pharmaceutical manufacturers, healthcare professionals, and patients [1-4]. In a busy health care work environment, drug products are often mistaken for other products because of similar names. Hundreds of articles have been published about „Look alike and sound alike’’ drugs. These papers listed the drugs with a similar names in each specialty and discussed the possible confusion which may result among them. The different strategies to tackle this confusion have been elaborated [1-5]. The similarities in the names, specially, of drugs in medical field, are a cause of confusion to the health care providers and hence a great source of risk to the patients. In dermatology, in particular, confusing dermatologic drug names do, also, exist [5]. Moreover, the problem of „look- or sound-alike’’ names is not limited to the drugs but also involve the names of the diseases and other terms in dermatology literature.i As a matter of fact, one can find a single term, for two different things. Medication errors contribute substantially to patient injury and death, with 25% of these errors attributed to drug names that look or sound alike [5]. Hutchinson’s sign is a clinical sign which may refer to two different things [6,7]. SIMILAR NAMES AND TERMS IN DERMATOLOGY; AN APPRAISAL Ahmad Al Aboud1, Khalid Al Aboud2 1Dermatology Department, King Abdullah Medical City, Makkah, Saudi Arabia 2Pathology Department, Wake Forest University, Winston-Salem, NC, USA Source of Support: Nil Competing Interests: None declared Our Dermatol Online. 2012; 3(4): 366-367 Date of submission: 26.06.2012 / acceptance: 21.07.2012 Corresponding author: Dr. Khalid Al Aboud amoa65@hotmail.com Competing Interests: None declared Cite this article: Ahmad Al Aboud, Khalid Al Aboud: Similar names and terms in dermatology; an appraisal. Our Dermatol Online. 2012; 3(4): 366-367 Corresponding author: Dr. Khalid Al Aboud Our Dermatol Online. 2012; 3(4): 366-367 Corresponding author: Dr. Khalid Al Aboud The term, pseudo-Kaposi sarcoma, is generally used Scientist Examples of diseases linked his name Remarks Abraham Buschke (1868-1943), German dermatologist Buschke-Löwenstein tumour Verrucous carcinoma of genital skin Buschke-Ollendorff syndrome Dermatofibrosis lenticularis disseminate Henri Gougerot (1881-1955), French dermatologist Gougerot-Blum disease Lichenoid type of pigmented purpura Gougerot-Carteaud papillomatosis Confluent and reticulate papillomatosis François Henri Hallopeau (1842- 1919), French dermatologist Acrodermatitis continua of Hallopeau Pustular eruption of the fingers and toes Hallopeau-Siemens syndrome Recessive dystrophic epidermolysis bullosa Josef Jadassohn (1863-1936), German dermatologist Jadassohn-Lewandowsky syndrome Pachonychia congenita Nevus sebaceous of Jadassohn Yellowish to orange or tan hairless plaquelike lesions, usually present at birth Table I. Examples of scientists whose names are eponymously linked to more than one condition in dermatology literature 10. Sweet RD: An acute febrile neutrophilic dermatosis. Br J Dermatol. 1964;76:349-56. 10. Sweet RD: An acute febrile neutrophilic dermatosis. Br J Dermatol. 1964;76:349-56. Corresponding author: Dr. Khalid Al Aboud Similarly, „Mali’’ in the term acroangiodermatitis of Mali [11,12], does not refer to Republic of Mali but for Dr Mali, who described it 1965, in 18 patients having mauve colored macules and papules predominantly over the extensor surface For instance, in medication orders that are communicated orally, whether in person or by telephone or other auditory www.odermatol.com synonymously with acroangiodermatitis of Mali, but is a broader term and includes both acroangiodermatitis of Mali and Stewart-Bluefarb syndrome [11]. of feet with underlying chronic venous insufficiency [11]. Similar name might be thought for and confused with another person, for example verrucous carcinoma of Ackerman is named after Lauren Vedder Ackerman (1905-1993) and not, A. Bernard Ackerman (1936-2008). We have also published that, there are 2 „Bart’s’’ in the eponyms of dermatology. Dr Bruce J Bart, who is behind „Bart syndrome’’, and Dr Robert Bart, who was one of the men behind „Bart-Pumphrey synrome’’ [15]. One may see also identical names for 2 different eponyms. For examples „Sjögren’’ in „Sjögren’s syndrome’’ (Sicca syndrome), is named after Henrik Samuel Conrad Sjögren (1899-1986), Swedish ophthalmologist. Whereas, „Sjögren’’, in „Sjögren-Larsson syndrome’’, is named after, Karl Gustaf Torsten Sjögren (1896-1974), Swedish physician, psychiatrist and inheritance researcher [13]. „Look-alike or sound-alike’’ eponyms are not rare. This is because there is extensive list of eponyms bearing the name of the same scientist [16,17]. In Table I, we listed examples of scientists whose names are eponymously linked to more than one condition in dermatology literature. Similarly, „Stewart’’ in „Stewart-Treves syndrome” [14], (a malignancy that arises within chronic lymphedema), is different from the one in’’ Stewart-Bluefarb syndrome’’. The latter is a type of acroangiodermatitis which was described independently by Stewart as well as by Bluefarb and Adams on the legs of patients with arterio-venous malformations [11]. The term, pseudo-Kaposi sarcoma, is generally used It goes without saying that consolidation of the nomenclature is needed in medicine. The concept of „re-naming’’ the similar names of drugs or diseases, to prevent possible confusion, has been debated over the years and there is a still controversy over this topic. Neverthless, healthcare providers need to be, at least, vigilant about the similarities in the names, in particular those which may potentially cause a patient harm. [11]. Copyright by Ahmad Al Aboud, et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. REFERENCES 11. Jindal R, De D, Dogra S, Saikia UN, Kanwar AJ: Acroangiodermatitis of Mali in a patient with congenital myopathy. Dermatol Online J. 2010;16:4. 1. Santell JP, Cousins DD: Medication errors related to product names. Jt Comm J Qual Patient Saf. 2005;31:649-654.i 2. Cohen MR: Medication errors. The suffix problem: will the F.D.A. solve it? Nursing. 1990;20:17. 12. Mali JW, Kuiper JP, Hamers AA: Acroangiodermatitis of the foot. Arch Dermatol. 1965;92:515-8. 3. Hoffman JM, Proulx SM: Medication errors caused by confusion of drug names. Drug Saf. 2003;26:445-52. 13. Al Aboud K, Al Aboud D: Karl Gustaf Torsten Sjögren and the Sjögren-Larsson syndrome. Dermatol Reports. 2011;3:74-e34. 4. Al Aboud A, Al Aboud K: From where did the names of dermatology drugs and brands come from? J Pak Asso of Dermatol. 2008;18:165-6. 14. Aguiar Bujanda D, Camacho Galán R, Bastida Iñarrea J, Aguiar 14. Aguiar Bujanda D, Camacho Galán R, Bastida Iñarrea J, Aguiar Morales J, Conde Martel A, et al: Angiosarcoma of the abdominal wall after dermolipectomy in a morbidly obese man. A rare form of presentation of Stewart-Treves syndrome. Eur J Dermatol. 2006;16:290-2. 5. Gremillion L, Hogan DJ: Dermatologic look- or sound-alike medications. J Drugs Dermatol. 2004;3:61-4. of presentation of Stewart-Treves syndrome. Eur J Dermatol. 2006;16:290-2. 6. Al Aboud K, Al Hawsawi K, Ramesh V, Al Aboud D, Al Githami A: Cutaneous signs. Skinmed. 2003;2:104-7. 15. Al Aboud A, Al Aboud K: Separating ‚’Bart’s’’ apart in dermatology eponyms. Our Dermatol Online. 2012;3:64-5. 7. van Ruth S, Toonstra J: Eponyms of Sir Jonathan Hutchinson. Int J Dermatol. 2008;47:754-8. 16. Al Aboud A, Al Aboud K: Josef Jadassohn (1863-1936), Felix Lewandowsky (1879-1921), and their syndrome. Clin Cosmet Investig Dermatol. 2011;4:179-82. 8. Al Aboud K, Al Hawsawi K, Ramesh V, Al Aboud D, Al Githami A: An appraisal of terms used in dermatology. Skinmed. 2003;2:151-3. 17. Al Aboud K: Rudolf Happle and the dermatology eponyms linked to his name. Our Dermatol Online. 2012;3:143-44. 9. Al Aboud K, Al Hawsawi K, Ramesh V, Al Aboud D, Al Githami A: Eponyms in dermatology. Skinmed. 2004;3:11-2. 9. Al Aboud K, Al Hawsawi K, Ramesh V, Al Aboud D, Al Githami A: Eponyms in dermatology. Skinmed. 2004;3:11-2. © Our Dermatol Online 4.2012 367
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Osteopontin plays a pivotal role in increasing severity of respiratory syncytial virus infection
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RESEARCH ARTICLE Viviana Sampayo-Escobar1,2, Ryan Green3, Michael B. Cheung1,2, Raminder Bedi1,2, Subhra Mohapatra1,3*, Shyam S. Mohapatra1,2* 1 James A Haley Veterans Affairs Hospital, Tampa, Florida, United States of America, 2 Department of Internal Medicine, Department of Molecular Medicine, University of South Florida Morsani College of Medicine, Tampa, Florida, United States of America, 3 Department of Molecular Medicine, University of South Florida Morsani College of Medicine, Tampa, Florida, United States of America * smohapa2@health.usf.edu (SM); smohapat@health.usf.edu (SSM) * smohapa2@health.usf.edu (SM); smohapat@health.usf.edu (SSM) a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Sampayo-Escobar V, Green R, Cheung MB, Bedi R, Mohapatra S, Mohapatra SS (2018) Osteopontin plays a pivotal role in increasing severity of respiratory syncytial virus infection. PLoS ONE 13(4): e0192709. https://doi.org/ 10.1371/journal.pone.0192709 Editor: Stephania A. Cormier, Louisiana State University System, UNITED STATES Received: September 26, 2017 Accepted: January 29, 2018 Published: April 20, 2018 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Citation: Sampayo-Escobar V, Green R, Cheung MB, Bedi R, Mohapatra S, Mohapatra SS (2018) Osteopontin plays a pivotal role in increasing severity of respiratory syncytial virus infection. PLoS ONE 13(4): e0192709. https://doi.org/ 10.1371/journal.pone.0192709 Editor: Stephania A. Cormier, Louisiana State University System, UNITED STATES Editor: Stephania A. Cormier, Louisiana State University System, UNITED STATES Received: September 26, 2017 Accepted: January 29, 2018 Published: April 20, 2018 Received: September 26, 2017 Accepted: January 29, 2018 Published: April 20, 2018 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Abstract The molecular mechanisms underlying susceptibility to severe respiratory syncytial virus (RSV) infection remain poorly understood. Herein, we report on the role of osteopontin (OPN) in regulation of RSV infection in human epithelial cells and how interleukin-1 beta (IL- 1β), a cytokine secreted soon after RSV infection, when persistently expressed can induce OPN expression leading to increased viral infection. We first compared OPN expression in two human epithelial cell lines: HEK-293 and HEp-2. In contrast to HEp-2, HEK-293 expresses low levels of pro-caspase-1 resulting in decreased IL-1β expression in response to RSV infection. We found a correlation between low IL-1β levels and a delay in induction of OPN expression in RSV-infected HEK-293 cells compared to HEp-2. This phenomenon could partially explain the high susceptibility of HEp-2 cells to RSV infection versus the mod- erate susceptibility of HEK-293 cells. Also, HEK-293 cells expressing low levels of pro-cas- pase-1 exhibit decreased IL-1β expression and delayed OPN expression in response to RSV infection. HEK-293 cells incubated with human rIL-1β showed a dose-dependent increase in OPN expression upon RSV infection. Also, incubation with rOPN increased RSV viral load. Moreover, HEp-2 cells or mice infected with a mucogenic RSV strain RSV-L19F showed elevated levels of OPN in contrast to mice infected with the laboratory RSV strain rA2. This correlated with elevated levels of OPN following infection with RSV-L19F com- pared to rA2. Together, these results demonstrate that increased OPN expression is regu- lated in part by IL-1β, and the interplay between IL-1β and OPN signaling may play a pivotal role in the spread of RSV infection. Data Availability Statement: All relevant data are within the paper. Funding: This research was supported by a VA Merit Review Awards #BX003685 and BX003413, and Research Career Scientist Awards to Dr. Shyam Mohapatra (IK6 BX003778) and Dr. Subhra Mohapatra (IK6BX004212). Viviana Sampayo was supported by Fulbright Scholarship (Becas Caldas 2011-COLCIENCIAS) and the Helen Hoxeng Fund. Though this report is based upon work supported in part by the Department of Veterans Affairs, Osteopontin plays a pivotal role in increasing severity of respiratory syncytial virus infection Viviana Sampayo-Escobar1,2, Ryan Green3, Michael B. Cheung1,2, Raminder Bedi1,2, Subhra Mohapatra1,3*, Shyam S. Mohapatra1,2* * smohapa2@health.usf.edu (SM); smohapat@health.usf.edu (SSM) Osteopontin and respiratory syncytial virus infection individuals throughout life because infection does not lead to a persistent immune memory response [1–5]. Healthy adults infected with RSV typically experience mild cold-like symp- toms. However, severe RSV infection commonly causes bronchiolitis in infants resulting in 120,000 hospitalizations annually in the US. Severe RSV infection constitutes a high risk for the development of childhood asthma [6–8]. Elderly persons also develop severe RSV-induced pneumonia that leads to increased morbidity and mortality in this age group causing >11,000 deaths annually in the US alone [9, 10]. Despite progress made towards understanding the biology of RSV infection, the molecular mechanism which determines the severity of RSV infection is not well understood [11–13]. Veterans Health Administration, Office of Research and Development, the contents of this report do not represent the views of the Department of Veterans Affairs or the United States Government. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. RSV infection induces a persistent inflammatory response which sometimes escalates beyond control [14–17]. This exaggerated inflammation complicates the disease outcome and leads to respiratory complications such as asthma exacerbation or recurrent wheezing, making it difficult to identify a treatment option [18–25]. Immune cells and tissues express pattern- recognition receptors (PRRs) capable of recognizing pathogen-associated molecular patterns (PAMPS), activating the innate immune response to release pro-inflammatory cytokines that facilitate pathogen clearance but also mediate disease pathology [26, 27]. RSV infection induces the expression of several pro inflammatory cytokines including IL-1β, IL6 and chemo- kines such as IL-8 and TNF-α that contribute to inflammation and the pathology of the infec- tion. However, whether this inflammation contributes to increased viral load and spread of infection is unclear. In an effort to dissect the molecular basis of severity of RSV infection, previously we con- ducted a microarray analysis and identified several genes whose expressions are influenced by both aging and RSV infection. Our previous studies in a murine model compared the progres- sion of RSV infection in aged vs. young mice. We showed that aged mice express higher levels of IL-1β and OPN prior to infection compared to their younger counterparts, and this pro- inflammatory state that comes with aging impairs the antiviral response in those mice when they are exposed to RSV infection [28]. Introduction Respiratory syncytial virus (RSV) is one of the most common causes of lower respiratory tract infections with a global disease burden estimated at ~34 million new cases and 160,000 deaths every year. RSV is one of the first pathogens encountered by the infant immune system and most infants have at least one RSV infection by two years of age. However, RSV may re-infect PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 1 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 Virus purification, infection and plaque assay Three different sources of RSV were used: rgRSV-A2 which expresses green fluorescent pro- tein, the mucogenic rA2-L19F (RSV-L19F) recombinant variant of the A2 strain with its fusion protein replaced with that of the line 19 RSV, and a version of the line 19 that expresses a red fluorescent protein (RSV-KL19F). The virus was propagated by infecting 60% confluent HEp- 2 cells with a multiplicity of infection (MOI) of 0.1 plaque forming units (pfu) per cell for two hours at 37˚C with gentle rocking every 15 minutes, after which the medium was replaced by fresh DMEM containing 5% FBS. Cells and media were collected when 70–80% of the cells showed cytopathological effects. RSV was pelleted through a layer of 30% glycerol (0.22 μm-fil- tered) in 0.1 M MgSO4 and 50 mM HEPES, pH 7.5. The viral particles were pelleted by centrifuging at 11,600 rpm in an SW28 rotor for 3 h at 4˚C. Supernatants were carefully aspi- rated without disturbing the viral pellets and the viral pellets were re-suspended in pre-cooled, 0.22 μm-filtered 50 mM HEPES, pH 7.5, 0.1 M MgSO4, 150 mM NaCl. This buffer was used as mock infection media in all experiments and the re-suspended pellet was aliquotted and stored at −80˚C until use. UV-inactivation of RSV was performed by irradiating aliquots of virus with 1200 mJ of UV for 20 mins using a Stratalinker. For all experiments, a monolayer of HEp-2 or HEK-293 cells at 80% confluence was infected with 0.1, 0.5, 1 or 10 MOI (as indicated in the figure legends). Cells were incubated with the viral inoculum in Opti-MEM (Life Technologies) containing 2% FBS for 2 hours at 37˚C. After this, the infectious medium was replaced by fresh DMEM with 5% FBS. At 24, 48 or 72 hours post infection (hpi) the cell supernatants and pellets were collected for viral plaque assay, RNA or protein analysis. WT or OPN KO mice were intranasally infected with 1 or 3 x 106 plaque forming units (pfu)/mouse of RSV-L19F or rgRSV-A2 and euthanized 1, 3 or 5 days post-infection (dpi). Lungs were collected for RNA or protein extraction. To determine the viral titers, HEp-2 cells were seeded in 24 well plates to 80% confluence and infected in duplicates with serial dilutions of cell supernatants for a period of 2 hours at 37˚C. Cell culture Human epithelial type 2, HEp-2 (CCL 23; American Type Culture Collection, Rockville, MD), and human embryonic kidney 293, HEK-293 (ATCC CRL-1573), cells were maintained in Dulbecco´s modified Eagle´s medium (DMEM, HyClone) supplemented with 5% fetal bovine serum (FBS, HyClone) and 1% penicillin-streptomycin (GIBCO). Cells were incubated at 37˚C in a humidified incubator with 5% CO2/95%. Mice Female osteopontin-deficient knockout mice (OPN KO) (strain B6.Cg-Spp1tm1Blh/J) and wild type (WT) C57BL/6 were purchased from Jackson Laboratory and intranasally infected with RSV when they were 6–8 weeks old. All animal work was approved by and performed in accordance with the policies of the University of South Florida Institutional Animal Care and Use Committee. Mice were acclimated for 7 days prior to the start of experiments. Mice were provided with standard rodent chow and water ad libitum. OPN is a secreted multifunctional protein also known as secreted phosphoprotein 1 (SPP- 1) and early T-lymphocyte activation-1 (Eta-1) factor [29]. Of note, OPN expression is regu- lated by mediators of acute inflammation such as IL-1β [30]. Although it was first identified in osteoclasts and is highly expressed in bone, OPN is secreted by a variety of cells and tissues including macrophages, smooth muscle cells, epithelial, and endothelial cells [29, 31–34]. OPN can have both anti- and pro-inflammatory activities; hence it has been associated with many physiological functions and pathological conditions [35–39]. Integrin αvβ3 and CD44 are receptors for OPN and their interaction modulates the immune response by facilitating neu- trophil and macrophage migration to sites of injury. Also, OPN promotes dendritic cell (DC) maturation and migration to the lymph nodes where DCs can present antigens to naïve T cells, thus serving as a bridge between the innate and the cell-mediated immune responses [33, 40–42]. Since OPN is up-regulated in other inflammatory lung diseases such as asthma and RSV has been implicated in asthma exacerbation, we reasoned that OPN might be involved in regu- lating susceptibility to RSV infection. To test this hypothesis, we investigated the role of OPN in RSV infection and determined that IL-1β expression correlated with the up-regulation of OPN during RSV infection. We also found increased OPN protein expression in human epi- thelial cells and mice infected with RSV-L19F, a strain that produces severe infection associ- ated with increased viral loads, compared to rgRSV-A2 which induces mild infection and moderated viral loads. This suggests that OPN expression is correlated with increased spread of the virus between cells and increased cell permissiveness to RSV infection. PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 2 / 24 Osteopontin and respiratory syncytial virus infection PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 Western immunoassay Cells were plated in 6-well culture plates the day before treatment/infection and harvested in lysis buffer containing 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 8.0 and a protease inhibitor cocktail (Thermo Fisher Scientific). After removal of cellular debris by centrifuga- tion, total protein concentration was measured at 660nm using protein assay reagent (Thermo Fisher Scientific) and 25 μg of total protein were separated in a precast 12% mini-PROTEAN TGX gel (Bio-Rad) and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was incubated with a rabbit polyclonal antibody to human OPN (Abcam, ab181440) and a mouse monoclonal antibody to β-actin (Sigma-Aldrich); proteins were detected by incubating with a secondary anti-mouse IgG-HRP and/or anti-rabbit IgG-HRP, followed by the ECL reagent kit (Pierce). Images were captured using a ChemiDoc XRS+ imaging system (Bio- Rad). Quantitative RT-PCR Total RNA was isolated from cells using TRIzol (Life Technologies). Samples were treated with recombinant DNase I (Life Technologies) to remove any contaminating DNA. 1 μg of RNA was reverse transcribed using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific) as described in manufacturer’s instructions. Quantitative real-time PCR (qPCR) was performed on the BioRad CFX384™Real-time PCR Detection system using DyNamo Color Flash SYBR master mix (Thermo Fisher Scientific). The sequences of all prim- ers used in this study are as follows (forward and reverse): RSV-N, 5’- CAT CTA GCA AAT ACA CCA TCC A-30 and 50-TTC TGC ACA TCA TAA TTA GGA GTA TCA A-30; human IL-1β (PrimeTime1 -qPCR primers-IDT), 5’- GAA CAA GTC ATC CTC ATT GCC-3’ and 5’-CAG CCA ATC TTC ATT GCT CAA G-3’; human OPN, 5’-TGG CCG AGG TGA TAG TGT G-3’ and 5’- CGG GGA TGG CCT TGT ATG-3’; human IFN-β, 5’-CAA CTT GCT TGG ATT CCT ACA AAG-3’ and 5’- TGC CAC AGG AGC TTC TGA CA-3’. All samples were run in four replicates and the data were analyzed using nor- malized gene expression (ΔΔCt). Expression of all genes was normalized to control hypoxan- thine-guanine phosphoribosyltransferase (HPRT): mouse HPRT, 50-GCT GAC CTG CTG GAT TAC ATT AA-30 and 50-TGA TCA TTA CAG TAG CTC TTC AGT CTG A-30; human HPRT, 5’- AGG AAA GCA AAG TCT GCA TTG TT-3’ and 5’- GGC TTT GTA TTT TGC TTT TCC A-3’. Osteopontin and respiratory syncytial virus infection protein (AbD Serotec). Plaques were visualized using an anti-mouse IgG horseradish peroxi- dase antibody (HRP)(Sigma) and developed with 4 CN peroxidase substrate (KPL); the dark purple spots were counted and each spot represented one plaque-forming unit (PFU). Virus purification, infection and plaque assay Infectious media were removed and cells were overlaid with 1 ml of 0.8% methylcellu- lose in DMEM supplemented with 5% FBS. 5 dpi, the cell monolayers were fixed overnight with 1 ml of 80% cold methanol. The next day, cells were rinsed with phosphate-buffered saline (PBS, Hyclone) and incubated with primary monoclonal antibody against RSV fusion PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 3 / 24 Immunofluorescence microscopy HEp-2 cells were plated in 8-well chamber slides the day before infection. Cells were mock infected or infected with 1 MOI of RSV strain RSV-L19F. Cells were fixed with cold 4% para- formaldehyde 24 or 48 hpi and stained with a goat polyclonal antibody against RSV (Millipore, ab1128) and a rabbit polyclonal antibody to OPN (ABCAM), followed by indirect immunoflu- orescence using secondary anti-goat IgG Alexa Fluor-555- and anti-rabbit IgG Alexa Fluor- 488-conjugated antibodies. Slides were mounted with 40, 6-diamidino-2-phenylindole (DAPI) containing anti-fade mounting media (Southern Biotech). A minimum of 10 images at 200X magnification were collected per slide with a DP72 digital camera on a BX51 Olympus fluores- cence microscope on the three different fluorescence channels. 4 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 Osteopontin and respiratory syncytial virus infection Enzyme-linked immunosorbent assay (ELISA) for OPN and IL-1β in lung homogenates Snap frozen lungs were homogenized in cold lysis buffer containing 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 10% Glycerol, 5 mM EDTA and a protease inhibitor cocktail. Tissue debris was pelleted by centrifugation at 4˚C for 10 min at 300×g and protein concentration in the supernatants was measured at 660 nm with protein assay reagent (Thermo Fisher Scien- tific). Mouse osteopontin ELISA kit (RayBiotech) and mouse IL-1β ELISA kit (BioLegend) were used per manufacturer’s instructions. Recombinant IL-1β and OPN treatment HEK-293 cells were infected with 1 MOI of RSV-L19F as described previously. Afterwards, the infectious media was replaced by fresh DMEM 5% FBS containing different concentrations (0–1 and 10 ng/ml) of human rIL-1β (PeproTech). Similarly, HEK-293 were treated four hours before infection with different concentrations (0–1–10–50–100 and 200 ng/ml) of human rOPN (PeproTech) diluted in fresh DMEM 5% FBS. Cells were infected with RSV-L19F at 0.1 MOI, and two hours after infection the media was replaced by fresh DMEM 5% FBS containing rOPN. Protein and supernatants were collected for viral titering and west- ern immunoassay. CD44 receptor-neutralization and RSV infection HEp-2 cells were seeded on 24-well plate the day before infection. On the infection day, cells were pre-treated at room temperature for 20 minutes with 10 μg broad spectrum rat-anti human CD44 antibody (clone A020) or normal rat IgG (control) (Millipore). After the pre- treatment, cells were infected with RSV-KL19F for two hours at 37˚C and infectious media was replaced with fresh growth media. After 24 hours, the percentage of RSV positive cells was determined by flow cytometry (BD FACS Canto II). Data was analyzed using BD FACS diva software. Caspase I inhibitor (Ac-YVAD-CHO) treatment HEp-2 cells were treated with 10 μM of caspase I inhibitor (Sigma-Aldrich) or DMSO (vehicle control) the two hours preceding the infection. Afterwards, cells were infected with 1 MOI of RSV-L19F, and two hours later the infectious media was replaced by fresh media containing 10 μM of caspase I inhibitor or DMSO. Protein and RNA were isolated 24 hpi. Flow cytometry assay for CD44 and RSV infected cells HEp-2 or HEK-293 cells were seeded on 6-well plates at an appropriate cell density to reach 70 to 80% confluence. On the day of the experiment, each set of cell cultures was infected with RSV-KL19F for 2 hours. 24 hpi, cells were washed with PBS, detached with accutase (Life Technologies) and stained with DAPI to determine cell viability, FITC mouse anti-human CD44 antibody (BD Bioscience) or isotype control antibody. The percentage of cells express- ing GFP (CD44 +) or RFP (RSV +) in each cell culture was determined by flow cytometry (BD FACS Canto II). Data was analyzed using BD FACS diva software. RSV infection upregulates IL-1β and OPN mRNA expression in epithelial cells Our previous study in a murine model of infection showed that higher basal levels of IL-1β and OPN in aged mice are associated with impaired antiviral response that leads to increased susceptibility to RSV infection. In vitro, to evaluate the correlation of IL-1β and OPN expres- sion with susceptibility to RSV infection we examined RSV infection in commonly used and highly susceptible HEp-2 cells and HEK-293 cells. HEK-293 cells were selected because they express low levels of endogenous pro-caspase1, which is essential for IL-1β expression; there- fore, they constitute a good model to compare viral infection and OPN expression in dimin- ished IL-1β expression [43]. We found a significant increase in expression (reported as fold expression compared to mock-infected cells) of RSV-N and IL-1β transcripts 24, 48 and 72 hpi in RSV-L19F-infected HEp-2 cells compared to RSV-L19F-infected HEK-293 cells (Fig 1A and 1B). We compared OPN expression levels obtained by qPCR performed on RNA extracted from HEp2 and HEK-293 cells infected with 1 MOI of RSV-L19F. In a similar pattern to that of IL-1β, OPN expression was significantly increased 48 hpi in RSV-L19F-infected HEp-2 cells. In contrast, HEK-293 cells showed decreased RSV infection, lack of IL-1β expression, and delayed OPN mRNA expression that was up-regulated 72 hpi (Fig 1C). These results sug- gest that increased RSV infection up-regulates both OPN and IL-1β expression. Also, lack of IL-1β expression in HEK-293 cells correlates with resistance to RSV infection and delays OPN expression. Statistical analysis All experiments were performed in triplicate and repeated at least twice. Statistical significance for each experiment was determined using Student’s t test and analysis of variance (ANOVA) with post-hoc Tukey’s multiple comparison test to find the differences among the groups, 5 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 Osteopontin and respiratory syncytial virus infection p<0.05. Calculations were performed and graphs produced using Prism 6.0 software (Graph- pad Software, San Diego, CA, USA). Graphs of results show the mean and error bars depicting the standard error of the mean, +/- SEM. RSV infection-induced IL-1β regulates OPN expression While the regulation of OPN expression is not fully understood, it is known that the expres- sion of OPN is regulated by different stimuli, depending on the cells and tissues where it is expressed [40, 41, 44]. To examine the role of IL-1β in the induction of OPN expression and to confirm that the production of OPN is not intrinsically impaired in HEK-293 cells, cells were Fig 1. IL-1β and OPN mRNA expression in RSV infected cells. HEp-2 and HEK-293 cells were mock infected or infected with 1 MOI of RSV-L19F. RNA was isolated at 24, 48 and 72 hpi. (A-C) Expression levels of RSV-N, IL-1β and OPN were determined by qPCR. Results are presented as fold-change in expression of RSV-N, IL-1β or OPN mRNA normalized to the control (HPRT). qPCR data are represented as means ±SEM. Experiments were performed in triplicate.  p < 0.01,  p < 0.001 p < 0.0001. Fig 1. IL-1β and OPN mRNA expression in RSV infected cells. HEp-2 and HEK-293 cells were mock infected or infected with 1 MOI of RSV-L19F. RNA was isolated at 24, 48 and 72 hpi. (A-C) Expression levels of RSV-N, IL-1β and OPN were determined by qPCR. Results are presented as fold-change in expression of RSV-N, IL-1β or OPN mRNA normalized to the control (HPRT). qPCR data are represented as means ±SEM. Experiments were performed in triplicate.  p < 0.01,  p < 0.001 p < 0.0001. https://doi.org/10.1371/journal.pone.0192709.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 6 / 24 Osteopontin and respiratory syncytial virus infection infected with 1 MOI of RSV-L19F, then the infectious medium was replaced with growth medium containing 1 or 10 ng/ml of human rIL-1β. Results showed that there was no OPN expression with RSV infection without rIL-1β treatment; however, incubation of HEK-293 cells with human rIL-1β led to increased OPN expression 24 hours after treatment. In addi- tion, the RSV-L19F-infected HEK-293 cells treated with rIL-1β showed higher levels of OPN expression compared to uninfected cells treated only with rIL-1β (Fig 2A). While rIL-1β increased the expression of OPN after mock or RSV-L19F infection, it also contributed to an increase in viral yield in RSV-infected cells (Fig 2B). RSV infection-induced IL-1β regulates OPN expression Since HEK-293 and HEp-2 cells are likely to have other genetic differences which may influence OPN expression or susceptibility to RSV infection, we treated HEp-2 cells with a caspase-1 inhibitor (Ac-YVAD-CHO) in order to mimic the reduced IL-1β expression seen in HEK-293 and establish that IL-1β is responsible for the observed differences in RSV infection and OPN expression between the two cell lines. Cells were treated with the inhibitor and subsequently infected with RSV-L19F (1 MOI). Our results showed that cells treated with caspase-I inhibitor showed decreased RSV-N and IL-1β mRNA levels (Fig 2C and 2D). Also, OPN protein expression was significantly reduced in those cells treated with the inhibitor as compared to control group (Fig 2E). These results dem- onstrate the regulatory effect of IL-1β and OPN expression in the progression of RSV infection. RSV replication is not required for increased OPN expression To assess baseline levels of OPN and subsequent changes in expression upon RSV infection, HEp-2 cells were infected with RSV-L19F at 1 MOI (1 PFU/cell) and the levels of OPN were confirmed by immunostaining of mock- or RSV-L19F-infected HEp2 cells; results showed ele- vated OPN expression in the virus-infected cells compared to mock-infected cells. There were comparatively fewer OPN-positive cells in the mock-infected than RSV-L19F-infected cells 48 hpi (Fig 3A). To examine the role of RSV replication during OPN induction, HEp-2 cells were infected with increasing doses (0.1, 1 and 10 MOI) of UV-inactivated RSV-L19F, native RSV-L19F or mock treatment. To evaluate the productivity of the infection, supernatants were used for RSV titration by plaque assay. As expected, the number of PFU increased in a dose-dependent man- ner from 0.1 to 10 MOI in RSV-L19F infected cells. There was no productive infection in the UV-attenuated virus (Fig 3B). The cells were lysed at 48 hpi for Western blot analyses. OPN expression was proportional to the MOI dose of the RSV—higher in 10 MOI-infected cells compared to 0.1 or 1 MOI-infected cells (Fig 3C). Furthermore, OPN expression was higher in RSV-L19F-infected cells than in UV-inactivated infected cells; however, at 10 MOI of UV- inactivated RSV there was up-regulation in the expression of OPN suggesting that some RSV proteins or nucleic acids in the inactivated form of RSV continued to contribute to OPN induction and expression. Osteopontin and respiratory syncytial virus infection Fig 2. RSV induced IL-1β regulates OPN expression during RSV infection. (A-B) HEK-293 cells were mock-infected or infected with 1 MOI of RSV-L19F. 2 hours after the infection cells were treated with increasing concentrations of human rIL-1β (0, 1 or 10 ng/ml). (A) Protein was harvested 24 hpi for western blots. 25μg of protein lysate was loaded in each lane. OPN is seen at 55 kDa and β-actin (loading control) at 42 kDa. (B) Plaque viral titers were obtained from the supernatants collected 24 hpi. (C-E) HEp-2 cells were pre-treated two hours prior to RSV-L19F infection with 10 μM of Ac-YVAD-CHO (caspase-I) inhibitor. Cells were infected with 1 MOI of RSV-L19F and infectious media was replaced with fresh media containing 10 μM of Ac-YVAD-CHO. RNA and protein were collected 24 hpi. (C and D) Expression levels of IL-1β and RSV-N were determined by qPCR. Results are presented as fold-change in expression of IL-1β or RSV-N mRNA normalized to the control (HPRT). (E) 25μg of protein lysate was loaded in each lane. OPN is seen at 55 kDa and β-actin (loading control) at 42 kDa. qPCR data are represented as means ±SEM. Experiments were performed in triplicate.  p < 0.0001. https://doi.org/10.1371/journal.pone.0192709.g002 Fig 2. RSV induced IL-1β regulates OPN expression during RSV infection. (A-B) HEK-293 cells were mock-infected or infected with 1 MOI of RSV-L19F. 2 hours after the infection cells were treated with increasing concentrations of human rIL-1β (0, 1 or 10 ng/ml). (A) Protein was harvested 24 hpi for western blots. 25μg of protein lysate was loaded in each lane. OPN is seen at 55 kDa and β-actin (loading control) at 42 kDa. (B) Plaque viral titers were obtained from the supernatants collected 24 hpi. (C-E) HEp-2 cells were pre-treated two hours prior to RSV-L19F infection with 10 μM of Ac-YVAD-CHO (caspase-I) inhibitor. Cells were infected with 1 MOI of RSV-L19F and infectious media was replaced with fresh media containing 10 μM of Ac-YVAD-CHO. RNA and protein were collected 24 hpi. (C and D) Expression levels of IL-1β and RSV-N were determined by qPCR. Results are presented as fold-change in expression of IL-1β or RSV-N mRNA normalized to the control (HPRT). (E) 25μg of protein lysate was loaded in each lane. OPN is seen at 55 kDa and β-actin (loading control) at 42 kDa. qPCR data are represented as means ±SEM. Experiments were performed in triplicate.  p < 0.0001. https://doi.org/10.1371/journal.pone.0192709.g002 Fig 2. RSV induced IL-1β regulates OPN expression during RSV infection. (A-B) HEK-293 cells were mock-infected or infected with 1 MOI of RSV-L19F. 2 hours after the infection cells were treated with increasing concentrations of human rIL-1β (0, 1 or 10 ng/ml). (A) Protein was harvested 24 hpi for western blots. 25μg of protein lysate was loaded in each lane. OPN is seen at 55 kDa and β-actin (loading control) at 42 kDa. (B) Plaque viral titers were obtained from the supernatants collected 24 hpi. (C-E) HEp-2 cells were pre-treated two hours prior to RSV-L19F infection with 10 μM of Ac-YVAD-CHO (caspase-I) inhibitor. Cells were infected with 1 MOI of RSV-L19F and infectious media was replaced with fresh media containing 10 μM of Ac-YVAD-CHO. RNA and protein were collected 24 hpi. (C and D) Expression levels of IL-1β and RSV-N were determined by qPCR. Results are presented as fold-change in expression of IL-1β or RSV-N mRNA normalized to the control (HPRT). (E) 25μg of protein lysate was loaded in each lane. OPN is seen at 55 kDa and β-actin (loading control) at 42 kDa. qPCR data are represented as means ±SEM. Experiments were performed in triplicate.  p < 0.0001. Fig 2. RSV induced IL-1β regulates OPN expression during RSV infection. (A-B) HEK-293 cells were mock-infected or infected with 1 MOI of RSV-L19F. 2 hours after the infection cells were treated with increasing concentrations of human rIL-1β (0, 1 or 10 ng/ml). (A) Protein was harvested 24 hpi for western blots. 25μg of protein lysate was loaded in each lane. OPN is seen at 55 kDa and β-actin (loading control) at 42 kDa. (B) Plaque viral titers were obtained from the supernatants collected 24 hpi. (C-E) HEp-2 cells were pre-treated two hours prior to RSV-L19F infection with 10 μM of Ac-YVAD-CHO (caspase-I) inhibitor. Cells were infected with 1 MOI of RSV-L19F and infectious media was replaced with fresh media containing 10 μM of Ac-YVAD-CHO. RNA and protein were collected 24 hpi. (C and D) Expression levels of IL-1β and RSV-N were determined by qPCR. Results are presented as fold-change in expression of IL-1β or RSV-N mRNA normalized to the control (HPRT). (E) 25μg of protein lysate was loaded in each lane. OPN is seen at 55 kDa and β-actin (loading control) at 42 kDa. rOPN increases RSV titers in a dose-dependent manner To better understand the effect of increased OPN expression on viral yield during RSV infec- tion and to validate that OPN expression is sufficient for the increase in viral titer seen in HEK-293 cells, we used varying concentrations of human rOPN (0, -1, 10, -50, 100 and 200 ng/ml) to pretreat HEK-293 cells four hours before infection with 0.1 MOI of RSV-L19F. After infection, the medium was replaced with fresh growth medium containing rOPN at the same concentrations. 24 hours after infection, there was a significant dose-dependent increase in viral titers in cells treated with rOPN, further confirming that OPN promotes RSV infection (Fig 4A). Additionally, to determine the effects of exogenous rOPN in modulating RSV PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 7 / 24 Osteopontin and respiratory syncytial virus infection Fig 3. RSV infection induces OPN expression in a dose dependent manner. HEp-2 cells were mock-infected or infected with RSV-L19F (1 MOI). (A) Images of Hep- 2 cells stained with polyclonal antibody against RSV and OPN at 48 hpi. Representative images of RSV positive (red) cells, OPN positive (green) cells, and DAPI (blue) nuclear staining at 200X magnification. (B) RSV titer by plaque assay of supernatants for increasing doses (0.1, 1 and 10 MOI) of UV-inactivated and native RSV-L19F at 48 hpi. (C) OPN protein (55 kDa) expression in HEp-2 cells at 48 hpi after mock, RSV and UV-inactivated RSV treatment in the indicated doses analyzed by western blots. β-actin (loading control) at 42 kDa was probed as the loading control. https://doi.org/10.1371/journal.pone.0192709.g003 Fig 3. RSV infection induces OPN expression in a dose dependent manner. HEp-2 cells were mock-infected or infected with RSV-L19F (1 MOI). (A) Images of Hep- 2 cells stained with polyclonal antibody against RSV and OPN at 48 hpi. Representative images of RSV positive (red) cells, OPN positive (green) cells, and DAPI (blue) nuclear staining at 200X magnification. (B) RSV titer by plaque assay of supernatants for increasing doses (0.1, 1 and 10 MOI) of UV-inactivated and native RSV-L19F at 48 hpi. (C) OPN protein (55 kDa) expression in HEp-2 cells at 48 hpi after mock, RSV and UV-inactivated RSV treatment in the indicated doses analyzed by western blots. β-actin (loading control) at 42 kDa was probed as the loading control. and cells were rinsed before infection; in the second group, rOPN was added during the two hours of infection, then media was aspirated from the cells and fresh media was added; in the third group, after the two hours of infection, the media was aspirated and replaced with fresh media containing rOPN. Our results show a significant increase in viral titers of cells treated with human rOPN during or after the infection (Fig 4B). As an additional test of rOPN effect during the infection, HEp-2 and HEK-293 cells were infected with 0.1 MOI of the clinical strain Line 19 that expresses a red fluorescent protein-RFP (RSV-KL19F) in the presence of rOPN. We evaluated the number of RSV infected cells using flow cytometry. We found that treatment with rOPN significantly increased the percentage of infected HEp-2 and HEK-293 cells by 23% and 20% respectively (Fig 4C). PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 qPCR data are represented as means ±SEM. Experiments were performed in triplicate.  p < 0.0001. Fig 2. RSV induced IL-1β regulates OPN expression during RSV infection. (A-B) HEK-293 cells were mock-infected infection, an intermediate OPN concentration (100 ng/ml) known to cause an effect on RSV infection was selected to treat the cells at three different time points: to one group of cells, rOPN was added to the media four hours before the infection, later the media was aspirated infection, an intermediate OPN concentration (100 ng/ml) known to cause an effect on RSV infection was selected to treat the cells at three different time points: to one group of cells, rOPN was added to the media four hours before the infection, later the media was aspirated PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 8 / 24 These findings suggest that OPN could facilitate the entry of the virus into the cells and increase cell permissiveness to RSV infection. PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 9 / 24 Osteopontin and respiratory syncytial virus infection CD44 modulates RSV infection CD44 i k t b t f OPN T l t th i f CD44 i k RSV Fig 4. Human rOPN increases RSV titers in a dose-dependent manner. (A) HEK-293 cells were treated with increasing concentrations of human recombinant OPN (rOPN) (0 to 200 ng/ml) four hours before infection. Cells were infected with 0.1 MOI of RSV-L19F and infectious media was replaced with fresh media containing the appropriate concentration of rOPN. Supernatants were collected 24 hpi and viral titers were determined by plaque assay. (B) HEK-293 cells were treated with 100 ng/ml at different time points: before, during or immediately after the infection. Supernatants were collected 24 hpi and viral titers were determined by plaque assay. Result of a representative experiment is shown.  p < 0.05,  p < 0.01,  p < 0.0001. (C) Hep-2 or HEK-293 cells were infected with 0.1 MOI of RSV-KL19F or RSV-KL19F in the presence of 100 ng/ml of rOPN. After 24 hours, the infected HEp-2 or HEK-293 cells were gated for RFP-expression (RSV + cells). https://doi.org/10.1371/journal.pone.0192709.g004 Fig 4. Human rOPN increases RSV titers in a dose-dependent manner. (A) HEK-293 cells were treated with increasing concentrations of human recombinant OPN (rOPN) (0 to 200 ng/ml) four hours before infection. Cells were infected with 0.1 MOI of RSV-L19F and infectious media was replaced with fresh media containing the appropriate concentration of rOPN. Supernatants were collected 24 hpi and viral titers were determined by plaque assay. (B) HEK-293 cells were treated with 100 ng/ml at different time points: before, during or immediately after the infection. Supernatants were collected 24 hpi and viral titers were determined by plaque assay. Result of a representative experiment is shown.  p < 0.05,  p < 0.01,  p < 0.0001. (C) Hep-2 or HEK-293 cells were infected with 0.1 MOI of RSV-KL19F or RSV-KL19F in the presence of 100 ng/ml of rOPN. After 24 hours, the infected HEp-2 or HEK-293 cells were gated for RFP-expression (RSV + cells). https://doi.org/10.1371/journal.pone.0192709.g004 Fig 4. Human rOPN increases RSV titers in a dose-dependent manner. (A) HEK-293 cells were treated with increasing concentrations of human recombinant OPN (rOPN) (0 to 200 ng/ml) four hours before infection. Cells were infected with 0.1 MOI of RSV-L19F and infectious media was replaced with fresh media containing the appropriate concentration of rOPN. Supernatants were collected 24 hpi and viral titers were determined by plaque assay. (B) HEK-293 cells were treated with 100 ng/ml at different time points: before, during or immediately after the infection. Supernatants were collected 24 hpi and viral titers were determined by plaque assay. Result of a representative experiment is shown.  p < 0.05,  p < 0.01,  p < 0.0001. (C) Hep-2 or HEK-293 cells were infected with 0.1 MOI of RSV-KL19F or RSV-KL19F in the presence of 100 ng/ml of rOPN. After 24 hours, the infected HEp-2 or HEK-293 cells were gated for RFP-expression (RSV + cells). Fig 4. Human rOPN increases RSV titers in a dose-dependent manner. (A) HEK-293 cells were treated with increasing concentrations of human recombinant OPN (rOPN) (0 to 200 ng/ml) four hours before infection. Cells were infected with 0.1 MOI of RSV-L19F and infectious media was replaced with fresh media containing the appropriate concentration of rOPN. Supernatants were collected 24 hpi and viral titers were determined by plaque assay. (B) HEK-293 cells were treated with 100 ng/ml at different time points: before, during or immediately after the infection. Supernatants were collected 24 hpi and viral titers were determined by plaque assay. Result of a representative experiment is shown.  p < 0.05,  p < 0.01,  p < 0.0001. (C) Hep-2 or HEK-293 cells were infected with 0.1 MOI of RSV-KL19F or RSV-KL19F in the presence of 100 ng/ml of rOPN. After 24 hours, the infected HEp-2 or HEK-293 cells were gated for RFP-expression (RSV + cells). https://doi.org/10.1371/journal.pone.0192709.g004 https://doi.org/10.1371/journal.pone.0192709.g004 CD44 modulates RSV infection CD44 is known to be a receptor for OPN. To evaluate the expression of CD44 in mock or RSV infected cells, HEp-2 and HEK-293 cells were infected with 0.5 MOI of RSV-KL19F. 24 hours after infection, cells were stained with a FITC-CD44 antibody and gated for CD44 (green) and PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 10 / 24 Osteopontin and respiratory syncytial virus infection Fig 5. CD44 expression mediates RSV infection. HEp-2 or HEK-293 cells were infected with 0.5 MOI of RSV-KL19F (red). After 24 hours, the cells were stained with FITC mouse anti-human CD44 antibody (green) and gated for RSV expression (red). (Top panel) Flow cytometry analysis of HEp-2 cells infected with RSV-KL19F. (Bottom panel) Flow c tometr anal sis of HEK 293 cells infected ith RSV KL19F Res lt of a representati e e periment is sho n E periments Fig 5. CD44 expression mediates RSV infection. HEp-2 or HEK-293 cells were infected with 0.5 MOI of RSV-KL19F (red). After 24 hours, the cells were stained with FITC mouse anti-human CD44 antibody (green) and gated for RSV expression (red). (Top panel) Flow cytometry analysis of HEp-2 cells infected with RSV-KL19F. (Bottom panel) Flow cytometry analysis of HEK-293 cells infected with RSV-KL19F. Result of a representative experiment is shown. Experiments were performed in triplicate. Fig 5. CD44 expression mediates RSV infection. HEp-2 or HEK-293 cells were infected with 0.5 MOI of RSV-KL19F (red). After 24 hours, the cells were stained with FITC mouse anti-human CD44 antibody (green) and gated for RSV expression (red). (Top panel) Flow cytometry analysis of HEp-2 cells infected with RSV-KL19F. (Bottom panel) Flow cytometry analysis of HEK-293 cells infected with RSV-KL19F. Result of a representative experiment is shown. Experiments were performed in triplicate. https://doi.org/10.1371/journal.pone.0192709.g005 https://doi.org/10.1371/journal.pone.0192709.g005 RSV (red) expression. We found higher cell surface expression of CD44 in the HEp-2 cell line (96.7%) than in HEK-293 (73.7%). We also found a significant difference in the number of RSV positive cells confirming that HEp-2 is more permissive to RSV infection (Fig 5). These results suggest that the earlier OPN expression in HEp-2 cells and the concomitant CD44 expression could be key mediators for OPN signaling during RSV infection that potentiates the infection of HEp-2 cells. To test whether CD44 expression is required for RSV infection, the CD44 receptor was neu- tralized with a broad-spectrum rat-anti human CD44 antibody. PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 CD44 modulates RSV infection Briefly, HEp-2 cells were pre- incubated during 20 minutes at room temperature and then infected with RSV-KL19F (0.5 MOI) for two hours, after which the infectious media was replaced with fresh growth media. 24 hpi we assessed the number of RSV positive cells (RFP +) by flow cytometry. We found ~ 52.4% of the cells were RSV positive in the cells pre-treated with CD44 antibody while there was 70.5% RSV positive cells in the control group. Our results showed a decrease in RSV posi- tive cells following treatment with anti-CD44 antibody prior to infection compared to normal rat IgG control (Fig 6). Together, these data suggest that CD44 may be involved in facilitating the RSV infection process. 11 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 Osteopontin and respiratory syncytial virus infection Fig 6. Neutralization of CD44 receptor reduces the number of RSV positive cells. Hep-2 cells were pre-treated with 10 μg of broad spectrum rat-anti human CD44 antibody (clone A020) or normal rat IgG (control). After the pre-treatment, cells were infected with RSV-KL19F (0.5 MOI) and infectious media was replaced with fresh growth media. After 24 hours, the percentage of RSV positive cells was determined by flow cytometry. Result of a representative experiment is shown. Experiments were performed in triplicate. Fig 6. Neutralization of CD44 receptor reduces the number of RSV positive cells. Hep-2 cells were pre-treated with 10 μg of broad spectrum rat-anti human CD44 antibody (clone A020) or normal rat IgG (control). After the pre-treatment, cells were infected with RSV-KL19F (0.5 MOI) and infectious media was replaced with fresh growth media. After 24 hours, the percentage of RSV positive cells was determined by flow cytometry. Result of a representative experiment is shown. Experiments were performed in triplicate. PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 OPN expression is a marker of high RSV loads To investigate the role of OPN in determining the severity of RSV infection, HEp-2 cells were infected with rgRSV-A2 or RSV-L19F. The latter is known to induce severe RSV infection and higher viral loads in a mouse model [45]. In vitro, infection with rgRSV-A2 and RSV-L19F resulted in similar plaque viral titers and RSV-N gene expression at 24 hpi (Fig 7A and 7B). However, at 48 hpi RSV-L19F-infected cells had a significantly increased number of viral pla- ques and a similar increase in RSV-N mRNA expression when compared to cells infected with rgRSV-A2 (Fig 7A and 7B). Following the same pattern, we found no significant difference in the gene expression of IL-1β at 24 hpi with the two RSV strains, but we noted a significant up- regulation of IL-1β mRNA expression 48 hpi with RSV-L19F compared to rgRSV-A2 (Fig 7C). Remarkably, IFN-β mRNA levels were significantly elevated 24 hpi in rgRSV-A2-infected cells compared to those infected with RSV-L19F. This may in part account for the difference in viral titers and RSV-N transcripts observed in cells infected with the different viral strains (Fig 7D). Western blot analysis revealed an increase in OPN protein expression at 24 hpi that remained high at 48 hpi in RSV-L19F-infected cells. Cells infected with rgRSV-A2 showed less OPN protein expression at 24 hpi and the expression started to return to baseline levels by 48 hpi (Fig 7E). 12 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 Osteopontin and respiratory syncytial virus infection RSV-N, IL-1β and IFN-β were determined by qPCR. Results are presented as fold-change in expression of RSV-N, IL-1β or IFN-β mRNA normalized to the control (HPRT). (E) Western blot analysis of OPN expression. 25μg of protein lysate was loaded in each lane. OPN is seen at 55 kDa and β-actin (loading control) at 42 kDa. Experiments were performed in triplicate.  p < 0.05,  p < 0.01,  p < 0.0001. https://doi.org/10.1371/journal.pone.0192709.g007 https://doi.org/10.1371/journal.pone.0192709.g007 RSV replication is diminished in mice lacking OPN (OPN KO) To determine the time points where IL-1β and OPN expression are induced and their effect during RSV infection, we infected OPN KO and WT mice intranasally with the mucogenic virus, RSV-L19F. Mice were euthanized 1, 3 and 5 dpi. As we reported before, RSV infection resolves much faster in OPN KO mice than WT. This was evidenced by a significant decrease in RSV-N transcripts at 5 dpi in the lungs of OPN KO mice as measured by qRT-PCR. There were no significant differences in RSV-N amplification 1 or 3 dpi, but the infection did not progress to 5 dpi in OPN KO mice as it did in WT mice (Fig 8A). Secretion of IL-1β was also measured upon infection since it was suspected to play a role in controlling OPN expression during RSV infection. We found a significant increase in IL-1β expression 1 dpi in both OPN KO and WT mice infected with RSV-L19F; yet there was no significant difference between the two strains of mice at 1 or 3 dpi. Nonetheless, at 5 dpi RSV infected WT mice had increased levels of IL-1β while in the OPN KO mice IL-1β levels returned to baseline (Fig 8B). The expression of OPN in RSV-L19F-infected WT mice was also determined and compared to mock-infected WT. OPN protein levels were measured by ELISA of lung homogenates. We observed a significant increase in OPN protein levels at 3 and 5 dpi in WT mice infected with L19F (Fig 8C). As expected, the levels of OPN were below the detection limit in RSV-L19F or mock-infected OPN KO mice (Fig 8C). Furthermore, we infected WT mice in increased doses of RSV-L19F (1 or 3 x 106) and found a significant increase in OPN protein levels that correlated with the increased viral con- centration used to infect those mice (Fig 9A). We also evaluated the effect of mild or severe RSV infection in vivo and how it influenced OPN expression. WT mice were infected with mock, RSV-L19F or rgRSV-A2. Similar to the results found in vitro, WT mice infected with RSV-L19F exhibited higher OPN protein levels compared to those infected with rgRSV-A2 (Fig 9B). These results suggest that increases in OPN expression levels are tightly associated with RSV viral loads. Osteopontin and respiratory syncytial virus infection Fig 7. OPN is a marker of high RSV loads in HEp-2 cells. HEp-2 cells were mock-infected or infected with 1 MOI of RSV-L19F or rgRSV-A2. RNA, supernatants and protein were isolated 24 and 48 hpi. (A) Plaque titers were obtained from supernatants of HEp-2 cells infected with RSV-L19F or rgRSV-A2. (B—D) Expression of Fig 7. OPN is a marker of high RSV loads in HEp-2 cells. HEp-2 cells were mock-infected or infected with 1 MOI of RSV-L19F or rgRSV-A2. RNA, supernatants and protein were isolated 24 and 48 hpi. (A) Plaque titers were obtained from supernatants of HEp-2 cells infected with RSV-L19F or rgRSV-A2. (B—D) Expression of Fig 7. OPN is a marker of high RSV loads in HEp-2 cells. HEp-2 cells were mock-infected or infected with 1 MOI of RSV-L19F or rgRSV-A2. RNA, supernatants and protein were isolated 24 and 48 hpi. (A) Plaque titers were obtained from supernatants of HEp-2 cells infected with RSV-L19F or rgRSV-A2. (B—D) Expression of PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 13 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 Osteopontin and respiratory syncytial virus infection Osteopontin and respiratory syncytial virus infection Fig 8. RSV replication is diminished in mice lacking OPN (OPN KO). C57BL/6 and OPN KO mice were mock- infected or infected with 3 x 106 RSV-L19F. Lungs were collected 1, 3 and 5 dpi for protein and RNA extraction. (A) RSV-N mRNA expression levels were determined by qPCR and results represented as fold-change in expression of RSV-N mRNA normalized to the control (HPRT). (B and C) Levels of OPN and IL-1β in lung homogenates were determined by ELISA. BD: Below detection limits. Lung homogenates were obtained from individual mice and not pooled (n>4 per group).  p < 0.01,  p < 0.0001. Fig 8. RSV replication is diminished in mice lacking OPN (OPN KO). C57BL/6 and OPN KO mice were mock- infected or infected with 3 x 106 RSV-L19F. Lungs were collected 1, 3 and 5 dpi for protein and RNA extraction. (A) RSV-N mRNA expression levels were determined by qPCR and results represented as fold-change in expression of RSV-N mRNA normalized to the control (HPRT). (B and C) Levels of OPN and IL-1β in lung homogenates were determined by ELISA. BD: Below detection limits. Lung homogenates were obtained from individual mice and not pooled (n>4 per group).  p < 0.01,  p < 0.0001. The induction of OPN is controlled by a variety of cytokines, growth factors and hormones [40, 46, 47]. A previous study showed that IL-1β dramatically increased OPN expression dur- ing pulmonary fibrosis through the activation of ERK1/2 but not by JNK pathway [30, 48]. Likewise IL-1β is one of the factors released at sites of injury that contributes to enhanced OPN expression [49]. RSV infection induces a substantial increase of IL-1β, a pro-inflamma- tory cytokine known to induce the expression of a plethora of downstream pro-inflammatory cytokines including OPN, thus resulting in magnification of the inflammatory process [43, 50–52]. We have explored the association between IL-1β and OPN up-regulation during RSV infection and our data shows that OPN and IL-1β expression leads to increased viral infection. We also show a delay in OPN mRNA expression levels in infected HEK-293 cells compared to HEp-2 cells, suggesting that the impaired production of IL-1β in HEK-293 cells partially con- tributes to the reduced expression of OPN in the infected HEK-293. Discussion and conclusions To our knowledge our lab was the first to determine the importance of OPN during RSV infec- tion, and the results from our in vivo and in vitro experiments in the present study show how OPN expression is linked with increased susceptibility to RSV infection. The salient findings of the present studies are as follows: i) RSV infection leads to increased IL-1β and OPN expres- sion, ii) IL-1β is involved in the regulation of OPN levels during RSV infection, iii) OPN on its own can enhance RSV infection and contribute to viral spread, and iii) OPN is a predictive marker of viral loads both in vitro and in vivo. A major finding of our study is that IL-1β is involved in up-regulating OPN expression lev- els during RSV infection in HEp-2 cells, but the same OPN up-regulation is not observed in HEK-293 cells which has disrupted expression of IL-1β. Our results suggest that OPN plays an important role in RSV infection and propagation in vivo and in vitro, and IL-1β amplifies the inflammatory response by inducing OPN expression, thus resulting in increased viral loads. Our results show that although RSV infection can induce significant OPN expression in the absence of IL-1β, in the presence of IL-1β it can enhance OPN expression and accelerate the infection process. PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 14 / 24 Osteopontin and respiratory syncytial virus infection 371/journal.pone.0192709 April 20, 2018 15 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 15 / 24 Osteopontin and respiratory syncytial virus infection leads to a decrease in IL-1β expression that also results in decreased OPN expression and decreased viral infection. In addition, we have found that OPN on its own can enhance RSV infection. OPN can have anti- and pro-inflammatory activity; hence it has been associated with physiological and path- ological conditions [47, 53]. To establish the mechanism for OPN up-regulation and also to verify that OPN was expressed in human epithelial cells infected with RSV, we infected HEp-2 cells with RSV-L19F and verified through immunostaining, Western blotting and qPCR that RSV triggers OPN expression. The result that infected cells were found to express more OPN indicates that RSV infection per se is a major trigger of OPN induction. Also, we found up-reg- ulation of OPN after infection with UV irradiated-RSV-L19F, while the UV inactivated virus led to lower expression of OPN protein levels compared to replicating RSV-L19F. We tested if UV—RSV would similarly affect IL-1β response and we did not find such a response, suggest- ing that RSV replication was needed for the IL-1β response (S1 Fig). This data also suggests that OPN is partially regulated by IL-1β during RSV infection. These results indicate that load- ing more viral proteins and/or nucleic acids potentiates OPN induction even in the absence of viral replication and suggest that viral replication is required for the overexpression of OPN. Nonetheless, in the absence of RSV replication a small increase in OPN was observed, which may be attributed to the ssRNA induced innate immune response. We have consistently observed an increase in OPN expression in cells and mice infected with RSV. In order to evaluate the feedback effect of high OPN levels on RSV infection we treated HEK-293 with human rOPN during RSV infection. Our results showed that HEK-293 cells treated with human rOPN displayed a dose-dependent increase in viral titers, suggesting that OPN has a regulatory effect on RSV infection. Moreover, the results of studies on timing of OPN action show the prominent effect of OPN during the infection process itself. Cells treated with rOPN during and immediately after the infection yielded higher viral titers, while cells pre-treated with OPN before the infection did not show a significant difference in viral titers when compared to RSV infected cells without rOPN treatment. Osteopontin and respiratory syncytial virus infection The increased early OPN expression in HEK-293 cells treated with rIL-1β proves that the production of OPN is not intrinsically impaired in HEK-293 cells but instead is dependent on IL-1β signaling. The corre- lation between increased OPN protein levels and increased RSV viral titers in HEK-293 cells treated with rIL-1β further confirms the role of these two pro-inflammatory cytokines in the regulation of RSV infection. Similarly, to rule out other differences between HEK-293 and HEp-2 cells, we inhibited caspase-I expression in HEp-2 cells and showed that this inhibition Fig 9. OPN expression is a marker of high RSV loads in vivo. (A) C57BL/6 mice were mock-infected or infected with 1 or 3 x 106 RSV-L19F. (B) C57BL/6 mice were mock-infected or infected with 3 x 106 RSV-L19F or rgRSV-A2. (A-B) Lungs were collected 3 dpi for protein isolation and levels of OPN in lung homogenates were determined by ELISA. Lung homogenates were obtained from individual mice and not pooled (n>4 per group).  p < 0.05,  p < 0.01,  p < 0.0001. https://doi.org/10.1371/journal.pone.0192709.g009 Fig 9. OPN expression is a marker of high RSV loads in vivo. (A) C57BL/6 mice were mock-infected or infected with 1 or 3 x 106 RSV-L19F. (B) C57BL/6 mice were mock-infected or infected with 3 x 106 RSV-L19F or rgRSV-A2. (A-B) Lungs were collected 3 dpi for protein isolation and levels of OPN in lung homogenates were determined by ELISA. Lung homogenates were obtained from individual mice and not pooled (n>4 per group).  p < 0.05,  p < 0.01,  p < 0.0001. https://doi.org/10.1371/journal.pone.0192709.g009 PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 16 / 24 This suggests that OPN mediates increased entry and/or assembly of the virus leading to a significant increase in pla- que viral titers. Further, cells treated with rOPN became infected with RSV at a significantly higher rate than untreated cells. Also, HEp-2 highly expressing the OPN receptor CD44 showed a higher percent of infected cells compared to HEK-293 cells whose expression of CD44 is lower. Although the basis of OPN regulation of RSV entry and/or assembly remains unclear, one pos- sibility is that OPN modulates the fusion process itself, thus aiding virus entry. This idea is sup- ported by the observation that the OPN receptor (CD44) co-localizes with RSV F protein and results in viral filament formation and syncytia formation which benefit the infectious process [54]. Additionally, the decrease we observed in RSV-positive cells after CD44 receptor neutral- ization evidenced that any manipulation of components of the lipid rafts which disrupts CD44 signaling could negatively affect the infection process; thus our results suggest that the interac- tion of proteins at the lipid rafts, like OPN and its receptor (CD44), could favor the infection process. Also, CD44 is expressed in a variety of immune cells, thus it is likely that the interac- tion of OPN-CD44 during RSV infection may play a role in modulating both innate and the adaptive immune response, which in turn plays a role in RSV clearance. Further, cells treated with rOPN became infected with RSV at a significantly higher rate than untreated cells. Also, HEp-2 highly expressing the OPN receptor CD44 showed a higher percent of infected cells compared to HEK-293 cells whose expression of CD44 is lower. Moreover, we found that OPN is a predictive marker of severe RSV infection. A compara- tive analysis of virus infection, IL-1β, OPN and IFN-β expression by two RSV strains (lab iso- late rA2 versus a mucogenic strain RSV-L19F) showed that OPN expression was significantly higher in RSV-L19F compared to rA2 infected cells. Also, our in vivo studies in a mouse model of RSV infection showed that IL-1β expression preceded OPN expression in WT mice after RSV infection. Importantly, we found a significant difference in infection at 5 dpi, where WT 17 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 Osteopontin and respiratory syncytial virus infection Fig 10. Model for the role of OPN during RSV infection. (Left panel) OPN-independent model. During RSV infection, the virus is recognized by the host cells through PRR (TLR-3, RIG-I, TLR-7); recognition of the virus leads to activation of innate immune response which leads to release of anti-viral and pro-inflammatory cytokines like IFN-β and IL-1β. Expression of IL-1β leads to up-regulation of OPN whereas expression of IFN-β down-regulates OPN expression. (Right panel) OPN- dependent model. Fusion of RSV is accelerated in the presence of OPN and its receptor (CD44), thus leading to increased number of infected cells which results in increased viral loads. Fig 10. Model for the role of OPN during RSV infection. (Left panel) OPN-independent model. During RSV infection, the virus is recognized by the host cells through PRR (TLR-3, RIG-I, TLR-7); recognition of the virus leads to activation of innate immune response which leads to release of anti-viral and pro-inflammatory cytokines like IFN-β and IL-1β. Expression of IL-1β leads to up-regulation of OPN whereas expression of IFN-β down-regulates OPN expression. (Right panel) OPN- dependent model. Fusion of RSV is accelerated in the presence of OPN and its receptor (CD44), thus leading to increased number of infected cells which results in increased viral loads https://doi.org/10.1371/journal.pone.0192709.g010 https://doi.org/10.1371/journal.pone.0192709.g010 infected mice exhibited increases in RSV-N gene amplification compared to OPN KO mice. This data is consistent with a previous study done by our lab where we found decreased viral titers from lung homogenates and fewer RSV-positive lung cells in the OPN KO mice infected with RSV compared to WT mice [28]. Further, these results are consistent with reports of increased levels of OPN positively correlated with severity of lung inflammation [55–59]. OPN up-regulation in serum and tissue samples from chronic rhinosinusitis and allergic patients also correlated with severity of disease [55, 60, 61]. In agreement with these studies, a recent paper found a positive correlation between increased levels of OPN in human serum and dis- ease severity in influenza patients [62]. Our in vitro finding suggests that IFN-β reduces the viral infection resulting in decreased IL-1 β and OPN expression and is consistent with previ- ous reports where RSV-A2 infection resulted in significantly higher expression of IFN-α than RSV-L19F in human epithelial cells and BALB/c mice [45, 63]. Additional studies have reported decreased OPN levels in serum samples from multiple sclerosis patients treated with IFN-β, supporting this IFN-β dependent mechanism of OPN regulation [64–66]. PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 Supporting information S1 Fig. IL-1β mRNA expression in UV-inactivated or native RSV-L19F HEp-2 infected cells. HEp-2 cells were infected with 1 MOI of UV-irradiated or native RSV-L19F. RNA was isolated at 24, 48 and 72 hpi. Expression levels of IL-1β were determined by qPCR. Results are presented as fold-change in expression of IL-1β mRNA normalized to the control (HPRT). (DOCX) Osteopontin and respiratory syncytial virus infection Proper control of the inflammatory response is required not only for effective viral clear- ance but also for prevention of subsequent complications caused by imbalanced immune response and exaggerated inflammation during RSV infection [70]. It has been suggested that the severity of the disease caused by RSV is not only controlled by replication of the virus but also by the host inflammatory response. Therefore, these two factors together could help us predict the severity of the disease and perhaps establish new treatments aiming to control infection and inflammation [71–73]. An accurate profiling of inflammatory mediators is a promising strategy to explore RSV physiopathology that could contribute to a better manage- ment of RSV disease. Together, the results of these studies have pointed to the role of OPN during the first cycle of RSV infection (OPN-independent cycle), where the viral nucleic acid is recognized by PRR, thus leading to IL-1β expression and subsequently to OPN up-regula- tion. Therefore, a second replication cycle of the virus would be OPN-dependent; the interac- tion of OPN and its receptor (CD44) would result in an increased viral fusion to subsequent host cells that could result in increased number of infected cells and therefore viral production (Fig 10). In conclusion, our studies suggest that IL-1β positively regulates OPN expression in the context of RSV infection. We also recognize that there could be other contributing factors like viral proteins or other cytokines found up-regulated after RSV infection that will control OPN expression. Also, increased levels of OPN protein are sufficient to increase viral loads, thus influencing the onset and severity of infection. Thus, we show that OPN could be used as a marker of severe infection or higher RSV loads. The knowledge acquired from this research could be used in the future to develop new drug targets for treatment and/or prophylaxis of RSV infection since it may lead to development of pharmacological strategies that allow for regulation of OPN expression during infection. Acknowledgments We would like to thank Dr. Martin L Moore (Emory University, Atlanta, Georgia, USA) and Dr. Mark Peeples (Nationwide Children’s Hospital, Columbus, Ohio, USA) for providing the RSV-L19F and rgRSV-A2. We also thank Dr. Gary Hellerman and Dr. Homero San Juan for a critical reading of the manuscript and suggestions. Appreciation is given to Dr. Sandhya Boya- palle and Dr. Terianne Wong for their advice and feedback. The authors would like to acknowledge Ms. Christen Bouchard for assistance in preparation of the manuscript. Also, an inverse relationship between IFN-β and OPN expression reported in recent data indicates that the fusion protein of the virus could directly modulate type I IFN expression and that the increase in viral loads after RSV-L19F infection lead to higher expression levels of viral non- structural proteins (NS1 and NS2) that are known to disrupt type I IFN response and therefore may act to increase OPN [45, 67–69]. 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Osteopontin Is Not Required for the Develop- ment of Th1 Responses and Viral Immunity. The Journal of Immunology. 2005; 175(9):6006–13. https://doi.org/10.4049/jimmunol.175.9.6006 PMID: 16237095 37. Kang CG, Im E, Lee HJ, Lee EO. Plumbagin reduces osteopontin-induced invasion through inhibiting the Rho-associated kinase signaling pathway in A549 cells and suppresses osteopontin-induced lung metastasis in BalB/c mice. Bioorganic & medicinal chemistry letters. 2017. Epub 2017/04/01. https:// doi.org/10.1016/j.bmcl.2017.03.047 PMID: 28359791. 38. West CE, Kvistgaard AS, Peerson JM, Donovan SM, Peng YM, Lonnerdal B. References Human Metapneumovirus Induces a Profile of Lung Cyto- kines Distinct from That of Respiratory Syncytial Virus. Journal of Virology. 2005; 79(23):14992–7. https://doi.org/10.1128/JVI.79.23.14992-14997.2005 PMID: 16282501 52. Blanco JCG, Richardson JY, Darnell MER, Rowzee A, Pletneva L, Porter DD, et al. Cytokine and che- mokine gene expression after primary and secondary respiratory syncytial virus infection in cotton rats. Journal of Infectious Diseases. 2002; 185(12):1780–5. https://doi.org/10.1086/340823 PMID: 12085325 53. Xanthou G, Alissafi T, Semitekolou M, Simoes DCM, Economidou E, Gaga M, et al. Osteopontin has a crucial role in allergic airway disease through regulation of dendritic cell subsets. Nat Med. 2007; 13 (5):570–8. https://doi.org/10.1038/nm1580 PMID: 17435770 54. McCurdy LH, Graham BS. Role of Plasma Membrane Lipid Microdomains in Respiratory Syncytial Virus Filament Formation. Journal of Virology. 2003; 77(3):1747–56. https://doi.org/10.1128/JVI.77.3. 1747-1756.2003 PMC140864. PMID: 12525608 22 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 Osteopontin and respiratory syncytial virus infection 55. Samitas K, Zervas E, Vittorakis S, Semitekolou M, Alissafi T, Bossios A, et al. Osteopontin expression and relation to disease severity in human asthma. European Respiratory Journal. 2011; 37(2):331–41. https://doi.org/10.1183/09031936.00017810 PMID: 20562127 56. Akelma AZ, Cizmeci MN, Kanburoglu MK, Bozkaya D, Catal F, Mete E, et al. Elevated level of serum osteopontin in school-age children with asthma. Allergologia et Immunopathologia. (0). http://dx.doi. org/10.1016/j.aller.2013.01.009. 57. Liu W, Xia W, Fan Y, Wang H, Zuo K, Lai Y, et al. Elevated serum osteopontin level is associated with blood eosinophilia and asthma comorbidity in patients with allergic rhinitis. Journal of Allergy and Clini- cal Immunology. 2012; 130(6):1416–8.e6. http://dx.doi.org/10.1016/j.jaci.2012.06.010. PMID: 22846382 58. Akelma AZ, Cizmeci MN, Kanburoglu MK, Bozkaya D, Catal F, Mete E, et al. Elevated level of serum osteopontin in school-age children with asthma. Allergologia et Immunopathologia. 2014; 42(04):275– 81. 59. Oh K, Seo MW, Kim YW, Lee DS. Osteopontin Potentiates Pulmonary Inflammation and Fibrosis by Modulating IL-17/IFN-gamma-secreting T-cell Ratios in Bleomycin-treated Mice. Immune network. 2015; 15(3):142–9. Epub 2015/07/04. https://doi.org/10.4110/in.2015.15.3.142 PMID: 26140046; PubMed Central PMCID: PMCPMC4486777. 60. Lu X, Zhang XH, Wang H, Long XB, You XJ, Gao QX, et al. Expression of osteopontin in chronic rhinosi- nusitis with and without nasal polyps. Allergy. 2009; 64(1):104–11. https://doi.org/10.1111/j.1398-9995. 2008.01829.x PMID: 19076536 61. Liu W, Zeng Q, Luo R. Correlation between Serum Osteopontin and miR-181a Levels in Allergic Rhinitis Children. Mediators of Inflammation. 2016; 2016:6. https://doi.org/10.1155/2016/9471215 PMID: 27199509 62. Zhu Y, Wei Y, Chen J, Cui G, Ding Y, Kohanawa M, et al. 73. Schwarze J, O’Donnell DR, Rohwedder A, Openshaw PJM. Latency and Persistence of Respiratory Syncytial Virus Despite T Cell Immunity. American Journal of Respiratory and Critical Care Medicine. 2004; 169(7):801–5. https://doi.org/10.1164/rccm.200308-1203OC PMID: 14742302 References Osteopontin Exacerbates Pulmonary Damage in Influenza-Induced Lung Injury. Japanese Journal of Infectious Diseases. 2015; 68(6):467–73. https:// doi.org/10.7883/yoken.JJID.2014.467 PMID: 25866117 63. Schlender J, Hornung V, Finke S, Gu¨nthner-Biller M, Marozin S, Brzo´zka K, et al. Inhibition of Toll-Like Receptor 7- and 9-Mediated Alpha/Beta Interferon Production in Human Plasmacytoid Dendritic Cells by Respiratory Syncytial Virus and Measles Virus. Journal of Virology. 2005; 79(9):5507–15. https://doi. org/10.1128/JVI.79.9.5507-5515.2005 PMID: 15827165 64. Chen M, Chen G, Nie H, Zhang X, Niu X, Zang YC, et al. Regulatory effects of IFN-beta on production of osteopontin and IL-17 by CD4+ T Cells in MS. European journal of immunology. 2009; 39(9):2525– 36. Epub 2009/08/12. https://doi.org/10.1002/eji.200838879 PMID: 19670379. 65. Comabella M, Pericot I, Goertsches R, Nos C, Castillo M, Blas Navarro J, et al. Plasma osteopontin lev- els in multiple sclerosis. Journal of Neuroimmunology. 158(1):231–9. https://doi.org/10.1016/j. jneuroim.2004.09.004 PMID: 15589058 66. Coclet-Ninin J, Dayer JM, Burger D. Interferon-beta not only inhibits interleukin-1beta and tumor necro- sis factor-alpha but stimulates interleukin-1 receptor antagonist production in human peripheral blood mononuclear cells. European cytokine network. 1997; 8(4):345–9. Epub 1998/02/12. PMID: 9459613. 67. Lo MS, Brazas RM, Holtzman MJ. Respiratory Syncytial Virus Nonstructural Proteins NS1 and NS2 Mediate Inhibition of Stat2 Expression and Alpha/Beta Interferon Responsiveness. Journal of Virology. 2005; 79(14):9315–9. https://doi.org/10.1128/JVI.79.14.9315-9319.2005 PMC1168759. PMID: 15994826 68. Boyapalle S, Wong T, Garay J, Teng M, San Juan-Vergara H, Mohapatra S, et al. Respiratory Syncytial Virus NS1 Protein Colocalizes with Mitochondrial Antiviral Signaling Protein MAVS following Infection. PLoS ONE. 2012; 7(2):e29386. https://doi.org/10.1371/journal.pone.0029386 PMID: 22383950 69. Ling Z, Tran KC, Teng MN. Human Respiratory Syncytial Virus Nonstructural Protein NS2 Antagonizes the Activation of Beta Interferon Transcription by Interacting with RIG-I. Journal of Virology. 2009; 83 (8):3734–42. https://doi.org/10.1128/JVI.02434-08 PMID: 19193793 70. Mejı´as A, Cha´vez-Bueno S, Rı´os AM, Saavedra-Lozano J, Fonseca Aten M, Hatfield J, et al. Anti- Respiratory Syncytial Virus (RSV) Neutralizing Antibody Decreases Lung Inflammation, Airway Obstruction, and Airway Hyperresponsiveness in a Murine RSV Model. Antimicrobial Agents and Che- motherapy. 2004; 48(5):1811–22. https://doi.org/10.1128/AAC.48.5.1811-1822.2004 PMC400529. PMID: 15105140 71. Varga SM, Braciale TJ. RSV-Induced Immunopathology: Dynamic Interplay between the Virus and Host Immune Response. Virology. 2002; 295(2):203–7. http://dx.doi.org/10.1006/viro.2002.1382. PMID: 12033778 72. DeVincenzo JP. A New Direction in Understanding the Pathogenesis of Respiratory Syncytial Virus Bronchiolitis: How Real Infants Suffer. Journal of Infectious Diseases. 2007; 195(8):1084–6. https://doi. PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 Osteopontin and respiratory syncytial virus infection References org/10.1086/512622 PMID: 17357042 23 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0192709 April 20, 2018 Osteopontin and respiratory syncytial virus infection Osteopontin and respiratory syncytial virus infection 24 / 24
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Measurement of the neutron capture resonances for platinum using the Ge spectrometer and pulsed neutron beam at the J-PARC/MLF/ANNRI
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Measurement of the neutron capture resonances for platinum using the Ge spectrometer and pulsed neutron beam at the J-PARC/MLF/ANNRI Koichi Kino1,a, Hiroyuki Hasemi2, Atsushi Kimura3, and Yoshiaki Kiyanagi4 1 National Institute of Advanced Industrial Science and Technology, Research Institute for Measurement and Analyt Instrumentation, 1-1-1 Umezono Tsukuba Ibaraki, Japan 1 National Institute of Advanced Industrial Science and Technology, Research Institute for Measurement and Analytical Instrumentation, 1-1-1 Umezono Tsukuba Ibaraki, Japan 2 Hokkaido University, Faculty of Engineering, Kita13 Nishi8 Kita-ku Sapporo, Japan 2 Hokkaido University, Faculty of Engineering, Kita13 Nishi8 Kita-ku Sapporo, Japan 3 Japan Atomic Energy Agency, 2-4 Shirakata Shirane Tokai Naka Ibaraki, Japan 4 Nagoya University, Graduate School of Engineering, Furo-cho Chikusa-ku Nagoya, Japa Abstract. The neutron capture cross-section for platinum was measured at J-PARC/MLF/ANNRI. The intense pulsed neutron beam was impinging on a natural platinum foil sample and the emitted prompt γ -rays were detected by a Ge spectrometer. The peak energies of the low energy resonances for natural platinum are consistent with those of the JEFF-3.1.2, RUSFOND2010 and next-JENDL data libraries except for the 20-eV resonance. The resonance cross-sections of the next-JENDL library do not contradict the present measurements within the uncertainty of the absolute value of the present work. We analysed the prompt γ -ray spectrum and found a clear 7921.93 keV peak that originates from the transition from the 196Pt compound state to its ground state. The neutron capture cross-section for 195Pt was obtained by choosing events of this peak. The peak energies of most of the low energy resonances are almost consistent with those of the RUSFOND2010 and next-JENDL libraries. However, there was a disagreement for the 20-eV resonance. DOI: 10.1051/epjconf/201714611024 DOI: 10.1051/epjconf/201714611024 EPJ Web of Conferences 146, 11024 (2017) ND2016 EPJ Web of Conferences 146, 11024 (2017) ND2016 1. Introduction other hand, JENDL aims at a complete library by adding elements whose importance is not high for nuclear energy applications. Very recently, K. Shibata published an article of the evaluation on platinum for the next version of the JENDL general-purpose file [8]. Platinum is one of the most important elements in our society and is widely used as a catalyst. For example, it is used to purify car exhaust gases and generate electricity in fuel cells. However, in car exhaust applications platinum’s activity as a catalyst deteriorates due to coupling to sulphur compounds. In the case of the fuel cell, deterioration occurs due to coupling to carbon monoxide. In order to investigate and improve these applications, non-destructive imaging methods may be useful. One such method is neu- tron resonance absorption spectroscopy (N-RAS) [1,2]. This method is based on the irradiation of a sample by a pulsed neutron beam. By using a position sensitive neutron detector, atomic density and temperature can be obtained as images. The purpose of this work is to measure the low energy resonances for platinum experimentally. As a first step, we performed a capture cross-section measurement using the Accurate Neutron-Nucleus Reaction Measurement Instrument (ANNRI) [9] in the Material and Life- science Experimental Facility (MLF) at the Japan Proton Accelerator Research Complex (J-PARC). © The Authors, published by EDP Sciences. This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). a e-mail: koichi.kino@aist.or.jp 2. Experiment and data analysis The dips near the resonance peaks, which are due to the dead time of the Ge spectrometer, are corrected. Figure 1. TOF spectrum without and with the dead time correction and live time spectrum. The dips near the resonance peaks, which are due to the dead time of the Ge spectrometer, are corrected. Figure 2. Neutron capture cross-section spectrum for natural platinum in a wide energy region. The cross section was normalized to the value (10.3 barn) reported by Mughabghab [4] at 25.3 meV. neutron source was 305 kW during the experiment. The measurement time of the platinum sample was 31 hours. Figure 3. Neutron capture cross-section spectrum for natural platinum in a narrow energy region. The spectra of JEFF-3.1.2 and RUSFOND2010 almost overlap in this energy region. p p The data analysis was conducted mainly by the following three steps. The first is the dead time correction. In order to obtain the live time spectrum as a function of TOF, we put pulse signals randomly with time at a rate of 1.4 kHz into the analogue signal lines of the Ge spectrometer. The data originating from these signals were distinguished by their pulse height [11]. The raw spectrum was divided by the live time spectrum. Figure 1 shows the TOF spectrum before and after the correction and the live time spectrum. This correction is important especially around the resonance peaks due to the high counting rate due to the large cross section of the capture reaction. The second step is a background subtraction. The background spectrum was obtained by the open beam data. Finally, the last step is the division by the neutron flux spectrum, which is a function of TOF. The flux spectrum was obtained using a boron sample. The size of the sample is 10 mm in diameter with a weight of 96.12 mg. The isotopic ratio of 10B is 90.4%. The neutron flux spectrum was calculated from the experimental data using the 478 keVγ -ray following the 10B(n,α)7Li reaction and the cross section of this reaction. The dependency of neutron beam on TOF was taken into account by the division with the neutron flux spectrum. Figure 3. Neutron capture cross-section spectrum for natural platinum in a narrow energy region. The spectra of JEFF-3.1.2 and RUSFOND2010 almost overlap in this energy region. resonance energy is clearly seen in Fig. 3. 2. Experiment and data analysis g The N-RAS needs resonance parameters of the nuclei included in the experimental sample. However, experimental data of resonance spectra for platinum is poor in the low energy resonance region. There are two data sets in the EXFOR library [3] of total cross sections for natural platinum. Both data sets show resonances at almost the same neutron energies. However, there are discrepancies in the absolute values of the resonances. For the capture cross section, no spectral data is available below 200 eV. For the nuclear data library, no library provides resonance parameters, although Mughabghab compiles the resonance parameters by referring the values in old papers [4]. JEFF-3.1.2 [5], JEFF-3.2 [6] and RUSFOND2010 [7] provide resonance spectra as a function of energy. On the We used the J-PARC/MLF/ANNRI [9]. An intense pulsed neutron beam was impinging on a natural platinum foil sample at a distance of 21.5 m from the neutron source. The size of the sample is 50 mm square with a thickness of 2.5 µm and a purity of 99.9%. The neutron beam is collimated by a rotary collimator [10] and has a diameter of 22 mm at the sample position. Prompt γ -rays following neutron capture at the sample were observed by Ge spectrometers set above and below the sample. The spectrometers consisted of 14 Ge crystals in total. The neutron energy of each event was determined by the time of flight (TOF) method between the neutron source and sample. In order to reduce γ -rays from the neutron source, a lead filter with a thickness of 37.5 mm was set on the neutron beam line. The proton beam power of the a e-mail: koichi.kino@aist.or.jp EPJ Web of Conferences 146, 11024 (2017) ND2016 DOI: 10.1051/epjconf/201714611024 Figure 1. TOF spectrum without and with the dead time correction and live time spectrum. The dips near the resonance peaks, which are due to the dead time of the Ge spectrometer, are corrected. Figure 2. Neutron capture cross-section spectrum for natural platinum in a wide energy region. The cross section was normalized to the value (10.3 barn) reported by Mughabghab [4] Figure 2. Neutron capture cross-section spectrum for natural platinum in a wide energy region. The cross section was normalized to the value (10.3 barn) reported by Mughabghab [4] at 25.3 meV. Figure 1. TOF spectrum without and with the dead time correction and live time spectrum. 2. Experiment and data analysis The spectrum above 200 eV is almost in agreement with the experimental data by R.C. Bloch. The cross sections at the low energy resonance peaks given by JEFF-3.1.2 and RUSFOND2010 were smaller than our experimental data in Fig. 3 even though the experimental data has a 10% uncertainty. On the other hand, the cross sections of the resonances by the next-JENDL did not contradict those of the present data. 3. Discussions The ANNRI has an advantage that the γ -ray energy is precisely determined by the Ge spectrometer. Therefore, we made use of it for selecting platinum isotopes in the capture reaction. There are six stable isotopes of platinum. The neutron separation energies are different for each isotope. Therefore, one can distinguish isotopes by selecting primary γ -ray on the basis of the decay from the compound state after neutron capture reaction to the ground state. Figure 4 shows the γ -ray energy spectrum in the energy region from 5500 to 8000 keV. There are many photoelectron peaks and the arrows in the figure indicate single-neutron separation energies for platinum isotopes. The peak corresponding to 196Pt is seen very clearly at 7921.93 keV. Some peaks for other isotopes are also seen although the signal to noise ratio is not high. Therefore, we applied the isotope separation only on 196Pt. This means that we select neutron capture reaction for 195Pt. The natural abundance of 195Pt is 33.78%. Figures 2 and 3 show the capture cross-section spectra for natural platinum in wide and narrow energy regions. The cross section was normalized to the value (10.3 barn) reported by Mughabghab [4] at 25.3 meV. The deviation from the 1/v law seen in Fig. 2 might be due to some kind of constant background and it is about 1 barn. This gives an uncertainty of about 10% for the absolute value of the cross section. Figure 2 shows that the energies of major low energy resonances are almost in agreement with those of JEFF- 3.1.2 and RUSFOND2010. The blue line is the spectrum converted at 300 K from the next-JENDL evaluation resonance parameters [8]. The resonance parameters of this evaluation are taken from the compilation work of Mughabghab. The experimental data showed agreement in resonance energies with the next-JENDL evaluation except for the resonance at 20 eV. The disagreement of the 20-eV 2 DOI: 10.1051/epjconf/201714611024 EPJ Web of Conferences 146, 11024 (2017) ND2016 EPJ Web of Conferences 146, 11024 (2017) Figure 4. Energy spectrum of prompt γ -rays in the energy region from 5500 to 8000 keV. The arrows indicate single neutron separation energies for platinum isotopes. given by the next-JENDL library. The resonances, whose energies are 46 and 95 eV for example, disappeared after choosing 195Pt. This is consistent with RUSFOND2010 and next-JENDL. 3. Discussions Disagreement of resonance energy for the 20-eV resonance is obtained between the present work and next-JENDL as shown in Fig. 6. In general, the decay schemes are different depending on the neutron capture states. This could be the reason for the disagreements of the cross sections between the present work and nuclear data libraries at the thermal neutron energy as seen in Fig. 5 and the 20-eV resonance as seen in Fig. 6. Figure 4. Energy spectrum of prompt γ -rays in the energy region from 5500 to 8000 keV. The arrows indicate single neutron separation energies for platinum isotopes. 4. Summary The neutron capture cross-section for platinum was measured at J-PARC/MLF/ANNRI. For natural platinum, most of the measured resonances were found at almost the same energies as the various nuclear data libraries except for the 20-eV resonance. The cross sections of the low energy resonances of the present work did not contradict those given by the next-JENDL library. We obtained the capture cross-section spectrum for 195Pt by choosing the prompt γ -rays for this isotope. Most of the resonance energies of the present work were consistent with those of RUSFOND2010 and next-JENDL libraries. However, the energy of the 20-eV resonance was different between the present work and the nuclear data libraries. Figure 5. Neutron capture cross-section spectrum for 195Pt in a wide energy region. This work was performed using the experimental instruments at the MLF at J-PARC and supported by the J-PARC staff. The authors would like to thank Dr. K. Shibata and Dr. N. Iwamoto for providing the nuclear data for the next version of JENDL. Figure 5. Neutron capture cross-section spectrum for 195Pt in a wide energy region. References Figure 6. Neutron capture cross-section spectrum for 195Pt in a narrow energy region. [1] H. Sato, T. Kamiyama, Y. Kiyanagi, Nucl. Instr. and Meth. A 605, 36 (2009) [2] H. Hasemi, M. Harada, T. Kai, et al., Nucl. Instr. and Meth. A 773, 137 (2015) [3] N. Otsuka, E. Dupont, V. Semkova, et al., Nucl. Data Sheets 120, 272 (2014) [4] S.F. Mughabghab, Atlas of neutron resonances (Elsevier, Amsterdam, 2006) [5] JEFF-3.1.2, http://www.oecd-nea.org/dbforms/ data/ eva/evatapes/jeff 31/JEFF312/ [6] JEFF-3.2, http://www.oecd-nea.org/dbforms/ data/eva/evatapes/jeff 32/ [7] RUSFOND2010, http://www.ippe.ru/podr/ abbn/english/libr/rosfond.php Figure 6. Neutron capture cross-section spectrum for 195Pt in a narrow energy region. g p p [8] K. Shibata, J. Nucl. Sci. Technol 54, 147, (2016) [9] M. Igashira, Y. Kiyanagi, M. Oshima, Nucl. Instr. and Meth. A 600, 332 (2009) Figures 5 and 6 show the capture cross-section spectra for 195Pt in wide and narrow energy regions. The normalization was performed by setting the cross section of the present work at 12-eV resonance to the value [10] K. Kino, M. Furusaka, F. Hiraga, Nucl. Instr. and Meth. A 626–627, 58 (2011) [11] T. Kin, K. Furutaka, S. Goko, et al., J. Korean Phys. Soc. 59, 1769 (2011) 3 3
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Izmenenija gruppovogo sostava bitumoidov s glubinoj po razrezu sverhglubokoj skvazhiny sredneviljujskaja-27
Perspektivnye materialy s ierarkhicheskoy strukturoy dlya novykh tekhnologiy i nadezhnykh konstruktsiy» i «Khimiya nefti i gaza» v ramkakh Mezhdunarodnogo simpoziuma «Ierarkhicheskie materialy: razrabotka i prilozheniya dlya novykh tekhnologiy i nadezhnykh konstruktsiy
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ТЕЗИСЫ ДОКЛАДОВ Секция А. Химия нефти и газа DOI: 10.17223/9785946217408/452 ИЗМЕНЕНИЯ ГРУППОВОГО СОСТАВА БИТУМОИДОВ С ГЛУБИНОЙ ПО РАЗРЕЗУ СВЕРХГЛУБОКОЙ СКВАЖИНЫ СРЕДНЕВИЛЮЙСКАЯ-27 Д К В Долженко К.В. Институт нефтегазовой геологии и геофизики им. А.А. Трофимука СО РАН, Новосибирск, Россия DolzhenkoKV@ipgg.sbras.ru Комплексом геохимических методов исследовано рассеянное органическое вещество (РОВ) верхнепалеозойских отложений центральной части Вилюйской синеклизы (на примере сверхглубокой скважины Средневилюйская-27). В ранее опубликованных автором работах показано распределение органического углерода, его пиролитические характеристики, уровень зрелости [1-2]. По этим параметрам в разрезе выделены толщи, способные к генерации углеводородов, установлена нижняя граница предполагаемой нефтегазоносности. В настоящей работе был проанализирован групповой состав битумоидов, осуществлено разделение на аллохтонные и автохтонные образцы, обнаружены следы первичной миграции, установлены различия группового состава зон апо- и мезокатагенеза, проведен термодинамический рубеж. Последовательное наблюдение изменения группового состава с глубиной позволило обнаружить две принципиально различные группы. Первая выражена на глубинах 3370-4853 м и относится к толщам, которые являются потенциально нефтегазогенерирующими (в них обнаружены аллохтонные битумоиды, а в пределах кюндейской свиты по изменению содержания углеводородной части предположена первичная миграция), и имеет усредненно равномерный состав по трем компонентам с небольшим увеличением смолистой компоненты к нижней границе. Вторая относится к глубинам 4853-6558 м, на которых толщи по ранее установленным данным являются истощенными, и имеет явное смещение в сторону компонент углеводородов и смол, с явным сокращением содержания асфальтеновой компоненты по мере увеличения глубины, вплоть до полного исключения их из состава на глубине 5482 м. Такое поведение можно объяснить наличием границы, при достижении которой крупные структуры (асфальтены) разлагаются. Ранее [3] термодинамический рубеж предполагался на глубине 5300 м. Что интересно, с этой границей связано предполагаемое высвобождение некоторых соединений, «запертых» в структуре асфальтенов [4]. Исследование выполнено при финансовой поддержке РФФИ в рамках научного проекта № 18-35-00337. Литература 1. Фомин А.Н., Долженко К.В., Меленевский В.Н. Прогноз зон генерации жидких и газообразных углеводородов в центральной части Вилюйской синеклизы (на примере сверхглубокой скважины Средневилюйская-27) // Интерэкспо ГЕО-Сибирь-2016. XII Междунар. науч. конгр.: Междунар. науч. конф.: Сборник материалов в 4 т.. – 2016. – Т. 1. – С. 29-34 2. Долженко К.В. Геохимическая характеристика органического вещества верхнепалеозойских отложений Вилюйской синеклизы (на примере сверхглубокой скважины Средневилюйская-27) // Проблемы геологии и освоения недр: Труды XX Международного симпозиума им. акад. М.А. Усова студентов и молодых ученых, посвящ. 120-летию со дня основания Томского политехнического университета. – 2016. – Т. I. – С. 313-315 3. Конторович А.Э., Полякова И.Д., Колганова М.М., Соболева Е.И. Превращения органического вещества в мезо- и апокатагенезе // Советская геология. - 1988. - №7. - С.26-36. 4. Каширцев В.А., Фомин А.Н., Шевченко Н.П., Долженко К.В. Новые моноароматические стероиды в органическом веществе зоны апокатагенеза // Доклады РАН. – 2016. – Т. 469. – № 4. – С. 465-469 2. Долженко К.В. Геохимическая характеристика органического вещества верхнепалеозойских отложений Вилюйской синеклизы (на примере сверхглубокой скважины Средневилюйская-27) // Проблемы геологии и освоения недр: Труды XX Международного симпозиума им. акад. М.А. Усова студентов и молодых ученых, посвящ. 120-летию со дня основания Томского политехнического университета. – 2016. – Т. I. – С. 313-315 4. Каширцев В.А., Фомин А.Н., Шевченко Н.П., Долженко К.В. Новые моноароматические стероиды в органическом веществе зоны апокатагенеза // Доклады РАН. – 2016. – Т. 469. – № 4. – С. 465-469 703
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Normal values ​​for ultrasound parameters of the ulnar nerve require homogeneous, healthy cohorts
Neurological research and practice
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Letter to the editor studies of the ulnar nerve. The exclusion of subclinical neuropathy is essential before normative data can be col­ lected for ultrasound.h With interest we read the article by Pandal-Fernandez et al. on a study of 76 ulnar nerves from 38 asymptom­ atic subjects by means of ultrasound [1]. It was found that the diameter of the ulnar nerve as well as the distal and proximal area were larger at the proximal part of the ulnar groove as compared to the distal portion, and even more so in older individuals [1]. In most of the elderly probands, a mild, non-significant reduction of the nerve conduction velocity of < 5 m/s was found at the elbow [1]. The study is excellent, but has limitations that are cause of concerns and should be discussed.h The method section also does not mention what is meant by “asymptomatic” [1]. Do the authors mean no symptoms related to the ulnar nerve or no symptoms related to comorbidities. Were probands with complica­ tions in the peripheral nervous system after SARS-CoV-2 infection or vaccination ruled out? No mention is made of current medications, which may also be neurotoxic and therefore could affect the data. f A second limitation of the study is that females pre­ dominated with a female-to-male ratio of 2.8:1 [1]. Nor­ mative data should be extracted from cohorts with an equal number of females and males. If there are differ­ ences between the two groups, different normal values must be generated for both sexes. The main limitation of the study is that prior to includ­ ing subjects in the study, asymptomatic neuropathy was not ruled out. Several neuropathies of the ulnar nerve can be asymptomatic. Subclinical affection of the ulnar nerve can occur with polyradiculitis affecting the 7th and 8th cervical nerve root, plexitis, sulcus ulnaris syndrome, and primary or secondary mono- or polyneuropathy. Therefore, it is crucial that all 38 enrolled subjects had undergone nerve conductions studies prior to enrolment in the study. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. (2023) 5:27 (2023) 5:27 Neurological Research and Practice Finsterer Neurological Research and Practice (2023) 5:27 https://doi.org/10.1186/s42466-023-00254-8 Finsterer Neurological Research and Practice https://doi.org/10.1186/s42466-023-00254-8 Open Access Open Access Normal values ​for ultrasound parameters of the ulnar nerve require homogeneous, healthy cohorts Josef Finsterer1* Keywords  Ultrasound, Ulnar nerve, Nerve conduction, Normal values Letter to the editor Probands included in the study should not only have a negative history for neuropathies, but should also have normal proximal and distal nerve conduction Other weaknesses of the study are the small sample size (reference limits are usually derived from a large sample representative of the population of interest), the lack of acceptable sample size estimation considering the vari­ ability of the parameters in different populations (age, sex, race, height, body weight, associated health condi­ tions etc.), and lack of evidence that the enrolled subjects were really healthy individuals. It should be also considered that sleeping habits of the included subjects could affect the evaluation. Those sleeping with a flexed position of the arms may have a propensity to ulnar nerve distension at the ulnar canal as *Correspondence: Josef Finsterer fifigs1@yahoo.de 1Postfach 20, Vienna, Europe 1180, Austria *Correspondence: Josef Finsterer fifigs1@yahoo.de 1Postfach 20, Vienna, Europe 1180, Austria Competing interests h h d l h The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. f Overall, the interesting study has limitations that call the results and their interpretation into question. Addressing these issues would strengthen the conclu­ sions and could improve the status of the study. Norma­ tive data on ultrasound parameters of the ulnar nerve should only be obtained from subjects who actually have normal ulnar nerves. Generation of such cohorts requires not only a medical history devoid of comorbidities asso­ ciated with neuropathy, but also the exclusion of neurop­ athy by nerve conduction studies and the exclusion of risk factors associated with neuropathy and atherosclerosis. Data Availability d h Additionally, ulnar nerve function and morphology may depend on the vascularisation and perfusion of the ulnar nerve, why it is crucial to know how many of the included patients had classical cardiovascular risk fac­ tors, such as arterial hypertension, hyperlipidemia, or smoking, Atherosclerosis of the nerve arterioles may affect function and morphology of a peripheral nerve. data that support the findings of the study are available from the corresponding author. Finsterer Neurological Research and Practice (2023) 5:27 Page 2 of 2 Page 2 of 2 Finsterer Neurological Research and Practice (2023) 5:27 Compliance with Ethics Guidelines This article is based on previously conducted studies and does not contain any new studies with human participants or animals performed by any of the authors. Received: 2 April 2023 / Accepted: 29 May 2023 Received: 2 April 2023 / Accepted: 29 May 2023 Funding compared to those sleeping in a position with stretched upper limbs. g no funding was received. References References 1. Pardal-Fernández, J. M., Diaz-Maroto, I., Segura, T., & de Cabo, C. Ulnar nerve thickness at the elbow on longitudinal ultrasound view in con­ trol subjects. Neurol Res Pract 2023 Jan 26;5(1):4. https://doi.org/10.1186/ s42466-023-00230-2. Acknowledgements f h ( ) Statement of Ethics: (a) The study was approved by the institutional review board (responsible: Finsterer J.) at the 4th November 2022. (b) Written informed consent was obtained from the patient for publication of the details of their medical case and any accompanying images. Author contribution Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. JF: design, literature search, discussion, first draft, critical comments, final approval.
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THE MOLECULAR REARRANGEMENT OF TRIARYLMETHYL-HYDROXYLAMINES.
Journal of the American Chemical Society
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2069 2069 REARRANGBMBNT OF TRI~RYLLlETHYltIYDROXYLAMINES. Dissertation work of James K. Senior, University of Chicago, completed in 1914. q g ( ) 8th Intern. Congr. Appl. Chem., 25, 443 (1912); Ber., 46, 2 1 4 7 (1913); THIS JOURNAL, 36, 272 ( 1 9 1 4 ) . THE MOLECULAR REARRANGEMENT OF TRIARYLMETHYL- HYDROXYLAMINES. BY BERT ALLEN STAGNER. Received August I . 1916. That the hydroxylamine derivatives of triphenylmethane undergo a rearrangement entirely analogous to the Lossen rearrangement of hydroxamic acids and the Beckniann rearrangement of oximes and under exactly similar conditions, was shown by the work of Stieglitz, Reddick and Leech.2 The rearrangement is effected by the action of phosphorus pentachloride on the hydroxylamine derivative or by the action of phos- phorus pentoxide on the hydrochloride of the base.3 The product of the rearrangement is a phenylimidobenzophenone. According to the theory of Stieglitz4 the action is formulated as follows: H g (C6H5)&.NHoH -sH (CsH5)3C.N ----f (C&,)zC : NCsH5 ( I ) (C6H5)&.NHoH -sH (CsH5)3C.N ----f (C&,)zC : NCsH5 ( I ) When a substituting group replaces hydrogen in one or more of the phenyl groups, as in (XC6H4) (CsH5)2C.NHOH, the rearrangement leads to the formation of two substituted phenylimidobenzophenones, part of the substance yielding the phenylimide of the substituted benzophenone, for instance (XC6H4)(C)&)C : XC&Ib, part forming a substituted phenyl- imido derivative of benzophenone itself, namely, (C6Hj)ZC : NCeHX. A study of the proportions in which the groups migrate in a series of such derivatives promises to shed light on a number of interesting ques- tions. In the first place, exactly the same rearrangements have been observed by Stieglitz and Vosburgh5 in the study of triarylmethylhalogen- amines, and, recently, by Stieglitz and Senior6 in the case of the triaryl- methyl azides. In all three series the assumption of the formation of intermediate univalent nitrogen derivatives as the actually rearranging substances gives lis, according to Stieglitz, the best explanation of ’the rearrangements. Then, corresponding compounds in the three series, such as (XCsH4) (CsH&C.h”OH, (XCsH4) (CGH&C.NH(Hal), (XCsH4)- (CsH&C.N (Ns) could well yield the identical rearranging compound (XC6H4) (CGH&C.?J. The conditions for the rearrangement in each of these series vary as to the temperature and other environment factors, The material presented here forms part of a dissertation in fulfilment of the re- quirements for the Ph.D. degree (1914). 8th Intern. Congr. Appl. Chem., 25, 443 (1912); Ber., 46, 2 1 4 7 (1913); THIS JOURNAL, 36, 272 ( 1 9 1 4 ) . 8th Intern. Congr. Appl. Chem., 25, 443 (1912); Ber., 46, 2 1 4 7 (1913); THIS JOURNAL, 36, 272 ( 1 9 1 4 ) . THE MOLECULAR REARRANGEMENT OF TRIARYLMETHYL- HYDROXYLAMINES. See the references to the literature, THIS JOURNAL, 36, 2 7 2 ( 1 9 1 4 ) . 2070 BERT ALLEN STAGNEER. but the rearrangement of the univalent nitrogen derivative, being intra- molecular, might very well be largely independent of environment. The determinations of the proportions in which the various radicals CsH6, XCsH4, migrate in the rearrangement of corresponding compounds in each of the three series would give us, therefore, the necessary data for quantitative comparisons of the reactions in the three series. q p In the second place, such quantitative determinations would show what differences, if any, are shown in the migration of aryl groups XC6H4 with various negative or positive substitution groups in them (X = H, C1, Br, NO2, CH3, etc.), thus giving a definite answer to the question whether certain groups are more easily lost by carbon and drawn to nitro- gen than others. In previous studies of this problem, conclusions have been based, either on the behavior of the groups in question in related but different substances (e. g., C6H5CONHBr and N02C6H4CONHBr1 or on the behavior of ketoximes, in which stereoisomerism is supposed to play the major role in determining the migration of a given radical. For the triphenylmethane derivatives this complication is eliminated and we have, furthermore, all the groups in question in exactly the same re- arranging molecule, e. g., (CeH6) (XCsH4) (YC6H4)C.N and, therefore, under strictly comparable conditions. The problem is thus reduced to its simplest terms2 Stieglitz and Leech having examined only p-chlorophenyldiphenyl- methylhydroxylamine in regard to the proportion in which the phenyl and the chlorophenyl groups migrate to the nitrogen, I took up, at the suggestion and under the direction of Professor Stieglitz, the further investigation of this problem at this point. For this purpose the rearrange- ment of p-bromophenyl-diphenylmethylhydroxylamine, of di-p-chloro- phenyl-phenylmethylhydroxylamine and finally of p-chlorophenyl-p- bromophenyl-phenylmethylhydroxylamine was studied by me. The interesting result was obtained that whereas bromophenyl-di- phenylmethylhydroxylamine, (BrC6H4)(C6H5)&NHOH, exactly as the corresponding chloro derivative studied by Leech, rearranges so that, roughly, in two-thirds of the rearranging molecules a phenyl group and in the remaining third the bromophenyl group migrates, the ratio is reversed when one subjects dichlorophenyl-phenylmethylhydroxylamine, (C1C&I&(C6H&"OH, to the rearrangement. In this case for two- thirds of the rearranging molecules it is the chlorophenyl group that migrates and in the remaining third the phenyl group. THE MOLECULAR REARRANGEMENT OF TRIARYLMETHYL- HYDROXYLAMINES. Furthermore, * ff d R h 6 a A further question which is being studied io that of the effect of the positions of the substituting groups (ortho, meta and para) on the migration. It is expected to produce experimental evidence concerning the nature of the charges on the carbon atoms holding the aryl groups to the methyl carbon atoms and thus to secure evidence on cer- tain of Fry's assumptions concerning the charges in the benzene derivatives.-J. S. a A further question which is being studied io that of the effect of the positions of the substituting groups (ortho, meta and para) on the migration. It is expected to produce experimental evidence concerning the nature of the charges on the carbon atoms holding the aryl groups to the methyl carbon atoms and thus to secure evidence on cer- tain of Fry's assumptions concerning the charges in the benzene derivatives.-J. S. REARRANGEMENT OF TRIARYLMETHYLHYDROXYL4MIN~S. 207 I in the case of chlorophenyl-bromophenyl-phenylmethylhydroxylamine (CIC6€14) (BrC6H4) (C6HS)CNHOH, roughly one-third of the rearranging molecules show a migration of the chlorophenyl group, the bromophenyl group and’ the phenyl group, respectively. This result certainly would indicate that there is no marked effect of the halogens on the tendency of the aryl group to migrate to the nitrogen.’ See Lobry de Bruyn, Rec. trau. chim., IO, 100 (1891). Lobry de Bruyn, loc. cit. THIS JOURNAL, 36, 3152 (191s). Experimental Part. This base was extracted with dry ether and the extract treated with an excess of dry, hydrogen chloride. The salt, (BrC6H4) (C6H5)~CNH~0HCI, separated at once as a white solid. The salt becomes slightly soluble in ether after a large excess of the acid is introduced. It was collected on a filter, washed several times with ether, and once quickly with cold dilute (about 3 molar) hydro- chloric acid to remove hydroxylamine hydrochloride. y y y From 3 g. of bromophenyl-diphenylmethylchloride, 1.8 g. of the puri- fied salt was obtained. This was analyzed by titration of the hydro- chloric acid with silver nitrate solution by the Volhard method. Subst. 0.1711, 0.1730; cc. 0.1 NAgNOa, 4.34, 4.37. Calc. for (BrC6H4) (CeH&CNH?OHCl: HCl, 9.33, Found: 9.25, 9.19. Subst. 0.1711, 0.1730; cc. 0.1 NAgNOa, 4.34, 4.37. Calc. for (BrC6H4) (CeH&CNH?OHCl: HCl, 9.33, Found: The salt reduces Fehling's solution when boiled, but not in the cold, melts at 144-5' with decomposition, is soluble in alcohol, but not in lig- roin and but slightly soluble in ether. Rearrangement of p-Bromophenyl-diphenylmethylhydroxy1dne.- The free base, (BrC6HI) (C6H5)2CNHOH, was obtained from the hydro- chloride just described by treatment of the latter with 20% sodium hy- droxide solution and extraction of the base with ether. The ether solu- tion was dried with potassium carbonate and the solvent evaporated in a current of dry air. The base thus obtained was a pale yellow viscous liquid or gum. Some of it (1.35 g.) was dissolved in 30 cc. of anhydrous ether and powdered phosphorus pentachloride (2.5 g.) was added to it. When the mixture was allowed to stand at room temperature for a few hours a trace of a yellow deposit collected on the phosphorus chloride. The mixture was then heated on an electric bath for two hours and a greater amount of the yellow deposit collected. The molecular rearrange- ment was expected to yield the two phenylimidobenzophenones, p-bromo- phenylimidobenzophenone, (C6H5)zC : NHCsH4Br and phenylimido- bromobenzophenone, (BrC6Hr) (C&)C : NH.C6H5. To insure that these bases should be present in the form of their salts, some ether (2 cc.) satu- rated with hydrogen chloride was added to the reaction product. The supernatant liquid was then decanted through a filter from the main resi- due and the residue was washed with ether containing some hydrogen chloride. Experimental Part. p - Bromophenyldiphenylmethylhydroxylamine, (BrCsH4) (CaHs)z- CNH0H.-This was prepared according to Gomberg2 from benzophenone- dichloride and bromobenzene by the Friedel and Craft reaction. The chloride was treated with hydroxylamine by a modification of the methods of Mothwurf3 and of Stieglitz and LeechS4 Many futile preliminary attempts by these methods showed that it is necessary to have the hydroxyl- amine solution very concentrated, for otherwise the principal reaction takes place between the triarylmethylchloride and the alcohol, forming the ether (C6HS)z(BrC6H4)COCH3. Even with this precaution some of the ether was always formed. A saturated solution of 3.2 g. of hydroxylamine hydrochloride in warm methyl alcohol was added to a solution of sodium methylate (I g. of sodium dissolved in the least quantity of methyl alcohol). It is desirable not to have any excess of the sodium methylate present and this was carefully determined by evaporation of a drop of the solution on a crucible lid and gentle ignition of any residue. If any alkaline substance remained, more hydroxylamine hydrochloride was added to the methylate solution until the test gave a negative result. The sodium chloride formed in the reaction was removed by filtration and the filtrate evaporated at 2 0 mm. pressure and 40’ to a volume of IO cc. This treatment gave a very con- centrated hydroxylamine solution and but little of the hydroxylamine e~caped.~ The approximate strength of the solution was determined by titration of 0.5 cc. of it with 0. I X hydrochloric acid, methyl orange being used as the indicator.6 Three grams of p-bromophenyl-diphenylmethyl chloride, dissolved in 30 cc. of anhydrous benzene, were added to the IO cc. of the alcohol solution of hydroxylamine. After the mixture had stood a few hours the small quantity of hydroxylamine hydrochloride which had separated was removed by filtration and the filtrate evaporated at 20 mm. pressure The discussion of minor differences in the migration of these radicals and the com- parison of the results obtained with corresponding hydroxylamines, chloroamines and azides will be presented in another paper by Professor Stieglitz. * Bey., 37, 1633 (1904). y Ibid., 37, 3152 (1904). THIS JOURNAL, 36, 3152 (191s). See Lobry de Bruyn, Rec. trau. chim., IO, 100 (1891). Lobry de Bruyn, loc. cit. BERT ALLEN STAGNER. 2072 and 50'. A yellow, gummy residue, consisting chiefly of the bromo- phenyl-diphenylmethylhydroxylamine, was left. 1 The presence of this salt was indicated by the hydrogen chloride content of the roduct when it was not purified in this way. Experimental Part. Since partial hydrolysis of some of the imidoketones by traces of moisture would form anilines and since aniline hydrochlorides are some- what soluble in ether containing an excess of hydrogen chloride, it was always possible that some of the aniline, if any were present here, might be dissolved by this treatment. For the recovery of any such substance, the ether filtrate was evaporated almost to dryness, a small amount of REARRANGElvfENT OF TRIARYLMETHYLHYDROXI’I.,ZAMINES. 1073 water added to the residue, and this warmed to decompose the phosphorus oxychloride. This aqueous solution and the trace of solid on the filter were then added to the main residue. This was then warmed with alcohol (IO cc.) and dilute hydrochloric acid (5 cc.) for an hour on a steam bath to completely hydrolyze the imidoketones. (BrCBHI)(C6H5)C = N.CoH5 + H20 -I- HC1 --f jC6H5)2C = NCeH4Br + HzO + HCI -+ (BrCaH4) (CsHdCO + C~HdW3CI ( 2 ) (C6&)&0 + BrC6H4NH3.C1. (3) (BrCBHI)(C6H5)C = N.CoH5 + H20 -I- HC1 --f jC6H5)2C = NCeH4Br + HzO + HCI -+ (BrCaH4) (CsHdCO + C~HdW3CI ( 2 ) (C6&)&0 + BrC6H4NH3.C1. (3) (C6&)&0 + BrC6H4NH3.C1. (3) The alcohol was removed by evaporation of the solution almost to dry- ness, followed by a second evaporation after some acidulated water had been added to the residue. Now, water (I j cc.) was added to the product and the solution twice extracted with 15 cc. of ether to remove the ketones. These ketones were identified as described further on. The aqueous solution contained the aniline and bromoaniline hydrochlorides. It was made alkaline, saturated with sodium chloride and the free anilines ex- tracted with two portions of 25 cc. each of ether. The extract was filtered into a weighed beaker and 2 to 3 cc. of ether saturated with hydrogen chloride were added to this filtrate to convert the anilines into hydrochlo- rides. Because of the partial solubility of these salts in such an acid- ether solution, the ether and the water held in solution by it were evap- orated in a current of dry air and the salts remaining were dried iiz vacuo. The dried salts were washed once with 10-15 cc. of absolute ether, dried again, and weighed. The p-bromophenyl-diphenylmethylhydroxylamine (1.35 g.) used in the rearrangement yielded 0.2075 g. of aniline hydro- chloride and bromoaniline hydrochloride. Two other quantitative rearrangements of the base were made by this method. ’ THIS JOURNAL, 35, 1162 (1913). 2 LOC. L i t . ’ THIS JOURNAL, 35, 1162 (1913). LOC L i t a bau s pu e b o oa e, e t g at 63 6 , * The reaction seems to take place slowly even when the mixture is heated only slightly, as shown by the fact that the yellow tint of the mixture grew deeper in color when it was pressed hard against the sides of the test tube with a glass rod. a Be?., 39, 3279 (1906). Ibid., 36, 3087 (1903). Experimental Part. In the first a yield of 0.2145 g. of aniline salts was obtained from 1.08 g. of the base and in the second the yield of the salts was 0.2865 g. when I . I 7 g. of the base was used. Theoretically I g. of the base could yield either 0.36 g. of aniline hydrochloride or 0.58 g. bromoaniline hydro- chloride. These mixtures of aniline and bromoaniline hydrochlorides were analyzed to as- certain the molar ratios of the aniline and bromoaniline. The bromination method of Curme’ was used. Subst. 0.0589, 0.0569, 0.0659; cc. 0.1 N KBr03, 21.65, 21.31, 24.63. Found: aniline hydrochloride, 74.73, 76.81, 76.58; bromoaniline hydrochloride, 25.27, 23.19, 2.3.42. Identification of Aniline and Bromoani1ine.-The method of separating aniline and chloroaniline used by Stieglitz and Leech2 proved satisfactory for separating and identifying the aniline and bromoaniline of this re- ’ BERT ALLEX STAGNER. 2074 arrangement. The hydrochlorides were converted into the oxalates and the greater solubility of the aniline oxalate in hot water permitted its separation from the bromoaniline oxalate. The former, in water solution and made alkaline, gave the violet color with bleaching powder solution; and the lat'ter, freed from the acid, gave crystals from ether solution, which melted at 62'. These when mised with pure p-bromo- aniline,' melted at 62-63'. Identification of Benzophenone and Bromobenzophenone.-The ether containing the ketones in solution was evaporated. The residue was quickly washed with a small amount of ligroin and then about seven- eighths of the residue was dissolved in warm ligroin (b. p. 70-80'). The small quantity of residue remaining melted at 81 ' (pure p-bromo- benzophenone melts at 82.5 '). The ligroin solution was evaporated to dryness and two-thirds of the residue dissolved. This treatment was repeated until about one-eighth of the original residue was in solution. A few crystals obtained from this solution melted at 46-47' and when they were mixed with pure benzophenone they melted at 47-48' (pure benzophenone melts at 48.5 '). Identification of Benzophenone and Bromobenzophenone.-The ether containing the ketones in solution was evaporated. The residue was quickly washed with a small amount of ligroin and then about seven- eighths of the residue was dissolved in warm ligroin (b. p. 70-80'). The small quantity of residue remaining melted at 81 ' (pure p-bromo- benzophenone melts at 82.5 '). The ligroin solution was evaporated to dryness and two-thirds of the residue dissolved. Experimental Part. This treatment was repeated until about one-eighth of the original residue was in solution. A few crystals obtained from this solution melted at 46-47' and when they were mixed with pure benzophenone they melted at 47-48' (pure benzophenone melts at 48.5 '). Rearrangement of p-Bromophenyl-diphenylmethylhydroxylamine Hy- drochloride by Means of Phosphorus Pentoxide.-Bromophenyl-diphenyl- methylhydroxylamine hydrochloride (0.8 g.) and phosphorus pentoxide (0.9 g.) were placed together in a test tube and gradually warmed in a metal bath. At 107' a rapid reaction took place and the mixture ex- panded to about three times its original volume into a brown gummy mass. The mass was kept at this temperature for 20 minutes and then cooled.2 The subsequent treatment was similar to that described above for the product of the rearrangement of the free base. Approximately 0.1 g. of the hydrochlorides of aniline and bromoaniline was obtained. The aniline gave the characteristic test with bleaching powder solution. No further attempts at effecting rearrangements by means of phosphorus oxide were made as the pentachloride seemed io give more satisfactory yields. y Di - p - chlorophenyl - phenylmethylhydroxylamine, (C1CBH4)2(C6Ha CNH0H.-This was made by Gomberg's method3 from dichlorobenzo- phenone by the Grignard reaction. The reagent was used according to the proportions given by H ~ u b e n , ~ i . e., 2.36 g. of specialmagnesium, 17.5 g. of bromobenzene, and 120 cc. of ether. After a few hours, .when the reaction between the magnesium and the bromobenzene was com- REARRANGEMENT OF TRIAKYLMETHYLHYDROXYLAMINES. 2075 plete, g g. of the solid di-pchlorobenzophenone were added. This ketone is only moderately soluble in ether but it dissolved at once in the Grig- nard reagent. It was warmed for three hours on a water bath and the magnesium compound was then decomposed with water. The excess of bromobenzene used in making the Grignard reagent and the benzene formed from it later were removed by steam distillation and the light colored insoluble gum remaining in the water was washed free from magnesium hydroxide with dilute hydrochloric acid. The gum was dissolved in ligroin an'd dried with fused calcium chloride. The solution was filtered and the solvent evaporated spontaneously in a desiccator over paraffin shavings. In three hours crystals of the carbinol began to form and 8.6 g. of the carbinol had separated out overnight. The carbinol melted at 85' without re- crystallization. Subst. 0.2287 0.2869; cc. 0.1 N AgNOI, 5.85, 7.29. Calc. for (C1CsH&(CeHs)CNH80HC1: HCI, 9.58. Found: 9.32, 9.26. Experimental Part. Gomberg did not succeed in obtaining this carbinol crystallized until he had taken it through a long troublesome method of purification. The preparation was twice repeated and the crystalliza- tion similarly obtained. The carbinol was converted into the chloride, (C1CsH4)2(CsHz)CCl, in the usual manner by dry hydrogen chloride passed into a carbon disulfide solution of the carbinol. The chloride thus formed was a gum at first and assayed 94% of the calculated amount of chlorine when it was hydrolyzed in alcohol solution with excess of alkali. This gum was used for the preparation of the hydroxylamine, but about 5 g. of it were left in a desiccator over solid potassium hydroxide and in a month's time crystals had formed on the sides of the beaker. These were pushed down into the gum and in several days the whole had become solid. The solution must be kept free from moisture. This impure solid melted at 43-jO'. Calc. for (ClCeH&(CsH6)CCl: C1, 10.21. Found: 9.50. Subst. 0.4333 g.; cc. 0.1 N KOH 11.58. The solidification is slow and time did not permit further eEorts at the puri- fication of the product. The chloride, as a gum, was allowed to act on a strong solution of hydroxylamine, in the way described above for bromo- phenyl-diphenylmethylchloride. The free base remaining after the al- cohol and benzene were removed, was extracted with ether and hydrogen chloride added to the ether extract. The hydrochloride, (CIC&&(C&,)- CNHZOHCI, separated at once as a white solid. The salt was removed by filtration and washed well with ether and once quickly washed with cold dilute hydrochloric acid. It is slightly soluble in ether, soluble in alcohol, but insoluble in ligroin and benzene. It reduces boiling Feh- ling's solution, and melts at 1zg-30~ into a pale yellow liquid which soon reddens when left at that temperature. Gomberg was not successful in getting this gum to solidify. 076 BERT ALLEN BERT ALLEN STAGNER. 2076 Rearrangement of Di-p-chlorophenyl-phenylmethylhydroxy1amine.- The free base was obtained from the hydrochloride described above by treatment of the salt with 20% alkali solution and extraction with ether. The ether solution was dried over potassium carbonate and the ether evaporated. The base was left as a pale yellow, syrupy liquid or gum which is soluble in ether, alcohol, ligroin, and benzene. The substance (1.47 g.) was dissolved in ether (30-40 cc.) and phosphorus pentachloride (2.5 g.)\was added to the solution. LOC. cit., p. 296. Experimental Part. - Di-p-bromophenyl-phenylme thylchloride was prepared by Gomberg's methoda2 The corresponding carbinol, (BrC6H4)z(C6H5)COH, which was first obtained, melted at I 13.5' when recrystallized from ligroin.' It melted at the same temperature when twice recrystallized. Gomberg gives the melting point as 110'. The chloride, (BrC6H4)2(C6H5)CC1, was dissolved in benzene and added to a solution of hydroxylamine in alcohol. The molecular proportions of substances, and the method of procedure used in the previous prepara- tions were repeated here. The alcohol and benzene were removed by distillation under diminished pressure and without the temperature rising above 60'. A gummy residue remained and consisted principally of the free base dibromophenyl-phenylmethylhydroxylamine. This base was extracted with ether and hydrogen chloride introduced into the solu- tion. A small quantity of white precipitate separated out at first but it redissolved as the solution became more strongly acid. The acid and ether were now evaporated and a mobile liquid was left. .Low boiling ligroin (b. p. 40-60') was added to it and the mixture stirred. The liquid, which was but little soluble in the ligroin, began to solidify im- mediately. The di-p-bromophenyl-phenylmethylhydroxylamine hydro- chloride thus formed is as soluble in methyl and ethyl alcohol and ether as is the dibromophenyl-phenylmethylchloride itself, but it is not soluble in ligroin, while the latter is. The salt was washed well with ligroin (b. p. 70-80') and once with cold dilute hydrochloric acid. It reduces boiling Fehling's solution and softens at about 75 '. Di - p - bromophenyl - phenylmethylhydroxylamine Subst. 0.2850, 0.3113; cc. 0.1 NAgN03, 5.73, 6.3 Subst. 0.2850, 0.3113; cc. 0.1 NAgN03, 5.73, 6.31. Calc. for (BTC~H~)~(C~H~)CNH~OHCI: HCI, 7.73. Found: 7,34, 7.40. Rearrangement of Di-p-bromophenyl-phenylmethylhydroxy1amine.- The free base, (BrCsH~)z(CsHs)CNHOH, was prepared from the hydro- chloride in the usual way. It was obtained as a yellow viscous liquid. Some of the base (I .6 g.) was dissolved in 30 cc. of ether and 3 g. of phos- phorus pentachloride added to the solution. The mixture was warmed at the boiling point of the ether for two hours. The imido-benzophenones appeared in that time as indicated by the yellow color of the mixture. The mixture was then subjected to hydrolysis and to the isolation of the ketones and the aniline hydrochlorides. The yield of the mixed aniline and bromoaniline hydrochlorides from the base (I .6 g.) was 0.4785 g. A second rearrangement of 1.11 g. base gave 0.3104 g. Experimental Part. The subsequent treatment was similar to that of the bromophenyl-diphenylmethylhydroxylamine as given above A yield of 0.4229 g. of mixed aniline hydrochlorides was obtained from 1-47 g. of the base. A second rearrangement of 1.21 g. of the base gave 0.354 g. of the anililie salts. A complete rearrangement would yield at most 0.37 g. of aniline hydro- chloride, or 0.47 g. of chloroaniline hydrochloride per gram of substance. A quantitative analysis of the aniline salts obtained Erom each of the rearrange- ments was made by the bromination method for their molar ratios. Subst. 0.0794, 0.0767; cc. 0.1 NKBrOa, 25.13, 23.93. Found: aniline hydrochloride, 38.41, 36.30; chloroaniline hydrochloride, 61.5 63.70. Identification of the Anilines.-A qualitative separation of the two anilines was made by the method of Stieglitz and Leech.' When separated, the aniline gave the violet color test with bleaching powder and the chloro- aniline gave crystals which melted at 68-69', and when they were mixed with pure chloroaniline they melted at 68-69' (pure chloroaniline melts at 69 '). Identification of Chlorobenzophenone and 3Dichlorobenzophenone.- The ether was evaporated from the p-chlorobenzophenone and di-p- chlorobenzophenone solution, leaving a large bulk of flaky crystals. Pre- liminary tests with a mixture of known mono- and di-chlorobenzophenones showed that the latter is the less soluble in ligroin and that the two could be separated by virtue of this difference. The ketones obtained from the rearrangement were dissolved in a small quantity of warm ligroin. When a few crystals had separated from the solution, as it cooled, they were collected in a filter. They melted at 139' and when they were mixed with pure p-dichlorobenzophenone they melted at 141-142 ' (pure p - dichlorobenzophenone melts at 143 '). The filtrate still containing the greater portion of the ketones, was evaporated to dryness and three- quarters of the residue again dissolved in ligroin. This process was re- peated until the last extraction contained about one-eighth of the original ketone residue. About one-half of this was crystallized from the solution and the melting point of these crystals determined. They melted at 79- LOC. cit., p. 296. REARRANGEMENT OF TRLARYI,METHYI,HYDROXnAMINES. 2077 REARRANGEMENT OF TRLARYI,METHYI,HYDROXnAMINES. 2077 80°, and when they were mixed with pure p-chlorobenzophenone' t melted at 78-80'. 80°, and when they were mixed with pure p-chlorobenzophenone' they melted at 78-80'. Hydrochloride, ( ( C6H5) C .NH20H. C1. * Kahlbaum's p-chlorobenzophenone melted at 77-78 '. Bey., 39, 3280 (1906). Experimental Part. of mixed aniline salts. Theoretically one gram of base could give 0.30 g. aniline hydro- * ' 2078 BERT ALLEN STAGNRR. chloride or 0.48 g. of bromoaniline hydrochloride. Each quantity of the mixed aniline hydrochloride was analyzed for the molar ratios of the anilines by the bromination method. chloride or 0.48 g. of bromoaniline hydrochloride. Each quantity of the mixed aniline hydrochloride was analyzed for the molar ratios of the anilines by the bromination method. chloride or 0.48 g. of bromoaniline hydrochloride. Each quantity of the mixed aniline hydrochloride was analyzed for the molar ratios of the anilines by the bromination method. Subst. 0.0513, 0.0439; cc. 0.1 N KBr03, 12.80, 11.04. Subst. 0.0513, 0.0439; cc. 0.1 N KBr03, 12.80, 11.04. Found: aniline hydrochloride, 30.25, 3 I .os ; bromoaniline hydrochloide, 69.76, 68. , ; , , ound: aniline hydrochloride, 30.25, 3 I .os ; bromoaniline hydrochloide, 69.76, 68.95. Identification of Aniline and Bromoani1ine.-The aniline and bromo - aniline were separated exactly as were the bases from the rearranged p-bromophenyl-diphenylmethylhydroxylamine. The aniline oxalate, after separation from the bromoaniline salt, was made alkaline and tested with bleaching powder solution. The violet color confirmed the identity of the aniline. The less soluble bromoaniline oxalate was freed from the oxalic acid and crystallized from ether. The crystals melted at 62-63', and, when they were mixed with pure bromoaniline, they melted at 63-64' (pure bromoaniline melts at 64'). Identification of Aniline and Bromoani1ine.-The aniline and bromo - aniline were separated exactly as were the bases from the rearranged p-bromophenyl-diphenylmethylhydroxylamine. The aniline oxalate, after separation from the bromoaniline salt, was made alkaline and tested with bleaching powder solution. The violet color confirmed the identity of the aniline. The less soluble bromoaniline oxalate was freed from the oxalic acid and crystallized from ether. The crystals melted at 62-63', and, when they were mixed with pure bromoaniline, they melted at 63-64' (pure bromoaniline melts at 64'). Identification of Bromobenzophenone and Dibromobenzophenone,- The ether solution of the ketones was evaporated to dryness in a current of air. Of the p-bromobenzophenone and p-dibromobenzophenone, the latter is the less soluble in ligroin (b. p. 70-80') and by fractional crystallization from that solvent some of it was isolated. It melted a t 167' and when it was mixed with synthetic dibromobenzophenone it melted at 167-168 ' (pure dibromobenzophenone melts at 172-173 '). Mackenzie, J. Ckem. Soc., 69, 987 (1896) 1 Gomberg, Bey., jg, 3278 (1906). 3 Overton, Bey., 26, 28 (1893); Peterson, Am. Chem. J., 46, 33a (1911). 1 Gomberg, Bey., jg, 3278 (1906). 3 Overton, Bey., 26, 28 (1893); Peterson, Am. Chem. J., 46, 33a (1911). Mackenzie, J. Ckem. Soc., 69, 987 (1896). 3 Overton, Bey., 26, 28 (1893); Peterson, Am. Chem. J., 46, 33 Experimental Part. The ligroin solution containing the more soluble portion of the ketones was evaporated to dryness and the process for separating monochloro- benzophenone from dichlorobenzophenone was followed. The crystals thus isolated melted at 78-80' (pure p-bromobenzophenone melts at 82.5'). Identification of Bromobenzophenone and Dibromobenzophenone,- The ether solution of the ketones was evaporated to dryness in a current of air. Of the p-bromobenzophenone and p-dibromobenzophenone, the latter is the less soluble in ligroin (b. p. 70-80') and by fractional crystallization from that solvent some of it was isolated. It melted a t 167' and when it was mixed with synthetic dibromobenzophenone it melted at 167-168 ' (pure dibromobenzophenone melts at 172-173 '). The ligroin solution containing the more soluble portion of the ketones was evaporated to dryness and the process for separating monochloro- benzophenone from dichlorobenzophenone was followed. The crystals thus isolated melted at 78-80' (pure p-bromobenzophenone melts at 82.5'). p-Chlorophenyl-p-bromophenyl-phenylcarbinol, (CICaHa) (BrCsHa) - (C6Hs) C0H.-This carbinol and the corresponding chloride required for the next experiment are not described in the literature and prob- ably have not been made. For the preparation of the carbinol, p- chlorobenzophenone was first obtained from benzoylchloride and chloro- benzene with the aid of the Friedel and Crafts reaction.' The chloro- benzophenone was converted into the dichloride by treatment with phosphorus pentachloride by the method used by Kekule and Franche- mont in converting benzophenone into benzophenone dichloride.2 The dichloride was fractionally di~tilled.~ Then, one molar proportion of the p-chlorobenzophenonedichloride and two to three of bromobenzene were placed on a steam bath in a round bottom flask with a reflux condenser attached, and to this solution 1.4 molar proportions of aluminum chloride were gradually added in portions of 5 to IO g. After REARRANGGMENT OF TRIARYLME?"YLHYDROXYL4MINES. 2079 all of the aluminum chloride had been added the mixture was heated two hours longer. It was then cooled and poured on ice and water to de- compose the aluminum compound. The excess of bromobenzene was removed by steam distillation and the residue, a dark gum insoluble in water, was well washed with dilute hydrochloric acid to remove the aluminum hydroxide. The gummy residue was extracted with carbon disulfide, the carbon disulfide solution filtered, the filtrate dried over fused calcium chloride, and the solvent evaporated. The dark colored gum remaining was dissolved in warm ligroin (b. p. 7-80') and the solution left in a cold place overnight. Experimental Part. It is soluble in alcohol, ether, and chloroform. It reduces boiling Fehling's solution and softens at 4j0, but has no true melting point. hydrochloric acid as it forms a soft, sticky mass as soon as brought into contact with the water. It is soluble in alcohol, ether, and chloroform. It reduces boiling Fehling's solution and softens at 4j0, but has no true melting point. Calc. for (Cl~HIINOCIBr)HCl: C1, 8.58%. Pound: 8.27. Subst. 0.4155; cc. 0.1 N AgNOa, 9.41. A yield of j g. of this salt was obtained from the chloride (7 g.) used. Rearrangement of p-Chlorophenyl-p-bromophenyl-phenylmethylhy- droxy1amine.-The free base of the salt just described was obtained as a yellow syrupy substance, which under low pressure began to expand as a kind of foam and to spread throughout the beaker as a viscous gum. This behavior is characteristic of all these bases. Some of the base (2.37 g.) was dissolved in 3-40 cc. of ether and phos- phorus pentachloride (4 g.) added to the solution. In twelve hours a slight change in color to yellow indicated a slow formation of the imido ketones at room temperature. The mixture was then heated for two hours at the temperature of boiling ether to complete the rearrangement. The mixture was next subjected to hydrolysis of the imido ketones,' and the isolation of the benzophenones and the anilines carried out in the manner described for the previous rearrangements. The yield of the mixed aniline, chloroaniline and bromoaniline hydro- chlorides from the 2.37 g. of base was 0.626 g. In a second rearrange- ment of I g. of base 0.1 j 9 j g. of the aniline salts was obtained. A com- plete rearrangement would give 0.33 g. of aniline hydrochloride, or 0.42 g. of chloroaniline hydrochloride or 0.54 g. of bromoaniline hydrochloride per gram of substance. A trace of the aniline mixture was made alkaline and treated with bleach- ing powder solution. -4 violet color was produced, proving the presence of the unsubstituted aniline. Another part of the aniline mixture was freed from hydrogen chloride and precipitated from ether solution with oxalic acid. The precipitate showed the presence of halogen when heated in a bunsen flame with copper oxide and also when fused with sodium and tested with silver nitrate solution. Three imido ketones were present, (C1C6H4) (BrC&)C = N(CeHd, (ClCaHd- (C6Hs)C = NCeHIBr, and (BrC6H4)(CeHs)C = NCsH421. 2 The reliability of this method of analysis was determined with a known mixture of the three hydrochlorides. For the Volhard method a larger quantity of the mixed salts was used than for the bromination method, as the data show. The method is reliable only to within 5%. Experimental Part. The carbinol separated out in light colored crystals. Recrystallization was twice repeated and the carbinol then melted at 91-93 '. 0.2161 g. of the carbinol gave 0..+850 g. COZ and 0.0760 g. HzO. Calc. for ClgH140ClBr: C, 61.03%; H, 3.77%. Found: C, 61.22%; H, 3.93'%, 0.2161 g. of the carbinol gave 0..+850 g. COZ and 0.0760 g. HzO. Calc. for ClgH140ClBr: C, 61.03%; H, 3.77%. Found: C, 61.22%; H, 3.93'%, p - Chlorophenyl - p - bromophenyl - phenylmethylchloride, (C1C6H4)- (BrC&) (C6H5)CCI.---'l'he carbinol was converted into the chloride, (C1C6H4) (BrC6H4) (C&)CC1, by a stream of hydrogen chloride passed into a carbon disulfide solution of the carbinol. After the acid was added, the disulfide solution mas dried over fused calcium chloride and filtered and the excess of acid and the carbon disulfide evaporated in vacuo. The chloride was left as a very viscous liquid. When it was hydrolyzed with excess of 0.1 N KOH and the excess of alkali was titrated with 0. I N HC1, it showed 93% of the calculated amount ol' chlorine. Attempts to crystallize the chloride were, at first, unsuccessful and for this reason the liquid chloride was used in making the hydroxylamine preparation. Later, however, the chloride crystallized from low boiling ligroin. The solid was twice recrystallized from higher boiling ligroin by inoculation of the solution with the crystals first obtained. It now melted at 69-72 O . Calc. for (CloH18ClBr)Cl: 9.0570 C1 (hydro- lyzable). Found: 8.67y0. Subst. 0.2229; cc. 0.1 N KOH, 5.44. pChlorophenyl-~-bromophenyl-phenylmethylhydroxylamine Hydro- chloride, (C1CsH4) (BrC&) (CGH~)CNH~.~HC~.--T~~ chlorophenyl-bromo- phenyl-phenylmethylchloride (7 g.) was dissolved in benzene (zoo g.) and condensed with a saturated alcoholic solution of hydroxylamine. The solvents were removed by evaporation. The gummy residue re- maining was extracted with ether and the ether solution made strongly acid with hydrogen chloride. The hydrogen chloride salt of the hydroxyl- amine derivation does not precipitate out from the ether solution. After the ether and exces acid were evaporated the triarylmethylhydroxyl- amine hydrochloride remained as a syrupy liquid; it was treated with low boiling ligroin and the mixture stirred with a glass rod. The salt began to solidify immediately. It is insoluble in ligroin and was purified by being washed well with that solvent. It cannot be washed with dilute, 2080 BERT ALLEN STAGNER. hydrochloric acid as it forms a soft, sticky mass as soon as brought into contact with the water. * The work reported on in this paper formed the basis of a dissertation submitted to the University of Chicago in part fulfillment of the requirements for the Ph.D. degree. Before bliss Vosburgh could come up for her final examinations, she was the victim of a fatal automobile accident, Dec. 4, 1914, near Mt. Holyoke, Mass. The present posthumous report, except for editing, is in the form in which it was written by Miss Vosburgh.-J. STIEGLITZ. * Hofmann, loc. cit.; Hoogewefl and van Dorp, Rec. trau. chim., 6, 373 (1887); 8, 173 (1889), etc.; Lengfeld and Stieglitz, Am. Chem. J., 15, 215, 504 (1893); Elizabeth Jeffreys, Ber., 30, 898 (1897); Am. Chem. J., 22, 14 (1899). Experimental Part. The mixed aniline hydrochlorides obtained from each of the rearrange- ments were analyzed volumetrically for the determination of the molar ratios of the three salts: the hydrochlorides of aniline, chloroaniline and bromoaniline. The bromination and the Volhard methods were used jointly to give sufficient data for the calculations.2 2 The reliability of this method of analysis was determined with a known mixture of the three hydrochlorides. For the Volhard method a larger quantity of the mixed salts was used than for the bromination method, as the data show. The method is reliable only to within 5%. 2 The reliability of this method of analysis was determined with a known mixture of the three hydrochlorides. For the Volhard method a larger quantity of the mixed salts was used than for the bromination method, as the data show. The method is reliable only to within 5%. IiBARRANGEMENT OF TRIPHENYLMETHYLHALOGENAMINES. 208 I 0.0754 g. of the mixed hydrochlorides required 2 1.68 cc. of 0.1 N KBr03; and 0.2 138 g. required 12.85 cc. 0.1 N AgNOa. Found: aniline hydrochloride, 38.0; chloroaniline hydrochloride, 28.8; and bromoaniline hydrochloride, 33.2. 0.0318 g. of the mixed hydrochlorides required 9.24 cc. of 0.1 N KBrOs, and 0.1276 g. of the same mixture required 7.85 cc. of 0.1 N AgN03. Found: aniline hydrochloride, 36.2 ; chloroaniline hydrochloride, 38.9; and bromoaniline hydrochloride, 24.9. I wish to express my indebtedness to Professor Stieglitz, not only for the constant interest that he has shown in this investigation and the many helpful suggestions, but also for the encouragement and his kindness toward me personally. CHICAGO, ILL. ’See Stieglitz, Reddick and Leech, 8th Intern. Congr. ApP1. Chem., a5, 444 (1912). See a preliminary report (abstract) by Stieglitz and Vosburgh, 8th Intern. Congr. AppZ. Chem., 25, 445 (1912); Ber., 46, 2151 (1913). * Beckmann, Ber., 19, 988 (1886), etc. g A. W. Hofmann, Ber., 14, 2 7 2 5 (1882); 15, 408 (1883), etc. * The analogy was pointed out by Hoogewerff and van Dorp, loc. cit.; and by tieglitz, Am. Chem. J., 18, 751 (1896). [CONTRIBUTION FROM THE KENT CHEMICAL LABORATORY OF THE UNIVERSITY OF CHICAGO. ] CONTRIBUTION FROM THE KENT CHEMICAL LABORATORY OF THE UNIVERSITY OF CHICAGO. ] METHYLHALOGENAMINES.‘ THE MOLECULAR REARRANGEMENT OF TRIPHENYL- BY ISABELLA VOSBURGH.* Received August 1. 1916. BY ISABELLA VOSBURGH.* Received August 1. 1916. The characteristic Hofmann rearrangement3 of acid halogen amides, RCONH(Hal), to derivatives of amines RNHz forms the basis of the well-known general method of preparation of amines.* The rearrange- ments is analogous in many important respects to the “Beckmann” re- arrangement of oximes.6 When it was found in this laboratory by Stieg- litz and Reddick’ that triphenylmethylhydroxylamine undergoes a re- arrangement quite as smoothly as any oxime, Professor Stieglitz proposed to me that I undertake under his direction a parallel investigation on the possibility of effecting rearrangements of the triphenylmethylhalogen- amines, Ar3CNH(Hal). The investigation of these particularly simple halogen amine derivatives promised to shed light on a number of ques- tions connected with the rearrangements: of those taken up in this in- See a preliminary report (abstract) by Stieglitz and Vosburgh, 8th Intern. Congr. AppZ. Chem., 25, 445 (1912); Ber., 46, 2151 (1913). *
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Impacto da pandemia de COVID-19 nos procedimentos dermatológicos no Brasil
Brazilian Medical Students
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3,873
RESUMO DISCUSSÃO: A pandemia da COVID- 19 reduziu o número de profissionais especializados atuantes nos ambulatórios, com o remanejamento da mão de obra e reestruturação do serviço para o atendimento aos infectados pela COVID-19, refletindo na redução de consultas e procedi- mentos, resultando no atraso do acompanhamento e resolução de doenças dermatológicas. CONCLUSÃO: Conclui-se, portanto, que o a realização de procedimentos dermatológicos foi fortemente influenciada pela pandemia da COVID-19, com efeito direto no funcionamento de serviços da especialidade. PALAVRAS-CHAVE: Dermatologia; SARS-CoV-2; Pandemias. ABSTRACT INTRODUCTION: Appointments in dermatology were hindered by the COVID-19 pandemic, resulting in delayed diagnoses and interrupted treatments. Patients at risk of skin cancer, for example, have been affected and metastasis risks increased. In this context, this study aimed to analyze the impact of the COVID-19 pandemic dermatological surgical procedures in Brazil. METHODOLOGY: The study is retroactive, descriptive, and revised. Data were extracted from DATASUS Health Information System (TABLET) records. “Small surgeries and open skin, subcutaneous tissue, and hospital mucosal surgeries of the SUS  RESUMO No período de 2016 a 2019 houve um aumento de 16,44% nos procedimentos realizados, totalizando 130.099 em 2019. Entretanto, no ano de 2020 houve queda de 38,59% em relação a 2019, com 79.887 procedimentos. Ademais, em todos os anos predomi- nou o caráter eletivo, totalizando 402.344 (72,5%). O aumento de procedimentos desse caráter foi de 25,2% no período de 2016 a 2019, mas em comparação a 2019, houve uma redução de 44,9% em 2020. DISCUSSÃO: A pandemia da COVID- 19 reduziu o número de profissionais especializados atuantes nos ambulatórios, com o remanejamento da mão de obra e reestruturação do serviço para o atendimento aos infectados pela COVID-19, refletindo na redução de consultas e procedi- mentos, resultando no atraso do acompanhamento e resolução de doenças dermatológicas. CONCLUSÃO: Conclui-se, portanto, que o a realização de procedimentos dermatológicos foi fortemente influenciada pela pandemia da COVID-19, com efeito direto no funcionamento de serviços da especialidade. INTRODUÇÃO: Atendimentos dermatológicos foram prejudicados pela pandemia da COVID-19, com diagnósticos atrasados e tratamentos interrompidos. Pacientes com câncer de pele, por exemplo, foram especialmente afetados, já que a progressão de lesões e o risco de metástase aumentam. Nesse contexto, o objetivo do estudo foi analisar o impacto da pandemia da COVID-19 na realização de procedimentos cirúrgicos em dermatologia no Brasil. METODOLOGIA: O estudo é retrospectivo, descritivo e quantitativo. Os dados foram obtidos a partir de registros do Sistema de Informações de Saúde (TABNET) do DATASUS. Foram analisadas “Pequenas cirurgias e cirurgias de pele, tecido subcutâneo e mucosa” entre os Procedimentos Hospitalares do SUS do período de janeiro de 2016 a dezembro de 2020, sendo consideradas as variáveis Região e Caráter de Atendimento. RESULTADOS: Foram realizadas 554.591 pequenas cirurgias e cirurgias de pele, tecido subcutâneo e mucosa entre 2016 e 2020 no Brasil. O Sudeste apresentou o maior número de registros (47,15%; n=261.477). No período de 2016 a 2019 houve um aumento de 16,44% nos procedimentos realizados, totalizando 130.099 em 2019. Entretanto, no ano de 2020 houve queda de 38,59% em relação a 2019, com 79.887 procedimentos. Ademais, em todos os anos predomi- nou o caráter eletivo, totalizando 402.344 (72,5%). O aumento de procedimentos desse caráter foi de 25,2% no período de 2016 a 2019, mas em comparação a 2019, houve uma redução de 44,9% em 2020. DOI: 10.53843/bms.v8i11.335 ESTUDO TRANSVERSAL RESUMO INTRODUÇÃO: Atendimentos dermatológicos foram prejudicados pela e tratamentos interrompidos. Pacientes com câncer de pele, por exemp de lesões e o risco de metástase aumentam. Nesse contexto, o objet COVID-19 na realização de procedimentos cirúrgicos em dermatologia descritivo e quantitativo. Os dados foram obtidos a partir de registros DATASUS. Foram analisadas “Pequenas cirurgias e cirurgias de pele, Hospitalares do SUS do período de janeiro de 2016 a dezembro de 20 de Atendimento. RESULTADOS: Foram realizadas 554.591 pequen mucosa entre 2016 e 2020 no Brasil. O Sudeste apresentou o maior n de 2016 a 2019 houve um aumento de 16,44% nos procedimentos rea ano de 2020 houve queda de 38,59% em relação a 2019, com 79.887 nou o caráter eletivo, totalizando 402.344 (72,5%). O aumento de pro 2016 a 2019, mas em comparação a 2019, houve uma redução de 44 19 reduziu o número de profissionais especializados atuantes nos am reestruturação do serviço para o atendimento aos infectados pela CO mentos, resultando no atraso do acompanhamento e resolução de portanto, que o a realização de procedimentos dermatológicos foi forte efeito direto no funcionamento de serviços da especialidade. PALAVRAS-CHAVE: Dermatologia; SARS-CoV-2; Pandemias. ABSTRACT INTRODUCTION: Appointments in dermatology were hindered by the and interrupted treatments. Patients at risk of skin cancer, for example In this context, this study aimed to analyze the impact of the COVID-19 METHODOLOGY: The study is retroactive, descriptive, and revised. D System (TABLET) records. “Small surgeries and open skin, subcutane INTRODUÇÃO: Atendimentos dermatológicos foram prejudicados pela pandemia da COVID-19, com diagnósticos atrasados e tratamentos interrompidos. Pacientes com câncer de pele, por exemplo, foram especialmente afetados, já que a progressão de lesões e o risco de metástase aumentam. Nesse contexto, o objetivo do estudo foi analisar o impacto da pandemia da COVID-19 na realização de procedimentos cirúrgicos em dermatologia no Brasil. METODOLOGIA: O estudo é retrospectivo, descritivo e quantitativo. Os dados foram obtidos a partir de registros do Sistema de Informações de Saúde (TABNET) do DATASUS. Foram analisadas “Pequenas cirurgias e cirurgias de pele, tecido subcutâneo e mucosa” entre os Procedimentos Hospitalares do SUS do período de janeiro de 2016 a dezembro de 2020, sendo consideradas as variáveis Região e Caráter de Atendimento. RESULTADOS: Foram realizadas 554.591 pequenas cirurgias e cirurgias de pele, tecido subcutâneo e mucosa entre 2016 e 2020 no Brasil. O Sudeste apresentou o maior número de registros (47,15%; n=261.477). IMPACTO DA PANDEMIA DA COVID-19 NOS PROCEDIMENTOS DERMATOLÓGICOS NO BRASIL Maria Eduarda Silveira Bührnheim 1*; Eric Pasqualotto 2; Amanda Carolina Fonseca da Silva 2; Beatriz Car- valho de Oliveira 2; Vítor Maurício Merlin Maschietto 2; Luiz Fernando Leite da Silva Neto 2; Geraldo Mendes de Araújo Junior 2; Carla Andrea Avelar pires 3 1. Universidade do Estado do Pará (UEPA), Acadêmica de Medicina. 2. Universidade Federal de Santa Catarina (UFSC), Acadêmico(a) de Medicina. 3. Universidade Federal do Pará (UFPA), Doutora em medicina pelo Núcleo de Medicina Tropical.  The Southeast had the highest regional records (47.15%; n=261,477). From 2016 to 2019, there was a 16.44% in- crease in the procedures performed, totaling 130,099 in 2019. However, in 2020 there was a decrease of 38.59% compared to 2019, with 79,887 procedures. Further, elective procedures were prevalent in all years, 402,344 (72.5%) in total. The numbers increased 25.2% from 2016 to 2019, but in 2020, compared to 2019, there was a 44.9% reduction. DISCUSSION: During the COVID-19 pandemic, the number of active healthcare professionals in clinics decreased due to workforce reloca- tion and services' restructuring, infected in the reduction of consultations and procedures, resulting in the delay of the follow- up and resolution of dermatological diseases. CONCLUSION: It was found that dermatological procedures were deeply in- fluenced by the COVID-19 pandemic, with a direct affection of dermatology services in Brazil. KEYWORDS: Dermatology; SARS-CoV-2; Pandemics. INTRODUÇÃO procura durante a pandemia e por novos pacientes que neces- sitarão iniciar tratamentos precoces6. Assim, espera-se encon- trar redução no número de procedimentos cirúrgicos dermato- lógicos no período da pandemia da COVID-19. Nesse con- texto, o presente estudo tem como objetivo geral analisar o im- pacto da pandemia da COVID-19 na realização de procedi- mentos cirúrgicos em dermatologia no Brasil, ademais, os ob- jetivos específicos são identificar o caráter de atendimento e distribuição regional dos procedimentos cirúrgicos em derma- tologia no período analisado. A pandemia de COVID-19, declarada em março de 2020 pela Organização Mundial da Saúde (OMS), impactou de maneira direta a saúde mundial, apresentando milhões de infectados e óbitos. Inúmeras medidas de restrição e isolamento, incluindo o distanciamento social foram implantadas, a fim de que a do- ença fosse controlada1,2.Nesse contexto, foram necessárias adaptações dos sistemas de saúde, incluindo a reorganização dos cuidados médicos, adiamento e cancelamento de consul- tas e procedimentos cirúrgicos eletivos1. Além da redução sig- nificativa do volume de consultas, também houve dificuldade na realização de programas de triagem em massa, procedi- mentos diagnósticos e tratamentos2. METODOLOGIA Desenho de estudo e coleta de dados O presente trabalho é retrospectivo, descritivo e com aborda- gem quantitativa. Os dados utilizados nesta pesquisa foram obtidos a partir de registros disponíveis no Sistema de Infor- mações de Saúde, mais especificamente na seção de Proce- dimentos Hospitalares do SUS e obtidos por meio eletrônico, através do site do Departamento de Informática do Sistema Único de Saúde do Brasil (DATASUS)7. Foram utilizados da- dos referentes aos Procedimentos Hospitalares do SUS, sub- grupo de procedimento 0401 (Pequenas cirurgias e cirurgias de pele, tecido subcutâneo e mucosa), do período de janeiro de 2016 a dezembro de 2020. A prática dermatológica foi significativamente afetada por essa situação, principalmente pela redução de suas consultas am- bulatoriais, o que gerou impactos negativos aos pacientes que não puderam dar prosseguimentos aos seus tratamentos ou que tiveram diagnósticos tardios de suas condições1. O retardo no diagnóstico dermatológico, sobretudo do câncer de pele, é preocupante, já que resulta na progressão de lesões, identifi- cação em estágios avançados e potencialização do risco de metástase, consequentemente, aumentando a morbidade e re- duzindo a sobrevida2,3,4,5. De acordo com a Sociedade Brasi- leira de Dermatologia, a pandemia afastou pacientes com cân- cer de pele dos ambulatórios, dificultando o diagnóstico e tra- tamentos precoces. Houve queda de 50% a 60% de procura por atendimento dermatológico, em quase todas as faixas etá- rias6. A aquisição de bibliografia, por sua vez, foi feita com buscas em diversas bases, como Biblioteca Virtual de Saúde, PubMed e Google Scholar.   DOI: 10.53843/bms.v8i11.335 from January 2016 to December 2020 were analyzed, focusing on the variables Region and Character of Care. RESULTS: 554,591 small surgeries and skin, subcutaneous tissue, and mucosal surgeries were performed between 2016 and 2020 in Brazil. The Southeast had the highest regional records (47.15%; n=261,477). From 2016 to 2019, there was a 16.44% in- crease in the procedures performed, totaling 130,099 in 2019. However, in 2020 there was a decrease of 38.59% compared to 2019, with 79,887 procedures. Further, elective procedures were prevalent in all years, 402,344 (72.5%) in total. The numbers increased 25.2% from 2016 to 2019, but in 2020, compared to 2019, there was a 44.9% reduction. DISCUSSION: During the COVID-19 pandemic, the number of active healthcare professionals in clinics decreased due to workforce reloca- tion and services' restructuring, infected in the reduction of consultations and procedures, resulting in the delay of the follow- up and resolution of dermatological diseases. CONCLUSION: It was found that dermatological procedures were deeply in- fluenced by the COVID-19 pandemic, with a direct affection of dermatology services in Brazil. DOI: 10.53843/bms.v8i11.335 from January 2016 to December 2020 were analyzed, focusing on the variables Region and Character of Care. RESULTS: 554,591 small surgeries and skin, subcutaneous tissue, and mucosal surgeries were performed between 2016 and 2020 in Brazil. The Southeast had the highest regional records (47.15%; n=261,477). From 2016 to 2019, there was a 16.44% in- crease in the procedures performed, totaling 130,099 in 2019. However, in 2020 there was a decrease of 38.59% compared to 2019, with 79,887 procedures. Further, elective procedures were prevalent in all years, 402,344 (72.5%) in total. The numbers increased 25.2% from 2016 to 2019, but in 2020, compared to 2019, there was a 44.9% reduction. DISCUSSION: During the COVID-19 pandemic, the number of active healthcare professionals in clinics decreased due to workforce reloca- tion and services' restructuring, infected in the reduction of consultations and procedures, resulting in the delay of the follow- up and resolution of dermatological diseases. CONCLUSION: It was found that dermatological procedures were deeply in- fluenced by the COVID-19 pandemic, with a direct affection of dermatology services in Brazil. from January 2016 to December 2020 were analyzed, focusing on the variables Region and Character of Care. RESULTS: 554,591 small surgeries and skin, subcutaneous tissue, and mucosal surgeries were performed between 2016 and 2020 in Brazil. Análise de dados Diante da duração dessas medidas no cenário nacional de sa- úde pública, é fundamental considerar o impacto que essa pa- ralisação causou no diagnóstico e no tratamento de pacientes com doenças, como o câncer de pele e melanomas, a fim de se traçar estratégias emergenciais de cuidado e assistência a esta demanda reprimida4. Ainda, é preciso ressaltar que há forte tendência de sobrecarga nos serviços dermatológicos de atendimento da rede pública, pelos pacientes que retardaram Nesse sentido, foram consideradas as variáveis Região e Ca- ráter de Atendimento para análise. Para realização da análise de dados, foram utilizadas as ferramentas de tabulação do TABNET e TABWIN, ferramentas de tabulação disponíveis no próprio site do DATASUS, além do Software SPSS 25 (Statis- tical Package for the Social Sciences 25) para análise estatís- tica, no qual foram feitas tabelas de frequências. Para a  DOI: 10.53843/bms.v8i11.335 elaboração dos gráficos, tabelas e textos pertinentes foram uti- lizados os softwares Microsoft Office Excel 2016 e Word 2016. RESULTADOS Foram realizados 554.591 procedimentos relacionados a pe- quenas cirurgias e cirurgias de pele, tecido subcutâneo e mu- cosa entre 2016 e 2020 no Brasil, com média anual igual a 110.918,2 e desvio padrão de 18.861,27. A região Sudeste apresentou o maior número de casos (47,15%; n=261.477), seguida da região Nordeste (28,58%; n=158.483), região Sul (15,72%; n=87.182), região Norte (4,31%; n=23.901) e região Centro-Oeste (4,25%; n=23.548), descrito no Gráfico 1. CONCLUSÃO Os procedimentos com caráter de atendimento eletivo aumen- taram 25,2% no período de 2016 a 2019. Entretanto, no ano de 2020, em comparação a 2019, houve redução de 44,9% nos procedimentos com caráter de atendimento eletivo, 19% em urgência, 9,3% em outros tipos de acidente de trânsito e 27,2% em outras lesões e envenenamento por agentes quími- cos e físicos. Tornou-se perceptível o impacto significativo da pandemia da COVID-19 em todas as regiões brasileiras na prática dermato- lógica no ano de 2020, visto que, em várias localidades geo- gráficas, houve a convocação de profissionais dessa especia- lidade para ajudar nos serviços de combate ao SARS-CoV-2. No contexto ambulatorial, é possível que tal fato tenha associ- ação com a diminuição da quantidade de dermatologistas dis- poníveis, resultando em um menor número de consultas mar- cadas, bem como na redução da realização de procedimentos que envolvem o manejo, o diagnóstico e o tratamento de do- enças. De forma consequente ao atraso do acompanhamento desses casos clínicos, há chance de piora da evolução da sin- tomatologia dos pacientes, especialmente aqueles que neces- sitam de visita contínua e periódica, bem como os indivíduos que possuem alto risco para uma determinada enfermidade. Aspectos éticos Essa pesquisa não necessitou de aprovação do Comitê de Ética em Pesquisa (CEP), estando de acordo com a Resolução nº 510 do Conselho Nacional de Saúde (CNS), de 7 de abril de 2016, artigo 1, inciso III que isenta pesquisa que utilize infor- mações de domínio público em Ciências Humanas e Sociais de registro no Comitê de Ética em Pesquisa da Comissão Na- cional de Ética em Pesquisa – sistema CEP/CONEP. GRÁFICO 1. Procedimentos dermatológicos por região entre 2016 e 2020. Fonte: Autoria dos pesquisadores. GRÁFICO 1. Procedimentos dermatológicos por região entre 2016 e 2020. Fonte: Autoria dos pesquisadores. No período de 2016 a 2019 houve um aumento de 16,44% no número de procedimentos dermatológicos realizados no Brasil, de modo que, enquanto em 2016 foram realizados 111.731 procedimentos, em 2019 foram 130.099. Os anos consecuti- vos apresentaram aumento de 0,71% entre 2016 e 2017, 6,95% entre 2017 e 2018 e 8,1% entre 2018 e 2019, como abordado no Gráfico 1. No período de 2016 a 2019 houve um aumento de 16,44% no número de procedimentos dermatológicos realizados no Brasil, de modo que, enquanto em 2016 foram realizados 111.731 procedimentos, em 2019 foram 130.099. Os anos consecuti- vos apresentaram aumento de 0,71% entre 2016 e 2017, 6,95% entre 2017 e 2018 e 8,1% entre 2018 e 2019, como abordado no Gráfico 1. janeiro e maio, o número de procedimentos aumentou 127,8% entre maio e novembro de 2020, como demonstrado no gráfico 2. 2. GRÁFICO 2. Procedimentos dermatológicos realizados no ano de 2020 no Brasil Fonte: Autoria dos pesquisadores. GRÁFICO 2. Procedimentos dermatológicos realizados no ano de 2020 no Brasil Entretanto, no ano de 2020 houve queda de 38,59% nos pro- cedimentos realizados em relação a 2019 e 27,28% em rela- ção à média do período de 2016 a 2019. Foram realizados 79.887 procedimentos dermatológicos em 2020. No período analisado, o desvio padrão mensal de procedimentos em 2020 foi maior (2403,7), comparado a 2016 (856,8), 2017 (894,3), 2018 (722,2) e 2019 (906,5). Os meses com mais procedi- mento realizados em 2020 foram janeiro (n=10.114), fevereiro (n=10.071) e março (n=8.787), enquanto, abril, maio e junho tiveram os menores registros, com 3.717, 3.579 e 3.888, res- pectivamente. Após a queda de 64,61% entre os meses de Fonte: Autoria dos pesquisadores. Fonte: Autoria dos pesquisadores. Aspectos éticos  DOI: 10.53843/bms.v8i11.335 pele, sendo necessário adiar procedimentos em lesões malig- nas de menor risco para priorizar quadros de alto risco, en- quanto casos de risco intermediário foram avaliados individu- almente - mas em sua maioria também adiados8,9. É válido res- saltar também, que, em diversas localidades, profissionais da dermatologia foram recrutados para atendimento de casos de COVID-19. Com isso, menor tempo para atendimento cirúrgico e ambulatorial puderam ser dedicados para a especialidade9. Em relação ao caráter de atendimento, em todos os anos pre- dominou o caráter eletivo, representando 70,4% (n=78.644) dos procedimentos em 2016, 72,4% (n=81.490) em 2017, 74,4% (n=89.497) em 2018, 75,7% (n=98.446) em 2019 e 67,9% (n=54.267) em 2020, totalizando 402.344 procedimen- tos (72,5%). Já o caráter de urgência foi o segundo maior (26,8%; n=148.876). Ele foi seguido de outras lesões e enve- nenamento por agentes químicos e físicos (0,4%; n=2.011), outros tipos de acidente de trânsito (0,2%; n=1.359) e 1 proce- dimento com caráter de atendimento de acidentes no local de trabalho ou a serviço da empresa, que juntos totalizaram 0,7% de todos os procedimentos, como descrito no gráfico 3. Em relação à distribuição regional de procedimentos dermato- lógicos nota-se uma redução global no ano de 2020 na procura devido à eclosão da pandemia de COVID-1910,11. No entanto, a tendência de redução acompanha a distribuição nacional existente, na qual a região sudeste concentra o maior número de profissionais da dermatologia ao mesmo tempo em que as regiões norte e nordeste apresentam as menores concentra- ções de especialistas e serviços. É importante pontuar, desta forma, que a pandemia agravou o problema de assistência mé- dica dermatológica reduzida nessas regiões, uma vez que nesse período também houve agravamento de doenças devido ao estresse e ansiedade, tendo assim um desequilíbrio entre número de profissionais especializados, demanda e casos de doença de pele12,13. Gráfico 3. Procedimentos dermatológicos por caráter de atendimento no Brasil de 2016 a 2020 Fonte: Autoria dos pesquisadores. Ademais, o presente estudo possui limitações. Os dados apre- sentados avaliam um período limitado de tempo na pandemia, considerando a indisponibilidade de dados integrais de perío- dos posteriores ao analisado. O desenho de estudo também impossibilita a geração de generalizações, sendo necessários estudos com metodologias mais rigorosas para tal. Fonte: Autoria dos pesquisadores. CONFLITOS DE INTERESSE Os pesquisadores afirmam que não há conflitos de interesse nesta pesquisa. 9. Gisondi P, Piaserico S, Conti A et al. Dermatologists and SARS‐CoV‐2: the impact of the pandemic on daily prac- tice. Journal of the European Academy of Dermatology and Venereology. 2020;34(6):1196–1201. FINANCIAMENTO 10. Conselho Federal de Medicina. Pandemia derruba quase 30 milhões de procedimentos médicos em ambulatórios do SUS [Internet]. 2021 [acesso em 2021 dez 1]. Disponí- vel em: https://portal.cfm.org.br/noticias/pandemia-der- ruba-quase-30-milhoes-de-procedimentos-medicos-em- ambulatorios-do-sus/ O financiamento deste trabalho foi realizado por meios pró- prios dos autores O financiamento deste trabalho foi realizado por meios pró- prios dos autores DISCUSSÃO No presente estudo, foi identificada notável redução no nú- mero de procedimentos dermatológicos eletivos no contexto pandêmico (Gráfico 2). Resultados similares foram encontra- dos em estudo de Felipe e colaboradores, no qual foi identifi- cada uma redução na disponibilidade de procedimentos eleti- vos e consultas em um serviço de referência em dermatologia1. Tal fato teve impacto negativo direto no seguimento do cui- dado, com possível agravamento de condições como conse- quência da ausência de acompanhamento. O presente estudo possibilita a identificação da variação nos procedimentos dermatológicos realizados, podendo servir como base para a realização de novos estudos em serviços de dermatologia brasileiros, possibilitando adaptações para me- lhor eficiência no atendimento e preparo para futuras situações de crise em serviços de saúde. Estudos internacionais apontam quadros semelhantes em ou- tros países, com redução de atendimentos dermatológicos e, em especial, pequenos procedimentos eletivos. As principais dificuldades foram encontradas no manejo de neoplasias de  DOI: 10.53843/bms.v8i11.335 [acesso em 2021 dez 1]. Disponível em: https://www.sbd.org.br/noticias/pandemia-dificulta-diag- nostico-precoce-de-cancer-de-pele-diz-sbd/ [acesso em 2021 dez 1]. Disponível em: https://www.sbd.org.br/noticias/pandemia-dificulta-diag- nostico-precoce-de-cancer-de-pele-diz-sbd/ Por fim, mais estudos são necessários para a avaliação da pro- gressão da realização de procedimentos dermatológicos em períodos posteriores aos avaliados pelo presente estudo, bem como estudos com metodologias mais robustas, para a reali- zação de correlações e generalizações sobre a temática. 7. DATASUS. tabnet.datasus.gov.br/tabnet/tabnet.htm. Dis- ponível em: <http://www.datasus.gov.br>. 8. Bhargava S, Negbenebor N, Sadoughifar R et al. Global impact on dermatology practice due to the COVID-19 pan- demic. Clinics in Dermatology. 2021;39(3):479-487. 8. Bhargava S, Negbenebor N, Sadoughifar R et al. Global impact on dermatology practice due to the COVID-19 pan- demic. Clinics in Dermatology. 2021;39(3):479-487. REFERÊNCIAS 1. Felipe CO, Medeiros ACTR, Queiroz MVR et al. Impactos do COVID-19 no ambulatório e residência médica em der- matologia. Revista Científica da Faculdade de Medicina de Campos. 2021;16(1):42–45. 11. Maia BR, Dias PC. Ansiedade, depressão e estresse em estudantes universitários: o impacto da COVID-19. Estu- dos de Psicologia (Campinas). 2020;37:e200067. 2. Sarmenghi IP, Tedesco CF, Almeida BV et al. IMPACTO DA PANDEMIA DA COVID-19 EM UM PROGRAMA DE TRIAGEM DE C NCER DE PELE NO ESPÍRITO SANTO. Brazilian Journal of Surgery and Clinical Research. 2021;36(1):12–16. 12. Melo MSB, Rocha NFL, Magalhães, SS et al. Influência de fatores emocionais nas doenças crônicas de pele: O es- tresse como gatilho para o desenvolvimento, reincidência ou agravamento da psoríase. ID on line REVISTA DE PSI- COLOGIA. 2019;13(46):584–608. 3. Canedo MIF, Martín, MT, Ruíz FR. Impact of the SARS- CoV-2 pandemic on the early diagnosis of melanoma. Med Clin (Barc). 2021;156(7):356–359. 13. Universidade de São Paulo. DEMOGRAFIA MÉDICA NO BRASIL [Internet]. 2020 [acesso em 2021 dez 1]. Disponí- vel em: https://www.fm.usp.br/fmusp/conteudo/Demogra- fiaMedica2020_9DEZ.pdf 4. Tejera-Vaquerizo A, Cañueto J, Toll A et al. Estimación del efecto en el tamaño y la supervivencia de los tumores cu- táneos debido al confinamiento por COVID-19: modelo ba- sado en un crecimiento exponencial. Actas Dermo-Sifili- ográficas. 2020;111(8):629–638. 4. Tejera-Vaquerizo A, Cañueto J, Toll A et al. Estimación del efecto en el tamaño y la supervivencia de los tumores cu- táneos debido al confinamiento por COVID-19: modelo ba- sado en un crecimiento exponencial. Actas Dermo-Sifili- ográficas. 2020;111(8):629–638. 5. Tejera-Vaquerizo A, Nagore E. Estimated effect of COVID‐ 19 lockdown on melanoma thickness and prognosis: a rate of growth model. Journal of the European Academy of Der- matology and Venereology. 2020;34(8):e351-e353. 6. Sociedade Brasileira de Dermatologia. Pandemia dificulta diagnóstico precoce de câncer de pele [Internet]. 2021 6. Sociedade Brasileira de Dermatologia. Pandemia dificulta diagnóstico precoce de câncer de pele [Internet]. 2021 
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Cytokine inhibition in chronic fatigue syndrome patients: study protocol for a randomized controlled trial
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Cytokine inhibition in chronic fatigue syndrome patients: study protocol for a randomized controlled trial Cytokine inhibition in chronic fatigue syndrome patients: study protocol for a randomized controlled trial Roerink, M.E.; Knoop, H.; Bredie, S.J.H.; Heijnen, M.; Joosten, L.A.B.; Netea, M.G.; Dinarello, C.A.; Meer, J.W.M. van der 2015, Article / Letter to editor (Trials, 16, (2015), article 439) Doi link to publisher: https://doi.org/10.1186/s13063-015-0971-z Cytokine inhibition in chronic fatigue syndrome patients: study protocol for a randomized controlled trial Roerink, M.E.; Knoop, H.; Bredie, S.J.H.; Heijnen, M.; Joosten, L.A.B.; Netea, M.G.; Dinarello, C.A.; Meer, J.W.M. van der 2015, Article / Letter to editor (Trials, 16, (2015), article 439) Doi link to publisher: https://doi.org/10.1186/s13063-015-0971-z Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/152096 Download date: 2024-10-24 * Correspondence: Megan.Roerink@radboudumc.nl 1Department of Internal Medicine, Radboud University Medical Center, Post Box 91016500 HB Nijmegen, The Netherlands Full list of author information is available at the end of the article Cytokine inhibition in chronic fatigue syndrome patients: study protocol for a randomized controlled trial Megan E. Roerink1*, Hans Knoop 2, Sebastian J. H. Bredie1, Michael Heijnen1, Leo A. B. Joosten1, Mihai G. Netea1, Charles A. Dinarello1 and Jos W. M. van der Meer1 * Correspondence: Megan.Roerink@radboudumc.nl 1Department of Internal Medicine, Radboud University Medical Center, Post Box 91016500 HB Nijmegen, The Netherlands Full list of author information is available at the end of the article © 2015 Roerink et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. © 2015 Roerink et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Chronic fatigue syndrome (CFS) is a medically unexplained syndrome for which no somatic or pharmacological treatment has been proven effective. Dysfunction of the cytokine network has been suspected to play a role in the pathophysiology of CFS. The disturbances of the cytokine network detected in CFS patients are highly variable, in part due to the lack of adequate controls in many studies. Furthermore, all studies have been performed on peripheral venous blood of patients. As cytokines mainly act in tissues, for example, the brain, the information that can be derived from peripheral blood cells is limited. The information regarding the possible role of cytokines in the pathophysiology could come from intervention studies in which the activities of relevant cytokines are reduced, for example, reducing interleukin-1, interleukin-6 or tumor necrosis factor. In this study, the clinical usefulness of anakinra, an IL-1 antagonist, will be assessed in patients with CFS. Methods/Design: A randomized placebo-controlled, double-blind trial will be conducted. Fifty adult female patients meeting the Centers for Disease Control (CDC) criteria for CFS and without psychiatric co-morbidity will be included. After inclusion, patients will be randomized between treatment with anakinra (recombinant human interleukin-1 receptor antagonist) or placebo. Each group will be treated for 4 weeks. Outcome measures will be assessed at baseline, after 4 weeks of intervention, and 6 months after baseline assessment. The primary outcome measure will be fatigue severity at 4 weeks, measured with the validated Checklist of Individual Strength (CIS). Secondary outcome measures are functional impairment, physical and social functioning, psychological distress, pain severity, presence of accompanying symptoms, and cytokine and cortisol concentrations. Discussion: This is the first randomized placebo-controlled trial that will evaluate the effect of interference with IL-1 on the experience of fatigue in patients with CFS. The results of this study may expand treatment options for patients with CFS, for whom graded exercise therapy and cognitive behavioral therapy are the only evidence-based interventions that exist at this moment. Trial registration: Clinicaltrials.gov: NCT02108210. Clinicaltrials.gov registration date: 8 April 2014. EudraCT: 2013-005466-19 Keywords: Chronic fatigue syndrome, Treatment, Protocol, Anakinra, Placebo, Interleukin-1, Cytokine © 2015 Roerink et al. Note: Note: To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). Roerink et al. Trials (2015) 16:439 DOI 10.1186/s13063-015-0971-z TRIALS Background This loss of gray matter might be caused by enhanced cytokine activity. p y p [ ] Several studies have been performed to investigate whether there is an excess of cytokines in CFS, but so far, findings are inconsistent [11, 12]. A recent sys- tematic review on circulating cytokines in CFS re- ported that the majority of studies performed during the past years did not find significantly increased con- centrations of proinflammatory cytokines [13]. A major problem is that many studies did not use ad- equate controls and used different methods to handle blood samples. Cytokine responses are under genetic control, but they are extremely vulnerable to other in- fluences, such as hormonal status, food, exercise, stress, behavior, drugs and vaccines [14]. Therefore, it is not easy to compose a good control group. An additional problem is that almost all studies have been performed on peripheral venous blood. As cyto- kines mainly act in tissues, with the brain being the most important target organ in CFS, information that can be derived from studying circulating cytokine concentrations (which are generally in the pg/ml range) is limited. The only information regarding a role of cytokines that is pathophysiologically relevant could come from intervention studies in which crucial cytokines in tissue are inhibited. A potentially relevant cytokine, which can be blocked in humans without severe side effects, is interleukin-1 (IL-1) [15]. Drugs that interfere with the proinflammatory cyto- kine IL-1 are commonly used nowadays for a variety of inflammatory disorders [26]. The recombinant IL-1 receptor antagonist (anakinra) reduces the activity of both IL-1α and IL-1ß by binding to the IL-1 receptor. This intervention is highly targeted and hence would allow investigators to draw conclusions regarding the pathophysiology of CFS, and the effect of reducing cytokine concentrations in CFS patients. Moreover, compared to blocking TNF-α or IL-6, blocking IL-1 with anakinra has a long safety recorded with respect to side effects, and is not associated with increased susceptibility to opportunistic infections such as Mycobacterium tuberculosis. The primary aim of this study is to assess the effect of anakinra on fatigue severity in patients with CFS. Fatigue is the most central and characterizing symp- tom of CFS, and in contrast to the accompanying symptoms, it is reported by all patients. It is also strongly related to the functional impairments re- ported by patients. Background 1. The complaints of patients with CFS are often described as that of a persistent flu. During infections like influenza, symptoms are generally ascribed to the action of cytokines (like IL-1, IL-6, tumor necrosis factor alpha (TNF) and interferons) [9]. Chronic fatigue syndrome (CFS) is a medically unex- plained syndrome characterized by severe disabling fatigue for a period of at least 6 months, which leads to considerable impairment in daily functioning [1]. Various accompanying symptoms may be present, such as headache, joint and muscle pain, sore throat, impaired memory and concentration and exercise intolerance. In the Netherlands, the prevalence of CFS is at least 27,000 persons [2]. So far, the cause for CFS is yet unclear [3]. Cognitive behavioral ther- apy (CBT) and graded exercise therapy (GET) are the only interventions that have shown positive re- sults in randomized controlled clinical trials for treating fatigue-associated CFS symptoms and dis- ability [4–8]. 2. Many disease states are accompanied by anorexia, loss of interest, somnolence and fatigue, a symptom complex coined as sickness behavior. The cytokines IL-1beta, TNF and IL-6 are thought to be responsible for it. Administration of either IL-1, IL-6, TNF or each of the interferons to humans and animals is accompanied by flu-like symptoms [16–18]. 3. Previously, it was reported that IL-8 and IL-10 were significantly elevated in the cerebrospinal fluid in patients with CFS, compatible with induc- tion of IL-1 [19]. 3. Previously, it was reported that IL-8 and IL-10 were significantly elevated in the cerebrospinal fluid in patients with CFS, compatible with induc- tion of IL-1 [19]. Cytokines are hormone-like proteins that convey messages between cells. Originally, they were thought to act only within the host defense system, but soon it be- came clear that they mediate an array of diverse effects in normal physiology and disease. Since proinflammatory cytokines play a key role in inflammation (for example, by causing fever, inducing muscle pain, fatigue, sleep and other flu like symptoms), they have been hypothe- sized to be responsible for the symptoms in CFS [9, 10]. 4. Beta amyloid precursor protein has also been found to be elevated in the cerebrospinal fluid of CFS patients [20]. Production of this protein is under control of IL-1 and TNF [21–23]. 5. Our group has previously established that patients with CFS have a significant loss of gray matter in the brain [24, 25]. Abstract Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Roerink et al. Trials (2015) 16:439 Page 2 of 8 Study population Fif b Fifty subsequent patients will be included and equally randomized between treatment arms. Inclusion criteria for participation comprise the CDC diagnosis of CFS [20, 31], fatigue duration ≤10 years or recent progres- sion of fatigue severity, female sex, age between 18 and 59 years old, a score ≥40 on the subscale fatigue severity of the Checklist of Individual Strength (CIS), and a score ≥700 on the Sickness Impact Profile (SIP). The ex- clusion criteria include females who are pregnant or nursing, intend to get pregnant during the study, use or have used psychotropic medication in the past month, received a live vaccine during the last 4 weeks, had sub- stance abuse in the past 3 months, have had symptoms more than 10 years, are taking any medication except oral contraceptives and/or paracetamol, have evident somatic comorbidity, have current engagement in CFS research, do not have the ability to understand the na- ture and the extent of the trial and the procedure re- quired, have psychiatric comorbidity (major depression, psychosis, eating disorders, anxiety disorders, bipolar disease and post-traumatic stress disorder) assessed with ‘The Mini-International Neuropsychiatric Interview’ (M.I.N.I.) [32], or are currently engaged in a legal pro- cedure with respect to disability claims. Study medication will be provided by the Swedish Or- phan Biovitrium (Sobi) and stored at the Department of Pharmacology of the RadboudUMC. Preparation and la- belling of anakinra and placebo will be done according to the current guidelines. This will be performed by the Clinical Trials Unit department of Clinical Pharmacol- ogy of the RadboudUMC. The anakinra and placebo sy- ringes will be identical in appearance; the placebo syringes will contain a mixture of sodium citrate, sodium chloride and polysorbate. Medication is used once daily, during a period of 4 weeks. The anakinra and placebo will be provided by the main investigator. On the first study day, patients will be instructed how to self-inject the study medication, as described by others [27]. Ad- ministration takes place in the subcutaneous tissue, most often the abdomen or the thighs. During the study, pa- tients will be advised to set their alarm clock daily at the same time to remind them to use the medication cor- rectly. Drug adherence will be evaluated after 1 week and after completion of treatment. Patients will return all used and unused syringes after 4 weeks. Background As a secondary study aim, we will assess the effect of anakinra on the level of functional impairment, physical and social functioning, pain se- verity, presence of accompanying symptoms and psy- chological distress. In this paper, we describe the protocol to evaluate the effects of anakinra. Other studies with anakinra or anti-IL-1ß revealed a de- crease in fatigue [27–30]. Although it is plausible that cytokines play a role in the pathophysiology of CFS, there is only indirect evidence for this theory: Roerink et al. Trials (2015) 16:439 Page 3 of 8 Methods/Design Study design number 2014/025). The study is registered at the Euro- pean Union Drug Regulating Authorities (EudraCT: 2013–005466–19) and will be conducted according to the Declaration of Helsinki, Good Clinical Practice (GCP) and Good Manufacturing Practice (GMP) guide- lines. The inclusion of patients started in July 2014. Written informed consent will be obtained from all pa- tients before participation in the trial. A randomized placebo-controlled trial (RCT) will be performed to determine whether interference with IL-1 is able to reduce fatigue and disabilities in CFS patients. Within each study arm, treatment will be double blind. The study will be performed in the Radboud University Medical Center, in the Department of Internal Medicine and in the Expert Center for Chronic Fatigue (ECCF), located in Nijmegen, the Netherlands. All female CFS patients visiting the outpatient clinic of the department of Internal Medicine or visiting the ECCF will be consid- ered for participation. Furthermore, patients connected to the “ME/CVS-stichting,” a Dutch foundation for CFS patients, will be asked to participate in the study. To in- crease homogeneity in our study population, we decided to only include female patients, as CFS is a disease that mostly affects women. After inclusion, each patient will receive an individual study code. Patients will be asked to bring a healthy, age-matched, neighborhood control to their first study visit. If patients decide not to partici- pate in this study, an attempt will be made to elucidate the reason for this, but patients are not obligated to explain their refusal. Study medication, randomization and follow-up Eligibility for participation of patients is determined at the pre-study visit. After giving informed consent, pa- tients will be screened for inclusion and exclusion cri- teria by means of the examinations listed in Fig. 1. Laboratory investigations performed earlier will be eval- uated for all patients and will be repeated if not per- formed recently, or when essential measurements, as recommended by others [31], are missing. Patients who qualify to be included will be randomized 1:1 to receive either anakinra (100 mg/day) or placebo. Randomization will be performed by the study pharmacist (Department of Clinical Pharmacology, Radboud University Medical Center), will be known only by the pharmacist and will be exposed only in case of emergency. If the code is broken, it will render the participant not eligible. When the study is completed, the randomization list will be made available by the study pharmacist. Study population Fif b During the intervention period, use of co-medication is only allowed when used for ≤14 consecutive days and on the condition that there are no known interactions with anakinra. Oral contraceptives and/or paracetamol can be used without limitation. During the follow-up period, there are no limitations regarding the use of Ethical approval The study is approved by the Medical Ethical Review Committee of the Radboud UMC Nijmegen (registration Roerink et al. Trials (2015) 16:439 Page 4 of 8 Fig. 1 Study-specific procedures Roerink et al. Trials (2015) 16:439 Page 5 of 8 medication. All co-medication will be registered and re- ported afterwards. 4. Pain severity. Pain severity will be assessed with a visual analog scale (VAS). This score can vary between 0 (no pain) and 10 (worst pain ever experienced). Since the anakinra arm is difficult to blind because of local reactions at the injection site, the research phys- ician and the principal investigator will not be informed of the side effects. After the injection instruction by the physician assistant (PA), the patient will be instructed to report adverse effects to the PA and not to the research physician. The PA will report all side effects to an inde- pendent physician. The independent physician will examine patients if needed and is mandated to stop treatment. To evaluate blinding of the treatment, pa- tients will be asked which medication they thought they were using during the trial, after they have completed the study. 5. Presence of accompanying symptoms. The presence of accompanying symptoms will be evaluated using the CDC criteria. The number of symptoms can vary between 0 and 8. 6. Cytokine concentration in blood. The cytokine concentrations in blood (plasma and blood in Pax- gene tubes) will be determined. Our study can pro- vide additional information regarding cytokine levels because we have the opportunity to compare cyto- kine concentrations with healthy neighborhood con- trols. Also we will compare pre-treatment concentrations with post-treatment concentrations. 7. Cortisol concentration in hair and saliva. Cortisol concentrations in hair and saliva will be assessed because of the possible role of the hypothalamus- pituitary-adrenal axis in CFS. For the baseline assess- ment, comparison will be made with cortisol con- centrations in matched neighborhood controls. All patients will collect saliva for 2 consecutive days at four different time points (directly after awakening, 30 min after awakening, 11 a.m., 8 p.m.), using the passive drool method. Saliva will be stored at -80 °C until further analysis. Study visits are carried out at week 0, week 4 and, if needed, after 6 months. After 1 week, patients will be contacted by telephone to evaluate the occurrence of any problems regarding the use of medication and drug utilization will be recorded. Secondary outcome measures Secondary outcome measures: 1. Level of functional impairment. The level of functional impairment will be measured with the Sickness Impact Profile (SIP8) total score. The SIP8 is a validated instrument to evaluate sickness-related dysfunction. The total score gives an indication of the experienced disabilities in all domains of functioning [35]. At 6 months, all questionnaires evaluating primary and secondary outcomes will be repeated to evaluate if the expected effects of the medication are maintained during the follow-up period of 5 months. Other study parameters collected at baseline will in- clude the following: demographic data, medical history, psychiatric history, serology results collected before in- clusion in the study, use of medication, smoking and the use of alcohol and drugs. 2. Physical and social functioning. Physical and social functioning will be assessed with the subscale physical functioning and subscale social functioning of the Short Form (SF)-36 [36]. 3. Level of psychological distress. The level of psychological distress will be assessed with the total score on the Symptom Checklist-90 (SCL-90). A high total score reflects psychological distress [37]. Ethical approval If there are serious side ef- fects, an additional study visit can be performed by the independent physician at any time. Between study visits, subjects will be asked to fill out web-based question- naires up to 6 months after their first study visit (Fig. 1). Outcome measures h 8. Microbiome determination in stools. This will give more insight into the microbial flora of the host, which is a new field of interest in a wide range of diseases. The availability of well-defined patients with CFS and matched controls is a great opportun- ity in an unexplored area of CFS research to assess whether the microbiome of CFS patients is peculiar. Patients will collect feces at home, and all samples will be stored at –80 °C until further analysis. 8. Microbiome determination in stools. This will give more insight into the microbial flora of the host, which is a new field of interest in a wide range of diseases. The availability of well-defined patients with CFS and matched controls is a great opportun- ity in an unexplored area of CFS research to assess whether the microbiome of CFS patients is peculiar. Patients will collect feces at home, and all samples will be stored at –80 °C until further analysis. The primary outcome measure is fatigue severity mea- sured with the subscale fatigue severity of the Checklist of Individual Strength (CIS) at 4 weeks, the primary end- point of the study. Scores on the CIS subscale range from 8 to 56 (8 items, 7-point Likert Scale). This ques- tionnaire has been validated extensively in patients with chronic fatigue syndrome [33, 34]. Patients will fill out this web-based questionnaire weekly during the course of the trial and monthly during the follow-up period (Fig. 1). All secondary outcome measures will be assessed at baseline and directly following the intervention (Fig. 1). Only the VAS pain scale will be filled in weekly (together with the CIS) during the trial. Discussion The primary analysis will be the comparison between the two different treatment groups (anakinra or placebo) at 4 weeks. Data will be analyzed on an ITT basis. Miss- ing values will be replaced using multiple imputation with fully conditional specification with at least five im- putations. The imputation method that will be used is predictive mean matching for missing data in primary and secondary outcome measures. Aside from condition, we will use the following variables assessed at baseline to generate the imputations: duration of symptoms, age, BMI and baseline values of the outcome measures. This study will be the first randomized placebo- controlled trial to evaluate the effect of blocking IL-1 on symptoms in patients with CFS. Earlier studies investi- gated cytokine production to be of relevance in CFS pa- tients, but conflicting results have been published [11, 13]. A possible explanation is that good controls have not been used in these studies, and cytokines were mea- sured in peripheral blood instead of in tissues. Blinding the study for anakinra is a difficult proced- ure because of the occurrence of local skin reactions in a significant amount of patients. To maintain the double-blind design of this trial, side effects will be evaluated by an independent PA. In earlier “double blind” trials, medication was injected in the presence of the main investigator [27]. The effect of anakinra will be measured up to 6 months after the start of treatment. In this manner, we can evaluate the long- term effect of blocking IL-1 for a short period. It will provide more insight into the best treatment for CFS patients when blocking IL-1 appears to be effective. The results will be analyzed with SPSS for Windows. To determine if there is a significant difference between the intervention arm and placebo condition, ANCOVA will be used with the outcome measure on the second assessment as the dependent measure, the baseline score as the covariate, and condition as the fixed factor [39]. We will test if significant differences exist between both groups in mean age and BMI at baseline, both known to influence circulating cytokine levels. If so, these variables will be entered as covariates in the ANCOVA. Adverse events d Adverse events (AE) are defined as any undesirable ex- perience occurring to a subject during the study, whether or not considered related to the investigational product. All adverse events reported spontaneously by the subject or observed by the investigator or his staff will be recorded. Anakinra is known as a very safe drug. Side effects are mainly related to irritation at the injec- tion site, these reactions usually wane with prolonged treatment. We have reported from our own experience with long-term use of anakinra in patients with Schnitz- ler syndrome that the risk for infection is not enhanced, even in this elderly group [38]. Nevertheless, the package insert warns against infectious complications in patients with underlying illness. Rarely, neutropenia develops during treatment, which is a reason to discontinue therapy. Withdrawal of individual participants Subjects can leave the study at any time for any reason if they wish to do so without any consequences. The inves- tigator or independent physician can decide to withdraw Roerink et al. Trials (2015) 16:439 Page 6 of 8 a subject from the study for urgent medical reasons. In general, noncompleters are not to be replaced. Subjects withdrawn from the study for a medical reason or ad- verse event will receive adequate follow-up. All analysis will be done according to the Intention-to-Treat principle (ITT). In case of discontinuation, efforts will be made to continue all study measurements. With- drawn patients, or patients who are still severely fatigued following the intervention, will be offered regular care, which is CBT at the ECCF. missing data. For the secondary outcome measures, the same analysis will be repeated, but the secondary out- come measures at the second assessment will serve as the dependent variable and the scores at baseline as the covariate. Power calculation h l The sample size is based on the fatigue subscale score of the CIS at 4 weeks. In the present study, the power cal- culation is based on results from comparable studies with CFS patients where fatigue severity was determined with the CIS-fatigue scale [40]. If interleukin-1 plays a central role in CFS symptomatology, we expect a consid- erable reduction of fatigue following treatment. Assum- ing a large controlled effect size of 0.85, an alpha of 0.05 and a power of 0.80, 23 patients are needed in each arm of the study. The number of patients can be further re- duced by using ANCOVA, with the outcome measure on the second assessment as the dependent measure, the baseline score as the covariate, and condition as the fixed factor. In this kind of trials, ANCOVA yields greater power compared to other statistical methods [39]. Based on the correlation between the pre- and post CIS fatigue scale, the sample size of 23 can be multiplied by 0.883 (1 to 0.3422). Including an assumed dropout rate of 20 percent, we will have to include 25 patients in each group to demonstrate a significant difference be- tween the medication group and placebo. Authors’ contributions JM initiated and participated in the design of the study as an expert on infectious diseases and chronic fatigue syndrome, obtained funding for the study, and will coordinate and supervise the study and data collection. MR participated in the design of the study and is responsible for data collection and analysis, and for drafting the manuscript. HK participated in the design of the study as an expert on chronic fatigue, and will help to coordinate and supervise the study. SB is involved as independent physician and will supervise the physician assistant. MH instructs patients on use of medication and will evaluate and report side-effects. MN participated in the design of the study as an expert on infectious diseases. LJ participated in the design of the study as an expert on proinflammatory cytokines. CD participated in the design of the study as an expert of infectious diseases and interleukin-1, and will help to coordinate and supervise the study. 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Cytokine-induced sickness behaviour: a neuroimmune response to activation of innate immunity. Eur J Pharmacol. 2004;500:399–411. 10. Lorusso L, Mikhaylova SV, Capelli E, Ferrari D, Ngonga GK, Ricevuti G. Immunological aspects of chronic fatigue syndrome. Autoimmun Rev. 2009;8:287–91. 10. Lorusso L, Mikhaylova SV, Capelli E, Ferrari D, Ngonga GK, Ricevuti G. Immunological aspects of chronic fatigue syndrome. Autoimmun Rev. 2009;8:287–91. 11. Lyall M, Peakman M, Wessely S. A systematic review and critical evaluation of the immunology of chronic fatigue syndrome. J Psychosom Res. 2003;55:79–90. 11. Lyall M, Peakman M, Wessely S. A systematic review and critical evaluation of the immunology of chronic fatigue syndrome. J Psychosom Res. 2003;55:79–90. 12. Neu D, Mairesse O, Montana X, Gilson M, Corazza F, Lefevre N, et al. Dimensions of pure chronic fatigue: psychophysical, cognitive and biological correlates in the chronic fatigue syndrome. Eur J Appl Physiol. 2014;114:1841–51. Acknowledgements 19. Natelson BH, Weaver SA, Tseng CL, Ottenweller JE. Spinal fluid abnormalities in patients with chronic fatigue syndrome. Clin Diagn Lab Immunol. 2005;12:52–5. We thank the Swedish Orphan Biovitrium for providing the study medication (anakinra and placebo). The authors wish to thank Judith de Natris and Lianne Vermeeren for their help in structuring the questionnaires.We thank Mirjam Tromp for instructing patients in absence of Michael Heijnen. We also thank Maren Blonk, the pharmacist working at the Clinical Pharmacy of the Radboud University Nijmegen Medical Center. This study was funded by an independent organization. We thank the Swedish Orphan Biovitrium for providing the study medication (anakinra and placebo). The authors wish to thank Judith de Natris and Lianne Vermeeren for their help in structuring the questionnaires We thank 20. Baraniuk JN, Casado B, Maibach H, Clauw DJ, Pannell LK, Hess SS. A Chronic Fatigue Syndrome - related proteome in human cerebrospinal fluid. BMC Neurol. 2005;5:22. Radboud University Nijmegen Medical Center. This study was funded by an independent organization. 21. Schmidt J, Barthel K, Wrede A, Salajegheh M, Bahr M, Dalakas MC. Interrelation of inflammation and APP in sIBM: IL-1 beta induces accumulation of beta- amyloid in skeletal muscle. Brain. 2008;131(Pt 5):1228–40. Competing interests Th h d l h The authors declare that they have no competing interests. The study sponsors have no role in the study design, data collection, data analysis, data interpretation, or writing the report. The authors declare that they have no competing interests. The study sponsors have no role in the study design, data collection, data analysis, data interpretation, or writing the report. 13. Blundell S, Ray KK, Buckland M, White PD. Chronic fatigue syndrome and circulating cytokines: A systematic review. Brain Behav Immun. 2015. doi:10.1016/j.bbi.2015.07.004. Author details 1 22. Goldgaber D, Harris HW, Hla T, Maciag T, Donnelly RJ, Jacobsen JS, et al. Interleukin 1 regulates synthesis of amyloid beta-protein precursor mRNA in human endothelial cells. Proc Natl Acad Sci U S A. 1989;86:7606–10. 1Department of Internal Medicine, Radboud University Medical Center, Post Box 91016500 HB Nijmegen, The Netherlands. 2Expert Centre Chronic Fatigue, Radboud University Medical Center, Nijmegen, The Netherlands. 23. Buxbaum JD, Liu KN, Luo Y, Slack JL, Stocking KL, Peschon JJ, et al. Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated alpha-secretase cleavage of the Alzheimer amyloid protein precursor. J Biol Chem. 1998;273:27765–7. Received: 28 October 2014 Accepted: 22 September 2015 Received: 28 October 2014 Accepted: 22 September 201 24. de Lange FP, Kalkman JS, Bleijenberg G, Hagoort P, van der Meer JW, Toni I. Gray matter volume reduction in the chronic fatigue syndrome. NeuroImage. 2005;26:777–81. Discussion When a statistically significant difference is found in the primary analysis, a sensitivity analysis will be performed on the basis of different assumptions about the values of In conclusion, this study will provide more insight into the pathophysiology and treatment of patients with CFS. If an effective treatment can be found, this will Page 7 of 8 Page 7 of 8 Roerink et al. Trials (2015) 16:439 Roerink et al. Trials (2015) 16:439 drastically improve the quality of life in patients with this disabling disease. drastically improve the quality of life in patients with this disabling disease. 5. Reid S, Chalder T, Cleare A, Hotopf M, Wessely S. Chronic fatigue syndrome. BMJ. 2000;320:292–6. 6. Whiting P, Bagnall AM, Sowden AJ, Cornell JE, Mulrow CD, Ramirez G. Interventions for the treatment and management of chronic fatigue syndrome: a systematic review. JAMA. 2001;286:1360–8. 6. Whiting P, Bagnall AM, Sowden AJ, Cornell JE, Mulrow CD, Ramirez G. Interventions for the treatment and management of chronic fatigue syndrome: a systematic review. JAMA. 2001;286:1360–8. Trial status 7. Larun L, Brurberg KG, Odgaard-Jensen J, Price JR. Exercise therapy for chronic fatigue syndrome. Cochrane Database Syst Rev. 2015;2:CD003200. doi:10.1002/14651858.CD003200.pub3. 7. Larun L, Brurberg KG, Odgaard-Jensen J, Price JR. Exercise therapy for chronic fatigue syndrome. Cochrane Database Syst Rev. 2015;2:CD003200. doi:10.1002/14651858.CD003200.pub3. Roerink et al. Trials (2015) 16:439 28. Omdal R, Gunnarsson R. The effect of interleukin-1 blockade on fatigue in rheumatoid arthritis–a pilot study. Rheumatol Int. 2005;25:481–4. 29. Alten R, Gomez-Reino J, Durez P, Beaulieu A, Sebba A, Krammer G, et al. Efficacy and safety of the human anti-IL-1beta monoclonal antibody canakinumab in rheumatoid arthritis: results of a 12-week, Phase II, dose- finding study. BMC Musculoskelet Disord. 2011;12:153. y 30. 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Jacobs HM, Luttik A, Touw-Otten FW, de Melker RA. The sickness impact profile; results of an evaluation study of the Dutch version. Ned Tijdschr Geneeskd. 1990;134:1950–4. 36. Ware Jr JE, Sherbourne CD. The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection. Med Care. 1992;30:473–83. 37. Derogatis LR, Savitz KL. Handbook of psychological assessment in primary care settings. Mahwah: Lawrence Erlbaum Associates; 2000. p. 297–334. 38. de Koning HD, Bodar EJ, Simon A, van der Hilst JC, Netea MG, van der Meer JW. Beneficial response to anakinra and thalidomide in Schnitzler’s syndrome. Ann Rheum Dis. 2006;65:542–4. 39. Van Breukelen GJ. ANCOVA versus change from baseline: more power in randomized studies, more bias in nonrandomized studies [corrected]. J Clin Epidemiol. 2006;59:920–5. 40. Knoop H, van der Meer JW, Bleijenberg G. Guided self-instructions for people with chronic fatigue syndrome: randomised controlled trial. Br J Psychiatry J Ment Sci:. 2008;193:340–1. References 1. Fukuda K, Straus SE, Hickie I, Sharpe MC, Dobbins JG, Komaroff A. The chronic fatigue syndrome: a comprehensive approach to its definition and study. International chronic fatigue syndrome study group. Ann Intern Med. 1994;121:953–9. 25. de Lange FP, Koers A, Kalkman JS, Bleijenberg G, Hagoort P, van der Meer JW, et al. Increase in prefrontal cortical volume following cognitive behavioural therapy in patients with chronic fatigue syndrome. Brain. 2008;131(Pt 8):2172–80. 2. Bleijenberg G, Vercoulen JH, Bazelmans E, Prins J. Chronisch vermoeidheidssyndroom, Psychologie en geneeskunde-behaviour medicine. Houten: Bohn Stafleu van Loghum; 2000. 26. Dinarello CA, Simon A, van der Meer JW. Treating inflammation by blocking interleukin-1 in a broad spectrum of diseases. Nat Rev Drug Discov. 2012;11:633–52. 3. Prins JB, van der Meer JW, Bleijenberg G. Chronic fatigue syndrome. Lancet. 2006;367:346–55. 27. Norheim KB, Harboe E, Goransson LG, Omdal R. Interleukin-1 inhibition and fatigue in primary Sjogren’s syndrome–a double blind, randomised clinical trial. PLoS One. 2012;7:e30123. 4. Price JR, Couper J. Cognitive behaviour therapy for adults with chronic fatigue syndrome. Cochrane Database Syst Rev. 2000;2:CD001027. doi:10.1002/ 14651858.CD001027. Page 8 of 8 Page 8 of 8 Roerink et al. Trials (2015) 16:439 Roerink et al. Trials (2015) 16:439 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission
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Older Australians Can Achieve High Adherence to the Mediterranean Diet during a 6 Month Randomised Intervention; Results from the Medley Study
Nutrients
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Older Australians Can Achieve High Adherence to the Mediterranean Diet during a 6 Month Randomised Intervention; Results from the Medley Study Courtney Davis 1,*, Jonathan Hodgson 2,3, Janet Bryan 4, Manohar Garg 5, Richard Woodman 6 and Karen Murphy 1 1 Alliance for Research in Exercise, Nutrition and Activity, School of Health Sciences, University of South Australia, GPO Box 2471, Adelaide, SA 5001, Australia; karen.murphy@mymail.unisa.edu.au 1 Alliance for Research in Exercise, Nutrition and Activity, School of Health Sciences, University of South Australia, GPO Box 2471, Adelaide, SA 5001, Australia; karen.murphy@mymail.unisa.edu.au 2 School of Medical and Health Sciences, Edith Cowan University, 27 Joondalup, WA 6027, Australia; jonathan.hodgson@ecu.edu.au School of Medical and Health Sciences, Edith Cowan University, 2 Joondalup, WA 6027, Australia; jonathan.hodgson@ecu.edu.au 3 School of Medicine and Pharmacology, University of Western Australia, 35 Stirling Highway, Perth, WA 6000, Australia 4 School of Psychology, Social Work and Social Policy, University of South Australian, GPO Box 2471 Adelaide, SA 5001, Australia; janet.bryan@unisa.edu.au 4 School of Psychology, Social Work and Social Policy, University of South Australian, GPO Box 2471, Ad l id SA 5001 A li j b @ i d j y 5 Nutraceuticals Research Program, School of Biomedical Sciences and Pha Callaghan, NSW 2308, Australia; manohar.garg@newcastle.edu.au j y 5 Nutraceuticals Research Program, School of Biomedical Sciences and Pharmacy, University of Newcas Callaghan NSW 2308 Australia; manohar garg@newcastle edu au 5 Nutraceuticals Research Program, School of Biomedical Sciences and Pharmacy, University of Newcastle Nutraceuticals Research Program, School of Biomedical Sciences and P Callaghan, NSW 2308, Australia; manohar.garg@newcastle.edu.au g Callaghan, NSW 2308, Australia; manohar.garg@newcastle.edu.au g g g 6 Flinders Centre for Epidemiology and Biostatistics, Flinders University, GPO Box 2100, g g g 6 Flinders Centre for Epidemiology and Biostatistics, Flinders University, Adelaide, SA 5001, Australia; richard.woodman@flinders.edu.au Adelaide, SA 5001, Australia; richard.woodman@flinders.edu.au * Correspondence: courtney.davis@mymail.unisa.edu.au; Tel.: +61-08-8302-1869 Received: 13 April 2017; Accepted: 19 May 2017; Published: 24 May 2017 Abstract: Adherence to a Mediterranean diet (MedDiet) is thought to be achievable in non-Mediterranean regions, but this has yet to be investigated. We aimed to determine if an older Australian population could adhere to a MedDiet for six months. We conducted a randomised, parallel dietary intervention trial with two dietary arms: the Mediterranean diet (MedDiet) group and the habitual diet (HabDiet) control group. A 15-point Mediterranean diet adherence score and food and nutrient intakes were estimated from three-day weighed food records collected at baseline, two and four months. Erythrocyte fatty acids, serum carotenoids and urinary metabolites were assessed at baseline, three and six months. Older Australians Can Achieve High Adherence to the Mediterranean Diet during a 6 Month Randomised Intervention; Results from the Medley Study We enrolled 166 participants; 152 commenced and 137 completed the study (70 in the MedDiet group, 67 in the HabDiet group). Adherence scores were significantly higher in the MedDiet group at two months (between group difference 2.2, 95% CI 1.3, 2.9) and four months (between group difference 2.6, 95% CI 1.9, 3.3). Consumption of vegetables, fruits, fish, legumes, nuts and olive oil significantly increased in the MedDiet group compared to the control, and discretionary food intake decreased (p < 0.01). Measures of compliance including serum β-carotene, lycopene and erythrocyte monounsaturated fatty acids were significantly higher in the MedDiet group at three and six months (p < 0.05). Our results indicate that a population of older Australians can adopt a Mediterranean diet over a six month period. Keywords: Mediterranean diet; adherence; Australia; nutrients; food intake; elderly nutrients nutrients www.mdpi.com/journal/nutrients 1. Introduction In the mid-20th century, evidence emerged that populations living in the Mediterranean region had lower incidence of cardiovascular disease (CVD) than those living in northern Europe and North America [1]. It was suggested that the distinguishing feature between these regions was diet, particularly the fat composition [2]. The Greek population was consuming relatively little processed foods, red meat and saturated fat, but consumed a larger amount of plant foods including olive oil Nutrients 2017, 9, 534; doi:10.3390/nu9060534 www.mdpi.com/journal/nutrients 2 of 20 Nutrients 2017, 9, 534 as the main culinary fat, providing monounsaturated fatty acids (MUFA), micronutrients, fibre and antioxidants [3]. This dietary pattern was termed the Mediterranean diet (MedDiet) [4]. The MedDiet has received much attention from researchers with the majority of studies reporting positive health outcomes when close adherence is achieved [5,6]. Large observational studies including the European Prospective Investigation into Cancer and Nutrition (EPIC), Finland, Italy, the Netherlands Elderly (FINE), and Healthy Ageing: a Longitudinal study in Europe (HALE) studies, the Survey in Europe on Nutrition and the Elderly (SENECA) study, and the Nurses’ Health Study have been meta-analysed by Sofiet al., most recently in 2014 [6–11]. With a database of over four million subjects, their results showed overall mortality risk was reduced by up to 32% when comparing lowest with highest MedDiet adherence level [6]. For every two-point increase in adherence score (maximum 9), the relative risk for CVD mortality/incidence was 0.90 (95% CI 0.87–0.92). The landmark Prevención con Dieta Mediterránea (PREDIMED) study is the largest Mediterranean diet randomised intervention trial completed to date, involving ~7500 Spanish adults with a median follow-up close to five years [12]. The primary outcome, risk of CVD (stroke, myocardial infarction or angina), was reduced by 30% after following the MedDiet, and risk of incident diabetes was reduced by 52% compared with a low fat control diet [13]. This evidence led the US Department of Health and Human Services and US Department of Agriculture to recognise and include the MedDiet as one of two alternative healthy dietary patterns in the 2015–2020 US Dietary Guidelines [14]. The US population, along with other Western nations including Australia and the UK has sub-optimal intakes of fruits and vegetables but higher intakes of discretionary foods than Mediterranean populations. These countries also suffer from a high prevalence of obesity, diabetes and CVD. 1. Introduction In Australia, discretionary foods contribute over 35% of energy intake and only 4% of the population consumes the recommended servings of vegetables [15]. Cardiovascular disease is the leading cause of death, responsible for 30% of mortality cases in 2012 [16]. Furthermore, with populations ageing in Western nations, rates of chronic disease are likely to increase, placing economic burden on health care systems [17]. Age is a leading risk factor of CVD and dementia, and so intervening with strategies to reduce the burden of these diseases is critical at the present time. Epidemiological evidence shows that non-Mediterranean populations with a priori measured high adherence to a MedDiet have lower risk of CVD [11,18]. Indeed, in the meta-analysis by Sofiet al., of 23 studies added to the 2014 analysis, 11 were from non-Mediterranean populations, nine were from Mediterranean populations and three were multicentre studies with both Mediterranean and non-Mediterranean countries. The MedDiet may offer a dietary strategy to reduce morbidity and mortality from chronic disease in non-Mediterranean countries. It has been suggested that the adaptability and variety of the diet makes it transposable to non-Mediterranean countries [18–20]. The limitation of the meta-analysis is the inclusion of observational studies only. There are few longer term intervention trials to assess how successfully non-Mediterranean populations can change their diets. Consequently, it remains unknown whether it is plausible for regions beyond the Mediterranean Sea to adopt the MedDiet. Barriers may include cultural beliefs, palatability, food access, cost, time for food preparation/shopping, and environment (for example proximity to fast food restaurants and access to discretionary foods) [21]. Whether the Australian population can adopt the MedDiet ad libitum over the longer term is unknown. We conducted a randomised controlled intervention trial in an older Australian population to determine how well this population could adopt the MedDiet in 6 months. The primary outcome for this study is adherence to the MedDiet measured via a 15-point score adapted from Trichopoulou et al. [22]. Secondary outcomes include self-reported food, nutrient, carotenoid and flavonoid intake from weighed food records and biomarkers of adherence. In addition, qualitative data are presented highlighting participant experiences with consuming the MedDiet. 2. Materials and Methods The Mediterranean diet for cardiovascular and cognitive health in the elderly study (MedLey) protocol has been published [23,24], and will be summarised. The study was a 6-month randomised 3 of 20 Nutrients 2017, 9, 534 controlled clinical dietary intervention trial. The primary aim was to assess the effect of the MedDiet on cognitive function after 6 months compared to the habitual Australian diet. Secondary aims included cardiovascular risk factors and the ability of Australians to adhere to the MedDiet. The results of the latter are presented here. Participants were recruited from metropolitan Adelaide between July 2013 and May 2014. Local television and newspapers (Messenger/Advertiser) were the primary methods of advertisement for the study. Interested volunteers left their details with the clinic, and were then called to confirm interest and sent a screening questionnaire via post or email. If eligible based on this questionnaire, volunteers attended a screening visit where blood samples, blood pressure, height, weight, waist circumference and cognitive function via DemTect were assessed to determine eligibility. Exclusion criteria included the following: persons considered by the investigator to be unwilling, unlikely or unable to comprehend or comply with the study protocol, age < 65 years, previous/current traumatic head/brain injury, neurological or psychiatric conditions, previous stroke, unstable use of anti–depressant medication (<6 months), use of medication to treat anxiety, current or recent (in the last 6 months) malignancy, major liver, kidney, respiratory or gastrointestinal disease, current cardiovascular disease or angina, uncontrolled hypertension (>170/100), current smoker, vegetarian (does not eat red meat, poultry or fish), actively undertaking weight loss program, use of appetite suppressants or Orlistat (Xenical), age and education adjusted scores of <13 on the DemTect, or body mass >135 kg (limit on dual energy X-ray absorptiometry (DEXA) scanner). Participants were stratified by age, gender and BMI and randomised into either the MedDiet group or the habitual diet (HabDiet) group by the process of minimisation. A chief investigator not involved in any participant contact performed the randomisation. Participants were listed by studyID, age, gender and BMI in an excel spreadsheet which was sent to the chief investigator. They then placed each ID into one of the two groups at random, so that age, BMI and gender averages remained similar between groups. 2. Materials and Methods We randomised 166 participants (n = 85 MedDiet group, n = 81 HabDiet group), and 14 withdrew before commencement leaving 152 (n = 80 MedDiet group, n = 72 control group) who commenced the study. Ten withdrew from the MedDiet group and 5 from the HabDiet group, thus 137 completed their 6 months visit (11% attrition from commencement). The study was conducted in 2 cohorts, the first (n = 69) conducted between August 2013 and April 2014, the second (n = 83) between May 2014 and February 2015. Those in the MedDiet group were prescribed a MedDiet for 6 months (energy balanced for individual requirements), while those in the HabDiet group followed their regular diets for 6 months in a parallel design. We aimed to assess dietary adherence through self-reported 3-day weighed food records (WFRs), daily checklists and also objective measures: fasting serum carotenoid levels, erythrocyte fatty acids and 24-h urinary metabolites. Dietary intake was assessed at baseline, 2 and 4 months. Urinary metabolites, erythrocyte fatty acids and serum carotenoids were assessed at baseline, 3 and 6 months. All clinic visits occurred at the Sansom Institute for Health Research, University of South Australia. Checklists were completed daily by those randomised to the MedDiet group only. Further to this, in an exit survey participants were asked to report on their experiences in relation to following the MedDiet. The study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects/patients were approved by the University of South Australia Ethics Committee (#31163). Written informed consent was obtained from all subjects/patients with an investigator present to answer and clarify any queries. Participants received $100 pro rata as an honorarium. The trial was registered with the Australia New Zealand 2.2. Intervention Strategy Participants were educated at baseline by a study dietitian. They received written resources detailing the basic principles of the MedDiet, a list of Mediterranean fruits and vegetables, a list of serving sizes for key foods, recommendations for number of servings to consume daily and weekly, suggestions for recipe modification and eating out, a recipe book with appropriate Mediterranean recipes and checklists to record their daily intake of servings. They were encouraged to eat a variety of foods, have cooked and raw vegetables, use olive oil in baking, cooking and as a dressing/sauce and consume 50% of their cereals from whole-grain sources. This education session lasted between 45 min to 1 h. Participants were provided with foods to aid adherence, including extra virgin olive oil (750 mL per fortnight), plain and flavoured low fat Greek yoghurt (1 kg per week), unsalted peanuts, almonds and walnuts (~350 g per week), canned legumes and canned tuna. Combined, this was 30–35% of estimated energy requirements. These foods were chosen because they were reasonably shelf-stable, somewhat expensive but important components of the diet, and were offered every 2 weeks. After the initial baseline session, participants attended a 30 min session with the dietitian biweekly to discuss the diet, return checklists, have body mass assessed and collect food. At these sessions, advice tailored to each individual was given to help participants adhere closely to the diet. We aimed to maintain body mass to avoid the possible confounding effects of weight loss on outcomes, such as lipids and blood pressure. If weight loss or gain was occurring, personalised advice was given to counteract this, by either increasing key foods such as olive oil, whole-grain cereals, fruits and vegetables, or decreasing certain foods such as discretionary foods. In addition, participants could call or email the dietitian at any time during the week with questions or concerns. Other than these supports, participants were required to adapt to the MedDiet in their own capacity. 2.1. The Mediterranean Diet The diet was based on a literature review to determine approximate food and nutrient content of the MedDiet [26] and was tested in a pilot study in a population group similar to that of the MedLey study [27]. The review included descriptive studies, observational and intervention diets, including data from the EPIC and PREDIMED studies [12,28]. A recommendation for daily and weekly servings of key foods and the resulting nutrient content was determined. Based on their 4 of 20 Nutrients 2017, 9, 534 energy requirements and in line with a traditional MedDiet, volunteers were asked to consume the following: 1–3 tablespoons of extra virgin olive oil, 5–6 servings of vegetables, 2–3 servings of fruit, 4–6 servings of grain foods, up to 1 white potato, up to 200 mL of red wine and up to 1 cup of skim milk per day, and 4–6 servings of nuts, 6 servings of Greek yoghurt, 3–4 servings of cheese, 1–3 servings of poultry/pork, 3 servings of fish, 3 servings of legumes, 1–2 servings of small goods, up to 1 serve of red meat, up to 6 eggs, and up to 3 servings of discretionary foods per week. To adjust for differences in estimated energy requirements, recommended servings of key foods was reduced to create 3 additional lower energy diets, which nonetheless retained a nutrient profile in line with MedDiet principles. The nutrient content of the four energy levels is published elsewhere (Davis et al. [23]). 2.3. Three-Day Weighed Food Records Weighed food records were undertaken by participants in the week preceding commencement of the intervention to assess baseline dietary intake. They were then repeated between weeks 9–11 (2 months) and weeks 19–21 (4 months) during the intervention phase. WFRs were administered at the biweekly appointments rather than 3 and 6 month clinic visits to reduce participant burden. Participants were asked to record all food and beverage consumption on consecutive week days and one weekend day, i.e., either Thursday, Friday and Saturday, or Sunday, Monday and Tuesday. All participants received detailed verbal and written instructions from trained staff, digital kitchen scales and a paper diary in which to write their food intake. Upon return these were checked for completeness with the participant. Recorded foods were analysed using FoodWorks Professional Version 7.0.3016 (Xyris Software Spring Hill, QLD, Australia), by a study dietitian and exported to Microsoft Access™(Microsoft Corporation, Redmond, WA, USA) for analysis. Total quantity of flavonoids and carotenoids was calculated for all available foods from the following databases: United States Department of Agriculture (USDA) Database for the Flavonoid Content of Selected Foods (Release 3.1 and 2.1), USDA-NCC Carotenoid Database for U.S. Foods, and the USDA Database for the Isoflavone Content of Selected Foods (Release 2.0). Where necessary, data were supplemented with information from the online phenol explorer. To assess adherence to the MedDiet, we used a 15-point 5 of 20 Nutrients 2017, 9, 534 scoring system modified from the 9-point MedDiet score (MDS) created by Trichopoulou et al. [22]. Total food intake as grams/MJ was divided into 15 predefined food groups. Using the baseline group specific means as cut-offs, each participant scored either 1 point or 0 points for each of the 15 food groups: 1 point was awarded for intakes above the mean for vegetables, fruits, nuts, legumes, fish, breads, cereals, olive oil, and 1 point for intakes below the mean for sugars, eggs, dairy, potato, meat and miscellaneous foods. For red wine, intakes between 0–200 mL/day received 1 point, while above 200 mL received 0 points. Maximum score (15) reflects highest adherence. 2.4. Weekly Checklists To monitor adherence throughout the duration of the trial, all participants randomised to the MedDiet group were asked to complete a semi-quantitative checklist. On a daily basis, participants were asked to record when foods were consumed, as half or whole servings using a checklist. Discretionary foods, small goods, red meat, eggs, red wine, skim milk and white potatoes were optional foods and had no minimum requirement. Total weekly servings for each food group were summed, and a percentage adherence for each food group was calculated by comparing actual consumption with recommended consumption. For foods where there was a minimum requirement, the equation was as follows: a ÷ r × 100 where a is the number of actual servings consumed and r is the number of recommended servings. For foods with a maximum requirement, the inverse equation was used: r ÷ a × 100. If the per cent adherence was greater than 100%, it was rounded down to 100%. Total weekly adherence was calculated by averaging the adherence for each food group, and overall adherence calculated by averaging the weekly adherence percentages. Thus all food groups were weighted equally in considering per cent adherence. 2.3. Three-Day Weighed Food Records Miscellaneous foods included non-Mediterranean food items such as muesli bars, soy products, protein bars and other special dietary products, tropical fruit, and discretionary foods including confectionary, chocolate, potato crisps, deep-fried foods, alcoholic beverages other than red wine, biscuits, cakes, muffins, cupcakes, low fibre breakfast cereals, cream, butter, margarine (other than olive-oil based margarine), ice cream, bakery products and milkshakes. Beverages including tea, coffee, water, soft drinks, fruit and vegetable juices, cordials and flavoured milks were not included in the score. The adjustments to the score were for the following reasons: evidence suggests foods such as nuts and sugars may be independently associated with health outcomes, thus we separated these groups to allow future sensitivity analysis; a large amount of foods come from non-core food groups in the modern Australian diet, thus we deemed it appropriate to include a “miscellaneous” food group; we used the mean rather than median after excluding outliers, because in some cases the median intake for key food groups such as legumes was 0, which did not reflect a traditional MedDiet; we did not want vegetable intake to include white flesh potato as this can displace other vegetables in the diet; and finally instead of using gender-specific values, we controlled for differences in energy requirements and intake by using g/MJ. We did not include beverages in the final score as they were not originally included, and other than tea, water, coffee and to a lesser extent juice, intakes of other beverages were minimal. However, we did record beverage intake as a 16th group to potentially investigate further in future. 2.5. Serum Carotenoids, Erythrocyte Fatty Acids and 24-h Urinary Metabolites Participants gave 8-h fasting blood samples for the analysis of erythrocyte fatty acids and serum carotenoids. Samples were taken via venepuncture, centrifuged and stored at −80 ◦C until analysis. Erythrocyte fatty acid composition was determined as an indicator of longer term fatty acid status using direct transesterification as described elsewhere [29]. Carotenoids were measured as an indication of fruit and vegetable consumption, from serum according to the method of Barua et al. [30] using high performance liquid chromatography. Participants collected a 24 h urine sample for analysis of excretion of key minerals magnesium, calcium, potassium and sodium at a NATA accredited laboratory. The 24 h collection period began with the second void of the day before, and continued until the first void on the day of the baseline, 3 and 6 months clinic appointment. Nutrients 2017, 9, 534 6 of 20 2.6. Exit Survey Which, if any, components of the MedDiet are you likely to continue with now that the study is finished? Box 1. Exit survey questions pertaining to dietary adherence. Question 9. The table below asks you about the foods you were asked to eat and restrict. Please tick the appropriate box: Did you . . . Yes all of the time Yes most of the time Yes some of the time None of the time (1) Enjoy the yoghurt? (2) Enjoy the legumes? (3) Enjoy the tuna? (4) Enjoy the olive oil? (5) Enjoy the nuts? (6) Manage the red meat restrictions? (7) Manage the “extras” restrictions? (8) Manage with the milk restrictions? Question 10. What was/were the most difficult thing(s) about following the Australianised MedDiet? Question 13/15. Which, if any, components of the MedDiet are you likely to continue with now that the study is finished? Question 10. What was/were the most difficult thing(s) about following the Australianised MedDiet? Question 13/15. Which, if any, components of the MedDiet are you likely to continue with now that the study is finished? 2.6. Exit Survey The MedLey exit survey questioned volunteers on their experiences within the trial, including aspects of following the intervention diet. Box 1 shows the three questions related specifically to the diet from the exit survey. Surveys were administered between the final two clinic appointments, which were spaced 1 week apart (i.e., during the final week of the trial). Between cohort 1 and cohort 2, two questions from the survey were removed, thus in cohort 1 the third question was number 15, and in cohort 2 it was 13. Responses to the exit survey were coded numerically for analysis. For question 9, the four possible answers were numbered 1–4 (1 = yes all of the time) and the count for each response was calculated for each food. For question 10, written answers were recorded, and based on the answers 11 categories were developed to capture the various responses. Each participants response was then grouped into one of the 11 categories: vegetables (1); attitudes of friends/family (2); restriction other specific foods or beverages (other than red meat or extras) (3); changing dietary habits (4); eating out/at friends/holidays/special occasions (5); nothing (6); olive oil (7); red meat restrictions (8); volume of food (9); white wine/beer restrictions (10); or extras restrictions (11). All answers given were coded in this way, thus some participants had multiple codes applied. For question 13/15, answers were coded as follows: 1 = all components, 2 = most components (at least 5 different aspects listed or “most components” indicated in the answer), 3 = 3–4 components listed, 4 = 1–2 components listed. The count for each code was then calculated. Box 1. Exit survey questions pertaining to dietary adherence. Box 1. Exit survey questions pertaining to dietary adherence. Question 9. The table below asks you about the foods you were asked to eat and restrict. Please tick the appropriate box: Did you . . . Yes all of the time Yes most of the time Yes some of the time None of the time (1) Enjoy the yoghurt? (2) Enjoy the legumes? (3) Enjoy the tuna? (4) Enjoy the olive oil? (5) Enjoy the nuts? (6) Manage the red meat restrictions? (7) Manage the “extras” restrictions? (8) Manage with the milk restrictions? Question 10. What was/were the most difficult thing(s) about following the Australianised MedDiet? Question 13/15. 2.7. Statistics Sample size for this study was based on the primary outcome measure of cognitive function and is explained in detail elsewhere [23]. We calculated that 128 subjects (two groups of n = 64) would provide 80% power to detect a significant (p < 0.05) 0.5 standard deviation change in cognitive outcomes. For this study, adherence score was the primary outcome. With a sample size of n = 128, based on results from our pilot study [27] we estimated we would have >80% power to detect a significant (p < 0.05) 1 unit difference in the 15-point adherence score between groups (standard deviation = 2). We expected an effect size of at least a 2-unit change. Raw data is available as online supplementary material (Table S1). Continuous variables are presented as mean ± standard deviation (SD), categorical variables as count (%). Residuals were checked for normality and any non-normal variables were log10 transformed before analysis. For dietary variables including nutrient and food intakes, outliers were identified from histograms and quantile-quantile plots. If these were >3 SD from the mean and affected the mean value for diet group and visit, they were removed. In this way extreme values for energy and other nutrients were identified and removed. To compare completers and non-completers for dietary, clinical and demographic characteristics at baseline, independent samples t-tests were used for normally distributed variables, and Mann-Whitney U test for non-normally distributed variables. To assess differences in adherence scores, nutrient and food intake, urinary metabolites, erythrocyte 7 of 20 Nutrients 2017, 9, 534 fatty acids and serum carotenoids, analyses were performed using intention-to-treat linear mixed effects models with a group × time interaction term to determine overall differences in effects across time and at each time point. Significant was set at 0.05 (two sided). For food intakes, gender was controlled for in the model. To control for energy intakes, all food and nutrients intakes were analysed per MJ of energy intake. In a sensitivity analysis, the 10-point MDS was applied to our data, to enable comparison to previous studies. Gender-specific median intakes at baseline were used as cut-offs for baseline, 2 month and 4 month intakes. One point was awarded if intakes of vegetables, legumes, fruits and nuts, cereals, fish and monounsaturated fatty acid:saturated fatty acid (MUFA:SFA) was above the median, and 1 point awarded if intakes of meat/poultry and dairy were below the median. 2.7. Statistics Additionally, 1 point was awarded for intakes between 10–50 g ethanol for males, and 5–25 g for females, so that minimum score was 0 and maximum was 9. To calculate adherence to the a priori MedDiet, we also calculated the MDS using cut-offs established from the Greek cohort enrolled in the EPIC study [22]. Linear mixed effects models were used to calculate between group differences for adherence score using these methods. The cut-offs for all three scoring methods are provided as online supplementary material (Table S2). Statistics were performed using IBM SPSS Statistics (version 21) and STATA (Version 13.0, StataCorp, College Station, TX, USA). 3. Results The first participants commenced in August 2013 and all had completed the 6-month period by February 2015. Of 152 participants who commenced, baseline data were complete for 150 participants for dietary intake and urinary metabolites, and 152 participants for erythrocyte fatty acids and carotenoids. All participants with baseline data were included in the analyses. Figure S1 (supplementary material) shows the CONSORT flow diagram for the MedLey study. One hundred thirty-seven completed the study (MedDiet = 70, HabDiet = 67). Groups were similar for age (71.0 ± 4.9 years in the MedDiet group, 70.9 ± 4.9 year in the HabDiet group) and BMI (26.7 ± 3.7 kg/m2 in the MedDiet group, 27.1 ± 4.1 kg/m2 in the HabDiet group) at baseline. Supplementary file 4 shows the CONSORT flow diagram including reasons for withdrawals. There were no significant differences between those who completed and those who withdrew for clinical and demographic characteristics, except that insulin was higher amongst withdrawals in the HabDiet group. For dietary intakes, there were no differences between completers and non-completers at baseline. Fifty eight per cent were female in the MedDiet group, 54% in the control group. Baseline demographic information has been published elsewhere [31]. Table 1 shows the baseline characteristics for carotenoids, erythrocyte fatty acids and urinary metabolites. 3.1. A Priori Adherence Score and Checklists Adherence scores increased in the MedDiet group significantly (p < 0.01), while they remained unchanged in the HabDiet group (Figure 1). Scores were significantly different between groups at two months (2.1, 95% CI 1.3, 2.9) and four months (2.6, 95% CI 1.9, 3.3), with a significant overall effect of diet on adherence (p < 0.001). The sensitivity analysis revealed similar changes when the 10-point MDS was applied using sex-specific baseline medians, although when the sex-specific medians from the Greek cohort of the EPIC study were used, adherence scores were lower (Table 2). According to the checklists, a mean of 91% of recommended servings were consumed, across all days and an adherence percentage of over 90% was achieved for milk, small goods, poultry, cheese, fruit, olive oil, fish, yoghurt and nuts. 8 of 20 Nutrients 2017, 9, 534 Table 1. Baseline serum carotenoids, erythrocyte fatty acids and urinary metabolites for participants enrolled in the MedLey study, by diet group allocation. Lycopene (ng/mL) 192.1 ± 133.0 191.2 ± 228.9 α-carotene (ng/mL) 92.0 ± 145.8 74.2 ± 128.7 β carotene (ng/mL) 1287 4 ± 1489 7 1028 3 ± 1785 9 Serum Carotenoids MedDiet (n = 80) HabDiet (n = 72) β-cryptoxanthin (ng/mL) 57.5 ± 55.8 52.7 ± 42.7 Lycopene (ng/mL) 192.1 ± 133.0 191.2 ± 228.9 α-carotene (ng/mL) 92.0 ± 145.8 74.2 ± 128.7 β-carotene (ng/mL) 1287.4 ± 1489.7 1028.3 ± 1785.9 Lutein:zeaxanthin 505.2 ± 376.0 513.2 ± 238.2 Erythrocyte fatty acids n = 80 n = 72 Total SFA (%tot) 43.3 ± 0.9 43.6 ± 1.1 Total trans fatty acids (%tot) 0.4 ± 0.1 0.4 ± 0.1 Total MUFA (%tot) 18.4 ± 0.9 18.5 ± 1.2 Total PUFA (%tot) 37.8 ± 1.1 37.2 ± 3.2 Total omega-3 fatty acids (%tot) 10.7 ± 2.6 10.4 ± 2.7 Total omega-6 fatty acids (%tot) 27.1 ± 2.7 27.1 ± 2.8 Omega-6:omega-3 2.7 ± 0.8 2.8 ± 0.8 Omega-3 index 7.4 ± 2.5 7.1 ± 2.5 Urinary metabolites n = 80 n = 70 Sodium (mmol/24 h) 115.2 ± 51.0 119.6 ± 78.3 Potassium (mmol/24 h) 79.4 ± 24.4 78.3 ± 23.8 Calcium (mmol/24 h) 3.6 ± 1.8 4.1 ± 2.0 Magnesium (mmol/24 h) 4.6 ± 2.2 4.3 ± 1.5 Values are mean ± standard deviation (SD). 3.1. A Priori Adherence Score and Checklists ( ) y, g diovascular disease in the elderly study; %tot, % of total erythrocyte fatty acids; SFA, satura y acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; Omega-3-Ind 7.1 9.7 10.6 7.4 7.7 7.9 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 Baseline 2 months 4 months Adherence score MedDiet HabDiet gure 1. Mean adherence score at each time point by diet group. Mean (error bars depict SEM edDiet adherence scores of Australian men and women (age ≥ 65) at baseline, and two and fo onths after following a MedDiet (n = 80) or their HabDiet (n = 70). MedDiet adherence score bas n intakes of 15 food groups calculated from 3-d WFRs, range 0–15 where 15 represents highe ossible adherence level. Mean scores at each time point presented. Linear mixed effects model wi ore as the dependent variable, diet and visit as factors, and unstructured covariance used mpare groups. Analyses were intention to treat. MedDiet, Mediterranean diet; HabDiet, habitu t WFR i h d f d d 7.1 9.7 10.6 7.4 7.7 7.9 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 Baseline 2 months 4 months Adherence score MedDiet HabDiet Figure 1. Mean adherence score at each time point by diet group. Mean (error bars depict SEM) MedDiet adherence scores of Australian men and women (age ≥65) at baseline, and two and four months after following a MedDiet (n = 80) or their HabDiet (n = 70). MedDiet adherence score based on intakes of 15 food groups calculated from 3-d WFRs, range 0–15 where 15 represents highest possible adherence level. Mean scores at each time point presented. Linear mixed effects model with score as the dependent variable, diet and visit as factors, and unstructured covariance used to compare groups. Analyses were intention to treat. MedDiet, Mediterranean diet; HabDiet, habitual diet; WFR, weighed food record. ure 1. Mean adherence score at each time point by diet group. Mean (error bars depict SE dDiet adherence scores of Australian men and women (age ≥ 65) at baseline, and two and fo nths after following a MedDiet (n = 80) or their HabDiet (n = 70). MedDiet adherence score bas intakes of 15 food groups calculated from 3-d WFRs, range 0–15 where 15 represents high ssible adherence level. Mean scores at each time point presented. 3.1. A Priori Adherence Score and Checklists MedLey, Mediterranean diet for cognitive and cardiovascular disease in the elderly study; %tot, % of total erythrocyte fatty acids; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; Omega-3-Index = sum of eicosapentaenoic acid and docosahexaenoic acid. β carotene (ng/mL) 1287.4 ± 1489.7 1028.3 ± 1785.9 Lutein:zeaxanthin 505.2 ± 376.0 513.2 ± 238.2 Erythrocyte fatty acids n = 80 n = 72 Total SFA (%tot) 43.3 ± 0.9 43.6 ± 1.1 Total trans fatty acids (%tot) 0.4 ± 0.1 0.4 ± 0.1 Total MUFA (%tot) 18.4 ± 0.9 18.5 ± 1.2 Total PUFA (%tot) 37.8 ± 1.1 37.2 ± 3.2 Total omega-3 fatty acids (%tot) 10.7 ± 2.6 10.4 ± 2.7 Total omega-6 fatty acids (%tot) 27.1 ± 2.7 27.1 ± 2.8 Omega-6:omega-3 2.7 ± 0.8 2.8 ± 0.8 Omega-3 index 7.4 ± 2.5 7.1 ± 2.5 Urinary metabolites n = 80 n = 70 Sodium (mmol/24 h) 115.2 ± 51.0 119.6 ± 78.3 Potassium (mmol/24 h) 79.4 ± 24.4 78.3 ± 23.8 Calcium (mmol/24 h) 3.6 ± 1.8 4.1 ± 2.0 Magnesium (mmol/24 h) 4.6 ± 2.2 4.3 ± 1.5 alues are mean ± standard deviation (SD). MedLey, Mediterranean diet for cognitive an rdiovascular disease in the elderly study; %tot, % of total erythrocyte fatty acids; SFA, saturate tty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; Omega-3-Ind sum of eicosapentaenoic acid and docosahexaenoic acid Values are mean ± standard deviation (SD). MedLey, Mediterranean diet for cognitive and cardiovascular disease in the elderly study; %tot, % of total erythrocyte fatty acids; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; Omega-3-Index = sum of eicosapentaenoic acid and docosahexaenoic acid. ( ) y, g diovascular disease in the elderly study; %tot, % of total erythrocyte fatty acids; SFA, satura y acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; Omega-3-In Values are mean ± standard deviation (SD). MedLey, Mediterranean diet for cognitive and cardiovascular disease in the elderly study; %tot, % of total erythrocyte fatty acids; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; Omega-3-Index = sum of eicosapentaenoic acid and docosahexaenoic acid. 3.2. Food and Nutrient Intake .2. Food and Nutrient Intake Figure 2 shows the mea Figure 2 shows the mean change in intake for 15 food groups. Based on Australian standard serving sizes [32], those in the MedDiet group increased their fruit, nut and vegetable intakes by approximately 1 serve per day and their dairy intake by 1 2 serve per day. Fish and legume consumption increased by 3 and 2.5 servings per week, respectively. Total meat consumption decreased by 25 g/day, approximately 0.25–0.3 servings. The largest change amongst both groups was a significant decrease in miscellaneous foods. Changes amongst the HabDiet group were otherwise minimal. Table S3 (supplementary material) shows the nutrient intakes, by diet group and gender at baseline and six months, compared to Australian guidelines [32]. Figure 2 shows the mean change in intake for 15 food groups. Based on Australian standar erving sizes [32], those in the MedDiet group increased their fruit, nut and vegetable intakes b pproximately 1 serve per day and their dairy intake by ½ serve per day. Fish and legum onsumption increased by 3 and 2.5 servings per week, respectively. Total meat consumptio ecreased by 25 g/day, approximately 0.25–0.3 servings. The largest change amongst both group was a significant decrease in miscellaneous foods. Changes amongst the HabDiet group wer therwise minimal. Table S3 (supplementary material) shows the nutrient intakes, by diet group an ender at baseline and six months, compared to Australian guidelines [32]. Figure 2. Changes in food intakes from baseline to four months, by four diet group. MedDiet (n = 80), HabDiet (n = 70): (A) changes between 10 and 50 g; (B) changes >50 g/day; and (C) changes <10 g per day. All changes based on the MedDiet group, with the exception of red wine for which the change was based on the HabDiet group. Miscellaneous foods included small goods, cakes, muffins, high fat crackers, pastries, sweet biscuits, high-sugar breakfast cereals, muesli bars, take away foods, chips, chocolate, confectionary <50% sugar, ice cream, beer, port, spirits, white wine. Sugar products included all foods comprising >50% sugars. MedDiet, Mediterraenan diet; HabDiet, Habitual diet. Figure 2. Changes in food intakes from baseline to four months, by four diet group. MedDiet (n = 80), HabDiet (n = 70): (A) changes between 10 and 50 g; (B) changes >50 g/day; and (C) changes <10 g per day. 3.1. A Priori Adherence Score and Checklists Linear mixed effects model w re as the dependent variable, diet and visit as factors, and unstructured covariance used mpare groups. Analyses were intention to treat. MedDiet, Mediterranean diet; HabDiet, habitu Figure 1. Mean adherence score at each time point by diet group. Mean (error bars depict SEM) MedDiet adherence scores of Australian men and women (age ≥65) at baseline, and two and four months after following a MedDiet (n = 80) or their HabDiet (n = 70). MedDiet adherence score based on intakes of 15 food groups calculated from 3-d WFRs, range 0–15 where 15 represents highest possible adherence level. Mean scores at each time point presented. Linear mixed effects model with score as the dependent variable, diet and visit as factors, and unstructured covariance used to compare groups. Analyses were intention to treat. MedDiet, Mediterranean diet; HabDiet, habitual diet; WFR, weighed food record. Nutrients 2017, 9, 534 Nutrients 2017, 9, 534 9 of 20 9 of 2 3.2. Food and Nutrient Intake .2. Food and Nutrient Intake Figure 2 shows the mea All changes based on the MedDiet group, with the exception of red wine for which the change was based on the HabDiet group. Miscellaneous foods included small goods, cakes, muffins, high fat crackers, pastries, sweet biscuits, high-sugar breakfast cereals, muesli bars, take away foods, chips, chocolate, confectionary <50% sugar, ice cream, beer, port, spirits, white wine. Sugar products included all foods comprising >50% sugars. MedDiet, Mediterraenan diet; HabDiet, Habitual diet. Figure 2. Changes in food intakes from baseline to four months, by four diet group. MedDiet (n = 80) HabDiet (n = 70): (A) changes between 10 and 50 g; (B) changes >50 g/day; and (C) changes <10 g pe day. All changes based on the MedDiet group, with the exception of red wine for which the change was based on the HabDiet group. Miscellaneous foods included small goods, cakes, muffins, high fa crackers, pastries, sweet biscuits, high-sugar breakfast cereals, muesli bars, take away foods, chips chocolate, confectionary <50% sugar, ice cream, beer, port, spirits, white wine. Sugar products included all foods comprising >50% sugars. MedDiet, Mediterraenan diet; HabDiet, Habitual diet. Figure 2. Changes in food intakes from baseline to four months, by four diet group. MedDiet (n = 80), HabDiet (n = 70): (A) changes between 10 and 50 g; (B) changes >50 g/day; and (C) changes <10 g per day. All changes based on the MedDiet group, with the exception of red wine for which the change was based on the HabDiet group. Miscellaneous foods included small goods, cakes, muffins, high fat crackers, pastries, sweet biscuits, high-sugar breakfast cereals, muesli bars, take away foods, chips, chocolate, confectionary <50% sugar, ice cream, beer, port, spirits, white wine. Sugar products included all foods comprising >50% sugars. MedDiet, Mediterraenan diet; HabDiet, Habitual diet. Figure 2. Changes in food intakes from baseline to four months, by four diet group. MedDiet (n = 80) HabDiet (n = 70): (A) changes between 10 and 50 g; (B) changes >50 g/day; and (C) changes <10 g per day. All changes based on the MedDiet group, with the exception of red wine for which the change was based on the HabDiet group. Miscellaneous foods included small goods, cakes, muffins, high fa crackers, pastries, sweet biscuits, high-sugar breakfast cereals, muesli bars, take away foods, chips chocolate, confectionary <50% sugar, ice cream, beer, port, spirits, white wine. Sugar products included all foods comprising >50% sugars. 3.2. Food and Nutrient Intake .2. Food and Nutrient Intake Figure 2 shows the mea MedDiet, Mediterraenan diet; HabDiet, Habitual diet. Figure 2. Changes in food intakes from baseline to four months, by four diet group. MedDiet (n = 80), HabDiet (n = 70): (A) changes between 10 and 50 g; (B) changes >50 g/day; and (C) changes <10 g per day. All changes based on the MedDiet group, with the exception of red wine for which the change was based on the HabDiet group. Miscellaneous foods included small goods, cakes, muffins, high fat crackers, pastries, sweet biscuits, high-sugar breakfast cereals, muesli bars, take away foods, chips, chocolate, confectionary <50% sugar, ice cream, beer, port, spirits, white wine. Sugar products included all foods comprising >50% sugars. MedDiet, Mediterraenan diet; HabDiet, Habitual diet. 10 of 20 Nutrients 2017, 9, 534 Table 2. Results of the sensitivity analysis to compare adherence scores using the Mediterranean Diet Score [22]. Table 2. Results of the sensitivity analysis to compare adherence scores using the Mediterranean Diet Score [22]. Adherence Score Adherence Scores p for Interaction Baseline 2 Months 4 Months MedDiet HabDiet MedDiet HabDiet MedDiet HabDiet 15-point score 7.1 ± 1.9 7.4 ± 2.4 9.7 ± 2.4 7.7 ± 2.2 10.6 ± 1.7 7.9 ± 2.5 <0.001 10-point MDS 1 4.2 ± 1.7 4.2 ± 1.6 5.9 ± 1.6 4.1 ± 1.7 6.2 ± 1.4 4.4 ± 1.5 <0.001 10-point MDS 2 2.8 ± 1.4 2.8 ± 1.5 3.7 ± 2.1 2.6 ± 1.5 4.7 ± 1.3 3.0 ± 1.3 <0.001 Values presented are mean ± SD. p for dietxvisit interaction derived from linear mixed effects models to compare MedDiet and HabDiet groups across time. 1 Using baseline sex-specific medians. 2 Using baseline sex-specific medians from the Greek cohort in the EPIC study, from Trichopoulou et al. [22] MDS, Mediterranean Diet Score; MedDiet, Mediterranean diet; HabDiet, Habitual diet. Values presented are mean ± SD. p for dietxvisit interaction derived from linear mixed effects models to compare MedDiet and HabDiet groups across time. 1 Using baseline sex-specific medians. 2 Using baseline sex-specific medians from the Greek cohort in the EPIC study, from Trichopoulou et al. [22] MDS, Mediterranean Diet Score; MedDiet, Mediterranean diet; HabDiet, Habitual diet. Table 3 shows the changes in nutrient intake between baseline and two and four months within and between groups. Table 4 highlights these changes for food intakes. 3.2. Food and Nutrient Intake .2. Food and Nutrient Intake Figure 2 shows the mea Within the MedDiet group, energy intakes did not change, nor did the contribution of protein or alcohol to total energy, vitamin C, folate, vitamin A, β-carotene, potassium, calcium, iron and α-linoleic acid intakes. Energy from saturated fat, cholesterol, sugar, sodium and zinc intakes decreased, while energy from total and MUFA, MUFA:SFA, fibre, vitamin E, linoleic acid and long chain omega-3 fatty acid intake increased significantly. There were no significant within-group differences for the HabDiet group between baseline and four months. At four months, the MedDiet group was consuming more total fat and MUFA, vitamin E, fibre and linoleic acid, and less SFA, sodium and zinc than the control group. While total flavonoid intake was not different between groups at four months, those in the MedDiet group were consuming more total carotenoids, and anthocyanidins were borderline significantly higher in the MedDiet group compared to the control (p = 0.05). Between groups, intakes of extra virgin olive oil, fruits, nuts, legumes, dairy, fish, meats and miscellaneous foods were significantly different at four months (diet*visit p < 0.05). Intakes of extra virgin olive oil, fruits, nuts, legumes, dairy, and fish were higher in the MedDiet group, while meat and miscellaneous food intakes were lower relative to the control group. Originally, 17 participants were allocated to energy levels 3 and 4, while 19 were allocated to energy level 2 and 27 to energy level 1. However, this was adjusted during the two and four week visits based on participant reports of satiety, and adherence levels. After the first month, 12 volunteers were allocated to energy level 1, 25 to energy level 2, 12 to energy level 3 and 31 to energy level 4. Mean energy intakes for energy levels 1, 2, 3 and 4 were 10 MJ, 9.6 MJ, 8.4 MJ and 7.8 MJ at four months, respectively. 3.3. Serum Carotenoids, Erythrocyte Fatty Acids and 24-h Urinary Metabolites Table 5 shows the changes within and between groups for objective markers of adherence, including urinary metabolites, carotenoids and erythrocyte fatty acids. After six months, serum β-carotene and lycopene increased significantly in the MedDiet group compared to the control group. MUFA as a percentage of total erythrocyte fatty acids increased, and SFA as a percentage of total erythrocyte fatty acids decreased. Magnesium excretion was significantly increased in the MedDiet group compared to the control at six months. There were no significant differences between groups for serum β-cryptoxanthin, α-carotene, lutein:zeaxanthin; erythrocyte content of omega-3, omega-6, omega-3:omega-6, and trans fats; or 24-h urinary excretion of sodium, potassium and calcium. For specific long chain fatty acids, there were no between group differences at 6 months, however docosahexaenoic acid (DHA) was significantly higher in the MedDiet group compared with the control at three months. 11 of 20 Nutrients 2017, 9, 534 Table 3. Daily nutrient and flavonoid intake assessed by three-day weighed food record, for the Mediterranean and habitual diet groups. 3.3. Serum Carotenoids, Erythrocyte Fatty Acids and 24-h Urinary Metabolites MedDiet (n = 80) HabDiet (n = 70) Between Group Difference at 4 Months 1 Nutrients 2 Baseline 4 Months p Value Baseline 4 Months p Value Diet*Visit Interaction Difference (95% CI) p Value Energy (kJ) 8847.2 ± 229.7 8777.8 ± 245.5 1.00 8773.9 ± 229.7 8380.1 ± 252.1 0.377 0.41 384.3 (−1070.6, 302.2) 0.27 kJ from protein (%) 19.3 ± 0.37 19.5 ± 0.4 1.00 19.3 ± 0.4 19.3 ± 0.4 1.00 0.96 0.2 (−0.9, 1.3) 0.74 kJ from fat (%) 33.5 ± 0.7 38.7 ± 0.8 <0.001 34.4 ± 0.7 35.6 ± 0.8 0.65 <0.001 3.1 (1.1, 5.2) <0.01 kJ from saturated fat (%) 12.1 ± 0.3 9.1 ± 0.3 <0.001 12.9 ± 0.3 13.1 ± 0.3 1.00 <0.01 −4.0 (−4.9, −3.1) <0.001 kJ from monounsaturated fat (%) 13.2 ± 0.4 19.7 ± 0.5 <0.001 13.2 ± 0.4 14.3 ± 0.5 0.24 <0.001 5.5 (4.2, 6.8) <0.001 kJ from carbohydrate (%) 42.3 ± 0.7 37.8 ± 0.8 <0.001 41.3 ± 0.8 40.3 ± 0.8 0.77 <0.001 −2.5 (−4.7, −0.3) 0.03 kJ from alcohol (%) 4.5 ± 1.1 2.7 ± 1.2 0.40 2.9 ± 1.2 3.4 ± 0.4 1.00 0.47 −0.6 (−3.2, 2.0,) 0.67 Fat as mono (%) 42.5 ± 0.7 54.3 ± 0.7 <0.001 41.3 ± 0.7 43.1 ± 0.7 0.09 <0.001 11.2 (9.3, 13.7) <0.001 Fat as saturated (%) 39.6 ± 0.8 25.3 ± 0.9 <0.001 41.0 ± 0.9 40.0 ± 0.9 1.00 <0.001 −14.7 (−17.1, −12.3) <0.001 Cholesterol (mg/MJ) 33.3 ± 1.4 25.5 ± 1.5 <0.001 34.7 ± 1.5 36.1 ± 1.5 1.00 <0.001 −10.5 (−14.7, −6.3) <0.001 Sugars (g/MJ) 12.5 ± 0.38 11.3 ± 0.36 <0.01 12.1 ± 0.4 11.6 ± 0.38 0.56 0.51 −0.3 (−1.3, 0.8) 0.62 MUFA:SFA 1.2 ± 0.1 2.2 ± 0.1 <0.001 1.1 ± 0.1 1.2 ± 0.1 0.50 <0.001 1.0 (0.9, 1.2) <0.001 Fibre (g/MJ) 3.4 ± 0.1 3.8 ± 0.1 <0.01 3.2 ± 0.1 3.1 ± 0.1 0.80 <0.01 0.7 (0.4, 1.0) <0.001 Vitamin C (mg/MJ) 17.6 ± 1.1 18.9 ± 0.97 1.00 15.9 ± 1.2 14.2 ± 1.0 0.53 0.21 4.7 (1.9, 7.5) <0.01 Vitamin E (mg/MJ) 1.2 ± 0.5 1.9 ± 0.6 <0.001 1.2 ± 0.6 1.3 ± 0.6 0.74 <0.001 0.6 (0.5, 0.8) <0.001 Total folate (µg/MJ) 54.4 ± 2.1 56.1 ± 1.2 1.00 49.8 ± 2.3 47.3 ± 2.0 1.00 0.51 8.8 (3.3, 14.4) <0.01 Total vitamin A equivalents (µg/MJ) 130.4 ± 6.8 140.0 ± 15.4 1.00 121.3 ± 7.3 131.4 ± 16.0 1.00 0.37 8.6 (−35.5, 52.6) 0.70 B-carotene equivalents (µg/MJ) 588.7 ± 40.4 687.4 ± 53.6 0.33 473.4 ± 43.2 467.4 ± 55.6 1.00 0.31 220.0 (67.4, 372.7) <0.01 Sodium (mg/MJ) 264.5 ± 8.8 202.3 ± 6.8 <0.001 267.7 ± 9.4 259.5 ± 7.0 1.00 <0.01 −57.2 (−76.6, −37.9) <0.001 Potassium (mg/MJ) 447.1 ± 10.6 442.5 ± 9.0 1.00 421.6 ± 11.3 403.1 ± 9.3 0.48 0.71 39.3 (13.8, 64.9) <0.01 Calcium (mg/MJ) 109.3 ± 4.0 105.5 ± 3.2 1.00 107.1 ± 4.3 103.9 ± 3.4 1.00 0.62 1.6 (−7.7, 10.8) 0.74 Magnesium (mg/MJ) 50.2 ± 1.3 44.2 ± 2.0 0.01 46.4 ± 1.4 41.8 ± 2.1 0.09 0.48 2.4 (−3.2, 8.1) 0.39 Iron (mg/MJ) 1.61 ± 0.4 1.57 ± 0.4 1.00 1.5 ± 0.4 1.5 ± 0.4 1.00 0.53 0.1 (−0.1, 2.1) 0.07 Zinc (mg/MJ) 1.4 ± 0.4 1.2 ± 0.5 <0.01 1.4 ± 0.4 1.5 ± 0.5 0.09 <0.001 −0.2 (−0.4, −0.1) <0.01 Total long-chain n3 (mg/MJ) 50.4 ± 6.6 89.0 ± 7.1 <0.001 52.9 ± 7.0 35.9 ± 7.3 0.27 <0.001 53.1 (32.9, 73.2) <0.001 Linoleic acid (g/MJ) 1.3 ± 0.6 1.7 ± 0.6 <0.001 1.3 ± 0.6 1.3 ± 0.6 1.00 <0.01 0.4 (0.2, 0.6) <0.001 A-linolenic acid (g/MJ) 0.16 ± 0.01 0.18 ± 0.01 0.68 0.17 ± 0.1 0.14 ± 0.1 0.17 0.07 0.0 (0.0, 0.1) 0.03 Total flavonoids (mg/MJ) 59.7 ± 5.7 64.9 ± 5.8 0.35 68.1 ± 6.0 63.0 ± 6.0 0.04 0.94 3.4 (−16.0, 22.8) 0.73 Anthocyanidins (mg/MJ) 7.8 ± 1.0 10.0 ± 1.0 0.02 7.8 ± 1.1 6.6 ± 1.1 0.20 0.01 3.3 (0.03, 6.66) 0.05 Flavan-3-ols (mg/MJ) 45.0 ± 4.7 47.3 ± 4.8 0.51 52.6 ± 5.0 51.7 ± 5.0 0.83 0.82 −2.1 (−16.3, 20.5) 0.82 Flavanones (mg/MJ) 3.0 ± 0.4 2.3 ± 0.3 0.05 2.6 ± 0.4 1.4 ± 0.3 <0.01 0.59 0.9 (−0.1, 1.8) 0.06 Flavones (mg/MJ) 0.3 ± 0.1 0.6 ± 0.1 0.10 0.4 ± 0.1 0.3 ± 0.1 0.50 0.36 0.6 (−0.2, 1.4) 0.14 Flavonols (mg/MJ) 3.3 ± 0.3 3.6 ± 0.3 0.16 3.2 ± 0.3 3.1 ± 0.3 0.56 0.18 0.5 (−0.2, 1.3) 0.18 Isoflavones (mg/MJ) 0.1 ± 0.0 0.2 ± 0.1 0.26 0.1 ± 0.0 0.1 ± 0.1 0.82 0.48 0.2 (−0.1, 0.4) 0.25 Carotenoids (mg/MJ) 1.6 ± 0.1 2.0 ± 0.1 0.03 1.4 ± 0.1 1.3 ± 0.2 0.46 0.03 0.8 (0.5 1.2) <0.001 Values are mean ± standard error of the mean (SEM). 3.3. Serum Carotenoids, Erythrocyte Fatty Acids and 24-h Urinary Metabolites 1 Linear mixed effects models with diet*visit interaction and unstructured covariance used to determine within and between group differences, α set to 0.05. For between group differences, HabDiet values were subtracted from the MedDiet values, hence negative values indicate MedDiet group had lower intake. 2 Nutrient intakes expressed as either % energy or per MJ (420 kcal) to control for energy intake. Table 3. Daily nutrient and flavonoid intake assessed by three-day weighed food record, for the Mediterranean and habitual diet groups. ke assessed by three-day weighed food record, for the Mediterranean and habitual diet groups. 12 of 20 Nutrients 2017, 9, 534 ups assessed by three-day weighed food record for the Mediterranean and habitual diet groups. Table 4. Daily intake of 15 food groups assessed by three-day weighed food record for the Mediterranean and habitual diet groups. Table 4. Daily intake of 15 food groups assessed by three-day weighed food record for the Mediterranean and habitual diet groups. 3.3. Serum Carotenoids, Erythrocyte Fatty Acids and 24-h Urinary Metabolites MedDiet (n = 80) HabDiet (n = 70) Between Group Difference at 4 Months Foods 1 (g/mJ/Day) Baseline 2 4 Months p Value Baseline 2 4 Months p Value Diet*Visit Interaction Difference (95% CI) p Value Extra virgin olive oil 0.3 ± 0.1 3.6 ± 0.2 <0.001 0.6 ± 0.1 0.8 ± 0.2 1.00 <0.001 2.9 (2.3, 3.4) <0.001 Vegetables 18.2 ± 1.4 25.7 ± 1.6 <0.001 19.0 ± 21.8 21.9 ± 1.6 0.40 0.02 3.8 (−0.6, 8.2) 0.09 White potato 4.8 ± 0.6 3.2 ± 0.6 0.09 5.5 ± 0.7 3.9 ± 0.6 0.09 0.09 −0.7 (−2.4, 0.9) 0.37 Fruits 30.6 ± 2.0 42.4 ± 2.3 <0.001 31.1 ± 2.1 35.6 ± 2.4 0.47 0.04 6.8 (0.2, 13.4) 0.05 Nuts 1.9 ± 0.3 5.6 ± 0.7 <0.001 1.7 ± 0.3 1.9 ± 0.7 1.00 <0.01 3.8 (1.9, 5.6) <0.001 Legumes 2.1 ± 0.5 5.2 ± 0.6 <0.001 1.7 ± 0.5 2.2 ± 0.6 1.00 <0.01 3.0 (1.3, 4.7) <0.01 Bread 8.3 ± 0.5 9.1 ± 0.6 0.26 9.5 ± 0.6 9.0 ± 0.6 1.00 0.22 0.05 (−1.6, 1.7) 0.95 Cereals 8.2 ± 0.8 7.4 ± 0.8 1.00 7.7 ± 0.9 8.3 ± 0.8 1.00 0.40 −0.8 (−3.1, 1.4) 0.46 Dairy (all types) 30.4 ± 2.5 37.5 ± 2.2 <0.01 32.2 ± 2.7 29.8 ± 2.2 0.94 0.01 7.7 (1.6, 13.8) 0.01 Fish and seafood 4.2 ± 0.7 9.2 ± 0.7 <0.001 6.5 ± 0.7 4..8 ± 0.7 0.17 <0.001 4.5 (2.4, 6.5) <0.001 Eggs 2.5 ± 0.4 2.2 ± 0.4 1.00 2.9 ± 0.4 3.2 ± 0.4 1.00 0.76 −1.0 (−2.1, 0.2) 0.09 Meats 8.0 ± 0.8 5.3 ± 0.8 0.03 8.8 ± 0.9 10.7 ± 0.9 <0.01 0.43 −5.4 (−7.8, −3.1) <0.001 Red wine 8.7 ± 1.8 10.7 ± 1.5 0.62 11.9 ± 1.9 7.5 ± 1.6 0.03 0.02 3.2 (−1.2, 7.5) 0.15 Sugars 1.3 ± 0.2 1.1 ± 0.2 0.9 1.3 ± 0.2 1.2 ± 0.2 1.00 0.01 −0.1 (−0.7, 0.5) 0.70 Miscellaneous 32.1 ± 2.1 15.6 ± 1.8 <0.001 34.5 ± 2.2 23.9 ± 1.8 <0.001 0.10 −8.2 (−13.3, −3.2) <0.01 Values are mean ± SEM. 1 Vegetables included all raw, cooked, canned and frozen vegetables including sweet potato. Fruits included all fresh, dried and canned fruit but excluded fruit juice/drinks. Cereals included all rice, pasta, couscous, quinoa, muesli, oats, low fat crisp breads and crackers, and wholegrain breakfast cereals. 3.3. Serum Carotenoids, Erythrocyte Fatty Acids and 24-h Urinary Metabolites Eggs included egg dishes such as quiche. Meats included all red meat, pork and poultry. Sugars included products at least 50% sugar (e.g., honey, jam, some confectionary, table sugar). Miscellaneous included confectionary and discretionary foods, small goods, soy products, special dietary products, muesli bars, sweet bakery products and take away foods. Beverages including water, tea, coffee, soft drink, cordials, milky drinks, fruit and vegetable juices, and alcoholic drinks other than red wine were not included. 2 Group specific baseline means used to calculate 15-point adherence score. Values are mean ± SEM. 1 Vegetables included all raw, cooked, canned and frozen vegetables including sweet potato. Fruits included all fresh, dried and canned fruit but excluded fruit juice/drinks. Cereals included all rice, pasta, couscous, quinoa, muesli, oats, low fat crisp breads and crackers, and wholegrain breakfast cereals. Eggs included egg dishes such as quiche. Meats included all red meat, pork and poultry. Sugars included products at least 50% sugar (e.g., honey, jam, some confectionary, table sugar). Miscellaneous included confectionary and discretionary foods, small goods, soy products, special dietary products, muesli bars, sweet bakery products and take away foods. Beverages including water, tea, coffee, soft drink, cordials, milky drinks, fruit and vegetable juices, and alcoholic drinks other than red wine were not included. 2 Group specific baseline means used to calculate 15-point adherence score. 13 of 20 Nutrients 2017, 9, 534 Table 5. Changes in serum carotenoids, erythrocyte fatty acids and urinary metabolites from baseline to six months1. 3.3. Serum Carotenoids, Erythrocyte Fatty Acids and 24-h Urinary Metabolites MedDiet Group (n = 82) HabDiet Group (n = 70) Between Group Difference 6 Months Baseline 6 Months p Value Baseline 6 Months p Value DietxVisit Interaction Difference (95% CI) p Value β-cryptoxanthin (ng/mL) 56.8 ± 5.6 52.7 ± 6.6 1.00 51.3 ± 5.9 49.5 ± 7.0 1.00 0.77 0.1 (−0.1, 0.2) 0.44 Lycopene (ng/mL) 184.0 ± 13.8 197.4 ± 13.4 1.00 162.1 ± 14.8 135.6 ± 13.7 1.00 <0.01 0.2 (0.1, 0.3) <0.01 α-carotene (ng/mL) 77.6 ± 10.2 125.1 ± 15.6 <0.01 61.6 ± 10.8 55.0 ± 16.1 0.50 <0.01 0.2 (−0.0, 0.4) 0.12 β-carotene (ng/mL) 1287.4 ± 141.4 1543.8 ± 136.1 0.10 858.7 ± 150.8 851.8 ± 136.3 1.00 <0.001 0.2 (0.1, 0.3) <0.001 Lutein:zeaxanthin 489.5 ± 30.6 524.6 ± 33.6 0.40 511.3 ± 32.2 521.8 ± 34.9 1.00 0.30 −0.0 (−0.1, −0.1) 0.65 Total erythrocyte saturated fat (%) 43.3 ± 0.1 42.8 ± 0.1 <0.001 43.5 ± 0.1 43.5 ± 0.1 1.00 <0.001 −0.7 (−1.0, −0.5) <0.001 Total erythrocyte trans−fat (%) 0.44 ± 0.02 0.37 ± 0.02 <0.001 0.4 ± 0.02 0.4 ± 0.02 0.34 <0.001 −0.0 (−0.1, 0.0) 0.08 Total erythrocyte MUFA (%) 18.4 ± 0.1 19.3 ± 0.1 <0.001 18.5 ± 0.1 18.6 ± 0.1 1.00 <0.001 0.8 (0.4, 1.1) <0.001 Total erythrocyte omega-3 (%) 10.8 ± 0.3 10.9 ± 0.3 1.00 10.5 ± 0.3 10.7 ± 0.3 0.30 0.24 0.2 (−0.6, 1.1) 0.62 Docosahexaenoic acid (22:6 n3) (%) 5.8 ± 0.14 6.1 ± 0.13 0.01 5.6 ± 0.14 5.7 ± 0.14 0.29 0.03 0.31 (−0.06, 0.68) 0.10 Eicosapentaenoic acid (20:5 n3) (%) 1.8 ± 0.13 1.8 ± 0.13 1.00 1.7 ± 0.14 1.8 ± 0.14 0.38 0.58 0.01 (−0.37, 0.38) 0.98 Docosapentaenoic acid (22:5 n3) (%) 3.0 ± 0.06 2.9 ± 0.06 <0.001 3.0 ± 0.07 3.0 ± 0.06 1.00 <0.001 −0.11 (−0.29, 0.06) 0.19 Total erythrocyte omega 6 (%) 27.1 ± 0.3 26.7 ± 0.3 0.07 27.2 ± 0.3 27.0 ± 0.3 1.00 0.50 −0.3 (−1.2, 0.5) 0.44 Omega-6:omega-3 from erythrocytes 2.7 ± 0.1 2.6 ± 0.1 0.05 2.8 ± 0.1 2.7 ± 0.1 0.15 0.12 −0.1 (−0.4, 0.1) 0.35 Omega 3 index 1.99 ± 0.3 2.0 ± 0.3 0.02 1.9 ± 0.3 2.0 ± 0.3 0.28 0.03 0.7 (−0.0, 0.2) 0.13 Sodium (mmol/24 h) 113.9 ± 5.4 107.8 ± 4.9 0.85 118.3 ± 5.7 111.5 ± 5.1 0.73 0.30 −5.5 (−20.4, 9.4) 0.47 Potassium (mmol/24 h) 79.7 ± 2.7 80.7 ± 3.0 1.00 77.9 ± 2.9 74.9 ± 3.1 1.00 0.63 5.0 (−3.2, 13.2) 0.24 Calcium (mmol/24 h) 3.6 ± 0.2 4.0 ± 0.2 0.16 4.1 ± 0.2 3.9 ± 0.2 0.99 0.13 −0.0 (−0.7, 0.6) 0.90 Magnesium (mmol/24 h) 4.4 ± 0.2 4.7 ± 0.2 0.17 4.3 ± 0.2 4.2 ± 0.2 1.00 0.12 0.6 (0.0, 1.1) 0.04 Values are mean ± SEM. 3.4. Exit Survey 3.4. Exit Survey 3.4. Exit Survey 3.4. Exit Survey Of 70 MedDiet group participants who completed the study, 68 completed the exit survey. The responses to question 9 are shown in Figure 3. The nuts and yoghurt were very well accepted, the majority of participants enjoying these foods most or all of the time. The restriction of “extra” foods was the least accepted dietary characteristic. Approximately 75% of the group enjoyed tuna and legumes all or most of the time, and they were the least enjoyed foods of the diet. In response to question 10, more than 20% of the sample suggested eating out or with friends, holidays or special occasions were the most difficult times to continue to adhere to the diet. Other top answers included restricting particular foods, e.g., bacon, butter or beer (17%), red meat restrictions (8%), the volume of food (8%) and meeting olive oil requirements (7%). Around 5% of the participants said meeting vegetable requirements was the most difficult aspect to following the diet, while approximately 12% of the sample suggested there were no difficult aspects. Almost half the sample (47%) suggested they would aim to continue with all components, and a further 30% suggested they would continue with most components. Sixteen per cent suggested they would continue with 3 or 4 key components, with 6% suggested they would keep up 1 or 2 components. One respondent did not answer this question. Of 70 MedDiet group participants who completed the study, 68 completed the exit survey. The responses to question 9 are shown in Figure 3. The nuts and yoghurt were very well accepted, the majority of participants enjoying these foods most or all of the time. The restriction of “extra” foods was the least accepted dietary characteristic. Approximately 75% of the group enjoyed tuna and legumes all or most of the time, and they were the least enjoyed foods of the diet. In response to question 10, more than 20% of the sample suggested eating out or with friends, holidays or special occasions were the most difficult times to continue to adhere to the diet. Other top answers included restricting particular foods, e.g., bacon, butter or beer (17%), red meat restrictions (8%), the volume of food (8%) and meeting olive oil requirements (7%). All of the time Most of the time Some of the time None of the time All of the time Most of the time Some of the time None of the time All of the time Most of the time Some of the time None of the time Figure 3. Self-reported enjoyment of requirements of the Mediterranean diet. Australian men’s and women’s (n = 68, age ≥ 65) answers to how well Mediterranean dietary recommendation were enjoyed/managed over a 6 month dietary intervention. Thus, an “all of the time” response indicates the dietary recommendation was enjoyed and/or well managed for the entire study duration. Yoghurt was recommended six times per week, legumes three times per week, tuna one time per week, olive oil (extra virgin) daily, nuts 4–6 times per week, red meat < 1/week, extras three serves or fewer per week and milk (skim) < 1 cup per day. Serving sizes were 170 g, 75 g, 95 g, 1 table spoon, 35 g, 100 g, 600 kJ and 250 mL for yoghurt, legumes, tuna, olive oil, nuts, red meat, extras and skim milk, Figure 3. Self-reported enjoyment of requirements of the Mediterranean diet. Australian men’s and women’s (n = 68, age ≥65) answers to how well Mediterranean dietary recommendation were enjoyed/managed over a 6 month dietary intervention. Thus, an “all of the time” response indicates the dietary recommendation was enjoyed and/or well managed for the entire study duration. Yoghurt was recommended six times per week, legumes three times per week, tuna one time per week, olive oil (extra virgin) daily, nuts 4–6 times per week, red meat < 1/week, extras three serves or fewer per week and milk (skim) < 1 cup per day. Serving sizes were 170 g, 75 g, 95 g, 1 table spoon, 35 g, 100 g, 600 kJ and 250 mL for yoghurt, legumes, tuna, olive oil, nuts, red meat, extras and skim milk, respectively. 3.3. Serum Carotenoids, Erythrocyte Fatty Acids and 24-h Urinary Metabolites 1 Linear mixed effects models with diet*visit interaction and unstructured covariance used to determine within and between group differences, α set to 0.05. For between group differences, HabDiet values were subtracted from the MedDiet values, hence negative values indicate MedDiet group had lower intake. Variables β-carotene, lycopene, α-carotene, β-cryptoxanthin and lutein/zeaxanthin and omega-3 index log10 transformed before analysis due to non-normal distribution. MedDiet, Mediterranean diet; HabDiet, habitual diet; MUFA, monounsaturated fatty acids. Table 5. Changes in serum carotenoids, erythrocyte fatty acids and urinary metabolites from baseline to six months1. hanges in serum carotenoids, erythrocyte fatty acids and urinary metabolites from baseline to six months1. Between Group Difference 6 Months Values are mean ± SEM. 1 Linear mixed effects models with diet*visit interaction and unstructured covariance used to determine within and between group differences, α set to 0.05. For between group differences, HabDiet values were subtracted from the MedDiet values, hence negative values indicate MedDiet group had lower intake. Variables β-carotene, lycopene, α-carotene, β-cryptoxanthin and lutein/zeaxanthin and omega-3 index log10 transformed before analysis due to non-normal distribution. MedDiet, Mediterranean diet; HabDiet, habitual diet; MUFA, monounsaturated fatty acids. 14 of 20 14 of 20 14 of 20 14 of 20 Nutrients 2017, 9, 534 Nutrients 2017, 9, 534 3.4. Exit Survey 3.4. Exit Survey 3.4. Exit Survey 3.4. Exit Survey Around 5% of the participants said meeting vegetable requirements was the most difficult aspect to following the diet, while approximately 12% of the sample suggested there were no difficult aspects. Almost half the sample (47%) suggested they would aim to continue with all components, and a further 30% suggested they would continue with most components. Sixteen per cent suggested they would continue with 3 or 4 key components, with 6% suggested they would keep up 1 or 2 components. One respondent did not answer this question. 0 10 20 30 40 50 60 70 80 90 100 Yoghurt Legumes Tuna Olive oil Nuts Red meat Extras Milk Percentage of respondnts (%) respectively 4. Discussion 4. Discussion According to a 15-point adherence score, self-reported food intakes and objective biological markers, older Australians were able to increase adherence to the MedDiet over a six-month intervention period. There was a significant increase in MUFA:SFA ratio measured from both dietary intake and erythrocyte fatty acids, and a significant increase in olive oil consumption. Serum differences in β-carotene and lycopene between groups indicated those in the MedDiet were consuming more carotenoids after four months. Carotenoid intakes and serum levels of α-carotene According to a 15-point adherence score, self-reported food intakes and objective biological markers, older Australians were able to increase adherence to the MedDiet over a six-month intervention period. There was a significant increase in MUFA:SFA ratio measured from both dietary intake and erythrocyte fatty acids, and a significant increase in olive oil consumption. Serum differences in β-carotene and lycopene between groups indicated those in the MedDiet were consuming more carotenoids after four months. Carotenoid intakes and serum levels of α-carotene both increased significantly in the MedDiet group, suggesting increased intake of fruits and vegetables, which was 15 of 20 Nutrients 2017, 9, 534 reflected by results from the WFRs. Previous studies including the EPIC and Women’s Health Study have shown serum carotenoids to correlate with fruit and vegetable intakes [33–36]. Exit survey results indicate that, while the diet was easy to adhere to, restrictions on discretionary foods were a potential barrier, as were special occasions, holidays and eating out. Nevertheless, the vast majority of volunteers in the intervention group intended to continue most aspects of the diet after finishing the study. y The average 15-point adherence score moved from a medium to high level in the MedDiet group. In a sensitivity analysis, we also calculated the 10-point MDS, using both our sex-specific median intakes, and those from the Greek cohort of the EPIC study. All three scoring methods showed a significant increase in score amongst the MedDiet group over time compared to the control group. Scores increased by 23% according to our score, 22% according to the MDS using our medians, and 21% using the Greek EPIC medians, thus increases were similar across the three systems. Adherence was lower at all three time points when using cut-offs from the Greek data, suggesting that, although adherence did increase, the dietary intake achieved by our participants is still lower than a Mediterranean population. respectively 4. Discussion This could have consequences for health outcomes, however in their meta-analysis, Sofiet al. [6] showed that a two-point increase in the MDS was associated with 10% reduced risk of CVD. In the PREDIMED study adherence scores increased from approximately 8.7 to 10.6 (out of a maximum score of 14) after one year [12], which is again similar to our results, suggesting our findings are clinically relevant. The authors of PREDIMED noted their findings may not be generalizable as the population studied was Spanish, who likely had some pre-existing Mediterranean dietary practices, as well as a culturally Mediterranean setting. Similarly, in the Lyon Diet Heart Trial, where adherence to the MedDiet was high long after the intervention period had ceased, the population were French, which may be culturally more similar to traditional Mediterranean countries than modern Western nations [37]. Epidemiological studies have confirmed a higher adherence to the dietary pattern does result in reduced risk of CVD in non-Mediterranean regions [6,11]. In US populations, closer adherence to the MedDiet has been shown to reduce the risk of all-cause, cardiovascular and cancer mortality in people with pre-existing CVD [20]. Kouris-blazos et al. [18] showed in an older Melbourne cohort of Anglo-Celtic participants, that those adhering more closely to a MedDiet had reduced risk of mortality after 5 years follow-up. Based on such evidence, it is suggested the benefits of the diet are transposable [38]. It is unknown whether the increase in adherence observed in our study will result in long term dietary change, and whether this would result in improved CVD outcomes. Further study is needed in Australia to determine long term ability to adhere to the MedDiet and effects on CVD outcomes. In general, objective data supported the self-reported nutrient intake data, with some exceptions: changes in dietary intakes and urinary excretion were similar except for magnesium, where urinary excretion increased while dietary intake reportedly decreased, although this did not reach significance. Although 24-h urinary excretion of sodium and potassium has been linked to disease outcomes [39], many factors other than diet affect excretion of urinary metabolites on an individual level, such as balance of electrolytes, degree of hydration, race, BMI and gender [40,41]. A recent study showed correlations between dietary sodium and potassium and 24-h urinary excretion were low [41]. respectively 4. Discussion Although there was a significant increase in fish intake and long chain omega-3 fatty acids reported, this was not reflected by change in the total per cent omega-3 within the erythrocyte fatty acids. There was however an increase in DHA intake amongst the MedDiet group, and a between group difference at three months for DHA, suggesting an increased intake of fish compared to the control group. Serra-Majem et al. [42] found in their review that red blood cell phospholipid DHA was a robust measure of intake of DHA. Very little omega-3 fats were contributed by α-linolenic acid; almost all were contributed by fish and seafood. The predominant changes in nutrient intakes appear to be due to increased olive oil intake as the main culinary fat, and reduced intake of discretionary foods. This was reflected by a significant change in MUFA:SFA, vitamin E and SFA intakes. There was also an increased intake of total n-6 fatty acids from plant foods, which may be due to increased nut intake. 16 of 20 16 of 20 Nutrients 2017, 9, 534 Nutrients 2017, 9, 534 This change was not reflected by change in erythrocyte fatty acid profile, where % total omega-6 was unchanged across groups and time. Although the MedDiet is typically moderate in dairy, we reported an increased intake. This could be a consequence of providing yoghurt to the participants, thereby encouraging greater consumption. In addition, baseline intake of dairy servings was below the amount recommended as part of the MedDiet. Thus, a rise in dairy intake could have been expected. Baseline dairy food intakes were similar to national averages obtained from the Australian Health Survey 2011–2012 (1.5 servings per day amongst those aged ≥2 years), which is well below national guideline recommendations [15,32]. Despite the reported increase in dairy consumption, calcium intakes did not significantly change, perhaps because of different types of dairy being consumed (e.g., white cheese instead of yellow cheese). One nutrient where a decreased intake may be of concern is zinc, which likely decreased due to decreased red meat intake. Notably however, both males and females were still meeting the estimated average requirement at four months, suggesting the diet provides adequate zinc [43]. Intake of Mediterranean foods including fruits, nuts, vegetables, legumes and fish increased, and non-core/non-Mediterranean foods decreased. As energy intakes did not change, core foods completely replaced the energy lost from discretionary foods. respectively 4. Discussion Across all four energy levels, energy intake was higher than the prescribed level, suggesting participants underestimated energy intake in their baseline WFRs. This is a common phenomenon with dietary recording [44]. In a literature review, we have previously determined that the “average” MedDiet provides a MUFA:SFA ratio of 2.1, 200 mg vitamin C, 500 µg folate, 3614 mg potassium and 31 g fibre daily—in four months participants in this study achieved a MUFA:SFA ratio of 2.2, vitamin C intake of 165 mg/day, folate intake of 487 µg/day, potassium intake of 3850 mg/day and a fibre intake of 33.5 g/day [26]. This suggests that, in most aspects, participants were able to achieve a diet nutritionally similar to a representative MedDiet by substituting with readily available Australian products. This is important when considering whether the MedDiet can be adopted in Australia. Estimations of total flavonoid intake on the MedDiet vary considerably [26]. In a recent estimation of a Mediterranean-Spanish diet which used similar calculation methods to the present study, cut-offs for the 4th and 5th quintiles were 512 and 670 mg/day, which is similar to the total flavonoid estimation we produced [45]. Interestingly only total carotenoid intakes increased significantly in the MedDiet group relative to the control, with no differences for other flavonoid subclasses. This was mainly driven by an increased intake of anthocyanidins, which are sourced primarily from red wine, although the increase in red wine intake did not reach significance when assessed based on self-reported intakes. Exit survey results indicate that while not all components of the diet were perceived as easy to adopt, the majority of volunteers intended to continue most aspects of the diet after finishing the study. The most difficult things about following the MedDiet were the influences of friends/family, eating out and while holidaying, and food restrictions such as red meat, beer and discretionary foods. Considering the MedDiet restricts intakes of these foods below the national average intake for men and women, this is not surprising [46]. Eating out is likely to challenge due to the abundant non-core food choices available, and lack of Mediterranean options. Special occasions, such as Christmas and holidays, are culturally orientated around particular foods which might explain why participants found these occasions difficult. However, small digressions for such occasions may be acceptable if at other times the MedDiet is followed. respectively 4. Discussion Education is likely required to ensure appropriate choices are made when eating out, or cooking instruction may be necessary to allow persons to cook within the home in accordance with MedDiet principles. Limitations to this study must be acknowledged. Use of a priori adherence scores in non- Mediterranean countries has been criticised, as they do not necessarily compare intakes to a traditional MedDiet, and often exclude important food groups such as nuts [47]. We developed a novel 15-point scoring system which includes six additional food groups compared to the traditional 9-point MDS [23] to help overcome this limitation. Our score is based on baseline mean intakes, thus focuses on movement of dietary intake towards or away from a traditional MedDiet from baseline, rather than 17 of 20 Nutrients 2017, 9, 534 comparing intakes directly to a traditional MedDiet per se. Our sensitivity analysis revealed that although adherence increased amongst the MedDiet group, when intakes were compared to those from a MedDiet country (Greece) adherence was still medium (4.8/9) at four months. The study was amongst older, metropolitan dwelling participants, thus results cannot be extrapolated to more rural or younger populations. Self-reported dietary intakes are subject to important biases, whereby participants may knowingly or unknowingly alter or misrepresent their food and beverage intakes. Three day WFRs do not necessarily capture complete diet, for example red meat intake may have been higher than that reported if participants were consuming it on days not recorded. However, repeat measures, consecutive days and the inclusion of weekend and week days combat these limitations. The subjective markers of adherence were supported by objective markers, suggesting dietary intake data were accurate. Participants were supported by visits with a dietitian held biweekly, which is essential for providing volunteers with tools and education and has been shown to enhance compliance and outcomes in dietary trials [48]. More data are needed to assess whether a free-living population could continue following the MedDiet without trial support. Six months may not be long enough to determine whether a population can permanently adopt substantial dietary changes. Follow-up data from this trial to determine any sustained dietary changes are underway. We did not assess in detail lifestyle factors that could impact adherence such as cost of the diet, only adherence itself. The monetary cost of adhering to the MedDiet in the past has been shown to be higher than consuming a Western diet. respectively 4. Discussion A systematic review included two Spanish studies which both concluded that the MedDiet is more expensive than Western diets [49]. However, in a North American study, while less energy dense, the MedDiet was not associated with increased food costs. In England, one small study (n = 23) found that money spent on foods before and after a three-week MedDiet intervention was not significantly different [19]. In Australia, a recently published article suggested the MedDiet would be cheaper than the typical diet of Australians [50]. The investigators collected seven-day WFRs from a subsample (n = 20) of participants with major depressive disorders. The estimated cost of food per week from the WFRs was $138 per person, compared to an estimated cost of a modified MedDiet at $112 per week per person [50]. Thus, cost may only be a perceived barrier in Australia and education can address this concern. References 1. Keys, A.; Mienotti, A.; Karvonene, M.J.; Aravanis, C.; Blackburn, H.; Buzina, R.; Djordjevic, B.; Dontas, A.; Fidanza, F.; Keys, M.H.; et al. The diet and 15-year death rate in the Seven Countries Study. Am. J. Epidemiol. 1986, 124, 903–915. [CrossRef] [PubMed] 1. Keys, A.; Mienotti, A.; Karvonene, M.J.; Aravanis, C.; Blackburn, H.; Buzina, R.; Djordjevic, B.; Dontas, A.; Fidanza, F.; Keys, M.H.; et al. The diet and 15-year death rate in the Seven Countries Study. Am. J. Epidemiol. 1986, 124, 903–915. [CrossRef] [PubMed] 2. Kromhout, D.; Menotti, A.; Bloemberg, B.; Aravanis, C.; Blackburn, H.; Buzina, R.; Dontas, A.S.; Fidanza, F.; Giampaoli, S.; Jansen, A.; et al. 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Nutr. 1989, 49, 889–894. [PubMed] 4. Willett, W.C.; Sacks, F.; Trichopoulou, A.; Drescher, G.; Ferro-Luzzi, A.; Helsing, E.; Trichopoulos, D. Mediterranean diet pyramid: A cultural model for healthy eating. Am. J. Clin. Nutr. 1995, 61 (Suppl. S6), 1402S–1406S. [PubMed] 4. Willett, W.C.; Sacks, F.; Trichopoulou, A.; Drescher, G.; Ferro-Luzzi, A.; Helsing, E.; Trichopoulos, D. Mediterranean diet pyramid: A cultural model for healthy eating. Am. J. Clin. Nutr. 1995, 61 (Suppl. S6), 1402S–1406S. [PubMed] 5. Roman, B.; Carta, L.; Marinez-Gonzalez, M.A.; Serra-Majem, L. Effectiveness of the Mediterranean diet in the elderly. Clin. Interv. Aging 2008, 3, 97–109. [PubMed] 5. Roman, B.; Carta, L.; Marinez-Gonzalez, M.A.; Serra-Majem, L. Effectiveness of the Mediterranean diet in the elderly. Clin. Interv. Aging 2008, 3, 97–109. [PubMed] 6. Sofi, F.; Macchi, C.; Abbate, R.; Gensini, G.F.; Casini, A. Nutrients 2017, 9, 534 Nutrients 2017, 9, 534 Author Contributions: K.M., J.M.H., J.B. and C.D. conceived and designed the experiments; C.D. performed the experiments; C.D., J.M. and R.W. analysed the data; M.G. contributed reagents/materials/analysis tools; and C.D. wrote the paper. All authors approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. 5. Conclusions In this study, a population of older, free-living Australians made substantial changes to their food and nutrient intake, resulting in changes in erythrocyte MUFA:SFA and serum carotenoids and an increased adherence to the MedDiet. From a feasibility perspective, our results suggest the MedDiet may be an option for dieticians and other health professionals to recommend to patients/clients with health issues. Importantly, data are still needed to confirm the health benefits of the MedDiet in Australia. Supplementary Materials: The following is available online at www.mdpi.com/2072-6643/9/5/534/s1, Table S1, MedLey raw data. Table S1 presented as excel spreadsheets. Spreadsheet contains the following tabs: (1) Food intakes as grams; (2) Food intakes as grams/MJ; (3) Nutrient intakes as grams; (4) Nutrient intakes as grams/MJ; (5) Carotenoids and erythrocyte fatty acids; (6) Urinary metabolites; and (7) Flavonoid intakes. All data presented in long format. Table S2, Cut-offs used to score adherence at baseline, and two and four months for the MedLey study. Table S3, Nutrient intakes by gender and diet group at baseline and four months, compared with Australian estimated average requirements/adequate intakes. Figure S1, The MedLey study CONSORT flow diagram. Acknowledgments: This study was funded by a National Health and Medical Research Council Grant (#APP1050949). The authors acknowledge the following personnel for their support in the recruitment, data collection and analysis during the trial: Louise Massie, Mark Cutting, Catherine Yandell, Crystal Grant, Alissa Knight, Nerylee Watson, Natalie Blanch and Kristina Petersen. We acknowledge Carlene Wilson for her advice regarding appropriate study design. We thank Kate Dyer for her work in analysing dietary intake data to calculate flavonoid intake. The authors acknowledge the following industry partners for donations of foods for the trial: Cobram Estate™; Peanut Company of Australia; The Grains & Legumes Nutrition Council TM; Simplot Australia Pty. Ltd.; Goodman Fielder Ltd; and The Almond Board of Australia. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Structure of the human activated spliceosome in three conformational states
Cell research/Cell Research
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Structure of the human activated spliceosome in three conformational states ofeng Zhang 1, *, Chuangye Yan 1, *, Xiechao Zhan 1, *, Lijia Li 1, Jianlin Lei 1, 2, Yigong Shi 1, 3 1Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sci- ences and School of Medicine, Tsinghua University, Beijing 100084, China; 2Technology Center for Protein Sciences, Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China; 3Institute of Biology, Westlake Institute for Advanced Study, Westlake University, Shilongshan Road No. 18, Hangzhou, Zhejiang 310064, China During each cycle of pre-mRNA splicing, the pre-catalytic spliceosome (B complex) is converted into the activated spliceosome (B act complex), which has a well-formed active site but cannot proceed to the branching reaction. Here, we present the cryo-EM structure of the human B act complex in three distinct conformational states. The EM map allows atomic modeling of nearly all protein components of the U2 small nuclear ribonucleoprotein (snRNP), includ- ing three of the SF3a complex and seven of the SF3b complex. The structure of the human B act complex contains 52 proteins, U2, U5, and U6 small nuclear RNA (snRNA), and a pre-mRNA. Three distinct conformations have been captured, representing the early, mature, and late states of the human B act complex. These complexes differ in the ori- entation of the Switch loop of Prp8, the splicing factors RNF113A and NY-CO-10, and most components of the Nine- Teen complex (NTC) and the NTC-related complex. Analysis of these three complexes and comparison with the B and C complexes reveal an ordered flux of components in the B-to-B act and the B act-to-B * transitions, which ultimately prime the active site for the branching reaction. Keywords: spliceosome; cryo-EM structure; activated spliceosome; B act complex; pre-mRNA splicing Cell Research (2018) 28:307-322. doi:10.1038/cr.2018.14; published online 23 January 2018 components, forming the activated spliceosome (Bact complex) [1]. These newly recruited proteins mainly constitute three classes: the NineTeen complex (NTC), the NTC-related (NTR), and the splicing factors [2-4]. Despite a well-formed active site, the B act complex still cannot catalyze the branching reaction due to spatial sep- aration of the BPS away from the 5′SS [5, 6]. Conversion of the B act complex into the catalytically activated B * complex by the ATPase/helicase Prp2 allows the branch- ing reaction to occur, generating a 5′-exon and an intron lariat-3′-exon intermediate. Structure of the human activated spliceosome in three conformational states The resulting catalytic step I spliceosome (C complex) is converted by Prp16 into the step II activated spliceosome (C * complex), which cata- lyzes the ligation of the 5′-exon with the 3′-exon. ORIGINAL ARTICLE ORIGINAL ARTICLE Cell Research (2018) 28:307-322. www.nature.com/cr *These authors contributed equally to this work. Correspondence: Yigong Shi a, Chuangye Yan b aE-mail: shi-lab@tsinghua.edu.cn bE-mail: yancy@mails.tsinghua.edu.cn Received 28 December 2017; revised 2 January 2018; accepted 8 January 2018; published online 23 January 2018 Introduction Pre-mRNA splicing is executed by a dynamic ribo- nucleoprotein complex known as the spliceosome [1]. The first assembled spliceosome is the pre-catalytic B complex, in which the 5-splice site (5′SS) and the branch point sequence (BPS) of the intron are recognized by U6 and U2 small nuclear ribonucleoproteins (snRNPs), respectively. The B complex lacks a functional active site and cannot proceed to the branching reaction. RNP remodeling of the B complex by the RNA-dependent ATPase/helicase Brr2 results in the dissociation of U1 and U4 snRNPs and the recruitment of about 20 protein Major mechanistic advances through structural biolo- gy have been achieved in the understanding of pre-mR- NA splicing in the past 2 years [7, 8]. The first structure of an intact spliceosome at a near-atomic resolution — that of the Schizosaccharomyces pombe (S. pombe) ILS complex at 3.6 Å [9, 10] — reveals a conserved overall *These authors contributed equally to this work. Correspondence: Yigong Shi a, Chuangye Yan b aE-mail: shi-lab@tsinghua.edu.cn bE-mail: yancy@mails.tsinghua.edu.cn Received 28 December 2017; revised 2 January 2018; accepted 8 January 2018; published online 23 January 2018 Structure of the human activated spliceosome 308 I, which has 1 464 033 particles [24] (Supplementary information, Figure S2). Following three parallel runs of multi-reference three-dimensional (3D) classification and subsequent local 3D classifications, 49 218 particles yielded a reconstruction of the human B act complex at an average resolution of 4.8 Å. A follow-up 3D classifica- tion with a soft mask in the RNF113A region led to the identification of two major conformational states at av- erage resolutions of 5.1 Å and 6.5 Å on the basis of the FSC value 0.143 (Supplementary information, Figures S2 and S4; Tables S1 and S2). As will be detailed later, the 5.1-Å and 6.5-Å reconstructions represent the mature and late human B act complexes, respectively. organization of the spliceosome and a conserved spatial arrangement of the splicing active site [11]. Subsequent cryo-EM structures of the Saccharomyces cerevisiae (S. cerevisiae) and S. pombe spliceosomes at different stag- es of the splicing cycle provide important mechanistic information [5, 6, 12-20]. In contrast to the yeast spliceo- some, structural information on the human spliceosome has been slow to emerge, in part due to its considerably more dynamic nature. Spliceosome isolation and electron microscopy Spliceosome isolation and electron microscopy p py An in vitro splicing assay was employed to assemble the human spliceosomes on an intact, synthetic pre-mR- NA. The spliceosomal sample (named sample I hereafter) were found to contain a mixture of the human B act, C, and C * complexes [24]. The cryo-EM structure of the human C complex was determined from this sample [24]. The RNP remodeling from the B to the B act complex involves flux of several dozen protein and RNA components and is thought to occur in distinct steps [20, 21]. To gain insights into this dynamic process, we deleted the 3′-exon from the synthetic pre-mRNA with only 19 nucleotides down- stream of the BPS such that Prp2 is unable to grab the RNA sequences for the B act-to-B * conversion [25]. Using the truncated pre-mRNA, we prepared a second batch of the cryo-EM sample (sample II, hereafter). Unlike sample I, sample II is predicted not to contain any spliceosomes beyond the B act complex and thus may yield information on the assembly of the human B act complex. Sample II was purified by affinity chromatography followed by glycerol gradient centrifugation (Supplementary infor- mation, Figure S1A). To maintain the structural integrity of the spliceosomes, chemical crosslinking by glutaral- dehyde was applied to the sample during centrifugation. After removal of the glycerol, sample II was examined by negative staining EM (Supplementary information, Figure S1B) and used for cryo-EM sample preparation. Micrographs were collected using the K2 Summit detec- tor mounted on a Titan Krios microscope (Supplementary information, Figure S1C). Introduction At present, we only have the struc- tures of the human B complex at 9.9 Å [21] and the hu- man C* complex at 5.9 Å [22] and 3.8 Å [23]. In this manuscript, we report the cryo-EM structures of the human B act complex in three distinct compositional and conformational states at resolutions of 4.9, 5.1, and 6.5 Å. These structures allow mechanistic understanding of the dynamic steps surrounding formation of the hu- man B act complex and its transitions from the B complex and to the B * complex. Next, we processed the data set from sample II, which yielded 629 472 particles (Supplementary information, Figure S3). As anticipated, only one dominant spliceo- somal complex — the B act complex — is present. Us- ing a similar data processing strategy, 96 523 particles yielded a reconstruction of the early B act complex at an average resolution of 4.9 Å (Supplementary information, Figures S3 and S4; Tables S1 and S2). Despite apparent differences among the three conformational states of the human B act complex (Supplementary information, Figure S5), they share the same structure in the SF3b region. Combining both data sets, we improved the local resolution to 4.2 Å in the SF3b region (Supplementary information, Figures S3, S4A and S6). Atomic modeling of the human B act complexes was facilitated by the local resolutions of about 4.0-4.5 Å at the core and the SF3b region of the B act complex (Supplementary information, Figures S4B and S6). In addition, the atomic coordinates of the yeast B act [5] and the human C * complex [23] greatly expedited modeling of the human B act complexes. Overall structure The final model of the human mature B act complex contains 15 479 amino acids from 52 proteins and 414 nucleotides from three snRNAs and the pre-mRNA (Figure 1A; Supplementary information, Tables S1 and S2), with a combined molecular weight of about 1.8 mega-Daltons. The 52 protein components include all 11 from U5 snRNP, 19 from U2 snRNP, five from the NTC, seven from the NTR, three from the retention and splicing (RES) complex (SNIP1, Bud13, and RBMX2), three splicing factors (SRm300, Cwc22, and RNF113A), two peptidyl prolyl isomerases (PPIs, NY-CO-10, and CypE), the ATPase/helicase Prp2, and the step II factor Prp17. The U2 snRNP includes all seven proteins of the SF3b complex (SF3b155, SF3b145, SF3b130, SF3b49, SF3b14a/p14, SF3b14b, and SF3b10), three proteins of the SF3a complex (SF3a120, SF3a66, and SF3a60), U2 snRNA, and nine proteins of the U2 snRNP core that We first processed the data set derived from sample SPRINGER NATURE | Cell Research | Vol 28 No 3 | March 2018 Xiaofeng Zhang et al. 309 Xiaofeng Zhang et al. 309 Xiaofeng Zhang et al. 309 et al. 309 g g 3 Figure 1 Cryo-EM structure of the human activated spliceosome (the B act complex). (A) Two views of the human mature B act com- plex. The protein and RNA components are color-coded and tabulated below the images. The structure of the mature B act com- plex shown here includes 52 proteins, three snRNAs, and one pre-mRNA, with a combined molecular mass of about 1.8 MDa. U2, U5, and U6 snRNAs are colored marine, orange, and green, respectively. Pre-mRNA is colored red. This coloring scheme is preserved throughout this manuscript. (B) Structural comparison between the human and yeast B act complexes [5, 6]. For protein components, only those that are unique in either spliceosome are colored. All shared protein components are shown grey. All structural images were created using PyMol [49]. 309 Figure 1 Cryo-EM structure of the human activated spliceosome (the B act complex). (A) Two views of the human mature B act com- plex. The protein and RNA components are color-coded and tabulated below the images. The structure of the mature B act com- plex shown here includes 52 proteins, three snRNAs, and one pre-mRNA, with a combined molecular mass of about 1.8 MDa. U2, U5, and U6 snRNAs are colored marine, orange, and green, respectively. Pre-mRNA is colored red. Overall structure Compared to the mature B act complex, the late B act complex no longer contains the splicing factors RN- F113A (Cwc24 in yeast) and NY-CO-10 (Cwc27 in yeast) (Figure 3B; Supplementary information, Figure S10). This structural finding is consistent with the bio- chemical observation that these two proteins only tran- siently associate with the spliceosome and are released during the B act-to-B * transition [27]. Intriguingly, these two splicing factors are strongly present in the early B act complex, suggesting a role of RNF113A in organizing the active site during spliceosome activation [28]. Care- ful examination reveals that, compared to the early B act complex, the EM density for these two splicing factors is already weakened in the mature B act complex (Supple- mentary information, Figure S10B and S10C). This anal- ysis further suggests that, during the B act-to-B * transition, the splicing factors RNF113A and NY-CO-10 are likely released ahead of all other components. In the early and mature B act complexes, the guanine base of G1 stacks closely against the aromatic side chains of Phe213 and Phe219 from RNF113A (Figure 3D). These two aromatic residues, together with Lys218 that also stabilizes the 5′SS, come from the zinc-binding domain of RNF113A and are invariant in the yeast orthologues (Figure 3E). Because RNF113A directly protects the 5′-end guanine base of the 5′SS, its release may signal the beginning phase of the B act-to-B * transition. Therefore, the late B act complex likely represents the state of the spliceosome just preceding its transition to the B * complex through the action of Prp2. Because the early B act complex was obtained using a shortened pre-mRNA, Prp2 may be required for the conversion of the early to the mature and late B act complexes. Intriguingly, the N-terminus of RNF113A is buried in the cleft between the endonucle- ase-like domain and the N-domain of Prp8 (Supplemen- tary information, Figure S10A and S10B); therefore the The RNA elements in the human mature Bact com- plex adopt a generally similar conformation as that in the yeast B act complex [5, 6] (Figure 2A). The similarity extends to the fine local conformations of the active site RNA elements (Figure 2B). One notable difference is the helix II of the U2/U6 duplex, which is bent by about 40° in the human B act complex relative to that in yeast (Figure 2A). Overall structure This coloring scheme is preserved throughout this manuscript. (B) Structural comparison between the human and yeast B act complexes [5, 6]. For protein components, only those that are unique in either spliceosome are colored. All shared protein components are shown grey. All structural images were created using PyMol [49]. www.cell-research.com | Cell Research | SPRINGER NATURE Structure of the human activated spliceosome 310 only interact with U2 snRNA (U2-A′, U2-B′′, and the heptameric U2 Sm complex) (Figure 1A). only interact with U2 snRNA (U2-A′, U2-B′′, and the heptameric U2 Sm complex) (Figure 1A). the mature and late B act complexes, but not in the early B act complex (Figure 3B; Supplementary information, Figure S8). In addition, four proteins of the NTC (Prp19, Syf1, Spf27, and Cdc5), two components of the NTR (PPIL1 and Aquarius), and the PPI protein CypE are ful- ly loaded in the mature and late B act complexes, but not in the early B act complex (Supplementary information, Figure S9). Perhaps most importantly, the Switch loop in Prp8 of the mature and late B act complexes is positioned identically as that in the C or C * complex [23, 24] and in- teracts with the splicing factor SRm300 (Cwc21 in yeast) (Figure 3C). In contrast, SRm300 is yet to be loaded into the early B act complex and the Switch loop remains flexible with no obvious EM density. Collectively, these structural differences unequivocally identify the prema- ture nature of the early B act complex. The overall appearance of the human mature B act complex closely resembles that of the S. cerevisiae B act complex [5, 6] (Figure 1B). Compared to the S. cerevi- siae complex [5], the structure of the human mature B act complex contains 16 additional protein components: SF3a120 and SF3a60 of the SF3a complex, SF3b14a/ p14 of the SF3b complex, nine proteins of the U2 snRNP core, U5-40K of the U5 snRNP, and three proteins of the NTR (Aquarius, RBM22, and PPIL1) (Figure 1B). Of these proteins, RBM22 appears to have arisen from a fusion event between the two yeast NTR proteins Cwc2 and Ecm2 [26]. The N-terminal zinc-binding domain and the C-terminal RRM domain of RBM22 share significant sequence homology with Ecm2 and Cwc2, respectively. Overall structure Another marked difference is an extra turn of the U6/intron duplex in the human B act complex beyond the U6/5′SS duplex, which results in the separation of the downstream human intron sequences away from those in S. cerevisiae by up to 40 Å (Figure 2C). In addition, sim- ilar to the human C * complex [23], the intron sequences are locked by RBM22 through a positively charged cen- tral cavity in the mature and late B act complexes, but not in the early B act complex (Supplementary information, Figure S7). Because the loading of RBM22 occurs in the transition from the early to mature B act complex, RBM22 must undergo partial unfolding to enclose the intron se- quences that are already bound in the early B act complex. Three conformational states of the human B act complex Three conformational states of the human B complex Despite nearly identical conformation of the snRNA elements and the pre-mRNA, the early, mature, and late B act complexes can be conclusively differentiated on the basis of their protein components (Figure 3A). First, the splicing factor Prp17 and the NTR proteins RBM22 and G10 (Bud31 in yeast) are fully loaded in the mature and late B act complexes, but not in the early B act complex (Figure 3A; Supplementary information, Figure S7). These three proteins — G10, Prp17, and RBM22 — help stabilize the active site RNA elements and are present in the human C and C * complexes [23, 24]. Their absence strongly suggests the premature nature of the early B act complex. Second, the N-terminal domain (NTD) of the SF3a component SF3a66 (Prp11 in yeast) is present in SPRINGER NATURE | Cell Research | Vol 28 No 3 | March 2018 Xiaofeng Zhang et al. 311 Figure 2 The RNA elements and the splicing active site of the human mature B act complex. (A) Structure of the RNA ele- ments in the core of the human mature B act complex. The color-coded RNA elements of the human B act complex are shown in the left panel, and their superposition with those of the S. cerevisiae B act complex [5] is displayed in the right panel. All yeast RNA elements are colored grey. The helix II of the U2/U6 duplex in the human B act complex is bent relative to that in the yeast complex. (B) Structural overlay of the active site RNA elements between the human and S. cerevisiae B act complexes [5]. (C) The U6/intron duplex in the human B act complex is considerably longer than that in the S. cerevisiae B act complex [5]. Figure 2 The RNA elements and the splicing active site of the human mature B act complex. (A) Structure of the RNA ele- ments in the core of the human mature B act complex. The color-coded RNA elements of the human B act complex are shown in the left panel, and their superposition with those of the S. cerevisiae B act complex [5] is displayed in the right panel. All yeast RNA elements are colored grey. The helix II of the U2/U6 duplex in the human B act complex is bent relative to that in the yeast complex. Three conformational states of the human B act complex (B) Structural overlay of the active site RNA elements between the human and S. cerevisiae B act complexes [5]. (C) The U6/intron duplex in the human B act complex is considerably longer than that in the S. cerevisiae B act complex [5]. domain and Bud13 have been dislocated (Figure 3F), and the endonuclease-like domain of Prp8 also undergoes a 45° rotation (Figure 3G). release of RNF113A requires conformational changes in Prp8, which is confirmed in the late B act complex. The compositional changes in the core of the spli- ceosome also cause pronounced conformational and positional shifts for the surrounding protein components. For example, in the mature B act complex, the RNaseH- like domain of Prp8 interacts with the endonuclease-like domain, which directly binds to NY-CO-10 (Figure 3F). The RNaseH-like domain also associates with Bud13 of the RES complex. With the dissociation of RNF113A and NY-CO-10 in the late B act complex, the RNaseH-like The SF3a and SF3b complexes The SF3a and SF3b complexes SF3a120 adopts an all-α-helical conformation and associates with U2-A′ (Lea1 in yeast) and the U2 heptameric Sm complex. Similar to that in the S. cerevisiae Bact complex [5], the N-terminus of SF3a66 reaches into the active site and directly contributes to the coordination of the G1 nucleotide of the 5′SS. previous assignment [34, 35], SF3b14a/p14 is located at the periphery, not the center, of the SF3b complex in the human B act complex and is surrounded by three extend- ed N-terminal helices and N-terminal HEAT repeats of SF3b155 (Figure 4C and 4D). ( g ) Other than SF3b14a/p14, the other components of the human SF3b complex are located in generally the same positions as those of the S. cerevisiae SF3b complex [5, 6, 34] (Figure 4C). Similar to its S. cerevisiae orthologue Hsh155, SF3b155 contains an N-terminal helix-loop-he- lix (N-HLH) and 20 HEAT repeats (Supplementary information, Figure S12A). The N-HLH domain is sand- wiched between the RT Finger/Palm and the Linker do- mains of Prp8, and interacts with SKIP and components of the RES complex (Supplementary information, Figure S12B). Compared to Hsh155, SF3b155 contains two ex- tra sequence elements: a Trp-rich motif and a p14-bind- ing motif (Supplementary information, Figure S12A). The extended p14-binding sequences also interact with SKIP, SNIP of the RES complex, and the RT Finger/ Palm domain of Prp8 (Supplementary information, Fig- ure S12C). The HEAT repeats, each comprising a pair of anti-parallel α-helices, constitute a left-handed super- helical structure and serves as the central scaffold of the SF3b complex by interacting with a number of protein and RNA components (Supplementary information, Fig- ure S12D). The SF3b complex directly recognizes the BPS and surrounding intron sequences [29-31]. In the human B act complex, all seven components of the SF3b are structur- ally resolved, including SF3b155 (Hsh155 in S. cerevisi- ae), SF3b145 (Cus1 in S. cerevisiae), SF3b130 (Rse1 in S. cerevisiae), SF3b49 (Hsh49 in S. cerevisiae), SF3b14a/ p14, SF3b14b (Rds3 in S. cerevisiae), and SF3b10 (Ysf3 in S. cerevisiae) [32]. These seven proteins assemble into a compact subcomplex (Figure 4C). Importantly, SF3b14a/p14 is unique to the human spliceosome and absent in S. cerevisiae B act complex [33]. In contrast to SF3b10 binds the C-terminal α-helices of SF3b155, whereas SF3b145 in an extended conformation stabilizes six HEAT repeats at the C-terminus of SF3b155 on the outside of the superhelical structure (Figure 4C). The SF3a and SF3b complexes SF3b49 Figure 3 The early, mature, and late B act complexes represent three different conformational states of the human spliceo- some. (A) Structural comparison between the early B act complex (left panel) and the mature B act complex (right panel). Shown here are only the RNA elements and the protein components that undergo dynamic changes in the core of the spliceosome in the transition of the early to mature B act complex. Compared to that of the early B act complex, the core of the mature B act complex contains four additional proteins: G10, Prp17, RBM22, and the N-terminal domain (NTD) of the SF3a component SF3a66. (B) Close-up views on the early (left panel), mature (middle panel), and late (right panel) B act complexes. Notably, the splicing factors RNF113A (Cwc24 in S. cerevisiae) and NY-CO-10 (Cwc27 in S. cerevisiae) are loaded in the early and the mature, but not the late, B act complexes. On the other hand, SF3s66 is loaded in the mature and late B act complexes, but not the early B act complex. (C) A close-up comparison of the Switch loop regions between the early and mature B act complex- es. The Switch loop of Prp8 is stabilized by an extended sequence (named 1135-loop) and the splicing factor SRm300 (Cwc21 in S. cerevisiae) in the mature B act complex (right panel). SRm300 is absent and the 1135-loop is shifted away in the early B act complex (left panel); consequently the Switch loop is flexible and remains unidentified. (D) A close-up view on the recognition of the guanine nucleotide (G1) at the 5-end of the 5′-splice site (5′SS) by the splicing factor RNF113A. The stacking of the guanine base against the aromatic rings of Phe213 and Phe219 of RNF113A is reminiscent of that in the yeast B act complex [5]. (E) Sequence alignment between the human RNF113A and its yeast orthologues Cwc24 (S. cerevisiae) and Cwf24 (S. pombe). The three key residues involved in recognition of G1 of the 5′SS (Phe213, Lys218, and Phe219) are highly con- served. (F) Close-up views on the role of the endonuclease-like domain and the RNaseH-like domain of Prp8 in the mature and late B act complexes. In the mature B act complex (left panel), both domains of Prp8 appear to stabilize the binding of RN- F113A and NY-CO-10 in the spliceosome. The RNaseH-like domain also binds Bud13 of the RES complex. The SF3a and SF3b complexes The SF3a and SF3b complexes are major constituents of the U2 snRNP. The SF3a complex plays an important role in the formation of the splicing active site in the B act complex and interacts with the SF3b complex and the U2 snRNP core (Supplementary information, Figure S11). Only one component of the SF3a complex — Prp11 in www.cell-research.com | Cell Research | SPRINGER NATURE Structure of the human activated spliceosome he human activated spliceosome 312 SPRINGER NATURE | Cell Research | Vol 28 No 3 | March 2018 Xiaofeng Zhang et al. 313 S. cerevisiae (SF3a66 in human) — was structurally resolved in the yeast spliceosome [5]. In the structure of the human B act complex, all three components — SF3a120, SF3a66, and SF3a60 — are unambiguously identified (Figure 4A). All three proteins exhibit extend- ed conformations, with SF3a60 bridging the gap between SF3a120 and SF3a66. Three α-helices at the N-terminal half of SF3a60 closely interact with the α-helices at one end of SF3a120, stabilizing its extended conformation. Subsequently, an α-helix of SF3a60 directly contacts the β-sandwich domain of SF3a66, with the ensuing extend- ed sequences of SF3a60 wrapping around the β-sandwich (Figure 4A and 4B). Notably, SF3a66 is the only SF3a protein that specifically recognizes an RNA element — the U2/intron duplex. SF3a120 adopts an all-α-helical conformation and associates with U2-A′ (Lea1 in yeast) and the U2 heptameric Sm complex. Similar to that in the S. cerevisiae Bact complex [5], the N-terminus of SF3a66 reaches into the active site and directly contributes to the coordination of the G1 nucleotide of the 5′SS. S. cerevisiae (SF3a66 in human) — was structurally resolved in the yeast spliceosome [5]. In the structure of the human B act complex, all three components — SF3a120, SF3a66, and SF3a60 — are unambiguously identified (Figure 4A). All three proteins exhibit extend- ed conformations, with SF3a60 bridging the gap between SF3a120 and SF3a66. Three α-helices at the N-terminal half of SF3a60 closely interact with the α-helices at one end of SF3a120, stabilizing its extended conformation. Subsequently, an α-helix of SF3a60 directly contacts the β-sandwich domain of SF3a66, with the ensuing extend- ed sequences of SF3a60 wrapping around the β-sandwich (Figure 4A and 4B). Notably, SF3a66 is the only SF3a protein that specifically recognizes an RNA element — the U2/intron duplex. The SF3a and SF3b complexes In the late B act complex (right panel), RNF113A and NY-CO-10 have been dissociated, leading to the dislocation of the RNaseH-like domain and Bud13. (G) Superposition of the endonuclease-like domain of Prp8 between the mature and late B act complexes. The core machineries of the two complexes are aligned. www.cell-research.com | Cell Research | SPRINGER NATURE 4 ucture of the human activated spliceosome Figure 4 Structures of the SF3a and SF3b complexes. (A) Structure of the SF3a and SF3b complexes in the context of key surrounding components. In the left panel, the SF3a and SF3b complexes are colored cyan and yellow, respectively. The U2 snRNA, the RES complex, and Prp2 are colored blue, green, and teal, respectively. The RNA elements are displayed for orientation. In the right panel, the individual components of the SF3a and SF3b complexes are color-coded and labeled. The SF3a complex consists of three proteins: SF3a60, SF3a66, and SF3a120. The SF3b complex comprises seven proteins SF3b10, SF3b14a/p14, SF3b14b, SF3b49, SF3b130, SF3b145, and SF3b155. (B) A close-up view on the SF3a complex and its interactions with the U2 snRNP subcomplex involving U2 Sm ring. (C) A close-up view on the SF3b complex and the interactions among its constituents. The structure is shown in the left panel and the cartoon representation is displayed in the right panel. (D) A close-up view on the components SF3b14b and SF3b14a/p14 of the SF3b complex. (E) A close-up view on the 3′-end sequences of the pre-mRNA and nearby protein components. The 3′-end sequences of the pre-mRNA are bound by RBMX2 of the RES complex. The dotted lines leading to the RNA-binding groove of Prp2 indicate the path of the RNA se- d t f th l t d d l tid i th t t Structure of the human activated spliceosome 314 Figure 4 Structures of the SF3a and SF3b complexes. (A) Structure of the SF3a and SF3b complexes in the context of key surrounding components. In the left panel, the SF3a and SF3b complexes are colored cyan and yellow, respectively. The U2 snRNA, the RES complex, and Prp2 are colored blue, green, and teal, respectively. The RNA elements are displayed for orientation. In the right panel, the individual components of the SF3a and SF3b complexes are color-coded and labeled. The SF3a complex consists of three proteins: SF3a60, SF3a66, and SF3a120. The SF3a and SF3b complexes The SF3b complex comprises seven proteins SF3b10, SF3b14a/p14, SF3b14b, SF3b49, SF3b130, SF3b145, and SF3b155. (B) A close-up view on the SF3a complex and its interactions with the U2 snRNP subcomplex involving U2 Sm ring. (C) A close-up view on the SF3b complex and the interactions among its constituents. The structure is shown in the left panel and the cartoon representation is displayed in the right panel. (D) A close-up view on the components SF3b14b and SF3b14a/p14 of the SF3b complex. (E) A close-up view on the 3′-end sequences of the pre-mRNA and nearby protein components. The 3′-end sequences of the pre-mRNA are bound by RBMX2 of the RES complex. The dotted lines leading to the RNA-binding groove of Prp2 indicate the path of the RNA se- quences downstream of the last ordered nucleotide in the structure. Figure 4 Structures of the SF3a and SF3b complexes. (A) Structure of the SF3a and SF3b complexes in the context of key surrounding components. In the left panel, the SF3a and SF3b complexes are colored cyan and yellow, respectively. The U2 snRNA, the RES complex, and Prp2 are colored blue, green, and teal, respectively. The RNA elements are displayed for orientation. In the right panel, the individual components of the SF3a and SF3b complexes are color-coded and labeled. The SF3a complex consists of three proteins: SF3a60, SF3a66, and SF3a120. The SF3b complex comprises seven proteins SF3b10, SF3b14a/p14, SF3b14b, SF3b49, SF3b130, SF3b145, and SF3b155. (B) A close-up view on the SF3a complex and its interactions with the U2 snRNP subcomplex involving U2 Sm ring. (C) A close-up view on the SF3b complex and the interactions among its constituents. The structure is shown in the left panel and the cartoon representation is displayed in the right panel. (D) A close-up view on the components SF3b14b and SF3b14a/p14 of the SF3b complex. (E) A close-up view on the 3′-end sequences of the pre-mRNA and nearby protein components. The 3′-end sequences of the pre-mRNA are bound by RBMX2 of the RES complex. The dotted lines leading to the RNA-binding groove of Prp2 indicate the path of the RNA se- quences downstream of the last ordered nucleotide in the structure. SPRINGER NATURE | Cell Research | Vol 28 No 3 | March 2018 Xiaofeng Zhang et al. 315 of the essential features of the B act-to-B * transition. The SF3a and SF3b complexes De- spite the flux of more than one dozen proteins, the over- all appearance of the human B act complex is similar to that of the C complex [24], particular in the core region and on the side of the NTC core and U5 snRNP. binds SF3b145 from the outside and interacts with the upstream sequences of the BPS, stabilizing the U2/BPS duplex (Figure 4C and 4D). The N-terminal and C-termi- nal WD40 domains of the Y-shaped SF3b130 sandwich SF3b10 and the C-terminal α-helix of SF3b155. This structural arrangement places the C-terminal WD40 domain of SF3b130 in direct contact with one end of SF3b145. SF3b14b is bound in the hollow center of the SF3b155 superhelical structure and directly interacts with the N-terminal WD40 domain of SF3b130. Due to the dramatic remodeling, components of the spliceosome have undergone major positional adjust- ment. Brr2, e.g., is rotated 90° and translocated by about 90 Å in the B-to-B act transition, and is swirled and shift- ed by approximately 190 Å in the B act-to-C transition (Figure 5D). The entire SF3b complex undergoes a 70° rotation followed by a 120-Å translocation in the B-to- B act transition (Figure 5E). Intriguingly, the sequences near the 3′-end of U2 snRNA form two short stems loop structures known as IIa and IIb in the B complex and remain unchanged in the B act complex; however, these sequences constitute a long stem loop known as IIc in the C complex (Supplementary information, Figure S13). This structural finding is consistent with the biochemical observation that U2 IIa promotes spliceosome assembly whereas U2 IIc facilitates the branching reaction [45-47]. The U2/BPS duplex is bound to SF3b155 through a lateral opening of its superhelical structure (Figure 4D). The RNA sequences downstream of the BPS traverse through the hollow center of the SF3b155 spiral, contact- ing residues from both SF3b14b and SF3b155, and come out of the other side of the spiral. The following intron sequences skim over the surface of RBMX2 (Snu17 in S. cerevisiae) of the RES complex (Figure 4E). The human RES complex, with a critical role in the splicing and re- tention of pre-mRNA [36, 37], consists of SNIP, RBMX2, and Bud13 [38] and closely interacts with the SF3b com- plex. The RNA sequences downstream of those bound by RBMX2 would presumably reach the RNA-binding groove of the ATPase/helicase Prp2 (Figure 4E). Discussion In S. cerevisiae, only about 4% of the protein-encod- ing genes contain introns [48]. In contrast, most of the protein-encoding genes in the human genome contain introns. Pre-mRNA splicing in human is considerably more complex than that in yeast and is subject to more stringent regulation. Accordingly, the human spliceo- some is compositionally and conformationally more dy- namic compared to the yeast spliceosome. In this study, using synthetic pre-mRNA in the absence or presence of the 3′-exon, we were able to obtain two samples for cryo-EM analysis. The sample prepared using the intact pre-mRNA gave rise to the Bact, C, and C * complexes [24], of which the B act complex represent the mature and late states. In contrast, the sample prepared using the 3′-exon-deleted pre-mRNA only yielded one dominant spliceosome species — the early B act complex. The B-B act-C transition The spliceosomal B-to-B act transition, driven by the ATPase/helicase Brr2 [39, 40], is particularly dramatic, involving dissociation of the tri-snRNP-specific proteins and the entire U4 snRNP and recruitment of the NTC and NTR proteins (Figure 5A and 5B). Consequently, the overall appearance of the human B act complex bears little resemblance to that of the human B complex [20, 21]. The B act-to-B * transition, propelled by the ATPase/heli- case Prp2 [41], is less dramatic compared to the B-to-B act transition but involves flux of considerably more proteins than the C-to-C * and P-to-ILS transitions, which is driven by the ATPase/helicases Prp16 and Prp22, respectively [42-44]. Virtually all components of the SF3a and SF3b complexes, along with Prp2 and the splicing factors RN- F113A and NY-CO-10, are dissociated in the B act-to-B * transition. Prp16 and the step I factors CCDC49 and CCDC94 are recruited into the B * complex. At present, the B * complex remains the only structurally unchar- acterized spliceosome during the splicing cycle. Fortu- nately, the structure of the B * complex is predicted to be nearly identical to that of the C complex except in the ac- tive site region surrounding the 5′SS and the BPS where the branching reaction occurs [12]. There is no change of protein components between the B * and C complexes. Therefore, structural comparison between the B act and C complexes (Figure 5B and 5C) should recapitulate many The definition for these three conformational states of the B act complex is justified not only by the method of spliceosome assembly but also by the actual compo- sitions of the spliceosome (Figure 3). Importantly, the Switch loop of Prp8 is positioned similarly as that of the human C complex [24] only in the mature/late, but not the early, B act complex. The splicing factor SRm300, which stabilizes the Switch loop, is loaded similarly as that of the human C complex [24] only in the mature/ late, but not the early, B act complex. Another structural observation is the presence of RBM22 in the mature/late, but not the early, B act complex. Consequently, the intron www.cell-research.com | Cell Research | SPRINGER NATURE Structure of the human activated spliceosome 316 Figure 5 Structural comparison among the B complex, the B act complex, and the C complex. (A) Structure of the human B com- plex [21]. Two perpendicular views are shown. The B-B act-C transition The tri-snRNP-specific proteins, and U2, U4, U5, and U6 snRNPs are colored pink, blue, orange, magenta, and green, respectively. (B) Structure of the human mature B act complex. Two perpendicular views are shown, and these two views are identical to those in C of the human C complex [24]. Ribonucleoprotein remodeling from the B to the B act complex is the most dramatic in the splicing cycle, involving dissociation of the U4 snRNP and tri-snRNP-specific proteins and recruitment of the NTC and NTR components along with several splicing factors and the ATPase/helicase Prp2. (C) Structure of the human C complex [24]. Compared to the B act complex, the SF3a/SF3b complexes along with Prp2 and the splic- ng factor RNF113A have been dissociated, and the exon junction complex (EJC) along with the step I factors CCDC49/CCDC94 and the ATPase/helicase Prp16 have been recruited (D) Movement of the ATPase/helicase Brr2 in the B B act C transition The Figure 5 Structural comparison among the B complex, the B act complex, and the C complex. (A) Structure of the human B com- plex [21]. Two perpendicular views are shown. The tri-snRNP-specific proteins, and U2, U4, U5, and U6 snRNPs are colored pink, blue, orange, magenta, and green, respectively. (B) Structure of the human mature B act complex. Two perpendicular views are shown, and these two views are identical to those in C of the human C complex [24]. Ribonucleoprotein remodeling from the B to the B act complex is the most dramatic in the splicing cycle, involving dissociation of the U4 snRNP and tri-snRNP-specific proteins and recruitment of the NTC and NTR components along with several splicing factors and the ATPase/helicase Prp2. (C) Structure of the human C complex [24]. Compared to the B act complex, the SF3a/SF3b complexes along with Prp2 and the splic- ing factor RNF113A have been dissociated, and the exon junction complex (EJC) along with the step I factors CCDC49/CCDC94 and the ATPase/helicase Prp16 have been recruited. (D) Movement of the ATPase/helicase Brr2 in the B-B act-C transition. The U5 snRNA molecules from the three human spliceosomal complexes are superimposed. (E) Movement of the SF3b complex in the B-to-B act transition. The U5 snRNA molecules from the human B and B act complexes are superimposed. Figure 5 Structural comparison among the B complex, the B act complex, and the C complex. The B-B act-C transition (A) Structure of the human B com- plex [21]. Two perpendicular views are shown. The tri-snRNP-specific proteins, and U2, U4, U5, and U6 snRNPs are colored pink, blue, orange, magenta, and green, respectively. (B) Structure of the human mature B act complex. Two perpendicular views are shown, and these two views are identical to those in C of the human C complex [24]. Ribonucleoprotein remodeling from the B to the B act complex is the most dramatic in the splicing cycle, involving dissociation of the U4 snRNP and tri-snRNP-specific proteins and recruitment of the NTC and NTR components along with several splicing factors and the ATPase/helicase Prp2. (C) Structure of the human C complex [24]. Compared to the B act complex, the SF3a/SF3b complexes along with Prp2 and the splic- ing factor RNF113A have been dissociated, and the exon junction complex (EJC) along with the step I factors CCDC49/CCDC94 and the ATPase/helicase Prp16 have been recruited. (D) Movement of the ATPase/helicase Brr2 in the B-B act-C transition. The U5 snRNA molecules from the three human spliceosomal complexes are superimposed. (E) Movement of the SF3b complex in the B-to-B act transition. The U5 snRNA molecules from the human B and B act complexes are superimposed. SPRINGER NATURE | Cell Research | Vol 28 No 3 | March 2018 Xiaofeng Zhang et al. 317 Syf3, and the RES complex — are recruited, forming the early B act complex (Figure 6). Remarkably, the majority of the NTC and NTR proteins remain unbound in the early B act complex. Formation of the early B act complex is presumably transient because it is absent in the cryo-EM sample that was prepared using the intact pre-mRNA. Despite its transient nature, the early B act complex was trapped through the use of a 3′-exon-deleted pre-mRNA. The inability for Prp2 to bind and pull the 3′-end of the pre-mRNA likely allows the accumulation of this other- wise transient B act species. This analysis further suggests that the ensuing steps after the early B act complex may require the action of Prp2. sequences are only interlocked by RBM22 in the ma- ture/late, but not the early, B act complex (Supplementary information, Figure S7). These mutually coherent struc- tural observations are fully consistent with the reaction coordinate of the spliceosome and the requirement of the splicing reaction. The B-B act-C transition We speculate that the flux of protein components in both steps may be great- ly facilitated by the ATP hydrolysis-propelled pulling, which likely allows the empty binding sites to be more accessible to the incoming proteins. In the fourth and last step, through the action of Prp2, the SF3a complex, the SF3b complex, and the RES complex are dissociated, leading to the release of Prp2. The vacated space like- ly allows the recruitment of the step I-specific factors CCDC49 and CCDC94, the NTC proteins Sfy2 and Isy1, the exon junction complex, and the PPIs PPWD1 and PPIG (Figure 6). teins, together with the NTD of SF3a66, the splicing fac- tors SRm300 and Prp17 and the PPI CypE, are recruited, forming the mature B act complex (Figure 6). In the third step, the splicing factors RNF113A and NY-CO-10 are released, leading to the late B act complex. As suggested earlier, the second and third steps may both require the binding of the pre-mRNA by Prp2. We speculate that the flux of protein components in both steps may be great- ly facilitated by the ATP hydrolysis-propelled pulling, which likely allows the empty binding sites to be more accessible to the incoming proteins. In the fourth and last step, through the action of Prp2, the SF3a complex, the SF3b complex, and the RES complex are dissociated, leading to the release of Prp2. The vacated space like- ly allows the recruitment of the step I-specific factors CCDC49 and CCDC94, the NTC proteins Sfy2 and Isy1, the exon junction complex, and the PPIs PPWD1 and PPIG (Figure 6). (MINX cmd1 & cmd2) that are complementary to the upstream sequence of the 5′SS. The resulting solution was quenched on ice and incubated with the amylose resin (NEB) for 2 h. After exten- sive washing with the HS150 buffer (20 mM HEPES-KOH, pH 7.9, 150 mM NaCl, 1.5 mM MgCl2, 4% glycerol), the spliceosome was eluted using 20 mM maltose. For cryo-electron microscopy (cryo-EM) study, the eluted spliceosomal complexes were loaded onto a 38.6-mL 10%-30% linear glycerol gradient in the G150 buffer (20 mM HEPES-KOH, 150 mM NaCl, 1.5 mM MgCl2) supplemented with 0%-0.1% EM- grade glutaraldehyde [51]. Crosslinking, in our case by glutaral- dehyde, is essential for maintenance of the human spliceosome integrity. EM sample preparation and data acquisition After removal of glycerol, the spliceosomal complexes were further concentrated to about 0.12 mg/mL for EM sample prepa- ration. Uranyl acetate (2% w/v) was used for negative staining. Briefly, the copper grids supported by a thin layer of carbon film (Zhongjingkeyi Technology Co. Ltd) were glow-discharged. A 4-µL aliquot of the sample was applied onto the grid for 1 min and stored at room temperature. Negative staining images were taken on an FEI Tecnai Spirit Bio TWIN microscope operating at 120 kV to examine the sample quality (Supplementary information, Figure S1B). The same grids were used for cryo-EM sample preparation. Cryo-EM grids were prepared using Vitrobot Mark IV (FEI Com- pany) at 8 °C and with 100% humidity. To increase the density of the spliceosomal particles and at the same time to reduce protein aggregation, a multiple-blotting method was adopted. Briefly, a 3-µL aliquot of the sample was loaded onto a glow-discharged copper grid coated with a thin carbon film. After 2 min, the protein solution was manually absorbed with the blotting paper and anoth- er 3-µl aliquot of the sample was loaded. These steps are repeated 3-4 times depending upon the sample concentration. Grids were then blotted by Vitrobot Mark IV (FEI Company) and rapidly plunged into liquid ethane cooled by liquid nitrogen. The B-B act-C transition After centrifugation at 4 °C for 13 h at 25 300 rpm in a SW32 rotor (Beckman Coulter), the sample was manually frac- tionated from top to bottom. The total RNA in each fraction was extracted and analyzed on an 8% denaturing polyacrylamide gel (Supplementary information, Figure S1A). Fractions containing the B act complex were pooled and concentrated using a 100-kDa cut- off centrifugation filter unit (Amicon Ultra) to a volume of 500 μL. Glycerol was removed by dialysis of the sample against the G150 buffer using a 10-kDa Mini-lyzer (Pierce) for at least 5 h. g In summary, structural determination of three con- formational and compositional states of the human Bact complex facilitates mechanistic understanding of the transitions from the B to B act complex and from the B act to B * complex. Compared to the S. cerevisiae B act com- plex, the 16 additional protein components in the human complex allows meaningful comparison and derivation of conclusions that are unique to higher eukaryotes. Such differences may empower future efforts that are designed to modulate the function of the spliceosome in potential therapeutic intervention of human genetic diseases. The B-B act-C transition Analysis of the three conformational states of the B act complex suggests an ordered transition from the pre-cat- alytic B complex to the B * complex (represented by the C complex) (Figure 6). In the first step, driven by the AT- Pase/helicase Brr2, the tri-snRNP-specific components, the U4 snRNP, and proteins of the U6 snRNP are dis- sociated from the B complex. About 10 proteins — Ad- 002, Cwc22, NY-CO-10, PRL1, Prp2, RNF113A, SKIP, In the second step, the remaining NTC and NTR pro- Figure 6 A structure-based model of the ribonucleoprotein remodeling from the B complex to the C complex. The B-to-B act transition represents the most complex transition in the pre-mRNA splicing cycle [1]. The ATPase/helicase Brr2 drives the formation of the early B act complex, where the NTC and NTR components are yet to be recruited. In the active site of the early B act complex, the splicing factor RNF113A and the PPI NY-CO-10 are already loaded but the N-terminal domain (NTD) of SF3a66 along with G10 and Prp17 are yet to be recruited. Next, components of the NTC and NTR, Prp17, along with the NTD of SF3a66, are recruited to form the mature B act complex. Figure 6 A structure-based model of the ribonucleoprotein remodeling from the B complex to the C complex. The B-to-B act transition represents the most complex transition in the pre-mRNA splicing cycle [1]. The ATPase/helicase Brr2 drives the formation of the early B act complex, where the NTC and NTR components are yet to be recruited. In the active site of the early B act complex, the splicing factor RNF113A and the PPI NY-CO-10 are already loaded but the N-terminal domain (NTD) of SF3a66 along with G10 and Prp17 are yet to be recruited. Next, components of the NTC and NTR, Prp17, along with the NTD of SF3a66, are recruited to form the mature B act complex. www.cell-research.com | Cell Research | SPRINGER NATURE Structure of the human activated spliceosome 318 teins, together with the NTD of SF3a66, the splicing fac- tors SRm300 and Prp17 and the PPI CypE, are recruited, forming the mature B act complex (Figure 6). In the third step, the splicing factors RNF113A and NY-CO-10 are released, leading to the late B act complex. As suggested earlier, the second and third steps may both require the binding of the pre-mRNA by Prp2. In vitro splicing reaction p g In vitro splicing with a shortened 3′-tail of the intron in the syn- thetic pre-mRNA allows assembly of the B act complex but not its catalytic activation [4, 25, 49]. To capture the B act complex, the 3′- exon in the pre-mRNA MINX-GG [23] was deleted to generate the MINX-15 pre-mRNA construct. The MS2-binding sites were po- sitioned 46 nucleotides downstream of the 5′-splice site (5′SS) and 52 nucleotides upstream of the BPS as previously described [23]. The M7G(5′)ppp(5′)G-capped pre-mRNA was synthesized in the T7 runoff transcription using a template generated from the PCR re- action; the DNA template was then digested by RNase-free DNase I (Promega) while the RNA was further purified by PCI-extraction and ethanol precipitation. Splicing-active nuclear extract was pre- pared from HeLa S3 cells as described [50]. In vitro splicing reac- tion was performed in the presence of 15 nM MINX-15 pre-mRNA and 40% nuclear extract in a buffer that contains 20 mM HEPES- KOH, pH 7.9, 2 mM ATP, 20 mM creatine phosphate, 70 mM KCl, 3.5 mM MgCl2, and was incubated at 30 °C for 2 h. Micrographs were collected using a Gatan K2 Summit detector (Gatan Company) mounted on a Titan Krios electron microscope (FEI Company) operating at 300-kV and equipped with a GIF Quantum energy filter (slit width 20 eV). Micrographs were re- corded (Supplementary information, Figure S1C) in the super-res- olution mode with a normal magnification of 105 000×, resulting in a calibrated pixel size of 0.669 Å. Each stack of 32 frames was exposed for 8 s, with an exposure time of 0.25 s per frame. The to- tal dose rate was about 8.2 counts/second/physical-pixel (~4.7 e −/s/ Å 2) for each stack. AutoEMation was used for the fully automated data collection [52]. All 32 frames in each stack were aligned and summed using the whole-image motion correction program Mo- tionCor2 [53] and binned to a pixel size of 1.338 Å. The defocus value of each image was set from 0.8 to 1.8 μm and was deter- mined by Gctf [54]. mined by Gctf [54]. Image processing and calculation Image processing and calculation For data set II, 629 472 particles were auto-picked using DeepPicker [55]. Similar to the processing of data set I, a guided multi-reference classification procedure was applied using RE- LION2.0 [56] (Supplementary information, Figure S3). The same set of seven references as used in the processing of data set I were used. To avoid the problem of discarding good particles, we simul- taneously performed three parallel multi-reference 3D classifica- tions. After the global classification, particles that belong to the B act complex served as the input for a follow-up local classification. After local classification, the first three classes (references of B act, C, and C *) converged and became the B act complexes. The parti- cles that belong to the first three B act complexes (7.4%/8.2%/5.6%, 6.3%/6.5%/7.0%, and 7.9%/8.1%/6.3% of the total particles in the three runs) were merged, and the duplicated particles were re- moved as described [5]. The remaining 186 780 particles represent 29.7% of the original particles in data set II, which gave rise to an average resolution of about 6.3 Å after auto-refinement with 2× binned particles (pixel size: 2.676 Å) (Supplementary information, Figure S3).i Two data sets (I and II) prepared from different samples were used for the calculation (Supplementary information, Figures S2 and S3). Data set I is the same data as that described in the manu- script that reports the cryo-EM structure of the human spliceoso- mal C complex [24]. The synthetic pre-mRNA contains a 5′-exon, an intron, and a 3′-exon. Only a small proportion of the spliceoso- mal particles in this sample (named sample I, hereafter) are the B act complex; the rest are the human C and C * complexes. As will be made clear later, the B act complexes in sample I have been identi- fied to be the mature and late B act complexes. Data set II is derived from the sample (named sample II hereafter) for which detailed purification procedure was described in Materials and Methods. The synthetic pre-mRNA in sample II is similar to that in sample I except that the 3′-exon has been deleted to prevent formation of any spliceosomes beyond the B act complex. Consistent with the rationale, the spliceosomes derived from sample II are exclusively the early B act complexes. mined by Gctf [54]. y p For data set I, 1 464 033 particles were auto-picked using the deep-learning program DeepPicker [55]. The convolutional neural network model for particle picking was trained using the previous data set of the ILS complex from S. pombe [9]. A guided multi-ref- erence classification procedure was applied to the full data set us- ing the program RELION2.0 [56, 57] (Supplementary information, Figure S2). Details of this modified procedure were detailed in the manuscript reporting the cryo-EM structure of the human C * com- plex [23]. Briefly, the generated 3D volumes of the human B act, C, and C * complexes and four bad classes were obtained from a pilot analysis of 157 388 particles and were used as initial references (Round 1) (Supplementary information, Figure S2). These seven references were low-pass filtered to 40 Å. To avoid the problem of discarding good particles, we simultaneously performed three parallel multi-reference 3D classifications. Then, the particles that belong to the B act, C, and C * complexes were combined and served as the input for a follow-up local classification. The particles that belong to the B act complex (4.9%/5.1%/5.0% of the total particles in the three runs) were merged, and the duplicated particles were removed as described [5]. The remaining 113 931 particles, repre- senting 7.8% of the original particles in data set I, gave an average resolution of 7.6 Å after auto-refinement with 2× binned particles (pixel size: 2.676 Å) (Supplementary information, Figure S2).i A second round (Round 2) of local 3D classification was per- formed on the remaining 186 780 particles. 2× binned particles (pixel size: 2.676 Å) were used for the classification (Supplemen- tary information, Figure S3). A total of 96 523 particles from the good class (representing 51.7% of the input particles or 15.3% of the total original particles) yielded a reconstruction of the human B act complex with an average resolution of 4.9 Å after auto-refine- ment using unbinned particles (pixel size: 1.338 Å) (Supplementary information, Figures S3 and S4A). As will be clear from atomic modeling, the B act complexes in sample I represent the mature and late states, whereas the B act com- plexes in sample II exhibit an early state. Nonetheless, these three compositionally different B act complexes share the same SF3b region. mined by Gctf [54]. By combining these B act particles from the two data sets (49 218 particles from data set I and 96 523 particles from data set II), we generated a larger date set of 145 741 particles. Following auto-refinement with a local mask on the SF3b region, the local resolution was improved to 4.2 Å (Supplementary information, Figures S3 and S4A; Table S2). In the 4.9-Å cryo-EM map of the early B act complex and the 4.8-Å cryo-EM map of the mature and late B act complexes, the local resolution reaches 4.0-5.0 Å in the core of the spliceosome (Supplementary information, Figure S4B). The angular distri- butions of the particles used for the final reconstruction of both human B act complexes are reasonable (Supplementary information, Figure S4C), and the refinement of the atomic coordinates did not suffer from severe over-fitting (Supplementary information, Figure S4D). The EM density maps of all three B act complexes display similar overall structural features but with important differences in a number of key regions (Supplementary information, Figure S5). The density maps exhibit clear features for the secondary structural elements of the human B act complex in the core region. The RNA elements and their interacting proteins are also reasonably well de- fined by the EM density maps and can be modeled with structural references from the human C [24] and C * [23] complexes and the yeast B act complex [5]. A second round (Round 2) of local 3D classification was per- formed for the remaining 113 931 particles. 2× binned particles (pixel size: 2.676 Å) were used for the classification (Supplemen- tary information, Figure S2). A total of 49 218 particles from the good class (representing 43.2% of the input particles or 3.4% of the total original particles) yielded a reconstruction of the human B act complex with an average resolution of 4.8 Å after auto-refine- ment using unbinned particles (pixel size: 1.338 Å). In the final round (Round 3), the remaining 49 218 particles were classified without alignment but with a soft mask on the RN- F113A region of the spliceosome. Two major classes, representing two different conformations, were identified. 27 405 particles (55.4% of the input particles) in one class yielded a reconstruction at an average resolution of 5.1 Å for the entire spliceosome, which was identified as the mature B act complex. www.cell-research.com | Cell Research | SPRINGER NATURE Purification and crosslinking of the spliceosomal complexes Purification and crosslinking of the spliceosomal complexes Purification and crosslinking of the spliceosomal complexes After spliceosome formation, the free pre-mRNA that had not been incorporated into the spliceosome was digested by endog- enous RNase H with the addition of two DNA oligonucleotides SPRINGER NATURE | Cell Research | Vol 28 No 3 | March 2018 Xiaofeng Zhang et al. 319 an average resolution of 6.5 Å for the entire spliceosome, which was identified as the late B act complex (Supplementary informa- tion, Figures S2 and S4A; Tables S1 and S2). an average resolution of 6.5 Å for the entire spliceosome, which was identified as the late B act complex (Supplementary informa- tion, Figures S2 and S4A; Tables S1 and S2). Acknowledgments We thank the Tsinghua University Branch of China National Center for Protein Sciences (Beijing) for the cryo-EM facility and the computational facility support on the cluster of Bio-Computing Platform. This work was supported by funds from the National Natural Science Foundation of China (31621092 and 31430020) and the Ministry of Science and Technology (2016YFA0501100 to JL). The crystal structure of the human SF3b core complex (PDB code: 5IFE [34]), including SF3b155, SF3b130, SF3b14b/PHF5A, and SF3b10, was docked into the density map guided by the yeast B act structure [5]. SF3b145 was generated from Cus1 of the yeast B act complex. The N-terminal domain of SF3b145 and the RRM domain of SF3b49 was generated from the crystal structure of Hsh49p in complex with Cus1p (PDB code: 5LSB [64]). Crystal structure of SF3b14a/p14 in complex with SF3b155 N-terminal fragments (PDB code: 2F9J [65]) was docked into the SF3b re- gion of the cryo-EM maps. The crystal structure of the human SF3a complex (PDB code: 4DGW [66]), which includes SF3a120, SF3a66, and SF3a60, was docked into the extra density around the Sm ring of U2 snRNP. Prp2 is docked into the cryo-EM map on the basis of the structure of the yeast B act complex [5]. CypE is docked into the map near the N-terminal HAT repeats of Syf1. No- tably, a patch of weak EM density is also located in the same place of the yeast B act complex as in the human B act complex, suggesting that CypE may be recruited into the yeast B act complex. i Competing Financial Interests Competing Financial Interests The authors declare no competing financial interests. The authors declare no competing financial interests. The authors declare no competing financial interests. Structure of the human activated spliceosome 320 0.143 criterion, and the FSC curves were corrected for the ef- fects of a soft mask on the FSC curve using high-resolution noise substitution [58]. Prior to visualization, all density maps were corrected for the modulation transfer function of the detector, and then sharpened by applying a negative B-factor that was estimated using automated procedures [59]. Local resolution variations were estimated using ResMap [60]. 0.143 criterion, and the FSC curves were corrected for the ef- fects of a soft mask on the FSC curve using high-resolution noise substitution [58]. Prior to visualization, all density maps were corrected for the modulation transfer function of the detector, and then sharpened by applying a negative B-factor that was estimated using automated procedures [59]. Local resolution variations were estimated using ResMap [60]. the refined model against the other map [69] (Supplementary in- formation, Figure S4D). The structure of the human B act complex was validated through examination of the Molprobity scores and statistics of the Ramachandran plots (Supplementary information, Table S1). Molprobity scores were calculated as described [70]. Distinguishing features of the cryo-EM maps among the three B act complexes are detailed for the SF3b complex (Supplementary in- formation, Figure S6), G10, Prp17, and RBM22 (Supplementary information, Figure S7), the N-terminal domain (NTD) of SF3a66 (Supplementary information, Figure S8), the NTC proteins (Sup- plementary information, Figure S9), RNF113A and NY-CO-10 (Supplementary information, Figure S10), and the SF3a and sur- rounding regions (Supplementary information, Figure S11). Accession code The atomic coordinates for the early, mature and late B act spli- ceosomes have been deposited in the Protein Data Bank with the accession code 5Z58, 5Z56 and 5Z57, respectively. The EM maps for the early, mature and late B act spliceosomes have been deposit- ed in EMDB with the accession code EMD-6891, EMD-6889 and EMD-6890, respectively. The atomic models of RNF113A and the N-terminal domain of SF3a66 in the human B act complex were generated from Cwc24 and the N-terminal domain of Prp11 in the S. cerevisiae B act com- plex [5] using CHAINSAW [63]. The backbone was manually adjusted using COOT [61]. The atomic coordinates of U6 snRNA, protein components of the U5 and U2 snRNPs, protein compo- nents of the NTC and NTR complex, Prp17, and Aquarius from the human C * complex (PDB code:5XJC [23]) were directly docked into the density maps of the human B act complex and were manual- ly adjusted using COOT [61]. Assignment of the 5-splice site (5′SS) and the duplex between U2 snRNA and the BPS was greatly aided by the structure of the yeast spliceosomal B act complex [5]. mined by Gctf [54]. 14 316 particles (29.3% of the input particles) in the other class yielded a reconstruction at Reported resolutions were calculated on the basis of the FSC www.cell-research.com | Cell Research | SPRINGER NATURE Author Contributions XiaofengZ and XiechaoZ purified the human spliceosomal complexes and prepared the cryo-EM samples. XiaofengZ, Xiech- aoZ, JL, and CY collected the EM micrographs and processed the data. CY calculated the cryo-EM map and built the atomic model. All authors contributed to project discussion and structure analy- sis. XiaofengZ and XiechaoZ contributed to manuscript prepara- tion. CY and YS wrote the manuscript. YS conceived and guided the project. Model building and refinement Due to a wide range of resolution limits for the various regions of the human B act complex, we combined homology modeling and rigid docking of components with known structures to generate an atomic model (Supplementary information, Table S2). Identifi- cation and docking of the components of the human B act complex were facilitated by the atomic models of the human C [24] and C * [23] complex and the S. cerevisiae B act complex [5]. The protein components that were derived from known structures of the pro- tein data bank (PDB) are summarized in Supplementary informa- tion, Table S2. These structures were docked into the density map using COOT [61] and fitted into density using CHIMERA [62]. References 1 Wahl MC, Will CL, Luhrmann R. The spliceosome: design principles of a dynamic RNP machine. Cell 2009; 136:701- 718. The final overall models of the early and mature B act complexes were refined against the overall 4.9-Å and 4.8-Å map, respec- tively, using REFMAC in reciprocal space [67], using secondary structure restraints that were generated by ProSMART [68]. The atomic model of the late B act complex was generated by removing the RNF113A, NY-CO-10, and RNase H like domain of Prp8 from the mature B act complex. Overfitting of the overall model was monitored by refining the model in one of the two independent maps from the gold-standard refinement approach, and testing 2 Chan SP, Kao DI, Tsai WY, Cheng SC. The Prp19p-associated complex in spliceosome activation. Science 2003; 302:279- 282. 3 Ohi MD, Gould KL. Characterization of interactions among the Cef1p-Prp19p-associated splicing complex. RNA 2002; 8:798-815. 4 Fabrizio P, Dannenberg J, Dube P, et al. 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Structure of a yeast step II catalytically activated spliceosome. Science 2017; 355:149-155. 33 MacMillan AM, Query CC, Allerson CR, Chen S, Verdine GL, Sharp PA. Dynamic association of proteins with the pre-mRNA branch region. Genes Dev 1994; 8:3008-3020. 15 Fica SM, Oubridge C, Galej WP, et al. Structure of a spliceo- some remodelled for exon ligation. Nature 2017; 542:377- 380. 34 Cretu C, Schmitzova J, Ponce-Salvatierra A, et al. Molecular architecture of SF3b and structural consequences of its can- cer-related mutations. Mol Cell 2016; 64:307-319. 16 Wan R, Yan C, Bai R, Lei J, Shi Y. Structure of an intron lar- iat spliceosome from Saccharomyces cerevisiae. Cell 2017; 171:120-132.e12. 35 Query CC, Strobel SA, Sharp PA. Three recognition events at the branch-site adenine. EMBO J 1996; 15:1392-1402. 17 Bai R, Yan C, Wan R, Lei J, Shi Y. Structure of the Post-cata- lytic spliceosome from Saccharomyces cerevisiae. 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Genes Dev 1991; 5:2521-2533. 46 Hilliker AK, Mefford MA, Staley JP. U2 toggles iteratively between the stem IIa and stem IIc conformations to promote pre-mRNA splicing. Genes Dev 2007; 21:821-834. 62 Pettersen EF, Goddard TD, Huang CC, et al. UCSF Chime- ra--a visualization system for exploratory research and analy- sis. J Comput Chem 2004; 25:1605-1612.i 63 Stein N. CHAINSAW: a program for mutating pdb files used as templates in molecular replacement. J Appl Crystallogr 2008; 41:641-643. 47 Perriman RJ, Ares M, Jr. Rearrangement of competing U2 RNA helices within the spliceosome promotes multiple steps in splicing. Genes Dev 2007; 21:811-820. 48 Bon E, Casaregola S, Blandin G, et al. Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns. Nucleic Acids Res 2003; 31:1121-1135. 64 van Roon AM, Oubridge C, Obayashi E, et al. Crystal struc- ture of U2 snRNP SF3b components: Hsh49p in complex with Cus1p-binding domain. 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EMBO J 2004; 23:4847-4856. 18 Liu S, Li X, Zhang L, et al. Structure of the yeast spliceoso- mal postcatalytic P complex. Science 2017; 358:1278-1283. 37 Wysoczanski P, Schneider C, Xiang S, et al. Cooperative structure of the heterotrimeric pre-mRNA retention and splic- ing complex. Nat Struct Mol Biol 2014; 21:911-918. 19 Wilkinson ME, Fica SM, Galej WP, Norman CM, New- man AJ, Nagai K. Postcatalytic spliceosome structure re- veals mechanism of 3′-splice site selection. Science 2017; 358:1283-1288. 38 Ohi MD, Link AJ, Ren L, Jennings JL, McDonald WH, Gould KL. Proteomics analysis reveals stable multiprotein complex- es in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs. Mol Cell Biol 2002; 22:2011-2024. 20 Plaschka C, Lin PC, Nagai K. Structure of a pre-catalytic spli- ceosome. Nature 2017; 546:617-621. 39 Raghunathan PL, Guthrie C. RNA unwinding in U4/U6 sn- RNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2. Curr Biol 1998; 8:847-855. 21 Bertram K, Agafonov DE, Dybkov O, et al. Cryo-EM struc- ture of a pre-catalytic human spliceosome primed for activa- tion. Cell 2017; 170:701-713.e711. 22 Bertram K, Agafonov DE, Liu WT, et al. Cryo-EM structure of a human spliceosome activated for step 2 of splicing. Na- ture 2017; 542:318-323. 40 Laggerbauer B, Achsel T, Luhrmann R. The human U5- 200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro. Proc Natl Acad Sci USA 1998; 95:4188-4192. 23 Zhang X, Yan C, Hang J, Finci LI, Lei J, Shi Y. An atomic structure of the human spliceosome. Cell 2017; 169:918-929. e914. 41 Kim SH, Lin RJ. Spliceosome activation by PRP2 ATPase prior to the first transesterification reaction of pre-mRNA splicing. Mol Cell Biol 1996; 16:6810-6819. 42 Schwer B, Guthrie C. A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis. EMBO J 1992; 11:5033-5039. 24 Zhan X, Yan C, Zhang X, Lei J, Shi Y. Structure of a human catalytic step I spliceosome. Science 2018; doi: 10.1126/sci- ence.aar6401. 43 Schwer B. A conformational rearrangement in the spliceo- 25 Cheng SC. Formation of the yeast splicing complex A1 and www.cell-research.com | Cell Research | SPRINGER NATURE Structure of the human activated spliceosome Structure of the human activated spliceosome Acta Crystallogr D Biol Crystallogr 1997; 53:240-255. 51 Kastner B, Fischer N, Golas MM, et al. GraFix: sample preparation for single-particle electron cryomicroscopy. Nat Methods 2008; 5:53-55. 68 Nicholls RA, Fischer M, McNicholas S, Murshudov GN. Conformation-independent structural comparison of macro- molecules with ProSMART. Acta Crystallogr D Biol Crystal- logr 2014; 70:2487-2499. 52 Lei J, Frank J. Automated acquisition of cryo-electron mi- crographs for single particle reconstruction on an FEI Tecnai electron microscope. J Struct Biol 2005; 150:69-80. 69 Amunts A, Brown A, Bai XC, et al. Structure of the yeast mi- tochondrial large ribosomal subunit. Science 2014; 343:1485- 1489. 53 Zheng SQ, Palovcak E, Armache JP, Verba KA, Cheng Y, Agard DA. MotionCor2: anisotropic correction of beam-in- duced motion for improved cryo-electron microscopy. Nat Methods 2017; 14:331-332. 70 Davis IW, Leaver-Fay A, Chen VB, et al. MolProbity: all-at- om contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res 2007; 35:W375-383. 54 Zhang K. Gctf: Real-time CTF determination and correction. J Struct Biol 2016; 193:1-12. (Supplementary information is linked to the online version of the paper on the Cell Research website.) (Supplementary information is linked to the online version of the paper on the Cell Research website.) 55 Wang F, Gong H, Liu G, et al. DeepPicker: A deep learning approach for fully automated particle picking in cryo-EM. J Struct Biol 2016; 195:325-336. 56 Kimanius D, Forsberg BO, Scheres SH, Lindahl E. Accelerat- ed cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 2016; 5:pii: e18722. This work is licensed under a Creative Commons Attribution 4.0 Unported License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http:// creativecommons.org/licenses/by/4.0/ 57 Scheres SH. RELION: implementation of a Bayesian ap- proach to cryo-EM structure determination. J Struct Biol 2012; 180:519-530. 58 Chen S, McMullan G, Faruqi AR, et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomi- croscopy. Ultramicroscopy 2013; 135:24-35. © The Author(s) 2018 59 Rosenthal PB, Henderson R. Optimal determination of parti- SPRINGER NATURE | Cell Research | Vol 28 No 3 | March 2018
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Editorial
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editorial editorial Se as relações entre literatura e cinema existem desde o momen- to em que este surgiu no horizonte cultural da modernidade, as formas de interação, interseção e diálogo com que ambos foram dinamizando esse campo de relações ao longo do século XX não podem ser circunscritas apenas ao trabalho de adaptação fílmica de textos literários ou à incorporação, por parte destes, de ele- mentos e estratégias oriundos do discurso cinematográfico. Cum- plicidades explícitas e implícitas, diálogos subliminares, contami- nações e provocações recíprocas, citações, evocações e “transcriações” nunca deixaram também de atravessar o espaço móvel da conjunção/disjunção entre literatura e cinema. Além disso, por receberem, ainda, os influxos de outras linguagens ar- tísticas e das demandas culturais do momento e do lugar em que se inscrevem, tais relações se tornam ainda mais complexas pela força de vários outros tipos de interseção e diálogo. Se as relações entre literatura e cinema existem desde o momen- to em que este surgiu no horizonte cultural da modernidade, as formas de interação, interseção e diálogo com que ambos foram dinamizando esse campo de relações ao longo do século XX não podem ser circunscritas apenas ao trabalho de adaptação fílmica de textos literários ou à incorporação, por parte destes, de ele- mentos e estratégias oriundos do discurso cinematográfico. Cum- plicidades explícitas e implícitas, diálogos subliminares, contami- nações e provocações recíprocas, citações, evocações e “transcriações” nunca deixaram também de atravessar o espaço móvel da conjunção/disjunção entre literatura e cinema. Além disso, por receberem, ainda, os influxos de outras linguagens ar- tísticas e das demandas culturais do momento e do lugar em que se inscrevem, tais relações se tornam ainda mais complexas pela força de vários outros tipos de interseção e diálogo. Foi exatamente considerando a complexidade, os matizes e a multiplicidade desses entrecruzamentos, as demandas da sociedade contemporânea e as mediações culturais do presente, que surgiu a idéia de contemplar, neste número especial da Revista Aletria, as relações possíveis (e impossíveis) entre literatura e cinema. Estudiosos de um campo e/ou de outro, advindos de países, instituições e áreas de atuação diferentes, e atentos a essas interfaces, vieram contribuir para o debate proposto, com artigos, intervenções e provocações que tratam da questão sob diferentes ângulos e perspectivas teóricas. editorial Tais contribuições foram organizadas, à luz de fragmentos da letra da canção “Cinema Novo”, de Caetano Veloso, em oito partes temáticas, sendo que a primeira conta especialmente com um ensaio do cineasta britânico Peter Greenaway, que atendeu gentilmente nosso convite para participar deste volume. A ele nossos agradecimentos pela oportunidade preciosa que nos deu de publicar um artigo de sua autoria. Neste, o diretor e roteirista de O livro de cabeceira não apenas busca desestabilizar, pelo viés da ironia, os lugares-comuns instituídos em torno da relação do cinema com o texto literário como mostrar formas alternativas de se “ultrapassar o eclipse” a que a imagem tem sido submetida ao ser usada como mero dispositivo ilustrativo do modelo narrativo do século XIX. Foi exatamente considerando a complexidade, os matizes e a multiplicidade desses entrecruzamentos, as demandas da sociedade contemporânea e as mediações culturais do presente, que surgiu a idéia de contemplar, neste número especial da Revista Aletria, as relações possíveis (e impossíveis) entre literatura e cinema. Estudiosos de um campo e/ou de outro, advindos de países, instituições e áreas de atuação diferentes, e atentos a essas interfaces, vieram contribuir para o debate proposto, com artigos, intervenções e provocações que tratam da questão sob diferentes ângulos e perspectivas teóricas. Tais contribuições foram organizadas, à luz de fragmentos da letra da canção “Cinema Novo”, de Caetano Veloso, em oito partes temáticas, sendo que a primeira conta especialmente com um ensaio do cineasta britânico Peter Greenaway, que atendeu gentilmente nosso convite para participar deste volume. A ele nossos agradecimentos pela oportunidade preciosa que nos deu de publicar um artigo de sua autoria. Neste, o diretor e roteirista de O livro de cabeceira não apenas busca desestabilizar, pelo viés da ironia, os lugares-comuns instituídos em torno da relação do cinema com o texto literário como mostrar formas alternativas de se “ultrapassar o eclipse” a que a imagem tem sido submetida ao ser usada como mero dispositivo ilustrativo do modelo narrativo do século XIX. Maria Esther Maciel & Marli Fantini Scarpelli editorial 5 2001 - A L E T R I A Assim, da exploração das potencialidades de assombro da ima- gem, passando pelos “jeitos do Brasil” inscritos no cinema que tam- bém se quer samba, até as “visões das coisas grandes e pequenas” que atravessam as manifestações verbais e visuais da contemporaneidade, vários outros tópicos se fazem presentes nes- ta coletânea de textos: a radicalidade do cinema e da literatura que se furtam à linearidade e aos paradigmas da narrativa con- vencional para buscar, na linguagem poética, vias alternativas para a criação da imagem e da palavra; as várias linhas de expressão que se cruzam tacitamente nas frestas, nas dobras e nas margens da indústria cinematográfica norte-americana; as tensões/ interações entre ordem e desordem na literatura e no cinema de expressão germânica; as múltiplas possibilidades de entrecruzamento cultural entre cinemas e literaturas de “todos e muitos” lugares; o exercício crítico-reflexivo sobre filmes em tem- pos de reprodutibilidade técnica e euforia tecnológica. Acreditamos que, através desta publicação, o CEL (Centro de Estudos Literários) e o POSLIT (Programa de Pós-Graduação em Estudos Literários) da Faculdade de Letras da UFMG vêm não apenas reafirmar seus compromissos com a prática sempre instigante da transdisciplinaridade como também estimular reflexões e interlocuções no âmbito da linha de pesquisa “Literatura e Outros Sistemas Semióticos”, abrindo-se, cada vez mais, ao debate contemporâneo sobre os trânsitos, travessias, trocas, traduções e diálogos no horizonte artístico e cultural deste início de milênio. Maria Esther Maciel & Marli Fantini Scarpelli 6 A L E T R I A - 2001
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Multi-algorithm approach for identification of structural behavior of complex structures under cyclic environmental loading
Structural health monitoring
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Multi-algorithm approach for identification of structural behaviour of complex structures under cyclic environmental loading Francesca Lanata, Franck Schoefs Francesca Lanata, Franck Schoefs To cite this version: Francesca Lanata, Franck Schoefs. Multi-algorithm approach for identification of structural behaviour of complex structures under cyclic environmental loading. Structural Health Monitoring, 2012, 11 (1), pp.51-67. ￿10.1177/1475921710397711￿. ￿hal-01007091￿ Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01007091 https://hal.science/hal-01007091v1 Submitted on 16 Jun 2014 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License y signal processing analysis, quay structures, cyclic loading, statistical analysis, damage-sensitive features y signal processing analysis, quay structures, cyclic loading, statistical analysis, damage-sensitive features GeM Institute for Research in Civil and Mechanical Engineering, University of Nantes, 2 rue de la houssinie´re, 44322 Nantes, France. Multi-algorithm approach for identification of structural behavior of complex structures under cyclic environmental loading Francesca Lanata and Franck Schoefs Abstract In the past years, an increasing number of structures have been equipped with permanent monitoring systems, able to record the structural response in terms of displacements and strains over very long periods of time, and theoretically, for the entire life of the structure (Structural Health Monitoring). Despite the high number of applications, to date, very few studies have been presented aimed at interpreting the data, in particular, when the spatial and behavioral complexity of the structure under investigation requires a non-model-based approach. This article shows that the interpretation of data from a long-term static monitoring can be very helpful for the comprehension of the structural behavior under complex interaction structure–environment. The discussion aims at underlining that a scheduled and unique procedure is very hard to define depending on the great variety of structures and applications and on the right identification of the parameters able to explain structural degradation evolutions or sudden changes. In these cases, a multi-step algorithm with different signal processing techniques applied in cascade seems to be the only reliable approach. The proposed procedure will be carried out using available data from the long-term monitoring of a quay wall in the Port of Genoa, in Italy, but it can be generalized to several structures under cyclic environmental loading (tides, temperature, etc.). The characterization of the structural behavior under temperature variations will be used here to define the represen- tative features of the quay. Once defined, these parameters will provide a means for detecting and localizing the insur- gence of damage or material degradation from the measurements. Corresponding author: Francesca Lanata, GeM Institute for Research in Civil and Mechanical Engineering, University of Nantes, 2 rue de la houssinie´re, 44322 Nantes, France Email: Francesca.Lanata@univ-nantes.fr Introduction plants, communication facilities, schools, hospitals, homeland defense, harbor installations, etc. The economical effort of building, replacing, or retro- fitting infrastructures is becoming more and more important in industrialized countries, where the existing infrastructure stock is subjected to aging and obsoles- cence. For example, the majority of the transportation infrastructures existing in Europe and in the United States have been built from the 1950s to the 1970s, when features of the traffic intensity and loads were significantly different from the current features and when some of the phenomena related to material degradation were not fully discovered or understood.1–3 Moreover, the problem is not confined to transporta- tion infrastructures, but also includes other types of infrastructures like energy production and distribution systems, water supplies and wastewater treatment For special classes of structures or for specific envi- ronmental situations, models used in design practices may not cover the wide range of problems that need to be solved when assessing the safety conditions of the structure as built. This is, particularly, true for large GeM Institute for Research in Civil and Mechanical Engineering, University of Nantes, 2 rue de la houssinie´re, 44322 Nantes, France. Corresponding author: Francesca Lanata, GeM Institute for Research in Civil and Mechanical Engineering, University of Nantes, 2 rue de la houssinie´re, 44322 Nantes, France Email: Francesca.Lanata@univ-nantes.fr Moreover, the problem is not confined to transporta- tion infrastructures, but also includes other types of infrastructures like energy production and distribution systems, water supplies and wastewater treatment 1 Recent literature16 has put into evidence that theo- retical and lab knowledge needs to be transferred to real structures to control static or dynamic parameters during in-service conditions. In particular, for large structural systems interacting with their environment, safety and operability of the system depend on phenomena involving not only structural behavior, but also coupled structure–environment responses. Moreover, due to the inherent geometrical and physical complexities of the problem, model-based diagnosis can be seldom invoked, and translation of measurement data into valuable information shall be based mostly on engineering judgment and on mathematical process- ing of the instrumentation signals. In addition, when monitoring systems are installed not at the beginning of the life-cycle of the structure but at a later stage (repair for instance), major modifications of the struc- tural system may have occurred because of the effect of loads and other external causes, or because of human intervention. Introduction This aspect has a great influence on data analysis and interpretation, because of the greater role that uncertainties do play in this case.17 structures in aggressive environments, like bridges in coastal or mountain areas and marine structures,4 or in the case of prevention of pollution loads discharged from distribution networks.5 In all these cases, observation of the structural behavior and/or of the environmental parameters with an adequate monitor- ing system can cover the gap between the reality and the theoretical models that have been developed for the design to obtain a picture of the structural state and evolution. structures in aggressive environments, like bridges in coastal or mountain areas and marine structures,4 or in the case of prevention of pollution loads discharged from distribution networks.5 In all these cases, observation of the structural behavior and/or of the environmental parameters with an adequate monitor- ing system can cover the gap between the reality and the theoretical models that have been developed for the design to obtain a picture of the structural state and evolution. The continuous development in sensor technology and data acquisition systems makes possible the reali- zation of complex permanent instrumental monitoring systems; they are able to monitor the most significant parameters of structural response under external loads and environmental conditions, enabling, in principle, a continuous evaluation of the actual safety conditions of such structures and finally a data-based reliability anal- ysis.6 Through these large-scale monitoring projects, a considerable amount of data can be available, so that data analysis and interpretation play a very important role in structural health monitoring (SHM) research. Thanks to these recent technologies, the field of con- tinuous monitoring is facing the research community since the last years, while the periodic monitoring of dynamic parameters has been largely investigated in the past.7 Usually, the measured structural features are used to construct a mechanical model that is later updated using monitoring data. The mechanical model can be used for: (a) reliability analysis and/or (b) damage identification. Recently, the integration of the reliability analysis with the long-term SHM has been attempted8–11 and improved by including the uncer- tainty propagation in the assessment process.12,13 On the other hand, the mechanical model is very useful also to extract the structural features (static or dynamic) that are sensitive to damage or material degradation. The monitoring program The San Giorgio pier is used for coal import and it has been subjected to a retrofitting program to increase the water depth of the nearby basin. The facility has been built in the 1920s and the vertical walls delimiting the quays are made of heavy concrete blocks. Dredging has required strengthening of the wall: the structure has been underpinned with jet-grouting columns, and the blocks have been connected by means of vertical steel rods. Stability has been improved with permanent active tendons installed along the entire length of the pier.38 The crown block of the San Giorgio pier was equipped in 1999 with an array of 67 SOFO fiber- optic strain sensors,39 located in a service tunnel along the top blocks, in such a way to have 3 longitu- dinal sensors in 24 measuring sections, named measuring blocks in the following. The generic block cross-section (left) and the schematic longitudinal view (right) of the equipped pier are represented in Figure 1. Each sensor has an active length of 10 m. The monitor- ing had the goal to detect possible distress caused by dredging activities in the adjacent basin and also to run a long-term health monitoring experience, as a first step of a larger project aimed at establishing a decision support system for maintenance operations of the Port facilities.36 In the section ‘The San Giorgio pier,’ the discussion on the complexity of data processing will be carried on by means of available data from the real long-term monitoring of a quay wall in the Port of Genoa, Italy.36 Section ‘Strategy for data analysis’ tries to give a generally valuable strategy for data processing through a multi-algorithm approach. Sections ‘Classification of block behavior,’ ‘Damage-sensitive feature for blocks of type 1,’ and ‘Damage-sensitive feature for blocks of type 2’ emphasize the preliminary steps aimed at understanding the mechanical response of such a complex structure under cyclic loads in terms of representative features of the quay. As a matter of fact, in the field of structures with a strong random behavior, it is necessary to develop a mechanical- probabilistic model through available data from SHM. This can be relatively easy if the structure is subjected to cyclic actions because its random behavior can be identified using the information from loading cycles. Introduction For example, regarding the inverse prob- lem of detecting a single open crack in an elastic straight beam in bending, dynamic identification tech- niques provide explicit expressions for the position and the severity of the crack; by the way, the methodology is valid only for small damages and for initially uniform beams under special boundary conditions (pinned– pinned, sliding–sliding).14 Other models reconsider the inverse problem of detecting a single crack in an elastic straight beam in bending from static measurements to provide explicit expressions for its position and its severity, which represent the exact solutions of the inverse problem.15 However, these are above all theo- retical approaches, because the meaning of data from a real monitoring is usually not clearly associated to any mechanical model of structural response, due to the particularity of the instrumented structures and their boundary conditions and loads. The automatic detection of damages from continu- ous static monitoring is still a challenge. The measured quantities during continuous monitoring are typically displacements or strains under operational and envi- ronmental loads. Practitioners have difficulties in choosing the best data processing approach for each application. Scientifically, the most successful methods are not yet identified, even though a great number of signal processing algorithms have been investigated. Moyo and Brownjohn18 proposed the detection of anomalous structural behavior using discrete wavelet analysis. Experimental studies on structural damage identification based on wavelet packet transform have been done with good results.19–21 In SHM, one of the most common model-free methods for damage detec- tion is based on autoregressive analysis. In their general version, these methodologies consist of expression of a time series as a linear function of its past values and the past values of exogenous variables. The estimation of the parameters is done continuously and damages are normally discovered if a significant variation in the parameters appears.22–27 The linear regression method has also attracted a notable interest because of its simplicity and wide applicability. However, this meth- odology may be prone to a harmful affect in case where data are used with outliers, a common problem observed during real monitoring. Outliers’ accommo- dation methods are well known by robust methods, where robustness is given by statistical procedures insensitive to outliers. The monitoring program In particular, in this article, focus will be placed on the monitoring of evolutionary phenomena, taking place in structures as a result of the interaction with environment and operational loads.37 Due to the large amount of instrumented blocks, in this applica- tion, the statistical analysis has proved to be successful for the identification of the typical response features. The second part of the proposed multi-algorithm anal- ysis will be aimed at giving information on the struc- tural health conditions. Using the previously identified response parameters as deterioration-sensitive features, section ‘Damage detection’ presents the linear regres- sion algorithm as an effective damage detection Acquisition campaigns were programmed four times a day to analyze the main variations of temperature during 24 h (by day and by night) and with greater reason, weekly and seasonal variations. The system was fully operational for 4 years (from November 1999 to October 2004), with the exception of some interruptions due to the maintenance on the system and other management reasons (Figure 2). This moni- toring time is a common lifetime for operational systems near the sea.37 Introduction In this framework, robust regres- sion analysis has been proposed and applied in the field of structural monitoring.28–30 The Correlation Anomaly Scores Analysis has been proposed for 2 change analysis of correlated multi-sensor systems using a neighborhood preservation principle.31 The goal of change analysis is to compute the anomaly score of each sensor when it is known that the system has some potential difference from a reference state. If the system is working normally, the neighborhood graph of each sensor (defined using the correlation between sensor measurements) is almost invariant against the fluctuations of experimental conditions. Deraemaeker et al.32 presented a complex methodology for damage detection under changing environmental conditions. The effects of environment are treated using factor analysis and damage is detected using statistical process control with the multi-variate Shewhart-T control charts. Posenato et al.33 proposed a methodology based on moving principal component analysis (MPCA); a comparative study with other signal processing algorithms has shown that for quasi-static monitoring of civil structures, the proposed methodology performs better than wavelet methods, Short Term Fourier Transform and Instance-Based Method, also in case of noise, outliers, data missing, and database with different sensor typologies.34,35 algorithm and applies it to the available data to successfully detect and localize the insurgence of damage or material degradation in the monitored quay. Understanding of mechanical behavior In order to choose the best approach for data interpre- tation, it is useful to understand if the structure has a well-defined mechanical behavior and if damage mech- anisms can be clearly described. The different layers of concrete blocks are linked each other, thanks to the gravity force. After the reinforcement works, the blocks should have a higher connection rate due to the distributed vertical steel rods. For these reasons, it 3 1 1 +2.90m +2.50m C8 C7 C6 C5 C4 C3 C2 C1 B8 B7 B6 B5 B4 B3 B2 B1 A8 A7 A6 0.00 m s.l.m. Section 1–1 1 2 3 80 80 Block type 2 Block type 1 Block type 1 Figure 1. Sensor installation along the quay for 19 measuring blocks (right) and block cross-section (left) with sensor position inside the service tunnel (measurements in cm). The thick vertical lines represent two possible locations of the dilatation joints along the pier (section ‘Understanding of mechanical behavior’). 1 1 +2.90m +2.50m C8 C7 C6 C5 C4 C3 C2 C1 B8 B7 B6 B5 B4 B3 B2 B1 A8 A7 A6 0.00 m s.l.m. Section 1–1 1 2 3 80 80 Block type 2 Block type 1 Block type 1 Figure 1. Sensor installation along the quay for 19 measuring blocks (right) and block cross-section (left) with sensor position inside the service tunnel (measurements in cm). The thick vertical lines represent two possible locations of the dilatation joints along the pier (section ‘Understanding of mechanical behavior’). 30/11/99 05/08/00 12/04/01 18/12/01 25/08/02 02/05/03 07/01/04 14/09/04 21/05/05 –1 –0.5 0 0.5 1 1.5 2 2.5 3 3.5 × 10–4 Sections A7 – A8 – B5 – C2 – C3 – C6 – C8 Strain (–) Monitoring period Figure 2. Example of time histories for some measuring blocks during the whole monitoring period. Figure 2. Example of time histories for some measuring blocks during the whole monitoring period. can be supposed that the structure behaves as a mono- lithic structure, so that great displacements interesting the underwater layers are transferred to the superior layers until the crown block. Indeed, the installed mon- itoring system is able to measure the crown block displacements. variations in the structural behavior or damages are expected to occur as a result of complex phenomena. Understanding of mechanical behavior The study of a reliable numerical model of the pier is very complex and it would require a lot of hypothesis and simplifications. Thus, data interpretation will have to resort to statistical algorithms for gathering some information on the structural behavior. On the other hand, the crown block can itself expe- rience small displacements induced by external temper- ature variations and sea level changes. The horizontal relative displacements between blocks are allowed, thanks to the presence of dilatation joints along the pier (Figure 1). However, the conditions and the posi- tion of joints are unknown because most of them are completely filled by coal dusts and it is not clear if they behave properly as dilatation joints. In addition, the crown block should not be allowed to expand freely with thermal variations due to the continuous support of the lower block. Key factors Fixed marine structures are primarily designed to with- stand environmental and operational forces. The main phenomena in the marine environment able to produce changes in the structural response and loss of function- ality can be: It can be concluded that the structure under investi- gation is strongly non-homogeneous due to the interac- tion between old structural elements and new strengthening elements. The structure is characterized by spatial and behavioral complexity and possible . corrosion of steel and degradation of concrete and other materials; . crane movements and operations that can cause overloads on the quays; 4 –4 –2 0 2 4 6 8 10 × 10–5 Strain C82 (–) 30/11/99 02/04/00 05/08/00 08/12/00 12/04/01 15/08/01 18/12/01 22/04/02 25/08/02 –10 –5 0 5 10 15 20 25 Temperature (°C) Monitoring period Temperature Sensor C82 Moored ship Figure 3. Strain time history of sensor C82 and temperature variations. Black crosses represent measurements performed with the moored ship. Figure 3. Strain time history of sensor C82 and temperature variations. Black crosses represent measurements performed with the moored ship. 5.5 6 6.5 7 7.5 8 8.5 9 9.5 × 10–5 Strain C82 (–) 31/07/00 05/08/00 10/08/00 15/08/00 20/08/00 25/08/00 30/08/00 20 21 22 23 24 25 26 27 28 Temperature (°C) Monitoring period Temperature Sensor C82 Moored ship Figure 4. Zoom of the black rectangle in Figure 3. Figure 4. Zoom of the black rectangle in Figure 3. . interaction with sea actions, for example tide and wave actions; . interaction with sea actions, for example tide and wave actions; phenomenon of interest in the Mediterranean area and for this reason they have not been considered as a key factor for the San Giorgio pier analysis. . moored ships, also in case of collision; The relationship between the measured strains and the presence of the moored ship has been here investi- gated. It is also related to the crane operations for the goods uploading and downloading. No clear correla- tion with the moored ships has been identified (Figures 3 and 4). . interaction with environmental parameters, such as temperature, solar radiation, and wind. Internal structures in a port environment, such as piers, docks, berths, dolphins, transportation facilities, and buildings, are not usually designed to withstand sea actions, because wave actions are absorbed by the protection facilities. Steps of the analysis A multi-step algorithm is here proposed for a generally valuable data processing. The proposed algorithm cannot be completely independent of key factors anal- ysis (section ‘Key factors’) and of the expert’s judgment as it is completely data driven and some starting assumptions are necessary. The focus issue regards the choice of: (a) one or more reference measuring blocks and (b) a training period. The reference measur- ing blocks can be selected by observing the stability and the regularity of the measured variables during the monitoring period. The reference measuring blocks should be, as most as possible, stationary with time. The training period is a time period that can be assumed as a reference period for comparison with further monitoring periods. When the parameter under observation has a cyclic trend, the training period should contain at least one complete cycle. As a consequence, the evaluation of the right training period depends on the governing load or action and on the corresponding periodicity of the measured vari- ables; as in the case of San Giorgio pier, when cyclic loads, for example temperature variations, play the most important role, at least 1 year of observation is required to make the analysis independent of the seasonal variations, as it will be demonstrated in the section ‘Measuring blocks of type 1’. Two mechanical behaviors can be identified from the data: . The behavior in the direction normal to the block cross-section mainly due to dilatation. It can be assumed that this behavior is homogeneous for all measuring blocks because sensors have the same ori- entation and position inside the blocks (Figure 1); . The bending behavior of vertical (own weight and operational loads) and horizontal (operational loads) axes. . The bending behavior of vertical (own weight and operational loads) and horizontal (operational loads) axes. In addition, the presence of dilatation joints has come to light from strains time histories, but their posi- tion along the structure cannot be exactly determined. Figure 1 shows two possible positions of dilatation joints: the first one is crossing a measuring block (block type 2), while the second one is located between two monolithic measuring blocks (named block type 1). The correlation indices between the time histories of strain and temperature have been studied as first step of the flow chart in Figure 5. Steps of the analysis Time dependence of corre- lation indices ðt0, tÞ between strain sensors located in the same position along the quay (all sensors in location 1 for examples), between strain sensors in the same measuring block (sensors in locations 1–3 for each mea- suring block, Figure 1), and between strain sensors and environmental temperature, are computed using the following formula: The flow chart in Figure 5 suggests a sequence of signal analysis algorithms to be applied in cascade for the continuous monitoring of a structure with charac- teristics similar to the pier under study, and the infor- mation that can be drawn out without using any numerical structural model. This flow chart is represen- tative of the processing steps performed for the San Giorgio pier and presented in the following sections. Three main steps are highlighted with bold type and listed on the right side of the flow chart: (1) classifica- tion of measuring blocks behavior, (2) identification of damage-sensitive features for the specific application, and (3) damage detection. A dashed rectangle groups the ensemble of the actions and tests to be performed for each step of the processing. The input data and the output results of the multi-algorithm flow chart are pointed out using a parallelogram. ðt0, tÞ ¼ corr Saðt0, tÞ, Sbðt0, tÞ ð Þ ¼ cov Saðt0, tÞ, Sbðt0, tÞ ð Þ aðt0, tÞbðt0, tÞ ð1Þ ð1Þ where Sa and Sb are the vectors of time histories strain– strain or strain–temperature from t0 (beginning of data acquisition) to time t, cov, a, and b the covariance and the standard deviations of the two time series, respec- tively. The variation with time of the correlation index has been computed using, for each instant time t, the vectors containing all the previous measurements. Two main classes of measuring blocks with the same structural behavior have been identified in function of the sign of the correlation index strain–temperature (Figure 5): Key factors In addition, tides are not a On the contrary, the time histories of Figure 3 and the zoomed Figure 4 show that a strong correlation 5 with thermal variations exists and the following analy- sis will be mainly focused on temperature actions. complete (only 603 data missing on a total of 3776 measurements). The measurement campaigns with some data missing have simply not been considered in the analysis and they are assumed not to alter the results. This assumption is rather consistent because data missing are not concentrated in the same period. Classification of block behavior (group 1 in Figure 5) . Type 1 – positive correlation indices between sensors in the same block and also with temperature that plays the most important role in the structural . Type 1 – positive correlation indices between sensors in the same block and also with temperature that plays the most important role in the structural The analysis has been focused on the first 2 years of measurements (Figure 2), because they are almost 6 START Block measurements Temperature T Correlation analysis Classification of block behavior Correlation index with T Type 1 Classical beam block Type 2 Specific block (joint) Reference block type 1 Expert Analysis Identification of: -damage sensitive features -service period for statistical analysis Which type? Reference block type 2 Type 1 Relation with temperature Type 2 Model Sensitivity parameters Joint opening Thermal strain Filtering Statistical analysis Which type? Type 1 Fourier analysis Type 2 Reliability analysis Damage detection Second-order strain Linear regression Threshold definition Training period Expert analysis Residual error Inside threshold? No damage Damage detected END Occurrence time Location > 0 < 0 Residual error Yes No END Validation of classification Group 1 Group 2 Group 3 • STEP 3 • STEP 2 • STEP 1 Figure 5. Flow chart of the data processing steps. Figure 5. Flow chart of the data processing steps. and it is not a monolithic block (for example, block type 2 of Figure 1). Six measuring blocks belong to type 2 (specific blocks with joint). response (fair bending behavior, monolithic blocks like blocks type 1 of Figure 1). Thirteen measuring blocks belong to type 1 (classical beam blocks). . Type 2 – positive correlation indices between sensors in the same block but negative correlation indices with temperature (out-of-phase behavior). The temperature plays the most important role in the structural response, but this block contains a joint For each class, the expert analysis has to identify the measuring block to be used as reference by taking into account the stability of the correlation index all over the monitoring time. 7 7 A detailed explanation of this choice can be found in Ref.40 At the generic time i, the strain is computed as: The analysis of the correlation index with time also allows the identification of two subclasses of measuring blocks: "i ¼ Li  L0 L0 ð3Þ ð3Þ . Classification of block behavior (group 1 in Figure 5) Sensors with persistent measurement errors have been removed from the analysis. It is useful to underline that the ratio of malfunctioning sensors for harbor structures is generally included in the range 30–50%.37 Several factors can explain this relatively large amount of malfunctioning sensors: connection to the structure (elec- trical continuity, waterproofness, etc.), malfunctioning of the sensor itself, and global electrical continuity. . Types 1B and 2B – good correlation indices between sensors in the same block for the first period, with some observed variations during the monitoring (gradual degradation in time or temporary transition toward a new value). On the contrary, the correla- tion indices with temperature seem stable in time (positive or negative): the cause of the variations could not be related to the temperature. The mechanical behavior of these blocks is not governed by temperature only. Seven measuring blocks have been identified as type 1B or 2B. The second step of the analysis consists in defining damage-sensitive features for measuring blocks of types 1 and 2 and it will be detailed in sections ‘Damage- sensitive feature for blocks of type 1’ and ‘Damage- sensitive feature for blocks of type 2.’ The identified damage-sensitive features will be the sensitivity param- eter and the joint opening, respectively, for measuring blocks of types 1 and 2. Statistical analysis on the sensitivity parameter (group 2 in Figure 5) As previously mentioned (section ‘Steps of the analy- sis’), a reference situation is needed, as the data processing is performed by means of statistical analysis on data supposed as representative of the normal struc- tural behavior under service life. Usually, the parame- ters representative of structural behavior are extracted by all sensor measurements during the training period in which the structure is assumed to behave normally, under the hypothesis of a spatial stationarity of the random process representing the parameters under investigation. In this application, two measuring blocks during the whole monitoring period have been considered as reference blocks to also consider poten- tial differences due to the spatial distribution of mea- suring blocks. For their regular trend with time, measuring blocks C8 and B2 have been a priori assumed to have a normal behavior during the whole monitoring period. Classification of block behavior (group 1 in Figure 5) Types 1A and 2A – good correlation indices between sensors in the same block for the first period, with some observed variations during the monitoring (gradual degradation in time or temporary transition toward a new value). The same variations also appear in the correlation indices with temperature (positive or negative); as a consequence, the temper- ature still plays the most important role. Five measuring blocks belong to type 1A or 2A. where Li is the current measured length of the sensor at time i and L0 the initial length of sensors measurement base, in this case equal to 10 m for all sensors. A unique value of strain "i (called actual strain in the following) has been used as representative of the global behavior of each measuring block; it has been considered as the true value of the axial strain and it is computed as the mean value from strains of all sensors contained in that mea- suring block at each measurement time. It has to be reminded that a measuring block contains only three lon- gitudinal sensors. A filter has been applied to data to cut the temperature variations inducing strain variations lower than sensor sensitivity (2 mm m1). Sensors with persistent measurement errors have been removed from the analysis. It is useful to underline that the ratio of malfunctioning sensors for harbor structures is generally included in the range 30–50%.37 Several factors can explain this relatively large amount of malfunctioning sensors: connection to the structure (elec- trical continuity, waterproofness, etc.), malfunctioning of the sensor itself, and global electrical continuity. where Li is the current measured length of the sensor at time i and L0 the initial length of sensors measurement base, in this case equal to 10 m for all sensors. A unique value of strain "i (called actual strain in the following) has been used as representative of the global behavior of each measuring block; it has been considered as the true value of the axial strain and it is computed as the mean value from strains of all sensors contained in that mea- suring block at each measurement time. It has to be reminded that a measuring block contains only three lon- gitudinal sensors. A filter has been applied to data to cut the temperature variations inducing strain variations lower than sensor sensitivity (2 mm m1). Computation of the sensitivity parameter (group 2 in Figure 5, left) Actual strain vs temperature for reference measuring block of type 1 (C8). 0 5 10 15 20 25 30 −4 −2 0 2 4 6 8 10 x 10 −5 Temperature (°C) Actual strain (–) Measuring block C8 First period Second period Third period Fourth period Fifth period m ( g ) 0 0.5 1 1.5 2 2.5 3 3.5 4 x 10−5 0 0.5 1 1.5 2 2.5 3 x 105 Sensitivity parameter Density C8 sensitivity data Exponential Extreme value Figure 7. Probability density function of the sensitivity variable for blocks of type 1. 0 5 10 15 20 25 30 −4 −2 0 2 4 6 8 10 x 10 −5 Temperature (°C) Actual strain (–) Measuring block C8 First period Second period Third period Fourth period Fifth period Figure 6. Actual strain vs temperature for reference measuring block of type 1 (C8). 0 0.5 1 1.5 2 x 10−5 0 0.5 1 1.5 2 2.5 3 3.5 4 x 105 Sensitivity parameter Density A7 sensitivity data Exponential Extreme value Figure 9. Probability density function of the sensitivity variable for blocks of type 2. 0 5 10 15 20 25 30 −5 −4 −3 −2 −1 0 1 2 3 4 5 x 10−5 Temperature (°C) Actual strain (–) Measuring block B2 First period Second period Third period Fourth period Fifth period Figure 8. Actual strain vs temperature for reference measuring block of type 2 (B2). 0 5 10 15 20 25 30 −5 −4 −3 −2 −1 0 1 2 3 4 5 x 10−5 Temperature (°C) Actual strain (–) Measuring block B2 First period Second period Third period Fourth period Fifth period Figure 8. Actual strain vs temperature for reference measuring block of type 2 (B2). 0 5 10 15 20 25 30 −5 −4 −3 −2 −1 0 1 2 3 4 5 x 10−5 Temperature (°C) Actual strain (–) Measuring block B2 First period Second period Third period Fourth period Fifth period Figure 6. Actual strain vs temperature for reference measuring block of type 1 (C8). Figure 8. Actual strain vs temperature for reference measuring block of type 2 (B2). Computation of the sensitivity parameter (group 2 in Figure 5, left) Computation of the sensitivity parameter (group 2 in Figure 5, left) The homogeneous dilatation of measuring blocks of type 1 can be computed in terms of the sensitivity of measured strain variations to temperature variations as follows: s ¼ "  T ð2Þ ð2Þ where " is the strain variation between two time instants and  T the mean temperature variation at the same time instants. As the structural response is time delayed due to its great thermal inertia, at each time step, the current temperature has been computed as the mean value on the last six temperature measure- ments, corresponding to a period of 1 day and a half. The amount of available data from the reference blocks is enough for performing a statistical analysis of the time distribution of the sensitivity parameter that is here considered as a random process indexed by the space at the measurement time. The stationarity of the random process with time has been verified by 8 having mean value and standard deviation 3.07 mstrain C1 for blocks of type 1 (Figure 7). plotting the strain variation versus the temperature variation for each seasonal period (summer–winter) of the monitoring period; it can be observed that the strain variations are not related to the observation period (Figure 6). As a consequence, measurements from the reference blocks allow us to conclude that the random process is stationary with time. The sensitivity param- eter has been further analyzed as a random variable of a stationary process and its probability distribution has been investigated. The estimated values of the sensitiv- ity are well described by the exponential distribution The sensitivity parameter has been also computed and plotted for measuring blocks of type 2 (Figure 8). The different data trend shown by blocks of type 1 and blocks of type 2 confirms the trend observed in the correlation indices: blocks of type 1 have a positive correlation between strain and temperature, while for blocks of type 2 the correlation is negative. Also for blocks of type 2, the estimated values of the sensitivity are well described by the exponential distribution having mean value and standard deviation 1.80 mstrain C1 (Figure 9). 0 5 10 15 20 25 30 −4 −2 0 2 4 6 8 10 x 10 −5 Temperature (°C) Actual strain (–) Measuring block C8 First period Second period Third period Fourth period Fifth period Figure 6. Computation of the sensitivity parameter (group 2 in Figure 5, left) 0 0.5 1 1.5 2 2.5 3 3.5 4 x 10−5 0 0.5 1 1.5 2 2.5 3 x 105 Sensitivity parameter Density C8 sensitivity data Exponential Extreme value 0 0.5 1 1.5 2 x 10−5 0 0.5 1 1.5 2 2.5 3 3.5 4 x 105 Sensitivity parameter Density A7 sensitivity data Exponential Extreme value Figure 9. Probability density function of the sensitivity variable for blocks of type 2. Figure 7. Probability density function of the sensitivity variable for blocks of type 1. 9 In a probabilistic framework, by considering the confidence intervals of a random variable exponentially distributed, the expected value of the sensitivity param- eter for measuring blocks of type 1 is between 2.92 mstrain C1 (S lower ð Þ) and 3.26 mstrain C1 (S upper ð Þ) with a probability of 99%. The sensitivity parameter is considered to give a valid interpretation of the mechanical behavior of the pier. Indeed, the mean assessed value of the sensitivity parameter (between 2.9 and 3.3 mstrain C1) is coherent but lower than the known dilatation coefficient in concrete structures (usually varying between 6 and 13 mstrain C1 depending on the relative humidity and the aggregates). This is rightly related to the boundary conditions of the monitored crown block that is continuously supported by the lower blocks and linked to them by means of distributed vertical steel bars, so that it is not allowed to freely expand with thermal variations. where S lower ð Þ and S upper ð Þ are, respectively, the lower and the upper values of the sensitivity parameter asso- ciated with a probability of 99%. Consequently, it is possible to remove (filtering, Figure 5), from the strain time histories (Equation (4)), the variations due to temperature that sometimes can induce strong varia- tions wrongly interpreted as anomalous behaviors. In this analysis, the estimated mean value of the sensi- tivity parameter has been used: "secondorder ¼ "  ~"thermal ¼ "  ~S Ti  T0 ð Þ ð7Þ ð7Þ Figures 10 and 11 show the total strain and the second-order strain after removing thermal strain for block C8 (reference measuring block) and for a generic measuring block. The plots show that the second-order strains are smoother than the total ones in particular during the summer, when higher temperature variations induce greater strains. Computation of the sensitivity parameter (group 2 in Figure 5, left) However, the second-order strains still have a cyclic and periodic behavior, probably because of secondary nonlinear effects of thermal strains with temperature variations. For their periodicity, the obtained second-order strains can be well fitted by a Fourier series, assessed by considering the data con- tained in the first year of measurements. The Fourier fitting, associated with its confidence intervals at 99%, also allows the validation of the classification of blocks behavior already performed with the correlation analysis in section ‘Classification of block behavior’ (Figure 5). Furthermore, the Fourier fitting gives a clearer Characterization of thermal and second-order strains (group 2 in Figure 5, left) When a change in temperature occurs, the thermo-elas- tic strains can be written as: " ¼ "thermal þ "secondorder ð4Þ ð4Þ where "thermal is the thermal strain in the linear the- ory and "secondorder the residual strain due to non-linear effects with the temperature and other mechanical effects. The strain caused by a change in temperature from T0 in the reference configuration (in this case the beginning of the monitoring) to T in the current configuration is called thermal strain and it can be expressed by the linear relation: 30/11/1999 02/04/2000 05/08/2000 08/12/2000 12/04/2001 15/08/2001 18/12/2001 22/04/2002 –6 –4 –2 0 2 4 6 8 10× 10–5 Measuring block C8 Monitoring period Actual strain (–) Prediction bounds Fourier Second–order strain Total strain Figure 10. Second-order strain after removing thermal strain and Fourier fitting (reference measuring block). Prediction bounds are the confidence interval at 99%. 30/11/1999 02/04/2000 05/08/2000 08/12/2000 12/04/2001 15/08/2001 18/12/2001 22/04/2002 –6 –4 –2 0 2 4 6 8 10× 10–5 Measuring block C8 Monitoring period Actual strain (–) Prediction bounds Fourier Second–order strain Total strain "thermal ¼  T  T0 ð Þ ð5Þ ð5Þ where  is here the computed sensitivity parameter. The probabilistic analysis on the sensitivity parame- ter (section ‘Statistical analysis on the sensitivity parameter’) allows the assessment of the range of variation of the thermal strains for the monolithic measuring blocks given by Equation (5). For each time i: S lower ð Þ Ti  T0 ð Þ ¼ "thermal lower ð Þ 5 "thermal 5 "thermal upper ð Þ ¼ S upper ð Þ Ti  T0 ð Þ Figure 10. Second-order strain after removing thermal strain and Fourier fitting (reference measuring block). Prediction bounds are the confidence interval at 99%. ð6Þ ¼ S upper ð Þ Ti  T0 ð Þ 10 visualization of the strain deviations in time, in terms of gradual degradation and/or instantaneous transition toward a new trend (Figure 11). The distance between the measured strains and their expected trend (and, in particular, its progression in time) can allow us to under- stand the possible causes of the strain deviation identi- fied in measuring blocks of types 1A, 2A, 1B, and 2B. Figure 12 reports a block of type 2 in the reference state (out-of-scale) that is used as a mechanical model for the block behavior’s study. Characterization of thermal and second-order strains (group 2 in Figure 5, left) L0 is the initial length of the block between two measurement points, in this case, 10 m. The two monolithic blocks across the joint have unknown length but the strain of the blocks of total length l  is supposed to be proportional to the strain measured in the reference measuring block of type 1; l0 is the initial opening of the dilatation joint (unknown). Characterization of dilatation joints (group 2 in Figure 5, right) Characterization of dilatation joints (group 2 in Figure 5, right) For measuring blocks of type 2, another parameter able to describe the mechanical behavior is the variation in the joint opening lj under temperature variations. This parameter can be estimated by knowing the strain mea- sure in the block with joint (measj ðtype2Þ) and assuming that the two integer blocks across the joint (block 2 on the right of the joint and block 2 on the left of the joint, Figure 1) have the same behavior of the blocks without joints. j For each measurement session j: l  ¼ "type 1  l  ð8Þ ð8Þ where "type 1 ¼ measj ðtype 1Þ L0 and l  ¼ L0  l0. From the previous relations, and knowing that L ¼ " type 2 ð Þ  L0, it is possible to obtain the variation in the joint opening: 30/11/1999 02/04/2000 05/08/2000 08/12/2000 12/04/2001 15/08/2001 18/12/2001 22/04/2002 –5 0 5 10 15 × 10–5 Measuring block A6 Monitoring period Actual strain (–) Prediction bounds Fourier Second–order strain Total strain Figure 11. Second-order strain after removing thermal strain and Fourier fitting (generic measuring block). Prediction bounds are the confidence interval at 99%. 30/11/1999 02/04/2000 05/08/2000 08/12/2000 12/04/2001 15/08/2001 18/12/2001 22/04/2002 –5 0 5 10 15 × 10–5 Measuring block A6 Monitoring period Actual strain (–) Prediction bounds Fourier Second–order strain Total strain ð9Þ lj ¼ measj ðtype 2Þ  measj ðtype 1Þ þ l0  "type 1 ð9Þ where measj(type 2) is the current measurement (in mm) in a generic block of type 2. The term related to the initial opening of the joint can be neglected because l   l0 and the relation can be simplified: ð10Þ lj ffimeasj ðtype 2Þ  measj ðtype 1Þ ð10Þ Damage-sensitive feature for blocks of type 2 Let us now consider an increase in temperature start- ing from this initial configuration. The block of initial length L0 will be subjected to a shortening L (as observed from measurements of blocks of type 2), while the monolithic blocks of total length l  will increase their length of l  as a block of type 1. As a consequence, the opening of the dilatation joint will decrease of the quantity lj. These length variations satisfy the relation L ¼ lj þ l . Reliability analysis The comparison of the computed joint opening varia- tion for the six measuring blocks of type 2 is reported in Figure 13. The plot shows that the joint opening trend is the same for all the considered blocks during the first Figure 11. Second-order strain after removing thermal strain and Fourier fitting (generic measuring block). Prediction bounds are the confidence interval at 99%. Figure 11. Second-order strain after removing thermal strain and Fourier fitting (generic measuring block). Prediction bounds are the confidence interval at 99%. |*/n L0 |0 |*/m Figure 12. Scheme of a measuring block containing a joint in the initial configuration. The ratio of n and m satisfies the following relation: 1/n + 1/m ¼ 1, and L0 ¼ l0 þ l . Figure 12. Scheme of a measuring block containing a joint in the initial configuration. The ratio of n and m satisfies the following relation: 1/n + 1/m ¼ 1, and L0 ¼ l0 þ l . 11 30/11/1999 02/04/2000 05/08/2000 08/12/2000 12/04/2001 15/08/2001 18/12/2001 22/04/2002 25/08/2002 –3 –2.5 –2 –1.5 –1 –0.5 0 0.5 1 1.5 2 Monitoring period Joint opening variation (mm) Opening B2 Opening B1 Opening B3 Opening B4 Opening B6 Opening C5 Figure 13. Joint opening variation for blocks of type 2. Table 1. Failure probability due to insufficient dilatation joint width Table 1. Failure probability due to insufficient dilatation joint width l0 ¼ 3 mm l0 ¼ 2 mm l0 ¼ 1 mm Pf <1010 8  109 1.70  101 Pf measured joint opening is lower than the initial joint opening l0 can be computed from the fitted density functions with the aim to obtain the failure probability of the pier due to an inadequate initial joint design (limit state). With reference to Figure 14, the probability of failure can be computed as: Pf ¼ P lj 5 l0   ¼ P lj 5 l0jwinter    P winter ð Þ þ P lj 5 l0jsummer    P summer ð Þ ð11Þ ð11Þ Figure 13. Joint opening variation for blocks of type 2. where the probability to have conditions like in winter or summer is related to the number of measurements in winter or summer in relation with the total number of measurements. Table 1 reports an example of the assessed failure probabilities related to different values of the initial joint opening l0. Reliability analysis –1.5 –1 –0.5 0 0.5 1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 PDF joint opening f(x) Opening variation Histogram of frequency Normal PDF summer Normal PDFwinter Figure 14. Probability density function of the joint opening variation for blocks of type 2. Damage detection (group 3 in Figure 5) Different damage-sensitive features have been identified from previous analysis: for measuring blocks of type 1, second-order strains have been tested as sensitive damage features, while for measuring blocks of type 2, the joint opening has revealed to be a good damage indicator. The second-order strains have been used as they are not fully correlated to thermal varia- tions and for this reason they are supposed to be more damage-sensitive than the total strains. Figure 14. Probability density function of the joint opening variation for blocks of type 2. g Different damage detection algorithms have been proposed in literature with similar data.41,42 In this application, the linear regression analysis will be presented and successfully applied to detect the insur- gence of anomalous behaviors in some measuring blocks. As regression analysis is a statistical tool for the investigation of relationships between variables,43 it has been used to analyze the measuring block behav- ior along the pier. As demonstrated in sections ‘Statistical analysis on the sensitivity parameter’ and ‘Reliability analysis,’ the identified damage-sensitive features have been considered as random variables of a stationary process with time in reference blocks. The spatial stationarity is here assumed and the damage- sensitive features in generic measuring blocks during the training period are considered as observations of the same random variables identified for reference blocks. monitoring period, with a divergence while increasing the time. The statistical analysis of the time distribution of the joint opening for the reference block B2 allows us to conclude that the joint opening variation can be also considered as a random variable of a stationary process with time (section ‘Statistical analysis on the sensitivity parameter’). In this case, the estimated joint width values are well described by a bimodal distribution and a reliabil- ity analysis on the joint width can be performed (Figure 5). To this purpose, two samples of data have been considered: the values registered during the summer (left) and the ones measured during the winter (right). The normal distribution is able to describe the distribution of the two samples well. Figure 14 shows the probability density function for measuring blocks of type 2. Damage detection (group 3 in Figure 5) The probability that the 12 30/11/1999 02/04/2000 05/08/2000 08/12/2000 12/04/2001 15/08/2001 18/12/2001 22/04/2002 25/08/2002 –5 0 5 Monitoring period Actual strain Time histories Section B5 Section C8 –5 –4 –3 –2 –1 0 1 2 3 4 X 10 –5 –5 0 5 Actual strain C8 Actual strain B5 Sections B5 vs C8 Training period Further measurements Linear fitting and conf bnds (99%) 30/11/1999 02/04/2000 05/08/2000 08/12/2000 12/04/2001 15/08/2001 18/12/2001 22/04/2002 25/08/2002 0 1 2 3 4 Monitoring period Residuals Residuals estimated–measured B5 Training period Further measurements Threshold (99%) × 10–5 × 10–5 × 10–5 Figure 15. Time histories of second-order strain, regression line, and residual associated to a training period of 1 year (measuring block B5). Figure 15. Time histories of second-order strain, regression line, and residual associated to a training period of 1 year (measuring block B5). The second subplot shows the relation between the two measuring blocks strains and the regression line assessed on the basis of a training period of 1 year, with the cor- responding 99% confidence interval. The training period measurements are in black points while measurements after the training period are plotted in grey crosses. The third subplot shows the time history of the residuals (absolute value) between the measured second-order strain of the block under investigation and the assessed regression line, associated with the threshold correspond- ing to the 99% confidence intervals. Also, in this subplot, grey crosses represent the measurements after the training period. Two anomalous overcoming phenomena of the threshold in the residual time history can be observed after the reference period, but both can be interpreted as transitory occurrences because after that the residuals get stable with time again. It can be concluded that in this measuring block no significant variations have been observed during the monitoring time. Measuring blocks of type 1 The relationship between sensors is someway changed but no other evolution in time is observed from the residual time history; this is also confirmed by the trend of the correlation index that reaches a new constant value (starting from December 2001) after a transition period related to the periodicity of the mobile window method. In this case, the detected phenome- non can be interpreted as a transition toward a new stable configuration, different from the initial one. This means that a new threshold and a new training period based on the new trend should be defined for further measurements. containing only a fixed number of last measurements has been used. The original aspect of this method is that the correlation index is computed only for a moving window of constant size. This means that after each measurement session, the correlation is calculated only with the mea- surements inside a moving window of 1 year. The use of a moving window has the advantages that it guarantees stability of average values, ensures rapid damage identi- fication, and reduces noise effects. Figures 16 and 17 show some examples of different sensor behaviors during the monitoring period. Figures 16 and 17 show some examples of different sensor behaviors during the monitoring period. 0 0.5 1 1.5 2 2.5 Residuals (–) 30/11/99 02/04/00 05/08/00 08/12/00 12/04/01 15/08/01 18/12/01 22/04/02 25/08/02 0 0.2 0.4 0.6 0.8 1 Correlation index C42–C82 (–) Residuals estimated–measured C42 Monitoring period × 10–4 Figure 16. Residual of second-order strains, threshold associ- ated to a training period of 1 year (line in black), and correlation index for sensors C42 and C82. Figure 17 illustrates a typical example of a progres- sive phenomenon observed in sensor A62. Around April 2001, the correlation index shows a decrease and at the same time the residuals overcome the thresh- old. Residuals remain over the limit for all the follow- ing measurements, showing also a divergence with time, and the correlation index does not reach a new stable value. Also in this case, the periodic trend in the correlation index is related to the mobile window procedure. Figure 16. Residual of second-order strains, threshold associ- ated to a training period of 1 year (line in black), and correlation index for sensors C42 and C82. Measuring blocks of type 1 A regression analysis has been performed on the second- order strain time histories by comparing the reference measuring block C8 with the other measuring blocks of type 1. The linear relationship between the reference block and the block under investigation is assessed during the reference period. The further observations of this latter block are forecasted using the linear rela- tion previously assessed. The difference between the measured and the forecasted second-order strains (resid- ual) gives some information on the persistency in time or on possible variations of the initial relationship. A threshold limit corresponding to a confidence interval of 99% has been evaluated taking into account the stan- dard deviation of the residuals computed during the training period. Two different training periods have been compared: the first one considers 6 months of mea- surements and the second one contains the first year of measurements. The analyses have demonstrated that a training period containing at least the whole periodicity of the parameter under study (in this case the yearly cycle) is more reliable for damage detection purposes. The analysis has been deepened using also the infor- mation from the correlation analysis (section ‘Classification of block behavior’). In normal conditions, the correlation index between sensors in the reference measuring block C8 and sensors in a generic measuring block of type 1 should be stationary with time under the assumption of the spatial stationarity. However, when damage or degradation occurs, correlations between sen- sors can change. In order to follow the strains time evo- lution more effectively, a window (a subset of time series) The plot in Figure 15 shows an example of damage detection results using the second-order strains as damage-sensitive parameter. The first subplot from the top shows the time histories of the reference measuring block (C8) and another generic block of type 1 under investigation (in this case measuring block B5). 13 Plots superimpose the time histories of the residuals computed from the previous linear regression analysis (black points) and the correlation index between the second-order strains of the same pair of sensors (green line on the secondary y-axis). The time histo- ries comparison should allow a better interpretation of the spatial evolution phenomena interesting the structure. Figure 16 reports the behavior of sensor C42. The decrease in correlation index after September 2000 is also detected by the overcoming of the threshold in the residuals. Measuring blocks of type 2 The same algorithm of linear regression analysis described in the previous section has been applied to the joint opening time histories of the reference measuring block (B2) and of a generic block of type 2 under investigation. Also in this case, a training period of 1 year has been considered. Figure 18 shows the analysis performed for the measuring block B4 that puts into evidence a degradation initiation and a grad- ual progression of the detected event with time. The discussion on the complexity of data processing has been carried on by means of available data from the real long-term monitoring of a quay wall in the Port of Genoa. This example wanted to put in evidence that an optimal and unique algorithm cannot be proposed depending on the variety of applications. A multi- algorithm approach has been considered to be the best tool for understanding the structural behavior and for defining response features. This article empha- sized the preliminary steps aimed at understanding the mechanical response of such a structure under opera- tional and environmental loads. It has been discussed that the complex interaction with environmental temperature had to be taken into account too. This initial phase helped to identify typical response features that have been used for further analysis of damage identification. Due to the huge amount of instrumented blocks, in this application, the statistical analysis has proved to be successful. The extracted response features have been used as damage-sensitive parameters. Measuring blocks of type 1 0 0.5 1 1.5 2 2.5 × 10–4 Residuals (–) 30/11/99 02/04/00 05/08/00 08/12/00 12/04/01 15/08/01 18/12/01 22/04/02 25/08/02 0 0.2 0.4 0.6 0.8 1 Correlation index A62–C82 (–) Residuals estimated–measured A62 Monitoring period Figure 17. Residual of second-order strains, threshold associated to a training period of 1 year (line in black), and correlation index for sensors A62 and C82. Figure 17. Residual of second-order strains, threshold associated to a training period of 1 year (line in black), and correlation index for sensors A62 and C82. 14 14 30/11/1999 02/04/2000 05/08/2000 08/12/2000 12/04/2001 15/08/2001 18/12/2001 22/04/2002 25/08/2002 –2 –1 0 1 Monitoring period Joint opening (mm) Time histories Section B2 Section B4 –1.2 –1 –0.8 –0.6 –0.4 –0.2 0 0.2 0.4 0.6 –0.5 0 0.5 Joint opening B2 (mm) Joint opening B4 (mm) Sections B4 vs B2 (REF C8) 30/11/1999 02/04/2000 05/08/2000 08/12/2000 12/04/2001 15/08/2001 18/12/2001 22/04/2002 25/08/2002 0 0.1 0.2 0.3 0.4 Monitoring period Residuals (mm) Residuals estimated–measured B4 Training period Further measurements Linear fitting and conf bnds (99%) Training period Further measurements Threshold (99%) Figure 18. Time histories of joint opening, regression line, and residual associated to a training period of 1 year for a measuring block presenting degradation (B4). Figure 18. Time histories of joint opening, regression line, and residual associated to a training period of 1 year for a measuring block presenting degradation (B4). measurements, while the data obtained from real cases often present large incompleteness, related to system maintenance problems or malfunctioning due to various causes. In addition, if the structure has not a well-defined structural behavior and if possible vari- ations in the structural behavior are expected to occur as a result of complex interaction phenomena, special algorithms and interpretation need to be applied. References 1. Mufti A. Guidelines for structural health monitoring, design manual no. 2. Winnipeg, Manitoba. Canada: ISIS, 2001. 17. Liu M, Frangopol DM and Kim S. Bridge system perfor- mance assessment from structural health monitoring: a case study. J Struct Eng 2009; 135(6): 733–742. 2. SAMCO. F09 report on bridge management, Final Report 2006, 2006. 3. Polder RB, Alonso MC, Cleland DJ, et al. 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Morassi A and Vestroni F. Dynamic methods for damage detection in structures. International Centre for Mechanical Sciences, Udine: Springer, 2009, p.221. 24. Omenzetter P and Brownjohn JMW. Application of time series analysis for bridge health monitoring. Conclusions This article aimed at giving a general procedure for data interpretation from a continuous monitoring of complex structures. This kind of monitoring system is able to produce time-histories that have proved to be very useful for the comprehension of the structural behavior under complex interaction structure–environ- ment. The interpretation of data from long-term static monitoring is generally faced on with different approaches, depending on the characteristics of the applications: model-based interpretation, statistical system identification, and signal processing methods for the recognition of typical response features. 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Computing correla- tion anomaly scores using stochastic nearest neighbors. Omaha, USA: ICDM, 2007. 39. Glisˇ icˇ B and Inaudi D. Fibre optic methods for structural health monitoring. England: John Wiley & Sons, 2007, p.262. 32. Deraemaeker A, Reynders E, De Roeck G and Kullaa J. Vibration-based structural health monitoring using output-only measurements under changing environment. 43. Berk RA. Statistical learning from a regression perspec- tive. New York: Springer-Verlag, 2008, p.358. References Mech Sys Sig Process 2008; 22: 34–56. 40. Lanata F. San Giorgio Pier data analysis: summary of significant results – GeM internal report (GeM-ECS- 1006ins01) – June 2009, Available on-line: http:// gem.ec-nantes.fr, poˆ le ‘‘Structures et Couplages’’, e´ quipe ‘‘Calcul des Structures’’, The` me 2, 2009, 17 pp. 33. Posenato D, Lanata F, Inaudi D and Smith IFC. Model- free data interpretation for continuous monitoring of complex structures. Adv Eng Inf 2008; 22(1): 135–144. 41. Lanata F. Damage detection algorithms for continuous static monitoring of structures, PhD Thesis, University of Genoa, 2005. 34. Berry M and Linoff G. Mastering data mining. New York: John Wiley & Sons, 2000. 35. Witten I and Eibe F. Data mining: practical machine learning tools and techniques, 2nd ed. San Francisco: Morgan Kaufmann, 2005. 42. Posenato D. Model-free data interpretation for continu- ous monitoring of complex structures, PhD Thesis, EPFL, n. 4481, 2009. 36. Del Grosso A, Lanata F, Brunetti G and Pieracci A. Structural health monitoring of harbour piers. In: SHMII-3 2007, Vancouver, November, 2007. 43. Berk RA. Statistical learning from a regression perspec- tive. New York: Springer-Verlag, 2008, p.358. 17 17
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Classification of Gene Samples Using Pair-Wise Support Vector Machines
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Engin Taş, Ph.D. * Assist. Prof, Department of Statistics, Faculty of Science and Literature, Afyon Kocatepe University, Afyonkarahisar, Turkey, engintas@aku.edu.tr The main problem in the classification problems encountered with gene samples is that the dimension of the data is high although the sample size is small. In such problems, the classifier to be used must be a classifier that allows the processing of high dimensional data and extracts maximum information from a small number of samples at hand. In this context, a classification methodology has been developed, which first transforms the problem of binary or multiple classification into separate pair-wise classification problems. To this end, an online classifier has been adapted to solve pair-wise binary classification problems. The resulting classifier performed better on most of the real problems compared to other popular classifiers. AJ ID: 2017.05.02.STAT.03 DOI: 10.17093/alphanumeric.345115 AJ ID: 2017.05.02.STAT.03 DOI: 10.17093/alphanumeric.345115 Received: October 19, 2017 Accepted: November 13, 2017 Accepted: November 13, 2017 p , Published Online: November 29, 2017 p Published Online: November 29, 2017 Available online at www.alphanumericjournal.com Available online at www.alphanumericjournal.com © 2013 -2017. Alphanumeric Journal The Journal of Operations Research, Statistics, Econometrics and Management Information Systems All rights reserved. alphanumeric journal p j The Journal of Operations Research, Statistics, Econometrics and Management Information Systems Volume 5, Issue 2, 2017 Received: October 19, 2017 Received: October 19, 2017 Accepted: November 13, 2017 ABSTRACT Tumor Classification, Pair-Wise Classification, Support Vector Machine, Kernel Methods 1. Giriş Kanser vakalarında kanserli tümörlerin hassas bir şekilde tespit edilmesi, zor olmasına karşın başarılı bir tedavi süreci için çok değerlidir. Bilgisayar teknolojisindeki son gelişmeler doğrultusunda elde edilen büyük boyutlu, yüksek verimli gen örneği çıkarma yöntemleri ve buna uygun tutarlı istatistiksel metotlar kullanılarak, biyomoleküler bilgi kanser tedavilerinde çok önemli bir konuma ulaşmıştır. Bu çalışmalardaki temel problem, mikrodizi deneyleri sonucu elde edilen veri kümelerinin binlerce gene ait örnekten oluştuğu için büyük boyutlu olması ve bunun yanında birkaç düzine mikrodizi örneğinden oluşmasıdır. Başka bir ifadeyle, gözlem sayısından çok daha fazla sayıda tahmin edici bağımsız değişken bulunmasıdır. Bu durum yeni istatistiksel problemlerin doğmasına ve bu problemlerin çözümüne yönelik yeni tekniklerin geliştirilmesine yol açmıştır. Mikrodizi verisine dayanarak kanserli hastaların tespit edilmesi problemi bir istatistiksel sınıflandırma problemidir. Bu problemin çözümü için literatürde diskriminant analizi, cezalandırılmış regresyon teknikleri ve en yakın komşu kuralı gibi klasik parametrik olmayan yöntemlerden, yapay sinir ağları ve destek vektör makineleri gibi modern yapay öğrenme tekniklerine kadar birçok yöntem bu problemlerin çözümünde kullanılmıştır. Bu çalışmaları gözden geçirmek için (Dudoit, Fridlyand, & Speed, 2003)'e bakılabilir. Bu çalışmada ise var olan yöntemlere alternatif olarak istatistiksel sınıflandırma problemine farklı bir açıdan yaklaşan bir sınıflandırma metodolojisi geliştirilmiştir. Şimdi ikili bir sınıflandırma problemini ele elalım. Elimizde belirli bir hastalığa ilişkin sağlıklı ve hasta insanlara ait gözlemlerden oluşan bir veri kümesi olsun. Aynı sınıftan gelen iki örnek arasında pozitif bir bağ, farklı sınıftan gelen iki örnek arasında negatif bir bağ olduğu düşünülebilir. Bu şekilde ele aldığımız bu problem, insanların varlıkları ve aynı sınıfa (sağlıklı/hasta) ait olup olmama bilgisinin de ilişkileri oluşturduğu bir ağ şeklinde temsil edilebilir. Bu sayede ikili sınıflandırma problemi ile bir ağdaki bağ tahmini arasında bir benzerlik kurulmuş olur. Diğer bir ifadeyle, ikili sınıflandırma probleminde yeni bir örneğin ilgili sınıfa atanması problemi, bir ağa yeni eklenen bir düğümün hangi düğümlerle bağlantı oluşturacağını tahmin etme problemi gibi düşünülebilir. Bu yaklaşımı pratikte gerçekleştirebilmek için mevcut verinin farklı bir şekilde temsil edilmesi gerekir. Örneğin, klasik sınıflandırma probleminde bir gözlem sadece bir kişiye ait özelliklerden oluşan bağımsız değişkenler ve bu kişinin ait olduğu sınıfı belirleyen ikili bir etiketten oluşurken, yeni yaklaşımda bir gözlem iki farklı kişiye ait özelliklerden oluşan bağımsız değişkenlerden ve bu iki kişinin aynı sınıfa ait olup olmadığını belirleyen bir etiketten oluşur. Destek Vektör Makinesi'nin (DVM) temel fikri, doğrusal olarak ayrılabilen veriler üzerinden optimal bir ayırma hiperdüzlemi oluşturmaktır (Boser, Guyon, & Vapnik, 1992). Gen Örneklerinin Eşli Destek Vektör Makinesi ile Sınıflandırılması Gen örnekleriyle ilgili karşılaşılan sınıflandırma problemlerinde en büyük sorun az sayıda örnek elde edilmesine karşın verinin büyük boyutlu olmasıdır. Bu tür problemlerde kullanılacak sınıflandırıcının büyük boyutlu verinin işlenmesine olanak sağlayan ve eldeki az sayıda örnekten maksimum bilgiyi çıkaran bir sınıflandırıcı olması gerekir. Bu kapsamda, öncelikle ikili/çoklu sınıflandırma problemlerini ayrı ayrı eşli ikili sınıflandırma problemlerine çeviren bir sınıflandırma metodolojisi geliştirilmiştir. Bunun için, çevrimiçi bir sınıflandırıcı eşli ikili sınıflandırma problemlerini çözecek şekilde tekrar düzenlenmiştir. Oluşan sınıflandırıcı gerçek problemlerin çoğu üzerinde diğer popüler sınıflandırıcılara göre oldukça iyi bir performans göstermiştir. ÖZ nahtar elimeler: Tümör Sınıflandırması, Eşli Sınıflandırma, Destek Vektör Makinesi, Çekirdek Yöntemler Tümör Sınıflandırması, Eşli Sınıflandırma, Destek Vektör Makinesi, Çekirdek Yöntemler Classification of Gene Samples Using Pair-Wise Support Vector Machines Taş 284 1. Giriş Aynı zamanda çekirdekleri ve yumuşak marj formülasyonlarını kullanarak doğrusal olarak ayrılamayan verilerde geniş marjlı bir hiperdüzlemi de öğrenebilir. Bununla birlikte, DVM başlangıçta ikili sınıflandırma için tasarlanmıştır ve DVM'yi çok sınıflı senaryoya genişletmek için iki ana yaklaşım vardır. Bir yaklaşım ikili algoritmayı çok sınıfa genelleştirmektir (Weston & Watkins, 1999, Mayoraz & Alpaydin, 1999), başka bir yaklaşım ise çok sınıflı sınıflandırma problemini bir dizi ikili problem haline dönüştürmektir. En eski ve en yaygın kullanılan uygulamalardan biri, her biri her sınıfı Alphanumeric Journal Volume 5, Issue 2, 2017 Gen Örneklerinin Eşli Destek Vektör Makinesi ile Sınıflandırılması Taş 285 diğerlerinden ayıran m ikili DVM sınıflandırıcılarını oluşturan, tümüne karşı tek yaklaşımıdır (Dietterich & Bakiri, 1995). İ. DVM, i. sınıfın tüm örneklerini pozitif etiketlerle ve diğer tüm örnekleri negatif etiketlerle ele alarak eğitilir. Eşli sınıflandırma ise, iki sınıflı problemlerin her birinden elde edilen eşli karşılaştırmalar göz önüne alınarak, çok sınıflı problemleri çözmek için alternatif bir tekniktir (Friedman, 1996). Test kümesinden bir örnek sınıflandırılırken, en çok eşli karşılaştırmayı kazanan sınıfa atanır. Bu çalışmada, çiftleri sınıflandırmada kullanılmak üzere her bir sınıf için eşli bir DVM modeli oluşturduk. Burada aynı sınıftan gelen iki yumurta pozitif bir çifti, farklı sınıftan gelen iki yumurta negatif bir çifti oluşturur. Bu eşli DVM modeli, herhangi bir çiftin pozitif bir çift olup olmadığını belirleyebilir. Diğer taraftan, eşli bir düzenlemede n örnek n^2 eşli örneğe karşılık gelir ve büyük ölçekli bir veri kümesi ile birlikte bir destek vektör makinesinin eğitilmesi çoğu durumda ciddi hesaplama maliyetleri getirir. Veriler toplu olarak işlendiğinde, DVM'ler her adımda amaç fonksiyonun hesaplanmasını gerektirir, ve bu temel olarak önceden tanımlanmış bir kayıp fonksiyonunun eğitilecek bir veri seti üzerinden hesaplanmasını gerektirir. Gradyana dayalı yöntemler, amaç fonksiyonunun her bir değerlendirmesinde sırasıyla gradyanı hesaplarken, Newton yöntemi ve eşlenik gradyan algoritması gibi standart sayısal optimizasyon teknikleri, amaç fonksiyonunun ikinci dereceden bilgisine ihtiyaç duyar. Mevcut veri setleri gittikçe büyüdükçe, bu tür klasik ikinci dereceden yöntemler neredeyse tüm durumlarda uygulaması pratik değildir. Buna karşın, algılayıcı (perceptron) (Rosenblatt, 1958, Minsky & Papert, 1969) ve varyantları (Freund & Schapire, 1999, Li & Long, 2002, Gentile, 2002, Anlauf & Biehl, 2007) gibi çevrimiçi gradyan tabanlı yöntemler , büyük ve tekrarlı veri kümelerinde büyük bir avantaja sahiptir. 1. Giriş Aslında, basit çevrimiçi gradyan düşümü yöntemleri (Bottou & LeCun, 2004, Shalev-Shwartz, Singer, & Srebro, 2007, Xu, 2011) genelde sofistike ikinci derece toplu algoritmalardan daha iyi performans sergiler, çünkü çevrimiçi yöntemlerin hesaplama gereksinimleri, eğitim verilerini tek tek örnekler veya küçük alt örnekler şeklinde işledikleri için oldukça düşüktür. Dolayısıyla bu çalışma, problemi farklı tanımlayarak ve buna uygun bir yöntemin seçimi ile ilgili iki temel fikre dayanır. İlk olarak, herhangi bir ikili veya çok sınıflı sınıflandırma probleminin eşli sınıflandırma problemine dönüştürülebileceğini biliyoruz. Bu, daha zengin bir veri kümesinden öğrenmenin avantajını getirir ve eşli model, örneklerden öğrendiğimizden daha fazlasını öğrenebilir. İkincil olarak, geleneksel öğrenme algoritmaları bu kapsamda daha verimsiz hale geldiğinden ve bazı durumlarda uygulanamadığı için (ör. toplu yöntemler), çevrimiçi DVM algoritmasını örnek çiftlerle çalışacak şekilde değiştirilmesini öneriyoruz. Bu yaklaşım, çiftleri tek tek işleme avantajını getirir ve daha büyük boyutlara sahip büyük ölçekli verilerden kaynaklanan zorlukların üstesinden gelir. 2. Gereç ve Yöntem 𝒳= (𝑥1, 𝑥2, . . . , 𝑥𝑚), ∀𝑥𝑖∈ℝ𝑛 şeklinde bir örnek kümemiz olsun. İki örneğin herhangi bir kombinasyonu 𝑝= (𝑥𝑖, 𝑥𝑗) ∈𝒫⊆𝒳2 çifti olarak düşünülebilir. Bu çiftlerden oluşan 𝑇= {(𝑝, 𝑦𝑝): 𝑝∈𝒫} dizisini ℤ= 𝒫× {+1,−1} olasılık dağılımınına sahip bir kitleden çekilmiş bir eğitim örneklemi olduğunu düşünelim. 𝑇'den gelen bir örnek, 𝑛-boyutlu bir sütün vektörü çifti ve bu iki örneğin aynı sınıftan gelip gelmediğini belirleyen bir 𝑦𝑝 (+1, −1) etiketinden oluşan bir üçlüdür. Amaç eğitim örnekleminden uygun bir 𝑓: 𝒫→ Alphanumeric Journal Volume 5, Issue 2, 2017 Classification of Gene Samples Using Pair-Wise Support Vector Machines Taş Taş 286 {+1,−1} fonksiyonunu öğrenmektir. Karar fonksiyonunun, 𝑓(𝑝) = ⟨𝑤, Φ(𝑝)⟩ şeklinde temsil edildiği doğrusal durumu ele alalım, burada 𝑤∈ℝ𝑛 eğitim örneklemi 𝑇'ye dayanarak tahmin edilmesi gereken parametre vektörüdür ve Φ ise çiftleri daha büyük bir uzaya gönderen bir özellik fonksiyonudur. {+1,−1} fonksiyonunu öğrenmektir. Karar fonksiyonunun, 𝑓(𝑝) = ⟨𝑤, Φ(𝑝)⟩ şeklinde temsil edildiği doğrusal durumu ele alalım, burada 𝑤∈ℝ𝑛 eğitim örneklemi 𝑇'ye dayanarak tahmin edilmesi gereken parametre vektörüdür ve Φ ise çiftleri daha büyük bir uzaya gönderen bir özellik fonksiyonudur. Optimal bir karar fonksiyonu ( en büyük marjine sahip optimal hiperdüzlem) özellik uzayında aşağıdaki amaç fonksiyonunu minimize ederek bulunur (Schölkopf & Smola, 2001):   2 1 ˆ 1 min 0 n i i i i i w i i i y y p w C with            (1) (1) (1) 𝜉= 𝜉1, 𝜉2, … , 𝜉𝑛 gevşek değişkenleri bazı çiftlerin marjinin yanlış tarafında yer almasına izin verir. Düzeltme parametresi 𝐶'nin büyük değerleri için, ayrılamayan çiftlere büyük bir ceza verilir ve daha fazla sayıda destek çifti oluşur. 𝐶'nin küçük değerleri bu cezanın etkisini yumuşatır ve gürültülü problemlerde daha iy sonuçlar elde etmemizi sağlar ama yetersiz uyuma sahip bir destek çifti modeli oluşturabilir. Bu konveks optimizasyon probleminin eşini (dual) maksimize etmek esas problemden daha basit bir konveks kuadratik programlama problemidir. 2. Gereç ve Yöntem DVM çekirdek genişlemesinin 𝛼𝑖 katsayıları aşağıdaki eş amaç fonksiyonunu    , 1 , 2 i i i j i j i i j W y K p p        (2) (2) tanımlayarak ve      0 max min 0, max 0, i i i i i i i i i A B with A Cy B Cy                  (3) tanımlayarak ve tanımlayarak ve      0 max min 0, max 0, i i i i i i i i i A B with A Cy B Cy                  (3) (3) DVM kuadratik programlama problemini çözerek bulunabilir. Orta büyüklükteki veri kümelerinde bile eşli öğrenme gerçekleştirdiğimizde çok büyük sayıda örnek çiftlerini işlememiz gerekir, bu nedenle etkin ve hızlı bir SVM sınıflandırıcısına ihtiyaç duyarız. Bu zorlukların üstesinden gelebilmek için çevrimiçi ve aktif öğrenme özellikleriyle çekirdeklerle çalışan hızlı bir sınıflandırıcı olan LASVM (http://leon.bottou.org/projects/lasvm) algoritması (Bordes, Ertekin, Weston, & Bottou, 2005) kullanılmıştır. Çevrimiçi yapısından dolayı eşli öğrenme kapsamında bu algoritma çeşitli avantajlara sahiptir. LASVM örnekleri tek tek işleyerek ve en çok bilgilendirici olan destek vektörlerini açılımında tutarak hesaplama karmaşıklığı ve maliyetiyle başa çıkabilir. Bu gerekli olan hesaplama miktarını ciddi anlamda düşürür. LASVM algoritmasının başka bir avantajı da seyrek veri kümelerinde kullanılmasına uygun olmasıdır zira veri boyutunun büyük olduğu çoğu veri kümesinde örnekler önemli derecede seyrek bir yapıya sahiptir yani örnek vektörünün bazı elemanları değer almaz. LASVM bu problemi bu örneklere uygun hızlı seyrek vektör çarpımları kullanarak bir avantaja dönüştürür. Bu sayede örneklerin ikili kombinasyonlarından oluşan çiftler tek tek işlenerek daha zengin bir veri kümesinden öğrenmenin avantajları değerlendirilmiş olur. LASVM ayrıca herhangi bir zamanda çekirdek Alphanumeric Journal Volume 5, Issue 2, 2017 Gen Örneklerinin Eşli Destek Vektör Makinesi ile Sınıflandırılması Taş 287 açılımında toplanan vektörlerin çevrimiçi süreçte açılımdan çıkarıldığı bir destek vektörü çıkarma adımına da sahiptir. Bu da o anda çekirdek açılımındaki etkinliğini yitirmiş olan destek vektörlerinin temizlenmesi anlamına gelir. Bu sayede çekirdek açılımı en etkin ve kompakt şekilde süreç boyunca korunmuş olur. LASVM algoritması aynı zamanda sıralı minimal optimizasyon (SMO) (Platt, 1999) algoritmasıyla ilişkilidir ve SVM kuadratik programlama probleminin çözümüne yakınsar. Diğer yandan, LASVM eşli öğrenme için uygun değildir. 2. Gereç ve Yöntem Çünkü, bu haliyle, çekirdek belleği eşler için hesaplanan çekirdek değerlerini tutar ve bu da hem bellek kapasitesi hem de hesaplama yükü olarak oldukça ciddi maliyetler oluşturur. Bu durum, algoritmayı büyük veri kümeleri için elverişsiz hale getirir. Ayrıca eşler için çekirdek değerlerini saklamak anlamsızdır, zira örnekler için çekirdek değerleri bir kez hesaplandığında herhangi bir çift için çekirdek değerleri hesaplamak daha sonra göreceğimiz üzere 3 basit aritmetik işlemden oluşur. Bu nedenle, bu çalışmada LASVM algoritmasını eşli öğrenme durumunda çalıştırabilmek için algoritmanın yapısında ciddi değişiklikler yapılmıştır. Oluşan algoritma eşli-LASVM olarak adlandırılmıştır. Bu çalışmanın birinci ana katkısı, aşağıda maddeler halinde özetlenen, eşli-LASVM algoritmasını oluşturabilmek için LASVM algoritmasında gerçekleştirilen temel değişikliklerdir.  Destek çiftlerinin indislerini ve karşılık gelen örneklerin indislerini tutmak için sırasıyla 𝑃 ve 𝑆 kümeleri tanımlanmıştır. Eşli-LASVM çekirdek açılımına bir çift eklediğinde (işleme), çiftin indisi 𝑃 kümesine eklenir ve aynı zamanda bu çifti oluşturan iki örneğin indisleri de 𝑆 kümesine eklenir. 𝑃 kümesi sadece çiftlerin indislerini tutmak için kullanılır, herhangi bir çekirdek önbelleği kullanılmaz. Çekirdek değerleri sadece ilgili çifti oluşturan örnekler için hesaplanır ve çekirdek önbelleğinde tutulur. Dolayısıyla, 𝑆 kümesiyle beraber örneklerin çekirdek değerlerini tutan bir çekirdek önbelleği kullanılır.  LASVM algoritmasında bir örneği işlemek için gerekli olan tüm yordamlar, bir çifti işleyecek şekilde yeniden düzenlenmiştir. Bu bir çift için ilgili gradyanın hesaplanması, maksimum gradyana sahip 𝜏-bozan dörtlünün (iki çift tarafında oluşturulan) belirlenmesi ve uygun adım yönlerinin belirlenmesini içerir.  Yeniden işleme bazı çiftleri 𝑃'den çıkartır. Buna karşılık 𝑆 kümesinden bu çiftle ilgili olarak iki örnek çıkartılır. Sonuç olarak sapma terimi 𝑏 ve 𝑃 kümesindeki en çok 𝜏-bozan dörtlünün gradyanı 𝛿 değerlerinin tümü hesaplanır. LASVM öncelikle çekirdek açılımına en az bir çift ekleyerek sürece başlar ve daha sonra o anki çekirdek açılımındaki var olan gereksiz destek çiftlerini arar. Çevrimiçi durumda bu, 𝑡 anında yeni bir çifti işlemek için kullanılabilir. Çekirdek açılımında herhangi bir 𝑡 anında αi ≠0 katsayısına sahip çiftler destek çiftleri olarak tanımlanır. Destek çiftlerine karşılık gelen çiftlerin indisleri 𝑃 kümesinde tutulurken, bu çiftlere karşılık gelen örneklerin indisleri 𝑆 kümesinde tutulur. Eğer 𝑖∉𝑆 ise karşılık gelen 𝛼𝑖'lerin değer almadığı varsayılır. Eşlerden öğrenme durumunda, eşli-LASVM'ın yapısı daha fazla önem arz eder, çünkü (işleme) ve (yeniden işleme) adımları daha fazla bilgiye sahip eşleri elinde tutarken, gerek duyulmayan önemini yitirmiş çiftleri de çekirdek açılımında çıkartarak bunlardan kurtulur. Bu çiftlerin sayısının kuadratik olarak büyüdüğü eşli öğrenme durumunda en kullanışlı durumdur. 2. Gereç ve Yöntem Diğer taraftan, çiftlerden bir model öğrenmek için, ortak bir özellik alanında bir çifti temsil edecek ek bir yapı türüne ihtiyaç duyarız. Basilico & Classification of Gene Samples Using Pair-Wise Support Vector Machines Taş 288 Hofmann (2004) kullanıcı derecelendirmelerini ve öğe özelliklerini ortak bir öğrenme mimarisinde birleştirmiş, farklı örnek çiftleri için uygun çekirdeklerin tasarımında iyi örnekler vermiştir ve çekirdeklerin tek bir çekirdeğe birleştirilmesinin birkaç yolunu göstermiştir. Birbirinden farklı özellik haritalarını basitçe birleştirmek için tensör çarpımlarını kullanmışlardır. Oyama & Manning (2004) eşli sınıflandırıcıları öğrenmede örnek çiftleri arasındaki özelliklerin kombinasyonlarını kullanmak için bir çekirdek önermiştir. Bu kapsamda, Ben-Hur & Noble (2005) proteinler arasındaki bir çekirdeği protein çiftleri arasındaki bir çekirdeğe çeviren tensör çarpım eşli çekirdeğini (TÇEÇ) önermiştir. Vert, Qiu, & Noble (2007) biyolojik ağların yeniden inşası için metrik öğrenme eşli çekirdeğini (MÖEÇ) geliştirmiştir. Kashima, Oyama, Yamanishi, & Tsuda, 2009) çevrimiçi öğrenme sürecini hızlandırmak için Kartezyen çekirdeğini kullanarak bu genel çerçevenin özel bir durumunu önermiştir. Kartezyen çekirdek TÇEÇ ve MÖEÇ' den daha seyrek bir yapıya sahiptir. Ayrıca çekirdek matrislerinin özdeğer analizine dayanarak iki farklı eşli çekirdek için genelleştirme sınırları verilmiştir. Bu çalışmada, eşleri temsil etmek için TÇEÇ kullanılmıştır. 𝑝1 = (𝑥1,𝑥2) ve 𝑝2 = (𝑥3, 𝑥4) şeklinde iki çift için TÇEÇ              1 2 3 4 1 3 2 4 1 4 2 3 , , , , , , , TPPK K x x x x K x x K x x K x x K x x   (4) (4)              şeklinde verilir. Bunun yanında örnekler arası RBF çekirdeği gibi bir çekirdek kullanarak, TÇEÇ çekirdeği çiftler arasındaki herhangi bir ilişkiyi öğrenen, evrensel olarak yaklaşan bir fonksiyonlar sınıfı H üretir. Bunun yanında, tüm denemelerimizde MÖEÇ'nin de performansını değerlendirmemize rağmen TÇEÇ'den anlamlı bir farklılık görülmemiştir. Bu nedenle, esas amaçtan uzaklaşmamak için uygulamada sadece TÇEÇ kullanılmıştır. şeklinde verilir. Bunun yanında örnekler arası RBF çekirdeği gibi bir çekirdek kullanarak, TÇEÇ çekirdeği çiftler arasındaki herhangi bir ilişkiyi öğrenen, evrensel olarak yaklaşan bir fonksiyonlar sınıfı H üretir. Bunun yanında, tüm denemelerimizde MÖEÇ'nin de performansını değerlendirmemize rağmen TÇEÇ'den anlamlı bir farklılık görülmemiştir. Bu nedenle, esas amaçtan uzaklaşmamak için uygulamada sadece TÇEÇ kullanılmıştır. 3. Sonuçlar Çalışmanın uygulaması Tablo 1'de verilen altı farklı problem üzerinde gerçekleştirilmiştir. Bu problemlerde temel özellik daha önce belirttiğimiz gibi değişken sayısının çok fazla olması ama bunun yanında gözlem sayısının çok az olmasıdır. Verilere uygulanan ön işlemlerden sonra, tüm gen örnekleri logaritma 10 tabanına dönüştürülmüş ve sıfır ortalamaya ve birim varyansa sahip olacak şekilde standartlaştırılmıştır. Veri kümesi Yayın n p M Bulgu Lösemi Golub vd. (1999) 72 3571 2 Lösemi'nin alt türleri Kolon Alon vd. (1999) 62 2000 2 Tümor/normal doku Prostat Singh vd.(2002) 102 6033 2 Tümor/normal doku Lenfoma Alizadeh vd. (2000) 62 4026 3 Lenfoma'nın alt türleri SRBCT Khan vd. (2001) 63 2308 4 Farklı tümör türleri Beyin A Pomeroy vd. (2002) 42 5597 5 Farklı tümör türleri Tablo 1. Çalışmada kullanılan veri kümeleri ve özellikleri Tablo 1. Çalışmada kullanılan veri kümeleri ve özellikleri Gözlemlerin üçte ikisinden dengeli bir eğitim kümesi oluşturulmuştur. Eğitim kümesi kullanılarak sınıflandırıcının ceza parametrelerinin ve çekirdek türünün belirlenmiştir. Tablo 2'de sınıflandırıcıların eğitimi aşamasında belirlenen en iyi ceza parametresi değerleri ve çekirdek türleri verilmiştir. Daha sonra nihai sınıflandırıcının eğitimi gerçekleştirilmiştir. Geriye kalan gözlemler test kümesi olarak kullanılmış ve bu gözlemlerin hangi sınıfa ait olduğu tahmin edilmiştir. Gerçek sınıf ile tahmini sınıf Alphanumeric Journal Volume 5, Issue 2, 2017 Gen Örneklerinin Eşli Destek Vektör Makinesi ile Sınıflandırılması Taş 289 üzerinde önerilen sınıflandırıcının yanlış sınıflandırma hatası hesaplanmıştır. Her bir problem için toplamda 50 deneme gerçekleştirilmiş ve hata tahminlerinin ortalaması Tablo 3'de verilmiştir. üzerinde önerilen sınıflandırıcının yanlış sınıflandırma hatası hesaplanmıştır. Her bir problem için toplamda 50 deneme gerçekleştirilmiş ve hata tahminlerinin ortalaması Tablo 3'de verilmiştir. Veri kümesi Sınıf C Kernel Lösemi 0 100 Doğrusal 1 100 Kolon 0 100 RBF 1 100 Prostat 0 10 Doğrusal 1 10 Lenfoma 0 10 Doğrusal 1 10 2 10 SRBCT 0 10 Doğrusal 1 10 2 10 3 10 Beyin Tek model 1000 Doğrusal Tablo 2. En iyi ceza parametresi değerleri ve çekirdek türleri problem için toplamda 50 d Tablo 3'de verilmiştir. Veri kümesi Sınıf C Kernel Lösemi 0 100 Doğrusal 1 100 Kolon 0 100 RBF 1 100 Prostat 0 10 Doğrusal 1 10 Lenfoma 0 10 Doğrusal 1 10 2 10 SRBCT 0 10 Doğrusal 1 10 2 10 3 10 Beyin Tek model 1000 Doğrusal Tablo 2. En iyi ceza parametresi değerleri ve çekirdek türleri Yanlış sınıflandırma oranları karşılaştırıldığında, önerilen eşli-LASVM sınıflandırıcısının 6 veri kümesinin 4'ünde diğer sınıflandırıcılara göre daha iyi performans göstermiştir. Tablo 3. Eşli-LASVM sınıflandırıcısı ile 7 farklı sınıflandırıcının 6 farklı mikrodizi veri kümesi üzerindeki hata oranlarının ortalamaları blo 3. Eşli-LASVM sınıflandırıcısı ile 7 farklı sınıflandırıcının 6 farklı mikrodizi veri kümesi üzerindeki hata o 3. Sonuçlar Lenfoma ve SRBCT veri kümelerinde ise daha kötü bir hata oranına sahiptir. Bu iki veri kümesinin ortak özelliği sınıflandırılacak sınıf sayısının diğer veri kümelerine göre daha fazla olmasıdır. Bu nedenle pw-LASVM tarafından oluşturulan ikili sınıflandırma problemi sayısı artar, m sınıflı bir sınıflandırma problemi için pw-LASVM m(m*1)/2 adet ikili SVM modeli oluşturur. Ayrıca, sınıflardaki örneklerin dengesiz dağılımı yine önerilen algoritmanın performansını düşürmüş olabilir. Bu gibi problemlerde, bu çalışmada önerilen yaklaşım çok da avantajlı olmayabilir. Diğer dört veri kümesinde ise önerilen yaklaşımın diğer sınıflandırıcılara göre daha üstün performans sergilediği açıktır. Lösemi Kolon Prostat Lenfoma SRBCT Beyin (%) (%) (%) (%) (%) (%) Eşli-LASVM 1.25 7.24 4.98 3.81 3.81 23.57 BagBoost 4.08 16.10 7.53 1.62 1.24 23.86 Boosting 5.67 19.14 8.71 6.29 6.19 27.57 RanFor 1.92 14.86 9.00 1.24 3.71 33.71 SVM 1.83 15.05 7.88 1.62 2.00 28.29 PAM 3.75 11.90 16.53 5.33 2.10 25.29 DLDA 2.92 12.86 14.18 2.19 2.19 28.57 k NN 3.83 16.38 10.59 1.52 1.43 29.71 Tablo 3. Eşli-LASVM sınıflandırıcısı ile 7 farklı sınıflandırıcının 6 farklı mikrodizi veri kümesi üzerindeki hata oranlarının ortalamaları Alphanumeric Journal Volume 5, Issue 2, 2017 Alphanumeric Journal Volume 5, Issue 2, 2017 Classification of Gene Samples Using Pair-Wise Support Vector Machines Taş 290 4. Tartışma Mikrodizi verisine dayanarak sınıflandırma problemlerinde temel amaç kanserli tümörlerin erken bir aşamada büyük bir doğrulukla tespit edilmesi ve bunun sonucunda ilgili tespite yönelik daha başarılı tedavilerin uygulanabilmesidir. Bu kapsamda, bu tür sınıflandırma problemlerinde karşılaşılan temel problem örnek sayısının az ama verini boyutunu büyük olmasıdır. Dolayısıyla büyük boyutlu gen örnekleriyle çalışabilen ve elde edilen az sayıdaki örneğin içerdiği bilgiden olabildiğince faydalanan sınıflandırma algoritmalarının geliştirilmesi gerekir. Bunun için, bu tür problemlerde karşılaşılan ikili/çoklu sınıflandırma problemlerini etkin bir şekilde yeniden düzenleyerek ikili eşli problemlere dönüştürüp daha başarılı bir şekilde sınıflandıran bir sınıflandırma metodolojisi geliştirilmiştir. Bu yaklaşım eldeki veriyi eşli hale dönüştürerek verinin genişletilmesine olanak sağlamıştır. Bu kapsamda, LASVM algoritması eşli veri kümleriyle çalışacak şekilde yeniden geliştirilmiş ve oluşan algoritma eşli-LASVM olarak adlandırılmıştır. Eşli-LASVM örnekleri çevrimiçi işleyerek oldukça hızlı bir şekilde eşli-SVM modelini kurabilir. Önerilen yaklaşımın gen örnekleriyle ilgili ikili/çoklu sınıflandırma problemlerinde oldukça başarılı bir performans gösterdiği gerçek veri kümeleri kullanılarak gösterilmiştir. Kaynakça Anlauf, J.K., & Biehl, M. (1989). The adatron: an adaptive perceptron algorithm. EPL (Europhysics Letters). 10(7): p. 687. Basilico, J., & Hofmann, T. (2004). Unifying collaborative and content-based filtering. In Proceedings of the twenty-first international conference on Machine learning. p. 9. ACM. Ben-Hur, A. & Noble, W.S. (2005). Kernel methods for predicting protein–protein interactions. Bioinformatics, 21(suppl 1): pp.i38-i46. Bordes, A., Ertekin, S., Weston, J., & Bottou, L. (2005). Fast kernel classifiers with online and active learning. Journal of Machine Learning Research. 6: pp.1579-1619. Boser, B.E., Guyon, I.M., & Vapnik, V.N. (1992). A training algorithm for optimal margin classifiers. In the Proceedings of the fifth annual workshop on Computational learning theory. pp: 144- 152. ACM. Bottou, L., & LeCun, Y. (2003). Large scale online learning. In NIPS. 30: p. 77. Dietterich, T., & Bakiri G. (1995). Solving multiclass learning problems via error correcting output codes, Journal of Artificial Intelligence Research, 2: 263-286. Dudoit, S., Fridlyand, J., & Speed, T. (2002). Comparison of discrimination methods for the classification of tumors using geneexpression data. J. Am. Stat. Assoc. 97: 77–87. Freund, Y., & Schapire, R.E. (1999). Large margin classification using the perceptron algorithm. Machine learning. 37(3): pp.277-296. Friedman J. (1996). Another approach to polychotomous classifcation, Technical Report, Technical report, Stanford University, Department of Statistics. Gentile, C. (2001). A new approximate maximal margin classification algorithm. Journal of Machine Learning Research. 2: pp.213-242. Kashima, H., Oyama, S., Yamanishi, Y., & Tsuda, K. (2009). On pairwise kernels: An efficient alternative and generalization analysis. In Pacific-Asia Conference on Knowledge Discovery and Data Mining. pp. 1030-1037. Springer Berlin Heidelberg. Li, Y., & Long, P.M. (2002). The relaxed online maximum margin algorithm. Machine Learning. 46(1- 3): pp.361-387. Mayoraz, E., & Alpaydin E. (1999). Support vector machines for multi-class classification. In the International Work-Conference on Artificial Neural Networks. Springer Berlin Heidelberg. Mayoraz, E., & Alpaydin E. (1999). Support vector machines for multi-class classification. In the International Work-Conference on Artificial Neural Networks. Springer Berlin Heidelberg. Minsky, M., & Papert, S. (1969). Perceptrons. Minsky, M., & Papert, S. (1969). Perceptrons. Minsky, M., & Papert, S. (1969). Perceptrons. Gen Örneklerinin Eşli Destek Vektör Makinesi ile Sınıflandırılması Taş 291 Oyama, S., & Manning, C.D. (2004). Using feature conjunctions across examples for learning pairwise classifiers. In European Conference on Machine Learning. pp. 322-333. Springer Berlin Heidelberg. Platt, J.C. (1999). 12 fast training of support vector machines using sequential minimal optimization. Advances in kernel methods. pp.185-208. Kaynakça Rosenblatt, F. (1958). The perceptron: A probabilistic model for information storage and organization in the brain. Psychological review, 65(6): p. 386. Shalev-Shwartz, S., Singer, Y., & Srebro, N. (2007). Pegasos: Primal estimated sub-gradient solver for svm. In Proceedings of the 24th international conference on Machine learning. pp. 807- 814. ACM. Schölkopf, B., & Smola, A.J. (2002). Learning with kernels: support vector machines, regularization, optimization, and beyond. MIT press. Vert, J.P., Qiu, J., & Noble, W.S. (2007). A new pairwise kernel for biological network inference with support vector machines. BMC bioinformatics, 8(10): p.S8. Weston, J., & Watkins, C. (1999). Support vector machines for multi-class pattern recognition. In ESANN. 99: pp. 219-224. Xu, W. (2011). Towards optimal one pass large scale learning with averaged stochastic gradient descent. arXiv preprint arXiv:1107.2490. Alphanumeric Journal Volume 5, Issue 2, 2017 Classification of Gene Samples Using Pair-Wise Support Vector Machines Taş Taş 292 Alphanumeric Journal Volume 5, Issue 2, 2017
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Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection of singlet oxygen in<i>Candida albicans</i>cells
Journal of biomedical optics
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1 Introduction hence the site of 1O2 generation, fluorescence microscopy is applied by exciting the respective PSs. Since the resolution of light microscopy is limited, this procedure should fail with small bacteria and fungus cells with a diameter of about 1 μm. The direct measurement of 1O2 luminescence at 1270 nm might be an alternative candidate to elucidate the cellular action of 1O2 because the rise and decay time of 1O2 luminescence depend critically on its adjacency.14,15 In addition, singlet oxygen lumi- nescence can provide information about the photodynamic proc- ess in bacteria during irradiation. The fast development of multiresistant patterns against antibiot- ics of many species of bacteria has led to novel antibacterial strategies like the antibacterial photodynamic therapy (aPDT).1,2 A lot of work has been done to develop molecular structures and their derivatives that are able to generate reactive oxygen species (ROS), which are the active agents for killing microorganisms.3–7 The search for photosensitizers (PSs) for aPDT has caused the synthesis of various porphyrin molecules, which have been investigated regarding their photophysics and antimicrobial activity.4,8,9 Naturally occurring porphyrins can be found endog- enously, e.g., the protoporphyrin IX that is in the prosthetic group of the hemoglobin or the chlorophylls based on the chlo- rine structure. Some endogenous porphyrins in bacteria are used to treat acne, where Propionibacterium acnes is a causative of the inflammatory processes.10 The porphyrin TMPyP has been frequently used for cell staining in order to investigate genera- tion and decay of 1O2.11–13 XF73 is a newly synthesized porphyrin molecule that already showed a high potential in antimicrobial PDT against gram-neg- ative and gram-positive bacteria.16,17 However, principal data are lacking regarding its use in 1O2 detection in vitro. Thus, it is the goal of the present study to investigate the photophysical proper- ties of XF73 and its potential to monitor photodynamic action in microorganisms. Exemplarily 1O2 luminescence detection was analyzed in vitro in Candida albicans cells. The well-known TMPyP was used for reference experiments. Different PSs are considered to localize in different compart- ments or regions in the eukaryotic or prokaryotic cell due to their number of positive charges and structure of the side chain. In order to determine the subcellular localization of PS and Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection of singlet oxygen in Candida albicans cells Ariane Felgenträger, Fernanda Pereira Gonzales, Tim Maisch, and Wolfgang Bäumler Regensburg University Hospital, Department of Dermatology, 93053 Regensburg, Germany Ariane Felgenträger, Fernanda Pereira Gonzales, Tim Maisch, and Wolfgang Bäumler Regensburg University Hospital, Department of Dermatology, 93053 Regensburg, Germany Abstract. Singlet oxygen (1O2) is an important reactive intermediate in photodynamic reactions, particularly in antimicrobial PDT (aPDT). The detection of 1O2 luminescence is frequently used to elucidate the role of 1O2 in various environments, particularly in microorganisms and human cells. When incubating the fungus, Candida albicans, with porphyrins XF73 (5,15-bis-[4-(3-Trimethylammonio-propyloxy)-phenyl]-porphyrin) or TMPyP (5,10,15,20-Tetrakis(1-methyl-4-pyridinio)-porphyrin tetra(p-toluenesulfonate)), the 1O2 luminescence sig- nals were excellent for TMPyP. In case of XF73, the signals showed strange rise and decay times. Thus, 1O2 gen- eration of XF73 was investigated and compared with TMPyP. Absorption spectroscopy of XF73 showed a change in absorption cross section when there was a change in the concentration from 1 × 10−6 M to 1 × 10−3 M indicating an aggregation process. The addition of phosphate buffered saline (PBS) substantially changed 1O2 luminescence in XF73 solution. Detailed experiments provided evidence that the PBS constituents NaCl and KCl caused the change of 1O2 luminescence. The results also indicate that Cl−ions may cause aggregation of XF73 molecules, which in turn enhances self-quenching of 1O2 via photosensitizer molecules. These results show that some ions, e.g., those present in cells in vitro or added by PBS, can considerably affect the detection and the interpretation of time- resolved luminescence signals of 1O2, particularly in in vitro and in vivo. These effects should be considered for any other photosensitizer used in photodynamic processes. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. [DOI: 10.1117/1.JBO.18.4.045002] Keywords: porphyrin; photodynamic; singlet oxygen; luminescence; aggregation. Paper 12512RR received Aug. 10, 2012; revised manuscript received Feb. 7, 2013; accepted for publication Mar. 14, 2013; published online Apr. 3, 2013. Keywords: porphyrin; photodynamic; singlet oxygen; luminescence; aggregation. R received Aug. 10, 2012; revised manuscript received Feb. 7, 2013; accepted for publication Mar. 14, 2013; published 2013. Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection of singlet oxygen in Candida albicans cells Ariane Felgenträger Fernanda Pereira Gonzales Tim Maisch Wolfgang Bäumler Journal of Biomedical Optics 18(4), 045002 (April 2013) Address all correspondence to: Ariane Felgenträger, Regensburg University Hospital, Department of Dermatology, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany. Tel: 0049‐941‐944‐9650; Fax: 0049‐941‐944‐8943; E- mail: ariane.felgentraeger@klinik.uni-regensburg.de Address all correspondence to: Ariane Felgenträger, Regensburg University Hospital, Department of Dermatology, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany. Tel: 0049‐941‐944‐9650; Fax: 0049‐941‐944‐8943; E- mail: ariane.felgentraeger@klinik.uni-regensburg.de 2.1 Chemicals Thecationicdiporphyrin-based5,15-bis-[4-(3-Trimethylammonio- propyloxy)-phenyl]-porphyrin (also referred to herein as XF73) with a molar mass of M ¼ 765.81 g∕mol, including the counter ion, was synthesized by Xiangdong Feng (Solvias Company, 045002-1 April 2013 • Vol. 18(4) Journal of Biomedical Optics Felgenträger et al.: Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection. . . Felgenträger et al.: Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection. . . Basel, Switzerland) and kindly provided by Destiny Pharma Ltd. (Brighton, United Kingdom). Basel, Switzerland) and kindly provided by Destiny Pharma Ltd. (Brighton, United Kingdom). where c the concentration of PS, l the length of light path through the solution, T the transmission in percentage, and NA the Avogadro constant. The 5,10,15,20-Tetrakis(1-methyl-4-pyridinio)-porphyrin tetra(p-toluenesulfonate) (also referred to herein as TMPyP) with a molar mass of M ¼ 1363.63 g∕mol, purity 97%, NaN3 sodium azide, Mannitol, NaCl, KCl, Na2HPO4, KH2PO4, and D2O have been purchased by Sigma Aldrich (Taufkirchen, Germany), and were used as received. The photosensitizers (PSs) were dissolved in bi-distilled water at a stock concentra- tion of 1 mM and stored at 4°C until use. Figure 1(a) shows the chemical structure of XF73 and TMPyP. 2.3 Photostability The PSs were irradiated with an incoherent broadband lamp (UV236; emission λ ¼ 380 to 480 nm) provided by Waldmann Medizintechnik (Villingen-Schwenningen, Germany). The maximal light intensity was 15.2 mW cm−2 at the level of the irradiated samples. The samples were irradiated for either 15 min (13.7 J cm−2) or 60 min (54.8 J cm−2). The emitted spectrum of the light source was recorded with a spectrometer (270 M, Jobin Yvon, Longjumeau, France) with 300 grid lines/ mm and a spectral resolution of approximately 0.4 nm [Fig. 1(b)]. The detection range was 350 to 650 nm. The recorded spectral data were corrected regarding the spectral sen- sitivity of the spectrometer. The emission spectrum of the Waldmann UV lamp was normalized to its maximum between 400 and 450 nm. Phosphate-buffered saline (PBS; PAA Laboratories GmbH, Pasching, Austria) at pH 7.4 has been used for aggregation experiments and contains NaCl (0.14 M), KCl (2.7 × 10−3 M), Na2HPO4 (1.0 × 10−2 M), and KH2PO4 (1.8 × 10−3 M). For the NMR spectroscopy, a parent solution of the PSs dissolved in D2O was made and a PBS solution for dilution containing D2O has been prepared by adding NaCl, KCl, Na2HPO4, and KH2PO4 with the accordant concentrations. 2.4 Cell Experiments Absorption spectra were recorded at room temperature with a spectrophotometer (DU640, Beckman Instruments GmbH, Munich, Germany) in a concentration range of 1 × 10−6 M to 2 × 10−3 M. The percentaged transmission has been measured and the absorption cross-section σðcm2Þ was calculated accord- ing to Eq. (1): The C. albicans strain ATCC-MYA-273 was used for the experi- ments. The planktonic cells of C. albicans were diluted to a number of 106. For the incubation of C. albicans, the PSs stock solution has been diluted with H2O. The cells were incu- bated with a PS concentration of 10−4 M in the dark for 15 min in H2O plus 50% PBS in falcons at slow rotation. The cells were rinsed twice with PBS to remove the not included or nonadher- ent PSs and afterward dissolved in pure H2O. For the singlet oxygen luminescence experiments, the planktonic cells were excited with a frequency doubled Nd:YAG-Laser (Photon- Energy, Ottensoos, Germany). σ ¼ −lnðT∕100Þ c · l · NA ; (1) σ ¼ −lnðT∕100Þ c · l · NA ; (1) Fig. 1 (a) Chemical structures of the porphyrins XF73 and TMPyP. (b) Normalized emission spectrum of the Waldmann-UV236 lamp. Absorption spectrum of XF73 and TMPyP with a concentration of 10−5 M each. (1) 2.5 Fluorescence Spectrophotometer The localization of XF73 in C. albicans was examined by fluorescence microscopy (Zeiss Vario-AxioTech, Goettingen, Germany) with an appropriate dual-band filter set for excitation and emission (Omega Optical, Brattleboro, Vermont) and a 63× magnification. Planktonic C. albicans were incubated 2 h with 10−4 M XF73 in PBS and were rinsed twice with PBS. 2.6 Singlet Oxygen Luminescence and Quantum Yield of 1O2 Formation (ΦΔ) IðtÞ ¼ C tR−1 −tD−1  exp  −t tD  −exp  −t tR  ; (2) (2) ð Þ On one hand, XF73 molecules were possibly localized at subcellular sites, where high quencher concentrations or low oxygen concentration affected the rise and decay of 1O2 lumi- nescence. On the other hand, the photophysical properties of XF73 could have been altered after the uptake of C. albicans cells. It is known for many porphyrin species that PS molecules can show stacking to J- (edge-to-edge) and H-aggregates (face- to-face) under certain conditions.22,23 Aggregation of porphyrin derivatives is influenced by concentration of inorganic salts, the polarity of the solvents, or the side chains of the porphyrins,24–26 whereas the results are still controversially discussed. Aggregation of PSs like TMPyP should not occur for concen- trations of less than 10−4 M.27–30 An overview of the discussions related to the aggregation of TMPyP is given by Vergeldt et al., who described adsorption onto surfaces or aggregation effects due to the impurity of the solvent.31 On one hand, XF73 molecules were possibly localized at subcellular sites, where high quencher concentrations or low oxygen concentration affected the rise and decay of 1O2 lumi- nescence. On the other hand, the photophysical properties of XF73 could have been altered after the uptake of C. albicans cells. It is known for many porphyrin species that PS molecules can show stacking to J- (edge-to-edge) and H-aggregates (face- to-face) under certain conditions.22,23 Aggregation of porphyrin derivatives is influenced by concentration of inorganic salts, the polarity of the solvents, or the side chains of the porphyrins,24–26 where C ¼ ½T1t¼0 kT1Δ ½3O2 was used to fit the singlet oxygen luminescence signal, describing the deactivation of the excited triplet state T1 of the photosensitizer by oxygen in its ground state (3O2).20 tR and tD are the rise and decay times, which is the excited triplet state decay time τT1 of the photosensitizer and the decay time of singlet oxygen τΔ. The attribution of τT1 and τΔ depends on the oxygen concentration in the system; at high oxygen concentrations, usually the decay time τD of the signal describes the decay time of singlet oxygen τΔ. In order to determine the rise and decay times, the Levenberg- Marquardt-algorithm of Mathematica (Wolfram Research, Champaign) was used. 2.6 Singlet Oxygen Luminescence and Quantum Yield of 1O2 Formation (ΦΔ) 2.6 Singlet Oxygen Luminescence and Quantum Yield of 1O2 Formation (ΦΔ) 2.6 Singlet Oxygen Luminescence and Quantum Yield of 1O2 Formation (ΦΔ) Solutions with PSs were filled in a cuvette (QS-101, Hellma Optik, Jena, Germany) and solutions of the planktonic cell sus- pension were investigated in acrylic cuvettes (SARSTEDT, Nümbrecht, Germany), both during magnetic stirring. The PSs were excited with a frequency doubled Nd:YAG-laser (PhotonEnergy, Ottensoos, Germany) with a wavelength λ ¼ 532 nm, power output P ¼ 50 mW, frequency of f ¼ 2 kHz, and therefore, energy per pulse of E ¼ 2.5 × 10−5 J. Every sam- ple was irradiated with 40,000 pulses. The C. albicans plank- tonic cells were excited with a frequency doubled Nd:YAG- laser (PhotonEnergy, Ottensoos, Germany) with a wavelength λ ¼ 532 nm, power output P ¼ 60 mW, frequency of f ¼ 5 kHz, and therefore, energy per pulse of E ¼ 1.2 × 10−5 J. Every sample was irradiated with 100,000 pulses. Fig. 1 (a) Chemical structures of the porphyrins XF73 and TMPyP. (b) Normalized emission spectrum of the Waldmann-UV236 lamp. Absorption spectrum of XF73 and TMPyP with a concentration of 10−5 M each. Direct detection as described in previous papers18–20 was done by time resolved measurements at 1270 nm (30 nm full width half maximum filter) in near-backward direction with April 2013 • Vol. 18(4) Journal of Biomedical Optics 045002-2 Journal of Biomedical Optics Felgenträger et al.: Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection. . . respect to the exciting beam using an infrared-sensitive photo- multiplier (R5509-42, Hamamatsu Photonics Deutschland GmbH, Herrsching, Germany) with using an additional 950 nm cut-off-filter. The luminescence intensity is given by cells produced a clear 1O2 luminescence signal with a rise time of tR ¼ ð1.77  0.2Þ μs and a decay time of tD ¼ ð6.74  0.7Þ μs [Fig. 2(a)]. In contrast to that, XF73 in C. albicans pro- duced completely different 1O2 luminescence signals showing no or a very short rise time, whereas the signal decayed in a multiexponentially manner. When starting the fit at 2 μs, the decay time was tD ¼ ð5.33  0.5Þ μs [Fig. 2(b)]. 3.1 Absorption Spectroscopy in Aqueous PS Solution Changes in the π-electron-system of porphyrin molecules can lead to the change of absorption cross-section σ and hence may affect 1O2 generation. TMPyP showed a constant absorp- tion cross-section in the range from 10−6 to 10−3 M (data not shown). In contrast to TMPyP, the absorption spectrum of XF73 in pure H2O clearly depended on XF73 concentration. The absorption cross-section decreased with increasing XF73 con- centration from 10−5 to 2 · 10−3 M and the absorption maxi- mum (Soret band) shifted to shorter wavelengths (∼7 nm) [Fig. 3(a)]. Both effects indicate aggregation of XF73 molecules. 2.6 Singlet Oxygen Luminescence and Quantum Yield of 1O2 Formation (ΦΔ) The luminescence signal was spectrally resolved using interference filters in front of the photomultiplier tube at wavelengths ranging from 1150 to 1400 nm or a mono- chromator (Horiba, Yobin Yvon Inc., USA) from 1200 to 1350 nm at 10 nm regular steps (XF73 in pure H2O). The values show the integrated luminescence signals detected at a certain wavelength and are normalized to the maximal value. A Lorentz-shaped curve has been fitted through the measurement points, with the maximum at λ ¼ 1270 nm, referring to the maximal value in H2O. Stacking of porphyrin molecules could occur at high photo- sensitizer concentrations or could be mediated by inorganic salts, which were particularly added with PBS to cells. Photosensitizer stacking may change the rate and rate constants for XF73 molecules and thereby affect the generation and decay of 1O2, which could be detected by time resolved detection of its luminescence. For the determination of ΦΔ of XF73 in H2O, it is compared with the ΦΔ of TMPyP, which is reported in literature being 0.7421 and 0.77  0.0412 in aqueous solution. Therefore, five probes of each PS of different concentrations (between 30% and 70% absorption at a wavelength of λ ¼ 532 nm) are irradi- ated and the emitted 1O2-photons are determined with the inte- gral over the luminescence curve, given with the fit routine mentioned. 3.1 Absorption Spectroscopy in Aqueous PS Solution 3.3 Photostability Also, the photostability and hence the change of absorption spectrum during irradiation may affect 1O2 luminescence. Therefore, the photostability of XF73 in solution containing PBS was investigated when illuminating the samples up to 54.8 J cm−2. No changes in the absorption spectrum of TMPyP were noticed within irradiation time of upto 60 min (data not shown). The XF73 in H2O and in 50% PBS þ H2O showed a decrease in absorption that was mainly detected in the spectral range of the Soret band (Fig. 4). Obviously, the presence of PBS, In the presence of Na2HPO4 or KH2PO4, the absorption cross-section showed no wavelength shift or new absorption maxima within given experimental accuracy (2 nm) when compared with pure H2O. The maximum value of absorption cross-section at (402  2) nm decreased from σmax ¼ 0.71× 10−15 cm2 (pure H2O) to σ ¼ 0.41 × 10−15 cm2 or σ ¼ 0.48 × 10−15 cm2 when Na2HPO4 or KH2PO4 was added, respectively. When adding PBS, σmax decreased from 0.71 × 10−15 cm2 to 0.25 × 10−15 cm2 and shifted to longer wavelengths (red shift) of 24  2 nm. In the presence of Na2HPO4 or KH2PO4, the absorption cross-section showed no wavelength shift or new absorption maxima within given experimental accuracy (2 nm) when compared with pure H2O. The maximum value of absorption cross-section at (402  2) nm decreased from σmax ¼ 0.71× 10−15 cm2 (pure H2O) to σ ¼ 0.41 × 10−15 cm2 or σ ¼ 0.48 × 10−15 cm2 when Na2HPO4 or KH2PO4 was added, respectively. Fig. 4 Photostability measurements with XF73 show a decrease of the absorption cross section with the time of illumination and therefore the applied energy. The light source was the Waldmann-UV236 lamp with an applied energy dose of 13.7 or 54.7 J cm−2, respectively. XF73 with a concentration of 10−5 M has been investigated in pure H2O and in PBS (50% in H2O). 2 4 2 4 p y When adding PBS, σmax decreased from 0.71 × 10−15 cm2 to 0.25 × 10−15 cm2 and shifted to longer wavelengths (red shift) of 24  2 nm. When adding NaCl or KCl to XF73 solution, σmax decreased to 0.25 × 10−15 cm2 for each. In addition, σmax shifted to the red by about 25  3 nm. 3 Results and Discussion 3.2 Absorption Spectroscopy in Aqueous XF73 Solution with PBS or PBS Constituents 3.2 Absorption Spectroscopy in Aqueous XF73 Solution with PBS or PBS Constituents effect needs several hours to develop. No light scattering effect in solutions was detectable by checking the absorption spectrum at shorter wavelengths. The PBS and cytosol of living cells contain various ions like Kþ, Naþ, Cl−, HCO3−, Mg2þ, Ca2þ, and HPO42−. As a first approximation to cellular environment, XF73 was dissolved in PBS solution. As XF73 was not easily soluble in PBS, the maximum concentration of PBS was 50% in H2O. Absorption spectra of XF73 (2 × 10−5 M) were recorded in pure H2O, in 50% H2O plus 50% PBS, and in 100% H2O adding single con- stituents of PBS such as KCl, NaCl, NaH2PO4, or KH2PO4, 0.1 M each [Fig. 3(b)]. The PBS and cytosol of living cells contain various ions like Kþ, Naþ, Cl−, HCO3−, Mg2þ, Ca2þ, and HPO42−. As a first approximation to cellular environment, XF73 was dissolved in PBS solution. As XF73 was not easily soluble in PBS, the maximum concentration of PBS was 50% in H2O. Absorption spectra of XF73 (2 × 10−5 M) were recorded in pure H2O, in 50% H2O plus 50% PBS, and in 100% H2O adding single con- stituents of PBS such as KCl, NaCl, NaH2PO4, or KH2PO4, 0.1 M each [Fig. 3(b)]. Journal of Biomedical Optics 3 Results and Discussion As a first experiment, cells of C. albicans were incubated with XF73 or TMPyP for 15 min using a concentration of 100 μM. The cells were washed twice, suspended in H2O solution, and subsequently excited with the laser at 532 nm. TMPyP in the Fig. 2 Singlet oxygen luminescence signal of planktonic solution of C. albicans cells incubated with 10−4 M of TMPyP (a) and XF73 (b) for 15 min in the dark. The cells were washed and are surrounded by pure H2O with a cell concentration of 106 cells per mL. Fig. 2 Singlet oxygen luminescence signal of planktonic solution of C. albicans cells incubated with 10−4 M of TMPyP (a) and XF73 (b) for 15 min in the dark. The cells were washed and are surrounded by pure H2O with a cell concentration of 106 cells per mL. Journal of Biomedical Optics 045002-3 April 2013 • Vol. 18(4) 045002-3 April 2013 • Vol. 18(4) Journal of Biomedical Optics 045002-3 045002-3 April 2013 • Vol. 18(4) April 2013 • Vol. 18(4) Journal of Biomedical Optics Felgenträger et al.: Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection. . . Fig. 3 (a) Absorption spectrum of XF73 with increasing PS concentration. A blue-shift of the absorption maxima of 7 nm was detected when increasing the concentration from 10−5 to 10−3 M. (b) Comparison of the influence of the single components of PBS on the absorption spectrum of XF73. A PS concentration of 2 × 10−5 M has been used and NaCl, KCl, KH2PO4, and Na2HPO4 had each a concentration of 0.1 M. The table shows the wave- lengths λmax of the absorption maximum and its value σmax for each component of PBS, for PBS and H2O. Felgenträger et al.: Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection. . . Fig. 3 (a) Absorption spectrum of XF73 with increasing PS concentration. A blue-shift of the absorption maxima of 7 nm was detected when increasing the concentration from 10−5 to 10−3 M. (b) Comparison of the influence of the single components of PBS on the absorption spectrum of XF73. A PS concentration of 2 × 10−5 M has been used and NaCl, KCl, KH2PO4, and Na2HPO4 had each a concentration of 0.1 M. The table shows the wave- lengths λmax of the absorption maximum and its value σmax for each component of PBS, for PBS and H2O. 3.3 Photostability At the same time, the absorption spectrum showed new absorption maxima within the spectral range of the Soret band. Addition of Cl−leads to a fundamental change of the absorption spectrum including a red shift. It is suggested that Cl−affects the tetrapyrrol ring system and enhances the aggre- gation, which was already reported for other porphyrin structures.32 Avisible precipitation of the solute started when using >10% PBS þ H2O. This effect was shown to be reversible by diluting the solution with pure H2O. As a consequence of this dilution, the absorption spectrum of XF73 in PBS changed back to the absorption spectrum in pure H2O (data not shown). The precipi- tation does not affect the absorption measurements because the probes are directly used after being diluted and the precipitation Fig. 4 Photostability measurements with XF73 show a decrease of the absorption cross section with the time of illumination and therefore the applied energy. The light source was the Waldmann-UV236 lamp with an applied energy dose of 13.7 or 54.7 J cm−2, respectively. XF73 with a concentration of 10−5 M has been investigated in pure H2O and in PBS (50% in H2O). April 2013 • Vol. 18(4) 045002-4 Felgenträger et al.: Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection. . . 3.5 1O2 Luminescence Experiments with PBS 3.5 1O2 Luminescence Experiments with PBS In light of the results above, 1O2 luminescence signals should be affected by molecule stacking, in particular when the photosen- sitizer in located in C. albicans cells [Fig. 2(b)]. Therefore, we investigated the PBS effect on time-resolved 1O2 luminescence generated by XF73 in air saturated solution at a concentration of 5 × 10−5 M, for which stacking due to PS concentration should be still minimal [Fig. 3(a)]. The results clearly show that 1O2 luminescence substantially changed with increasing PBS con- centration [Fig. 5(a)–5(c)]. From 0% to 50% PBS in H2O, the rising part of 1O2 luminescence signal disappeared, whereas the decaying part shortened. Now, the luminescence signals at high PBS concentrations [Fig. 5(c)] were similar to those recorded for XF73 in C. albicans cells [Fig. 2(b)] yielding again a multiexponential decay. In light of the results above, 1O2 luminescence signals should be affected by molecule stacking, in particular when the photosen- sitizer in located in C. albicans cells [Fig. 2(b)]. Therefore, we investigated the PBS effect on time-resolved 1O2 luminescence generated by XF73 in air saturated solution at a concentration of 5 × 10−5 M, for which stacking due to PS concentration should be still minimal [Fig. 3(a)]. The results clearly show that 1O2 luminescence substantially changed with increasing PBS con- centration [Fig. 5(a)–5(c)]. From 0% to 50% PBS in H2O, the rising part of 1O2 luminescence signal disappeared, whereas the decaying part shortened. Now, the luminescence signals at high PBS concentrations [Fig. 5(c)] were similar to those recorded for XF73 in C. albicans cells [Fig. 2(b)] yielding again a multiexponential decay. When changing the concentration of O2 in the solution at a constant concentration of ½XF73 ¼ 5 × 10−5 M, the meaning of the rates KΔ and KT1 at ½3O2 ¼ 1.1 × 10−4 M changed accord- ing to the decay paths of 1O2 and T1 [Fig. 6(a)].20 This change occurs at a crossing point of t1−1 and t2−1, which was about ½3O2 ¼ ð0.11  0.02Þ 10−3 M for XF73. By extrapolating t2−1, KT1 (ð½O2 ¼ 0 MÞ ¼ 0.03 μs−1 was determined yielding a lifetime of the triplet T1-state of ð33  5Þ μs in aqueous sol- ution without oxygen quenching. 3.5 1O2 Luminescence Experiments with PBS The quenching rate constant kq for quenching of the excited triplet state of XF73 by oxygen is therefore kq ¼ 2.3 × 109 s−1 M−1 resulting from the Stern- Volmer-plot in Fig. 6(a), where the oxygen concentration was varied and the triplet decay of XF73 was determined. When adding 1O2 quencher NaN3 36,37 to the 20% PBS sol- ution up to a high concentration of 2 × 10−3 M NaN3, the 1O2 luminescence signal almost disappeared. The residual signal should not originate from 1O2 luminescence [see Fig. 5(e)]. The same residual signal was detected in solutions without NaN3 and without oxygen (data not shown). 1O2 luminescence was also spectrally resolved for PBS 0% and 50% in H2O [Fig. 5(d) and 5(f)]. A Lorentz-shaped curve has been fitted through the measurement points and the values were normalized to the maximal value. Without PBS, the fit shows a clear maximum at 1270 nm that confirms the generated 1O2.38 At 50% PBS, the maximum at 1270 nm almost disap- peared, the baseline moved for wavelengths <1270 nm, and the signal-to-noise ratio decreased, which indicates a substantial decrease of 1O2 generation. As a next step, XF73 concentration was varied from ½XF73 ¼ 10−6 to 5 × 10−3 M at [½3O2 ¼ 5.6 × 10−5 M [Fig. 6(b)]. The value of t2−1 increased with increasing concen- tration that indicated a clear self-quenching effect of the excited triplet-T1-state for [XF73] up to about 2 × 10−4 M. Above this concentration, the quenching effect decreased and reached a pla- teau at t2−1 ¼ 0.205 μs−1, which is equivalent to a decay time of the triplet-T1-state of tT1 ¼ 4.9 μs [Fig. 6(b)]. According to the absorption spectroscopy data, a stacking of XF73 molecules occurred, which is easily detectable for XF73 concentration higher than 1 × 10−4 M [Fig. 3(a)]. Obviously, the stacking process had already led to the formation of dimers or oligomers of XF73 molecules at this concentration. Besides a different absorption cross-section, these aggregates also show different Comparable to absorption spectroscopy, the changes of time- and spectral resolved 1O2 luminescence signals, induced by PBS, could be simply reversed by diluting the used solutions with H2O and hence reducing the PBS concentration. A high degree of dilution of PBS concentration yielded time- and spec- trally resolved 1O2 luminescence signals comparable with Fig. 5(a) and 5(d). 3.4 1O2 Luminescence Experiments without PBS i.e., its ions, can additionally reduce radiation absorption of XF73. These effects may also affect the use of XF73 when applied for photodynamic inactivation of microorganisms. Incubation of bacteria or human cells with XF73 and subsequent irradiation yielded effective cell killing by means of 1O2 gener- ation, which was confirmed by adding 1O2 quencher NaN3 that significantly reduced the cell toxicity.16 Since detailed studies on 1O2 generation of the novel porphyrin molecule XF73 were In case of 1O2 experiments (see below), XF73 solutions were irradiated with 1 J of laser energy (532 nm). It is expected that σ values do not significantly change under these experimental conditions. values do not significantly change under these experimental conditions. g y y 1O2 generation of the novel porphyrin molecule XF73 were Fig. 5 (a)–(c) 1O2 luminescence signals of ½XF73 ¼ 5 × 10−5 M with different PBS concentrations in H2O with an oxygen concentration of ½3O2 ¼ 2.7 × 10−4 M. (d) Spectroscopically resolved 1O2 luminescence signal, generated by XF73 in H2O with an oxygen concentration of ½O2 ¼ 2.7 × 10−4 M. A Lorentz-shaped curve has been fitted through the measurement points. (e) 1O2 luminescence generated by XF73 in H2O þ 20%PBS at 1270 nm with 2 × 10−3 M NaCl in solution. (f) Spectroscopically resolved 1O2 luminescence signal, generated by XF73 in 30% PBS þ H2O with an oxygen concentration of ½O2 ¼ 2.7 × 10−4 M. A Lorentz-shaped curve has been fitted through the measurement points. Journal of Biomedical Optics 045002-5 April 2013 • Vol. 18(4) Fig. 5 (a)–(c) 1O2 luminescence signals of ½XF73 ¼ 5 × 10−5 M with different PBS concentrations in H2O with an oxygen concentration of ½3O2 ¼ 2.7 × 10−4 M. (d) Spectroscopically resolved 1O2 luminescence signal, generated by XF73 in H2O with an oxygen concentration of ½O2 ¼ 2.7 × 10−4 M. A Lorentz-shaped curve has been fitted through the measurement points. (e) 1O2 luminescence generated by XF73 in H2O þ 20%PBS at 1270 nm with 2 × 10−3 M NaCl in solution. (f) Spectroscopically resolved 1O2 luminescence signal, generated by XF73 in 30% PBS þ H2O with an oxygen concentration of ½O2 ¼ 2.7 × 10−4 M. A Lorentz-shaped curve has been fitted through the measurement points. 045002-5 April 2013 • Vol. 18(4) Journal of Biomedical Optics 045002-5 April 2013 • Vol. 3.4 1O2 Luminescence Experiments without PBS 18(4) 045002-5 enträger et al.: Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection. . Felgenträger et al.: Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection. deactivation of triplet T1-state as compared with XF73 mono- mers [Fig. 6(b)]. deactivation of triplet T1-state as compared with XF73 mono- mers [Fig. 6(b)]. missing, we investigated XF73 in pure aqueous solution accord- ing to previous studies on other photosensitizers.27 missing, we investigated XF73 in pure aqueous solution accord- ing to previous studies on other photosensitizers.27 After dissolving ½XF73 ¼ 5 × 10−5 M in air saturated (½3O2 ¼ 2.7 × 10−4 M), pure H2O, the rise and decay part of the time resolved signals could be assigned to the decay time τΔ of 1O2 and the decay time τT1 of PS, respectively. Experiments yielded τT1 ¼ 1.6  0.2 μs and decay time τΔ ¼ 3.5  0.3 μs [Fig. 5(a)]. The decay time is in good corre- lation with the lifetime of 1O2 in pure water.33–35 The spectrally resolved 1O2 luminescence revealed a peak at 1270 nm, which clearly confirmed the generation of 1O2 [Fig. 5(d)]. The 1O2 quantum yield ΦΔ of XF73 was determined in air saturated, pure H2O, using TMPyP as reference. The ΦΔ values of TMPyP are 0.7421 and 0.77  0.04.13 Using the previously reported technique,21 XF73 showed a value of ΦΔ ¼ 0.57  0.06. 3.5 1O2 Luminescence Experiments with PBS Scattering of photons within solution might also cause a 1O2 luminescence signal equal to the one in Fig. 5(e), and might be Fig. 6 (a) Rates t1−1 and t2−1 of the time resolved 1O2 signal depending on the concentration of O2. The meaning of the two rates changes at the crossing point of the curves. (b) The rate t2−1 characterizes the decay time of the triplet-T1-state and changes with the XF73 concentration; here the oxygen concentration is kept constant at ½3O2 ¼ 5.4 × 10−5 M. Fig. 6 (a) Rates t1−1 and t2−1 of the time resolved 1O2 signal depending on the concentration of O2. The meaning of the two rates changes at the crossing point of the curves. (b) The rate t2−1 characterizes the decay time of the triplet-T1-state and changes with the XF73 concentration; here the oxygen concentration is kept constant at ½3O2 ¼ 5.4 × 10−5 M. 045002-6 April 2013 • Vol. 18(4) 045002-6 April 2013 • Vol. 18(4) Journal of Biomedical Optics Felgenträger et al.: Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection. . . Fig. 7 Fluorescence image of C. albicans; the cells were incubated 2 h with 10−4 M XF73 in PBS and rinsed twice. An attachment of XF73 to the cells can be seen. aggregation of XF73 occurs in cells such as C. albicans. The time-resolved detection of the 1O2 luminescence in a solution of planktonic C. albicans cells incubated with XF73 and sur- rounded by pure H2O has been done [Fig. 2(b)]. In fact, the lumi- nescence signal is similar to the signal of XF73 generating 1O2 in 30% PBS [Fig. 5(c)] showing a multiexponential decay. This sig- nal indicates a surrounding of XF73 within C. albicans cells whose ionic concentration is similar to that of >30% PBS. Usually, the rise and decay times of luminescence provides infor- mation about the localization of 1O2 and hence of the photosen- sitizer applied due to the short diffusion length of 1O2 in cells. As the molecule XF73 is strongly influenced by the salts of the phos- phate buffer PBS, such interpretations could be misleading at the moment. This problem may also occur for any other PS that undergoes stacking in the presence of ions such as Cl−. Despite the results with XF73, the 1O2 luminescence detec- tion in cells is a great tool to elucidate photodynamic processes. 4 Conclusions The detection of singlet oxygen by its luminescence is a great tool to show the action of singlet oxygen even in cells or bac- teria. In this context it is important to have a detection procedure that provides reliable data from inside such cells, in particular when knowing that cellular constituents can substantially affect singlet oxygen luminescence. The interaction of porphyrins with C. albicans is controversially discussed that ranges from no uptake to tight binding or even internalization.39–43 Many por- phyrins are lipophilic and hence should accumulate in cellular membranes but the high water-solubility of XF73 suggests localization in the cytoplasm as well. Fluorescence microscopy showed the overall attachment of XF73 to the cell after washing; however, the low spatial resolution of optical microscopy impedes the evaluation of the subcellular photosensitizer locali- zation (Fig. 7). Thus, it would be of importance to gain addi- tional insight by evaluating the 1O2 luminescence data. Aggregation effects influence also the fluorescence of a dye, which has recently been described by López-Chicón et al. with an investigation of Hypericin in different species of Candida.46 The grade of aggregation depends on the surrounding and the fluores- cence is low or not existent at a high PS aggregation, which occurs in H2O-environment. Upon incubation of different species of Candida with Hypericin, one can draw a conclusion about the localization of the PS by monitoring the radiative decay, here the fluorescence that depends on the aggregation status. However, XF73 showed substantial stacking of molecules that affected light absorption as well as the generation and decay of 1O2. Stacking already occurred in pure H2O along with the increase of the PS concentration. The stacking is addi- tionally forced by the ionic pressure of Cl−. Such ions are either present in cells or are usually added in cell experiments in vitro via PBS to protect the cells from osmosis. Therefore, it is impos- sible to exclude such ions when investigating photosensitizers in cell experiments. Recently, with an optimized experimental setup singlet oxygen generation in C. 3.5 1O2 Luminescence Experiments with PBS The porphyrin TMPyP showed neither stacking in the investi- gated range of concentration nor interference with the salts of PBS. After attached to or taken up by C. albicans, the generated 1O2 could be easily detected by its luminescence with clear rise and decay components. The decay time of the 1O2 luminescence in Fig. 2(a) of tD ¼ ð6.74  0.5Þ μs, which is clearly longer than in pure water (3.5 μs) and can be most likely attributed to the decay time of the T1-state of TMPyP. If so, a triplet state decay time of 6.74 μs suggests an oxygen concentration of its sur- rounding of ½O2 ¼ 8 × 10−5 M, which is 30% compared with the oxygen concentration of ½O2sat ¼ 2.7 × 10−4 M of air saturated water. Fig. 7 Fluorescence image of C. albicans; the cells were incubated 2 h with 10−4 M XF73 in PBS and rinsed twice. An attachment of XF73 to the cells can be seen. originating from precipitation due to the stacking of the porphyr- ins. To exclude any scattering effects, the scattering agent SiO2 was added to aqueous solutions containing 5 × 10−5 M XF73 or TMPyP. No effect on the shape of the 1O2 luminescence signal and no change of the rise and decay times were detected for both photosensitizers. Additionally there was no scattering effect vis- ible in the absorption spectrum of XF73 in H2O þ 50% PBS. Nevertheless, the striking phototoxic effect of XF73 in bacteria was demonstrated.16 In vitro experiments showed a sub- stantial reduction of bacteria (∼8 log10 steps), which were incubated very small XF73 concentrations (10−8 M) for 5 min and subsequently irradiated with 13.7 J cm−2. The action of 1O2 was proven with the addition of the 1O2 quencher NaN3; however, the photodynamic effect could not be completely inhibited by the quencher. In addition, the rather small XF73 concentration in the range of 0.01 to 10 μM in those bacteria experiments could have minimized the stacking effect and there- fore maximized phototoxicity by an effective singlet oxygen generation. Journal of Biomedical Optics References ing affinities and modes,” Chem. Commun. 1, 23–24 (2000). 1. C. A. Hart and S. Kariuki, “Antimicrobial resistance in developing countries,” BMJ 317(7159), 647–650 (1998). 27. R. F. Pasternack et al., “On the aggregation of meso-substituted water- soluble porphyrins,” J. Am. Chem. Soc. 94(13), 4511–4517 (1972). 2. R. Wise et al., “Antimicrobial resistance. Is a major threat to public health,” BMJ 317(7159), 609–610 (1998). 28. K. Kano et al., “Evidence for stacking of cationic porphyrin in aqueous- solution,” Chem. Lett 12, 1867–1870 (1983). 3. M. 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Depending on the uptake mechanisms and the chemical struc- ture, a PS localizes in cellular membranes or in the cytoplasm close to any cellular structures.44,45 Cytoplasm shows a similar concentration of Cl−like PBS; therefore, it is very likely that Since the phototoxic efficacy depends on the localization and also on the aggregation status of the photosensitizer, which is influenced by ions, further investigations and comparative 045002-7 April 2013 • Vol. 18(4) April 2013 • Vol. 18(4) Felgenträger et al.: Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection. . . Felgenträger et al.: Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection. . . studies on the change of the singlet oxygen luminescence in dif- ferent species of microorganisms should lead to better insights about the change of the decay times due to the localization. 25. N. C. 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Nitzan, “Mechanistic aspects of photoinactivation of Candida albicans by exogenous porphyrins (dagger),” Photochem. Photobiol. 88(3), 604–612 (2012). 20. J. Baier et al., “Theoretical and experimental analysis of the lumines- cence signal of singlet oxygen for different photosensitizers,” J. Photochem. Photobiol. B 87(3), 163–173 (2007). 44. R. F. Pasternack et al., “A spectroscopic and thermodynamic study of porphyrin/DNA supramolecular assemblies,” Biophys. J. 75(2), 1024– 1031 (1998). 21. F. Wilkinson, W. P. Helman, and A. B. Ross, “Quantum yields for the photosensitized formation of the lowest electronically excited sin- glet-state of molecular-oxygen in solution,” J. Phys. Chem. Ref. Data 22(1), 113–262 (1993). 45. P. Kubat et al., “Interaction of novel cationic meso-tetraphenylporphyr- ins in the ground and excited states with DNA and nucleotides,” J. Chem. Soc.-Perkin Trans. 1, 6, 933–941 (2000). 22. F. Ricchelli, “Photophysical properties of porphyrins in biological mem- branes,” J. Photochem. Photobiol. B, 29(2–3), 109–118 (1995). 46. P. Lopez-Chicon et al., “On the mechanism of Candida spp. photoinac- tivation by hypericin,” Photochem. Photobiol. Sci. 11(6), 1099–1107 (2012). 23. E. Zenkevich et al., “Photophysical and photochemical properties of potential porphyrin and chlorin photosensitizers for PDT,” J. Photochem. Photobiol. B-Biol. 33(2), 171–180 (1996). 47. A. Eichner et al., “Dirty hands: photodynamic killing of human patho- gens like EHEC, MRSA and Candida within seconds,” Photochem. Photobiol. Sci. 12(1), 135–147 (2012). 24. K. M. Kadish, K. M. Smith, and R. Guilard, The Porphyrin Handbook, Academic Press, San Diego (2000). April 2013 • Vol. 18(4) April 2013 • Vol. 18(4) Journal of Biomedical Optics 045002-8
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On Communicative Competence : Its Nature and Origin
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Portland State University Portland State University PDXScholar PDXScholar Portland State University Portland State University PDXScholar PDXScholar Portland State University Portland State University PDXScholar PDXScholar Dissertations and Theses Dissertations and Theses On Communicative Competence : Its Nature and On Communicative Competence : Its Nature and Origin Origin Mary Lou Emerson Portland State University Follow this and additional works at: https://pdxscholar.library.pdx.edu/open_access_etds Part of the Applied Linguistics Commons Let us know how access to this document benefits you. Part of the Applied Linguistics Commons i Part of the Applied Linguistics Commons Let us know how access to this document benefits you. Recommended Citation Recommended Citation Emerson, Mary Lou, "On Communicative Competence : Its Nature and Origin" (2000). Dissertations and Theses. Paper 6400. htt //d i /10 15760/ td 3545 https://doi.org/10.15760/etd.3545 https://doi.org/10.15760/etd.3545 This Thesis is brought to you for free and open access. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of PDXScholar. Please contact us if we can make this document more accessible: pdxscholar@pdx.edu. THESIS APPROVAL The abstract and thesis of Mary Lou Emerson for the Master of Arts in Teaching English to Speakers of Other Languages were presented May 30, 2000, and accepted by the thesis committee and the department. COMMITTEE APPROVALf: ~m Dieterich, Chair tte DeCarrico -c..::: Donald Moor, Representative of the Office of Graduate Studies DEPARTMENT APPROVAL: Je~ue DeCarrico, Chair Department of Applied Linguistics The abstract and thesis of Mary Lou Emerson for the Master of Arts in Teaching English to Speakers of Other Languages were presented May 30, 2000, and accepted by the thesis committee and the department. g p g g p y , , accepted by the thesis committee and the department. COMMITTEE APPROVALf: ~m Dieterich, Chair tte DeCarrico -c..::: Donald Moor, Representative of the Office of Graduate Studies DEPARTMENT APPROVAL: Je~ue DeCarrico, Chair Department of Applied Linguistics Donald Moor, Representative of the Office of Graduate Studies Donald Moor, Representative of the Office of Graduate Studies DEPARTMENT APPROVAL: Je~ue DeCarrico, Chair Department of Applied Linguistics ABSTRACT An abstract of the thesis of Mary Lou Emerson for the Master of Arts in Teaching English to Speakers of Other Languages presented May 30, 2000. Title: On Communicative Competence: Its Nature and Origin. The purpose of this thesis is to trace the lineage of the term communicative competence (CC) and to provide a framework for understanding the term CC, a controversial term introduced by Hymes (1972). This pager argues that the term CC is only meaningful if it includes competence in the same sense as Chomsky (1980) defines it--underlying knowledge of the rules of language severed from ability. Although Chomsky discusses competence in terms of grammar, he suggests that there may be underlying knowledge of language use-pragmatic competence. In the end, I will attempt to demonstrate the possibility that there is a competence of communication­ pragmatic competence, like Chomsky's grammatical competence, and that both these competencies should be integrated into a theory of CC. This paper focuses on the theories of four linguists-Hymes (1972), Halliday (19TT), Savignon (1983), and Bachman (1990). Hymes and Halliday represent the empirical approach to CC--language is learned through experience with a speaker/hearer's native language. Savignon and Bachman represent the rational approach to CC--language is acquired through the interaction of grammatical competence with the speaker/hearer's native or second language. Savignon {1983) and Bachman (1990) maintain a distinction between competence and performance; and in this respect, I believe, they provide a more adequate theory of CC by including Chomsky's grammatical competence. Bachman (1990) theorizes that competence includes grammatical and pragmatic competence. Neurolinguistic studies by Ross (1981) and others support the theory that we possess underlying knowledge of pragmatics-­ communicative competence. Based on the above theories of CC and neuropsychological findings, I present a deductive model of CC which illustrates the intersection of UG, grammatical and pragmatic knowledge of language with Hymes and Halliday's theories of CC. Chomsky's UG has been expanded to include universal rules of pragmatics--Grice's Maxims {1975). UG combined with grammatical and pragmatic knowledge of language underlies the orderly processing of primary linguistic data and the rhythmic production of comprehensible speech. The organizing properties of pragmatic and grammatical competence rule out chaos. TABLE OF CONTENTS Page LIST OF FIGURES............................................................................iii CHAPTER 1 Reaching an Understanding of Chomsky's Grammatical Competence ............... 1 I. Chomsky's Grammatical Competence ......................................................2 II. Chomsky's Universal Grammar. .................................................................. ? 111. Chomsky and Others on Pragmatic Competence .................................12 IV. Communicative Competence ....................................................................16 CHAPTER 2 The Empiricists and the Notion of Communicative Competence .........................17 I. Hymes Argues Against the Chomskyan Perspective ............................22 IL Hymes' Ethnographic Perspective ............................................................24 Ill. Possible Compromise Between Chomsky and Hymes ........................24 IV. Halliday's Functional Theory Language Acquisition ............................29 V. Instrumental, Regulatory, and lnteractional Models of Language......30 VI. Personal, Heuristic, and Representational Models of Language.......31 Vil. Linguistic Structures Depend on Social Functions ..............................32 VIII. Alternatives to the Term Communicative Competence ......................43 CHAPTER 3 Psycholinguistic View Versus Sociolinguistic View: Chomsky's Defense....... .47 I. Empiricism Versus Nativism, Piaget Chomsky Debates .......................50 II. Chomsky's Reply to Piaget.. ....................................................................... 54 Ill. Syntactic Structures are Bound by Principles and Parameters.........55 CHAPTER 1 ii TABLE OF CONTENTS IV. Phonology is Governed by Abstract Universal Rules ..........................58 V. Chomsky Refutes Behaviorism and Constructivism .............................59 VI. Fodor Argues Against Constructivism .....................................................61 CHAPTER 4 Integrative Theories of Communicative Competence ...........................................64 I. Savignon's Theory and Model of Communicative Competence ........64 II. Bachman's Theory and Model... .............................................................. 76 of Communicative Language Ability CHAPTER 5 Deductive Model of Communicative Competence .................................................95 I. Recent Studies Show Evidence that Grammatical Competence .......99 Affects Pragmatic Competence IL Pragmatics Are Dominated by the Right Hemisphere ........................102 Ill. Pragmatic Competence Mirrors Grammatical Competence ..............105 IV. Language is an Interactive Process Between Knowledge ................ 109 Structures and the Environment V. Conclusion..................................................................................................112 REFERENCES...............................................................................113 CHAPTER 4 iii LIST OF FIGURES LIST OF FIGURES Page Figure 1. Nigel at 19 months: part of the instrumental component 38 Ability Figure 2. Nigel at 19 months: part of the regulatory component 39 Figure 3. Nigel at 19 months: part of the interactional component 41 Figure 4. Savignon's Components of Communicative Competence 75 Figure 5. Bachman's Components of Communicative Language 78 Figure 6. Bachman's Components of Language Competence 80 Figure 7. A Model of Language Use 91 Figure 8. Deductive Model of Communicative Proficiency 97 2 On Communicative Competence theories of the use of language are not new (he draws from Roman Jacobson), he develops his own models in reaction to Chomsky's theories of competence and performance. It is the contention of this paper and of many linguists that Hymes and other linguists not only misinterpreted Chomsky, but misappropriated the linguistic term competence as defined by Chomsky. In order to understand the controversy surrounding the term communicative competence, it is necessary to present Chomsky's theory of grammatical competence in detail. 2 On Communicative Competence theories of the use of language are not new (he draws from Roman Jacobson), he develops his own models in reaction to Chomsky's theories of competence and performance. It is the contention of this paper and of many linguists that Hymes and other linguists not only misinterpreted Chomsky, but misappropriated the linguistic term competence as defined by Chomsky. In order to understand the controversy surrounding the term communicative competence, it is necessary to present Chomsky's theory of grammatical competence in detail. 1 On Communicative Competence Chapter 1 Reaching an Understanding of Chomsky's Grammatical Competence 1 The purpose of this paper is to trace the lineage of the term communicative competence (CC), and to provide a framework for understanding the term CC, a controversial term introduced by Hymes (1972). This paper argues that the term CC is only meaningful if it includes competence in the same sense as Chomsky defines it--underlying knowledge of the rules of language severed from ability. Although Chomsky (1980) discusses competence in terms of grammar, he suggests that there may be underlying knowledge of language use--pragmatic competence. In the end, I will attempt to demonstrate the possibility that there is a competence of communication--pragmatic competence, like Chomsky's grammatical competence, and that both these competencies should be integrated into the theory of CC. In the process of tracing the idea for the term communicative competence back to Roman Jakobson, a Russian linguist whose work was not translated until the 1960s, I discovered an argument among linguists regarding the derivation of the term and a question as to whether the term was appropriately chosen. Hymes, who is credited with the term communicative competence (CC), discusses his model of CC at great length in a report presented at the Research Planning Conference on Language Development Among Disadvantaged Children in 1966 at Yeshiva University (1972, p. 269). In his discussion of CC, Hymes takes issue with Chomsky's theories of grammatical competence and performance. Although Hymes' Chomsky's Grammatical Competence Chomsky defined linguistic competence in terms of innate knowledge of language and tacit knowledge of a particular language that everyone possesses who acquires a native language (1965). Chomsky severed the term competence from "ability" (1980, p. 59 ). Thus, the term competence as defined by Chomsky is a linguistic term which includes innate knowledge of language as well as emergent knowledge of language. Competence is concerned with UG--nthe principles that specify the range of possible human grammarsn and tacit knowledge of '1he system of rules and principles that we assume have, in some manner, been internally represented by the person who knows a language and that enable the speaker, in principle, to understand an arbitrary sentence and to produce a sentence expressing. his thoughr (Chomsky, 1980, p. 201 }. Universal Grammar is knowledge apriori; it constitutes "the 'initial state' of the language faculty prior to any linguistic experience· (Chomsky, 1986, p. 4). This language acquisition device (LAD} generates a grammar and affords everyone the opportunity to make sense of 3 On Communicative Competence linguistic input or primary linguistic data (Chomsky, 1965, p. 27). "In particular, it allows pre-linguistic children to make sense of the linguistic input' (Dieterich, 1999). 3 Although Chomsky does not expound on the creative aspects of language use--the ability of a speaker to devise novel sentences appropriate to new situations, like Jakobson and Hymes, among others, he does not see life as irrelevant. Rather, Chomsky focuses on knowledge of the mind, and through the study of language or more specifically generative grammar, he purports to establish the existence of universal rules and innate representations of the human mind. Chomsky's perspective is as follows: Thus, linguistics is taken to be the field that relies on informant judgments, elicited material, whatever limited use can be made of an actual corpus, and so on, to try to determine the nature of grammar and universal grammar. Its concern is competence, the system of rules and principles that we assume have, in some manner, been internally represented by the person who knows a language and that enable the speaker, in principle, to understand an arbitrary sentence and to produce a sentence expressing his thought; and its further concern is universal grammar, the principles that specify the range of possible human grammars. (Chomsky, 1980, p. Chomsky's Grammatical Competence 201) Chomsky's theory of competence is founded in r~tionalist theories of the existence of innate ideas and generative grammar. His theory of innate ideas is in part rooted in seventeenth century rationalist philosophy. Descartes (1970) asserts that ideas arise from the f acuity of the mind, and that some ideas are innate. Lord Herbert (1624) and Cudworth (1731) expressed the same view that innate ideas and principles exist and remain latent when corresponding objects are not present (Chomsky, 1965, p. 49). Later Leibniz (1949) states a similar view-that the senses are necessary for our actual behavior, but the principles upon which our actio~s 4 On Communicative Competence are based are innate. Therefore, ideas and and innate truth are discovered through experience. "Thus it is that one possesses many things without knowing it ..." (Leibniz, 1949, p. 74). 4 On Communicative Competence are based are innate. Therefore, ideas and and innate truth are discovered through experience. "Thus it is that one possesses many things without knowing it ..." (Leibniz, 1949, p. 74). Applying this rationalist view to the special case of language learning, Humboldt (1836) concludes that one cannot really teach language but can only present the conditions under which it will develop spontaneously in the mind in its own way. Thus the form of the language. the schema for its grammar, is to a large extent given, though it will not be available for use without appropriate experience to set the language-forming process into operation. Like Leibniz, Humboldt reiterates the Platonistic view that, for the individual, learning is largely a matter of "Wierderezeugung," that is, drawing out what is innate in the mind. (Chomsky, 1965, p. 51) These rationalist views contrast sharply with the empiricist notion that the structure of language does not rely on innate principles. The empiricist assumes "that the procedures and mechanisms for the acquisition of knowledge constitute innate property of mind" (Chomsky, 1965, p. 51 ). On the other hand, Chomsky assumes that our linguistic abilities are based on mental structures of rules and representations, not procedures and mechanisms. In the empiricist tradition, the form of knowledge is not fixed in the same sense as it is for the rationalist philosophers. While it is important to understand the two views of language acquisition, it's also important to consider where these two seemingly divergent theories converge. Chomsk~ himself says, ..It is not, of course, necessary to assume that the empiricist and rationalist views can always be sharply distinguished and that these currents cannot cross paths" (Chomsky, 1965, p. 52). However, Chomsky distinguishes the two arguments specifically to reach "explicit hypotheses about the acquisition of knowledge, in particular, about the innate structure of a language acquisition device" (Chomsky, 1965, 5 On Communicative Competence p. 52). Thus, Chomsky admits there are points of intersection of the two theories. This seems to be where the argument lies among the different 5 On Communicative Competence p. 52). Thus, Chomsky admits there are points of intersection of the two theories. Despite the differences in theories, there are points of agreement and one theory does overlap the other. It is impossible for them not to overlap given the fact that both deal with human language. Somewhere along the continuum, language input intersects with the human mind. What is ctear is that the human mind is endowed with innate knowledge either in terms of grammatical competence (absolute knowledge of grammar), or in terms of procedural competence (relative knowledge of language). Chomsky defines knowledge of language "as a certain state of the mind/brain, a relatively stable element in transitory mental states once it is attained; furthermore, as a state of some distinguishable f acuity of the mind--the language faculty--with its specific properties, structure, and organization, one "module" of the mind (1986, pp. 12-13). One of the goals of this paper is to demonstrate that this innate fixed nucleus or 'module' of mind/brain--the language faculty--should be considered the foundation of language acquisition, and therefore, one of the integral components of communicative competence. Chomsky admits that there are different ways in which primary linguistic data may be necessary for language learning. In addition, he admits that normal language learning in some way requires the use of language in real-life situations {Chomsky, 1965, p. 33). However, the fact that sociological factors play a role in language acquisition, still doesn't show how language is acquired once the innate mechanism "is put to work and the task of language learning is undertaken by the chikf {Chomsky, 1965, p. 33). This seems to be where the argument lies among the different 6 On Communicative Competence linguists. Chomsky distinguishes between the innate language acquisition device which is defined in terms of a fixed grammar, and ability for use; whereas others don't make this distinction, in part because they aren't defining knowledge of the mind as a state or product, but rather as a process (Taylor, 1988, p. 153). Thus, what Chomsky maintains is that the language acquisition device selects grammars which are compatible with primary linguistic data. The selected grammar then provides the device with a method for interpreting linguistic input: method for interpreting linguistic input: The theory that the device has now selected and internally represented specifies its tacit competence, its knowledge of the language. The child who acquires a language in this way of course knows a great deal more than he has 'learned.' His knowledge of the language, as this is determined by his internalized grammar, goes far beyond the presented primary linguistic data and is in no sense an 'inductive generalization' from these data." (Chomsky, 1965, pp. 32­ 33) This theory parallels the view of Leibniz (1949)--that we possess more than we know. Innate knowledge becomes available to us given corresponding external stimuli or environmental reality that stimulates a particular mental reality. It is where these signals, or external stimuli meet the mind that there is agreement and common ground among linguists of differing opinions with respect to innate knowledge and how it interacts wit~ primary linguistic data. That is to say that there is universal agreement that the mind intersects with primary linguistic data; although how it intersects is a matter of debate. Chomsky necessarily separates competence from performance because knowledge of the rules of a language do not provide a model for th~ language user. However. UG does form the basis for understanding 7 On Communicative Competence language acquisition, and is therefore, a critical component of some of the major theories of language acquisition which will be discussed below. If one does not admit the existence of a language acquisition device, it would seem one has to acknowledge the existence of innate processes and procedures of the mind. Piaget (see pp. 50-54) and Halliday discuss innate mechanisms for language learning in terms of functional processes and procedures, not in terms of structure, or a fixed nucleus of the mind/brain. In order to adequately explain Chomsky's position regarding language acquisition, It is essential to look at examples of Chomsky's UG. The foltowing section describes a few of the principles and parameters of UG. Chomsky's UG The theory of UG holds that the speaker knows a set of principles that apply to all languages, and in the process of language acquisition s/he learns how to apply these principles to his /her native language. In addition, the speaker knows certain parameters that vary within clearly defined limits from one language to another, and s/he applies these to his/her native language. The theory of UG does not make vague or unverifiable statements about properties of the mind; instead, specific statements are made about innate properties of the human mind based on specific evidence. For example, the principle of structure-dependency which appears to apply to all languages, asserts that knowledge of language relies on the structural relationships of the sentence rather than the linear sequence of words. That is, in order to know which element of the sentence to move to form a well-formed sentence, one must know its underlying structure On Communicative Competence On Communicative Competence 8 (something that is not taught). (something that is not taught). For example, in order to construct a passive sentence out of the active sentence: They elected John president. ey e ected Jo p es de t one must choose the other NP and not the direct object to form the passive sentence: John was elected president. Interestingly, a native speaker of English never produces the following ill­ formed sentence: Interestingly, a native speaker of English never produces the following ill formed sentence: President was elected John. Equivalent sentences in Greek and German also demonstrate the principle of structure-dependency. In the German passive sentence: Hans wurde von Marie gesehen. (Hans was by Mary seen) Hans was seen by Mary (Cook & Newson, 1996, p. 10). it is the object NP, Hans, that moves to subject position, not any other NP or any other word. In the Greek passive sentence: o giatros didachtike Aglika apo ton Peter. (the doctor was taught English by Peter) The doctor was taught by Peter (Cook & Newson, 1996, p. 11 ). the NP, o giatros, was moved to the subject position. Chomsky gives the following example of question formation in Spanish: Esta' el hombre, que esta' contento, en la casa? President was elected John. Equivalent sentences in Greek and German also demonstrate the principle of structure-dependency. In the German passive sentence: Hans wurde von Marie gesehen. (Hans was by Mary seen) Hans was seen by Mary (Cook & Newson, 1996, p. 10). it is the object NP, Hans, that moves to subject position, not any other NP or any other word. In the Greek passive sentence: o giatros didachtike Aglika apo ton Peter. (the doctor was taught English by Peter) The doctor was taught by Peter (Cook & Newson, 1996, p. 11 ). the NP, o giatros, was moved to the subject position. The doctor was taught by Peter (Cook & Newson, 1996, p. 11 ). the NP, o giatros, was moved to the subject position. Chomsky gives the following example of question formation in Spanish: Chomsky gives the following example of question formation in Spanish: Esta' el hombre, que esta' contento, en la casa? 9 p "Is the man who is happy at homer (Chomsky, 1988, p. On Communicative Competence 43) where esta (is) has moved from the main VP, 'the man is at home,' not the relative clause VP, 'who is happy.' Thus, rules for the formation of passives and questions in all languages are structure-dependent, and not based on the linear order of elements. This principle of structure-dependency is just one example of a language universal that Chomsky discovered about the nature of human languages; it is a property of human language in general, and reflects the internal structure of the mind. Contrary to Hymes' (1996) claim that Chomsky's focus was English only, Chomsky's goal has always been to discover the universal aspects of language which reflect the properties of the human mind. "Real progress in linguistics consists in the discovery that certain features of given languages can be reduced to universal properties of language, and explained in terms of these deeper aspects of linguistic form" (Chomsky, 1965, p. 35). Furthermore, Chomsky's theory of UG claims that such principles and parameters are inherently impossible to learn; and if they are not learned, they must be part of the human mind--part of our biological endowment. While the principle of structure-dependency seems common to all languages, languages differ in many ways, and knowledge of language also differs. For example, the head parameter captures the variations between languages. As always, it is Chomsky's aim to express generalizations about the phrase structure of all languages rather than features that are idiosyncratic to a single language. One way in which language differs is where the essential element in each phrase. the head, occurs in On Communicative Competence 1O relationship to other elements of the phrase, called complements. English is a head-first language where the head of a phrase is first, or to the left of its complement. In the NP: food for the soul, the noun food appears first as is true for the VP: ran to the store, where the verb ran is first or to the left of its complement. Similarly, the preposition to appears first in the PP: to the store. On the other hand, Japanese is different. In the sentence: E wa kabe ni kakatte imasu (picture wall on is hanging) (picture wall on is hanging) The picture is hanging on the wall. (Cook and Newson, 1996, p. 14) "the head Verb kakatte imasu occurs on the right of the Verb complement kabe ni, and the postposition ni (on) comes on the right of the PP complement, kabe. Thus, Japanese is a head-last language where the head occurs to the right of its complement. A single generalization regarding the head of a phrase is that according to the head parameter, a language is either head-first or head-last. Again, Chomsky expresses his interest in all languages and the universal aspects of language: "Ideally, we hope to find that complexes of properties differentiating otherwise similar languages are reducible to a single parameter, fixed in one or another way" (1981, p. 6.) In addition to the syntactic principles discussed above, principles and parameters theory integrates the syntactic description of the sentence with the words of the language via the Projection Principle, which requires the syntax to accommodate the properties of each lexical item (Cook & Newson, 1996, p. 17). In other words, the lexical entry for each verb in the dictionary must specify whether on not it is followed by a NP--whether it is transitive or intransitive. Certain verbs (transitive) subcategorize NPs, while others prefer Verb, L NP] prefer Verb, L NP] Other verbs such as want subcategorize a NP, and can be followed by a phrase starting with to : I want money. I want to go. Speakers know how verbs may be used in sentences and need not be concerned with a rule such as VP --> V (NP), meaning that the VP consists of a verb and an optional NP. Instead a native speaker knows that certain lexical items subcategorize particular syntactic forms. This is known as the Projection Principle which Cook and Newson define as "the properties of lexical entries [that] project onto the syntax of the sentence" (1996, p. 20). Chomsky states that "structure must be represented subcategorically at every syntactic level" (1986, p. 84). The Projection Principle is another example of UG; all languages integrate the syntax with the properties of lexical items. Since there is no logical explanation for this particular characteristic of language, nor is it obvious how a child might acquire the Projection Principle, it is considered to be an innate property of the human mind. These are but a few of the principles and parameters of UG which demonstrate language universals--those principles and parameters that are common to all languages and explain language knowledge in terms of properties of the human mind. Again, Chomsky's primary goal is to discover what constitutes language knowledge common to everyone despite On Communicative Competence language differences. Chomsky's focus is on language universals and encompasses all languages, not just the English language. 12 Similarly, Canale and Swain argue that it is reasonable to assume Chomsky and Others on Pragmatic Competence Chomsky makes a clear distinction between competence and performance. His performance model is limited by memory, time and access (1965, p.10). In his later writing Chomsky admits the limitations of his performance model and adds a pragmatic component to his model of performance (1980, p. 59). Although Chomsky does not investigate pragmatic competence, he suggests that it may be domain-specific, like syntactic knowledge. In Rules and Representations, Chomsky defines pragmatic competence as the underlying ability to use grammatical knowledge combined with the conceptual system to achieve certain ends or purposes. He states the following: " It might be that pragmatic competence is characterized by a certain system of constitutive rules represented in the mind, as has been suggested in a number of studies" (1980, p. 59). Chomsky distinguishes grammatical competence from pragmatic competence. The first is restricted to "the knowledge of form and meaning", and the second is restricted to "knowledge of conditions and manner of appropriate use, in conformity with various purposes" (Chomsky, 1980, p. 224). Chomsky suggests that gg ( p ) Chomsky distinguishes grammatical competence from pragmatic competence. The first is restricted to "the knowledge of form and meaning", and the second is restricted to "knowledge of conditions and manner of appropriate use, in conformity with various purposes" (Chomsky, 1980, p. 224). Chomsky suggests that [p]ragmatic competence may include what Paul Grice has called a "logic of conversation." We might say that pragmatic competence places language in the institutional setting of its use, relating intentions and purposes to the linguistic means at hand. (1980, p. 225) 224). Chomsky suggests that [p]ragmatic competence may include what Paul Grice has called a "logic of conversation." We might say that pragmatic competence places language in the institutional setting of its use, relating intentions and purposes to the linguistic means at hand. (1980, p. 225) On Communicative Competence 13 "that there are rule-governed, universal, and creative aspects of sociolinguistic competence just as there are of grammatical competence" (1980, p. 6). Brown and Levinson develop a theory of pragmatic competence within the framework of Grice's Maxims, "namely, that there is a working assumption by conversationalists of the rational and efficient nature of talk" (1987, p. 4). They argue that politeness is rooted in rational and efficient modes of communication. Chomsky and Others on Pragmatic Competence However, Brown and Levinson develop their own universal theory of politeness based on the notion of 'face.' Regarding their theory, they state the following: 13 while the content of face will differ in different cultures (what the exact limits are to personal territories, and what the publicly relevant content of personality consists in), we are assuming that the mutual knowledge of members' public self-image or face, and social necessity to orient oneself to it in interaction, are universal. (Brown and Levinson, 1987, p. 61) Brown and Levinson believe that a speaker approaches any conversation with two "face wants:" "negative face:" the want to act unimpeded by others, and "positive face:" the want that his desires be liked and respected (1987, p. 61 ). For example, in conversation we maintain a 'negative face' when we make a request to the listener, do not want to impose, and yet we expect the request to be granted. We maintain a 'positive face' when we compliment someone or defer to someone in an effort to be understood, or approved of, or admired. Brown and Levinson study three language--English (from both sides of the Atlantic); Tzeltal, a Mayan language spoken in Chiapas; and South Indian Tamil. They argue that by studying three languages from unrelated cultures. and showing evidence for convergence. that their theory of On Communicative Competence 14 universals lies far beyond chance. However, Wierzbicka (1991) argues that their theory is flawed, because it is based on an Anglo-Saxon value of individualism--t~t the notion of 'face' relates to 'self;' and there are many cultures that are not individually oriented, or self-oriented, but rather are community oriented, such as Chinese, Japanese, and Polish. Brown and Levinson discuss universal politeness with respect to indirect speech acts. For all three languages studied they determined that in making requests all speakers employ the more 'polite' forms such as, 'Can you,' 'Would you like to,' and 'Do you want to.' From these results, they concluded that this is a universal rule of politeness. However, Wierzbicka claims that use of this indirect form in Polish would seem "particularly odd," and would be interpreted by the hearer as "naive hypocrisy" (1991, p. 34). In Polish a request would not be made unless the hearer can cooperate. Chomsky and Others on Pragmatic Competence In general, for Polish it is polite to use the direct form for making a request-- 'Please do X.' Furthermore, Yu (1999) who studied the use of requests in Chinese, also concluded that Chinese speakers use more direct forms in making requests. Because of their community orientation, this is considered a polite form or socially appropriate. Although Yu (1999) does not rule out pragmatic universals, he concluded that the use of direcVindirect speech was culture­ specific. Both Yu and Wierzbicka argue that Brown and Levinson's pragmatic theory of universality, based on Anglo-Saxon tradition which places specific emphasis on the rights and autonomy of every individual does not work when applied to many other cultures. It is clear. that further investigation of pragmatic u~iversals needs to be It is clear. that further investigation of pragmatic u~iversals needs to be On Communicative Competence 15 pursued. It seems likely, as Chomsky suggests, that there are pragmatic universals, such as Grice's Conversational Maxims (1975) which underlie language use. It is a matter of studying performance models in more depth. "[E]vidence about the actual organization of behavior may prove crucial to advancing the theory of underlying competence. Study of performance and study of competence are mutually supportive" (Chomsky, 1980, p. 226). Chomsky considers the distinction between psychology, which is concerned· with performance, and linguistics, which is concerned with competence, to be senseless: On Communicative Competence 15 pursued. It seems likely, as Chomsky suggests, that there are pragmatic universals, such as Grice's Conversational Maxims (1975) which underlie language use. It is a matter of studying performance models in more depth. "[E]vidence about the actual organization of behavior may prove crucial to advancing the theory of underlying competence. Study of performance and study of competence are mutually supportive" (Chomsky, 1980, p. 226). Delineation of disciplines [linguistics and psychology] may be useful for administering universities or organizing professional societies, but apart from that, it is an undertaking of limited merit. A person who happens to be interested in underlying competence will naturally be delighted to exploit whatever understanding may be forthcoming about process models that incorporate one or another set of assumptions about linguistic knowledge. Furthermore, it seems evident that investigation of performance will rely, to whatever extent it can, on what is learned about the systems of knowledge that are put to use. Chomsky and Others on Pragmatic Competence (1980,p.202) However, Chomsky does not focus on performance models because he seeks to explain how innate knowledge of grammar or universal grammar (UG) accommodates the creative aspect of language. Although Chomsky does not focus on performance, he does discuss it in terms of 'acceptability' (1965, p. 11). Communicative aspects of language include performance and acceptability. Chomsky differentiates between acceptability and grammaticality. Acceptability belongs to performance and grammaticality belongs to competence (1965, p.11 ). "Grammaticalness is only one of the many factors that interacts to determine acceptability" (1965, p. 11). Thus, Chomsky does not deny the sociological aspects of language production; On Communicative Competence 16 however, his interest lies in determining the nature of grammar and universal grammar of an ideal speakerJhearer. He nonetheless agrees with Lenneberg ( 1967) that the rules of grammar enter into the processing mechanisms of language in context. Chomsky states that if we accept Lenneberg's contention that "the rules of grammar enter into the processing mechanisms, then evidence concerning production, recognition, recall, and language use in general can be expected (in principle) to have bearing on the investigations of rules of grammar, on what is sometimes called 'grammatical competence' or 'knowledge of language'" (Chomsky 1980, p. 200-202). 16 Communicative Competence Hymes (1972), proposes a broader notion of competence-­ communicative competence, which includes not only grammatical competence (tacit knowledge of the rules of grammar), but also pragmatic competence, (knowledge of the rules of language use). Although Hymes (1996) reveres Chomsky's "brilliant work," he does not make a distinction between competence and performance. The following discussion in Chapter 2 attempts to demonstrate why Hymes' theory is not a competence theory. Instead, it is a performance theory, because Hymes is not investigating language universals or knowledge of language that unde~lies pragmatics. Since Hymes' theory of communicative competence is formulated in reaction to Chomsky's theory of grammatical competence, it seems appropriate to begin by discussing Hymes' socio-cultural theory of language acquisition. The following is a detailed discussion of the evolution of Hymes' theory of CC. On Communicative Competence Chapter 2 17 The Empiricists and the Notion of Communicative Compet 16) Thus, by extending the idea of competence, Hymes has changed the narrow meaning of the linguistic term put forth by Chomsky (1965). As stated above Chomsky severed the term competence from ability, and chose to do so in order to disentangle it from the many meanings of 'knowledge' (Chomsky,1980, p. 59). Taking Hymes' new definition of compet9nt?8 , the term communicative competence makes sense. However, this is where the terminological confusion begins, because there are many linguists who define competence according to Chomsky's definition-knowledge of The Empiricists and the Notion of Communicative Compet Theories of language acquisition are based on different philosophies or psychological theories. The two main views related to language acquisition are either based on rationalist theories or empiricist theories of learning. As stated above, the rationalist theories of Leibniz (1949) and Lenneberg (1967) form the basis of Chomsky's theory of language acquisition--that humans possess innate knowledge of language in the form of principles and parameters. The philosophical theories of John Locke (1975), an empiricist, and the psychological theories of Piaget (1980), a constructivist, form the foundation of the empiricist's theories--that there are no innate principles. Hymes (1972) and Halliday (1970) represent the empiricist view of language acquisition. Although Hymes acknowledges innate knowledge of language as defined by Chomsky, his emphasis is on the sociolinguistic and anthropological aspects of language. Since Hymes redefines competence to suit his perspective on language acquisition, I juxtapose Hymes with Chomsky in order to elaborate further on the theory of competence. For Hymes, grammatical knowledge is a resource, not an abstract cognitive configuration. How knowledge gets realized as use is a central issue and a component of communicative competence (CC). Chomsky's theoretical notion of the ideal speaker/hearer is ·unilluminating from the standpoint of the children we seek to understand and help" (Hymes, 1971, p. 4). Hymes admits that Chomsky's theoretical standpoint is necessary, but not sufficient on its own, ·because this idealized conception becomes On Communicative Competence 18 inadequate as soon as it is confronted with real children in a particular environment" (Hymes, 1971, p. 4). Hymes explains the value in Chomsky's theory: A theoretical perspective is essential, and the perspective afforded by transformational generative grammar is particularly valuable because it gives us the concept of an idealized learner, with built-in propensities for language. This does away with the notion of genetic racial inferiority of any particular group, or sub-group. (1971, p. 4) However, Hymes objects to the lack of socio-cultural factors in linguistic theory, and believes that in order for language to be adequately studied the notion of competence needs to be extended. He states the following: I should therefore take competence as the most general term for the speaking and hearing capabilities of a person. This choice is in the spirit of present linguistic theory, if at present against the letter. Competence is understood to be dependent upon two things: (tacit) knowledge and (ability for) use. (Hymes, 1971, p. On Communicative Competence 18 inadequate as soon as it is confronted with real children in a particular environment" (Hymes, 1971, p. 4). Hymes explains the value in Chomsky's theory: 18 A theoretical perspective is essential, and the perspective afforded by transformational generative grammar is particularly valuable because it gives us the concept of an idealized learner, with built-in propensities for language. This does away with the notion of genetic racial inferiority of any particular group, or sub-group. (1971, p. 4) However, Hymes objects to the lack of socio-cultural factors in linguistic theory, and believes that in order for language to be adequately studied the notion of competence needs to be extended. He states the following: I should therefore take competence as the most general term for the speaking and hearing capabilities of a person. This choice is in the spirit of present linguistic theory, if at present against the letter. Competence is understood to be dependent upon two things: (tacit) knowledge and (ability for) use. (Hymes, 1971, p. 16) Thus, by extending the idea of competence, Hymes has changed the narrow meaning of the linguistic term put forth by Chomsky (1965). As stated above Chomsky severed the term competence from ability, and chose to do so in order to disentangle it from the many meanings of 'knowledge' (Chomsky,1980, p. 59). Taking Hymes' new definition of compet9nt?8 , the term communicative competence makes sense. However, this is where the terminological confusion begins, because there are many linguists who define competence according to Chomsky's definition-knowledge of language severed from ability, and draw the distinction between competence (a state) and performance (a process). Hymes agrees with Chomsky that competence must be the central concern. •aut arbitrary restriction of the domain of underlying knowledge can . On Communicative Competence 19 be ended; the methodological spirit of generative grammar can be extended to the whole sphere of the abilities manifest in speech" (Hymes, 1971, p.11 ). Hymes claims that what is important now is "the relation between rules of grammar and rules of use" (1971, p.11 ). In 1968 the Centre for Advanced Study in the Developmental Sciences with the Ciba Foundation held a Study Group on Language Development in England. Hymes was one of the participants who presented a formal paper which was later edited for publication. In the discussion following Hymes' presentation on competence and performance, Bever points out that Hymes does not resolve the problem of the relationship between a linguistic grammar and speech performance (1971, p. 25). On Communicative Competence 18 inadequate as soon as it is confronted with real children in a particular environment" (Hymes, 1971, p. 4). Hymes explains the value in Chomsky's theory: Furthermore, inherent in Chomsky's theory of competence is that there is more to language than grammar, since grammaticality is seen as a psychological notion. Later in the ensuing discussion, Cazden asks Hymes if his notion of competence includes knowledge of rules of style and rules of social interaction, etc. Hymes answers, "Yes, and this may lead to different kinds of competence in people who grow up in different cultural contexts" (1971, p. 26). In this respect, Hymes' theory of competence relates more to a theory of pragmatic competence , since pragmatic competence directly relates to use. In this regard, his term communicative competence becomes more meaningful. The underlying rules, then, which relate to pragmatic behavior, would be different depending on cultural differences. However, the problem with Hymes' extended definition of competence, still remains, because Chomsky discusses grammatical competence and pragmatic competence in terms of language universals as well as emergent On Communicative Competence 20 knowledge of language. While each culture may have particular grammatical and pragmatic differences, Chomsky's purpose in investigating language is to discover universals which are general to all languages. Although Chomsky does not investigate pragmatic universals, he does investigate grammatical universals which are discussed under the heading of UG. Hence, the distinction between Chomsky's narrow definition of the term competence and performance remains of critical importance. 20 In an effort to attain a more global understanding of language, Hymes helped to clarify the domain of performance, but he muddied the domain of competence in his attempt to refocus the study of language from the intrinsic to the extrinsic. Hymes' model of CC contains four parameters: possibility, feasibility, appropriateness, and attestedness in actual performance. Below is an explanation of these four parameters: 1) Whether (and to what degree) something is formally possible. 2) Whether ( and to what degree) something is feasible in virtue of the means of implementation. 3) Whether (and to what degree) something is appropriate ( adequate, happy, successful) in relation to a context in which it is used and evaluated. 4) Whether (and to what degree) something is in fact done, actually performed, and what its doing entails. (Hymes, 1972, 285-286) There are two aspects to each parameter-(tacit) knowledge and (ability for) use, which make eight elements. Contrary to Chomsky, Hymes does not separate competence from performance. As stated above he redefines competence to include knowledge and use. On Communicative Competence 18 inadequate as soon as it is confronted with real children in a particular environment" (Hymes, 1971, p. 4). Hymes explains the value in Chomsky's theory: However, Hymes does make a distinction between knowledge and competence . "This distinction allows one to deal with differential competence due to differential On Communicative Competence 21 knowledge" (Hymes, 1971, p.16). If knowledge is differential then it is not a state, but rather a process related to performance. Hymes associates both terms, competence and knowledge, with ability, and white he says he is interested in the underlying rules of use or pragmatic competence, he continues to discuss competence in terms of ability for use. As stated above, in Rules and Representations Chomsky recognized pragmatic competence which he conceived of as underlying the ability to make use of the knowledge characterized as grammatical competence (1980, p. 59}. Even with respect to the term pragmatic competence, Chomsky draws the distinction between competence and performance. Chomsky only has two elements--competence and performance. As stated earlier Chomsky's concern is competence: the system of rules and principles that we assume have, in some manner, been internally represented by the person who knows a language and that enable the speaker, in principle, to understand an arbitrary sentence and to produce a sentence expressing his thought; and its further concern is universal grammar, the principles that specify the range of possible human grammars. (Chomsky, 1980; p. 201) So it seems that Chomsky's original theory of competence should remain intact, and while Hymes' theory of performance is important in its own right, it needs to be discussed in terms of a theory of language use. It is not a competence theory. Although Hymes talks about underlying rules of language use, he does not investigate pragmatic universals, just as Chomsky does not investigate or develop performance models. If Hymes changed his terminology and referred to his theory as one of performance, then Hymes and Chomsky's theories could complement each other. The following section on Hymes further demonstrates why Hymes' The following section on Hymes further demonstrates why Hymes' On Communicative Competence 22 theory is not a competence theory, but rather a theory of performance. Although in 1971, Hymes argued that Chomsky's theory of competence needed to be extended to include the concept of underlying competence for use, he does not explore the rules of use or pragmatic competence. Rather, Hymes discusses language fram a socio-cultural standpoint. His later work reveals his sociolinguistic perspective on language acquisition. 22 Hymes Argues Against the Chomskyan Perspective In Ethnography, Linguistics. Narrative, and Inequality Hymes (1996) discusses his ethnographic perspective on the study of language. He argues against the "Chomskyan perspective" of formal linguistics or abstract representations of the mind and UG, and elaborates on his theory of ethnography of speaking which seeks to reach an understanding of languages and all their dialects and varieties in terms of a socially constituted linguistic theory: *that verbal means and the social matrices in which they exist are interdependent; *that the organization of verbal means must be viewed from the vantage point of social matrices; *that one must discover ways in which verbal means are organized by virtue of social matrices (using 'social matrices' here as a general term for activities, institutions, groups, etc.) (Hymes, 1996, p. 102) For Hymes, "[t]he true scope of a socially constituted study of language is thus the study of speech styles within culturally constituted ways of speaking" (Hymes, 1996, p.102). Although Hymes admits that grammar can account for phonological, morphological and syntactic elements and their relationships, and can be abstracted from social context, he maintains that it On Communicative Competence 23 is not possible to account for speech styles in abstraction from social context. He suggests that both the formal properties and meaning of speech styles may lie in the relations among social contexts, not in the relations of the linguistic features themselves (Hymes, 1996, p. 102). It seems as though Hymes contradicts himself here, because if grammar can be abstracted from social context then theoretically a language faculty could exist that possesses specific properties, structure and organization. This is why I consider Hymes a straddler, because he never categorically states that there is no such module of the mind/brain called the language faculty. As stated above, Hymes reveres Chomsky's "brilliant work," yet he does not embrace it. Hymes does not investigate the formal properties of language; he merely criticizes Chomsky for creating a perspective on the study of language that diverted other linguists from properly pursuing the ethnography of speaking. Hymes asserts the following regarding Chomsky: Chomsky's conception of linguistics is the bringing to perfection of the trend to focus on formal models, while investing formal models with the ultimate significance of being avenues to human mind and nature, the only general goal worthy of a linguist . Hymes Argues Against the Chomskyan Perspective Chomsky's conception of linguistics is the bringing to perfection of the trend to focus on formal models, while investing formal models with the ultimate significance of being avenues to human mind and nature, the only general goal worthy of a linguist . The unintended consequence of the success of this brilliant work was to disable linguists from study of the social and to reinforce assumptions in American life prejudicial to understanding the place of language in it. (Hymes, 1996, p. 95) Chomsky's conception of linguistics is the bringing to perfection of the trend to focus on formal models, while investing formal models with the ultimate significance of being avenues to human mind and nature, the only general goal worthy of a linguist . The unintended consequence of the success of this brilliant work was to disable linguists from study of the social and to reinforce assumptions in American life prejudicial to understanding the place of l i it (H 1996 95) The unintended consequence of the success of this brilliant work was to disable linguists from study of the social and to reinforce assumptions in American life prejudicial to understanding the place of language in it. (Hymes, 1996, p. 95) Hymes holds Chomsky responsible for the following injustices with ect to language learning: Hymes holds Chomsky responsible for the following injustices with ect to language learning: respect to language learning: respect to language learning: On Communicative Competence 24 formal written English. Dialects and vernaculars are assumed to be superficial variations. p 4 English is enough. See 1 and 3. 5 Right or Wrong. Most Americans assume that there is a single standard. Chomskyan linguists were quick to apply a sign of exclusion, the asterisk, to sentences they judged impossible in English 6 Fluency and style are suspect [A] good many of the utterances set aside as ungrammatical prove acceptable in sufficient context, such as a poetic one; intonation, which is inseparable from the effect and acceptability of utterances, is ignored. {Adapted from Hymes, 1996, pp. 95-96) respect to language learning: 1 Only English. Most linguistic theory and analysis under his aegis focused on English. 2 Bilingualism is suspect. Chomsky's focus on his own language set bilingualism aside as secondary. A necessary simplifying assumption of the theory is the ideally fluent speaker/hearer in a homogeneous speech community. 3 Learn literary standard. Chomskyan linguistics was essentially On Communicative Competence 24 formal written English. Dialects and vernaculars are assumed to be superficial variations. Hymes' Ethnographic Perspective Hymes asserts that there were two stages in the development of the ethnography of speaking. He credits Sapir, Burke, and Jakobson (1960) for the first stage because they discussed language from a cross-cultural perspective, and their ideas laid the foundation for a discipline based on the social constitution of language in both structure and function. Jakobson argued that language must be investigated in terms of its many varieties and functions. "The verbal structure of a message depends primarily on the predominant function" {Jakobson, 1966, p. 353). "The second stage has been the undertaking of field studies explicitly devoted to questions of the structure and function of means of speech" (Hymes, 1996, p. 103). The third stage, which is in the developmental stages, has the task of comparing and contrasting case studies leading to a sharpening of terminology and a better understanding of the social constitution of language and how it relates to structure and function. Possible Compromise Between Chomsky and Hymes However, Hymes admits that: Possible Compromise Between Chomsky and Hymes However, Hymes admits that: However, Hymes admits that: Grammar in the usual sense contributes analysis of many of the On Communicative Competence 25 resources of speech styles, but not all. The grammar of discourse has additional properties. Two crucial properties are these: 1) a speech style involves selection and grouping of features across the usual levels of linguistic analysis, coordinating them in a novel way independent of such levels; 2) a socially significant speech style often involves only a portion of the occurring features. Now a grammarian is used to seeking total accountability at each level of analysis--all of the message form is referable to phonological elements and relationships, to morphological elements and relationships, to syntactic and semantic elements and relationships. This kind of accountability can be pursued to a fair degree in abstraction from social context. (Hymes, 1996, p. 102) To a certain degree, Hymes can embrace Chomsky's viewpoint--that the formal aspects of language can be pursued in abstraction from social context And to a certain degree, Chomsky can embrace Hymes' perspective stated below: It is not possible to pursue accountability of speech styles in abstraction from social context, and much of the interest, both in formal properties and in meaning, of speech styles may lie in the relations among social contexts, not in the relations of the linguistic features themselves. (Hymes, 1996, p.102} Chomsky would at least agree that speech styles or "special uses of language" cannot be abstracted from social context; however, I believe he would argue that the formal properties of speech styles are governed by tacit knowledge of underlying rules, and therefore, can and should be investigated independent of social context. Nonetheless, Chomsky (1986) is keenly interested in how knowledge of language is put to use--how the language faculty yields a particular language through interaction with presented experience such as specific speech styles. Hymes believes we should break away from the do~ination of the formal models of the investigation of grammar and look critically at language On Communicative Competence 26 from an ethnographic perspective. The question arises: Why not look at both aspects of language--the abstract and the concrete, as suggested by Spolsky (1989), Chomsky, and Widdowson (1989), who describes language in terms of 'usage' and 'use?' of 'usage' and 'use?' Hymes claims that: It is increasingly accepted that linguistic elements should be analyzed and explained in terms of their functional relevance, meaning not only their function within a grammar as such, but their relevance to needs and interests of users of language in conveying and processing information, making discourse coherent, managing social relationships. (1996, p. 88) Chomsky would be the first to admit that the investigation of the abstract principles of the mind/brain with respect to language does not say anything specific about cultural patterns or institutional constraints. However, in order to study the innate properties of the mind, one must look at language of the ideal speaker/hearer taken out of social context. In Language and Mind Chomsky states: "The most challenging theoretical problem in linguistics is that of discovering the principles of universal grammar that interweave with the rules of particular grammers to provide explanations for phenomena that appear arbitrary and chaotic" (1972, p. 48). Chomsky's theory is based on the Platonistic conception that knowledge of a particular language grows and matures along a course that is in part intrinsically determined, similar to the visual system or other bodily organs that develop along a course determined by genetic instructions under the triggering and shaping effects of environmental factors (1986, p. 2). Hymes reacts to Chomsky's theory of grammatical competence because he wants to emphasize the socio-cultural aspects of language, and Hymes reacts to Chomsky's theory of grammatical competence because he wants to emphasize the socio-cultural aspects of language, and On Communicative Competence 27 change the focus of linguistics from grammar to use. Hence, he redefines competence to include {tacit) knowledge and {ability for) use Hymes does not make a distinction between competence and performance because, unlike Chomsky, competence is not severed from ability for use. On the other hand, Chomsky makes the distinction, because knowledge of the rules of grammar does not tell us how a person might proceed in conversation. Furthermore, a speaker/hearer may possess full grammatical competence, but because of disabilities may not be able to employ his knowledge. While Chomsky is not interested in behavior and its products, he recognizes a relationship between knowledge of language and language acquisition. Several applied linguists suggest a compromise between Chomsky and Hymes. The following is a summary of of their perspectives. On Communicative Competence 29 theories. Chomsky and Bialystok agree that learners have structured representations of their knowledge and these representations are used systematically to produce utterances. On Communicative Competence 29 theories. Chomsky and Bialystok agree that learners have structured representations of their knowledge and these representations are used systematically to produce utterances. 29 29 Hymes' theory of communicative competence evolved over time. Originally, Hymes (1971) stated his goal to develop the concept of underlying knowledge for use from a socio-cultural standpoint. Later, Hymes (1996) argues against the "Chomskyan perspective" of formal linguistics or abstract representations of the mind and UG. Instead of a competence theory, he developed a theory of speech performance which is rooted in a functional theory of language use and acquisition. To support his theory, Hymes (1996) refers to Halliday's (1977) theory of the functional basis of language acquisition. Halliday's theory is discussed below. Hymes claims that: Widdowson (1989) also acknowledges a relationship between knowledge and acquisition, and he reformulates Hymes' parameters into two competencies--grammatica/ competence {the parameter of possibility) and pragmatic competence {all the other parameters). Canale and Swain adopt "the term 'communicative competence' to refer to the relationship and interaction between grammatical competence, or knowledge of the rules of grammar, and sociolinguistic competence, or knowledge of the rules of language use" {1980, p. 6). They also make a counterpoint to Hymes' earlier statement: "Just as Hymes {1972) was able to say that there are rules of grammar that would be useless without rules of language use, so we feel that there are rules of language use that would be useless without rules of grammar" {Canale and Swain, 1980, p. 5). In this same vein of thought, Spolsky {1989) asserts that grammatical competence is critical to any On Communicative Competence 28 performance model. Spolsky and Canale and Swain agree that there is a relationship between competence and performance (Spolsky, 1989, p.139). Spolsky proposes that these two independent measures of competence and performance overlap (1989, p.139). Bialystok maintains that competence theories and processing theories are derived from Chomsky's definition of competence and performance (Bialystok, 1990, p. 637). Most linguists discuss theories of communication either in response to Chomsky's theory of grammatical competence or in reaction to Chomsky's theories of generative grammar. Whether there is agreement or disagreement with regard to Chomsky's theory of grammatical competence, his theory remains a pivotal point for linguists that study language acquisition. Bialystok states that while Chomsky elevates competence and relegates performance to use, it is not what he intended, but is a result of his focus on knowledge of the mind rather than the whole of language which includes sociolinguistics. pragmatics, and language strategies. Still, Bialystok is committed to Chomsky's views. She quotes from Macnamara, a psychologist who extends Chomsky's distinction between competence and performance beyond linguistics (1990): "Is there understanding of the mind in its logical aspects without access to the mind's logical ideas?" (Macnamara 1990, p. 19). Bialystok agrees that competence theories by their very nature must be idealized because they are based on mental structures and representations. Furthermore, performance is an indirect and imperfect reflection of knowledge structures (1990, p. 643). Bialystok predicts that metatheoretical distinctions should be able to reconcile differences between competence theories and performance Halliday's Functional Theory of Language Acquisition There are other linguists who reject the Chomskyan perspective outright, and who consider it 'incomprehensible' that there is a language acquisition device, an innate component of the human mind that yields a particular language through interaction with presented experience. Hymes looks to the work of Halliday who has always defined language in terms of its functional relevance. In this respect, Halliday's theory of the functional basis of language is aligned with Piaget's (1980) constructivist philosophy of language--that there are no innate principles with respect to language, and that language learning is a process of building on experiences with a particular language. Halliday rejects the whole notion of competence and. performance and the distinction between them. On Communicative Competence 30 For Halliday, a child's language is what s/he internalizes as a result of his/her own experiences. In his book, Explorations in the Functions of Language, Halliday states the following: 30 The child knows what language is because he knows what language does. . . . He has used language in many ways--for the satisfaction of material and intellectual needs, for the mediation of personal relationships, the expression of feelings and so on. Language in all these uses has come within his own direct experience, and because of this he is subconsciously aware that language has many functions that affect him personally. Language is, for the child, a rich and adaptable instrument for the realization of his intentions; there is hardly any limit to what he can do with it. (Halliday, 19TT, p. 2) Halliday emphasizes the many-sidedness of a child's linguistic experience and identifies models of language with which the normal child is endowed by the time s/he is five. In other words, the child internalizes complex models of language through experience with the language as a functional system. Instrumental, Regulatory, and lnteractional Models of Language The simplest of the child's models of language is called the 'instrumental' model-- an internalized awareness that language provides a means to a desired end. Closely related to the instrumental mode of language is the 'regulatory' model of language--an internalized awareness of language used to control the behavior of others. Bernstein (1971) discusses different types of regulatory language used specifically to control the behavior of others. Based on Bernstein's work, Halliday gives the following examples of regulatory language that a child experiences and then internalizes, adding this model of language to his/her repertoire. In various On Communicative Competence On Communicative Competence 31 situations a mother may say: you mustn't take things that don't belong to you (control through conditional prohibition based on a categorization of objects in terms of a particular social institution, that of ownership); that was very naughty ( control through categorization of behaviour in terms of opposition approved/ disapproved); if you do that again I'll smack you (control through threat of reprisal linked to repetition of behaviour); you'll make Mummy ve,y unhappy if you do that (control through emotional blackmail); that's not allowed (control through categorization of behaviour as governed by rule) and so on. (1977, p. 5) Thus, the child applies these awarenesses or internalized models of language use to achieve a desired end (instrumental), and based on his/her experience with the language of rules and instructions (regulatory), he/she learns to control others. Closely related to the regulatory model is the third model called the 'interactional' model, which refers to the use of language in maintaining relationships. In this case, language is used to "define and consolidate the group, to include and to exclude, showing who is 'one of us' and who is not; to impose status, and to contest status that is imposed; and humour, ridicule, deception, persuasion, all forensic and theatrical arts of language are brought into play" (Halliday, 1977, p. 6). Thus, while the child is listening and involved in complex interactions, s/he is internalizing models of language that perform a function. onal, Heuristic, and Representational Models of Language The fourth model of language use internalized by the child is called the 'personal' model which refers to the child's awareness of language as a form of his own individuality; language plays an essential role in the development of a child's personality-- "his awareness of himself is closely On Communicative Competence 32 bound up with speech: both with hearing himself speak, and with having at his disposal the range of behavioural options that constitute language" (Halliday, 1977, p. 6). Fifthly, the child has a 'heuristic' model of language derived from his knowledge of how language has facilitated his investigation of reality. The child demonstrates this awareness of the 'heuristic' model of language by asking questions in order to gain information and explain facts. By the age of five many children already possess a metalanguage for the heuristic function--"they know what a 'question' is, what an 'answer' is, what knowing and 'understanding' mean, and they can talk about these things without difficulty" (Halliday, 1977, p. 7). Another aspect of 'metalanguage' or 'language about language' is how the child uses language to create his own environment. This is called the 'imaginative' model of language; "and this provides some further elements of the metalanguage, with words like story, make up, and pretend" (Halliday, 1977, p. 7). The sixth and final model of language is called the 'representational' model. This refers to the child's awareness that language is a means of communicating about something, or expressing propositions. "The child is aware that he can convey a message in language, a message which has specific reference to the processes, persons, objects, abstractions, qualities, states and relations of the real world around him" (Halliday, 1977, p. 8). Linguistic Structure Depends on Soclal Function Thus, the functional approach to the study of language investigates how the character of language has been shaped and determined by use. On Communicative Competence 33 The social function of language or 'diatypic' varieties, or 'registers' in language is governed by the range of uses that language is put to in a particular culture. This leads us to the fundamental question of the relation between the functions of language and the nature of the linguistic system. Halliday poses the question: "Is the social functioning of language reflected in linguistic structure--that is, in the internal organization of language as a system" (Halliday, 1977, p. 15)? 33 To answer this question, Halliday refers to the work of Malinowski {1923) who conducted ethnographic research among some Melanesian tribes of Eastern New Guinea. In general, what Malinowski attempts to make clear through his analysis of 'primitive' text is that language is essentially rooted in the reality of the culture, and that it cannot be explained without reference to the context of situation, an expression that he coined. From his analysis of the Trobriand language, a Melanesian language used by the natives of the Trobriand Islands, N. E. New Guinea, Malinowski concludes the following: The study of any form of speech used in connection with vital work [other than fishing] would reveal the same grammatical and lexical peculiarities: the dependence of the meaning of each word upon practical experience, and of the structure of each utterance upon the momentary situation in which it is spoken. Thus the consideration of linguistic uses associated with any practical pursuit, leads us to the conclusion that language in its primitive forms ought to be regarded and studied against the background of human activities and as a mode of human behaviour in practical matters. {Malinowski, 1949, p. 312) Malinowski goes on to say that meaning is directly related to use--that there is a direct connection with the meaning of the word and the object or prototype it represents. He states the following: On Communicative Competence 34 The meaning of a word arises out of familiarity, out of ability to use, out of the faculty of direct clamouring as with the infant, or practically directing as with primitive man. A word is used always in direct active conjunction with the reality it means. The word acts on the thing and the thing releases the word in the human mind. (1949, p. Linguistic Structure Depends on Soclal Function 322-323). However, we know this to be fa!se because many words have more than one meaning or associations; and many words refer to abstract ideas or intangible objects. Nonetheless, Malinowski maintains that "there can be no definition of a word without the reality which it means being present" (1949, p. 325). In the final section of Malinowski's article, he addresses the problem of the structure of language. He asserts that linguistic structure is not the result of the rules of human thought, nor is it the case described by H. Sweet--that every grammatical category is the expression of some logical category. While Malinowski believes real categories exist on which the grammatical divisions are based, these real categories are not derived from a primitive philosophic system. Instead, Malinowski claims the following: Language in its structure mirrors the real categories derived from practical attitudes of the child and of primitive or natural man·to the surrounding world. The grammatical categories with all their peculiarities, exceptions, and refractory insubordination to rule, are the reflection of the makeshift, unsystematic, practical outlook imposed by man's struggle for existence in the widest sense of this word. (1949, p. 327-328) Malinowski claims that each category closely corresponds with the part of speech. This part of speech (noun, verb, pronoun, etc.) is "rooted in active modes of behaviour and in active uses of speech" (1949, p. 332). So that the category of substance corresponds to the category of 'noun­ substantives,' and this connection between category and part of speech is On Communicative Competence 35 made by the child who observes active modes of behaviour and active uses of speech. In other words, Malinowski concludes that all linguistic processes including the grammatical structure of language, "derive their power only from real processes taking place in man's relation to his surroundings" (1949, p. 336). structure of language, Malinowski admits that all human languages show, in spite of great divergences, a certain fundamental agreement in structure and means of grammatical expression. It would be both preposterous and intellectually pusillanimous to give up at the outset any search for deeper forces which must have produced these common, universally human features of Language. (1949, p. 327) It is interesting to note that despite Malinowski's observation that language universals appear to exist in "structure and grammatical expression," he still maintains the close relationship between the structure of language and language use. Linguistic Structure Depends on Soclal Function "This adaptation, this correlation between language and the uses to which it is put, has left its traces in linguistic structure" (Malinowski, 1949, p. 327). It is interesting to note that despite Malinowski's observation that language universals appear to exist in "structure and grammatical expression," he still maintains the close relationship between the structure of language and language use. "This adaptation, this correlation between language and the uses to which it is put, has left its traces in linguistic structure" (Malinowski, 1949, p. 327). For Halliday also, language learning is learning the uses of language and meanings associated with their uses. He states the following: If language development is regarded as the development of a For Halliday also, language learning is learning the uses of language and meanings associated with their uses. He states the following: If language development is regarded as the development of a meaning potential it becomes possible to consider the Malinowskian thesis seriously, since we can begin by looking at the relation between the child's linguistic structures and the uses he is putting language to. (1949, p. 16) However, Halliday does not endorse Malinowski's original view that ancestral types of language were 'primitive.' When linguistic research had demonstrated that there was no such thing as a 'primitive language,' On Communicative Competence 36 Malinowski modified this view. Halliday agrees with Malinowski's later view-­ "all adult speech represented the same highly sophisticated level of linguistic evolution" {Halliday, 1977, p.16). Although Halliday acknowledges the linguistic movement of the 1960's--the "psycholinguistic view (the so-called 'nativist' view of the language learning faculty)," he asserts that this view is not relevant to his present perspective {Halliday, 1977, p. 16). Halliday defines language as a linguistic system where there is a set of options--that is choosing the correct vocabulary and saying it when it is appropriate. In other words, choice of words and meaning depend on 'context of situation,' to use Malinowski's term. The ability to say the right thing in the right place is what Hymes defines as communicative competence. Halliday does not make a distinction between meaning and function. He believes separating meaning from context is an artificial distinction. There are merely different contexts and the meaning of an expression within a particular context is different from its meaning elsewhere. Linguistic Structure Depends on Soclal Function Rather than characterize language subjectively as the ability, or competence, of a speaker in the way Hymes does, Halliday characterizes language objectively as a 'meaning potential,' a set of alternatives. He proposes "the concept of 'meaning potential,' which is what the speaker/hearer can {what he can mean, if you like), not what he knows" (Halliday, 1977, p. 17). In contrast, Chomsky does make a distinction between knowledge of language and the social function of language because his focus is on what the ideal speaker/hearer inherently knows about language. While Chomsky acknowledges the social functions of language. he does not focus on it because his goal is to discover and On Communicative Competence 37 establish universal rules and representations of language which allow us to communicate in a social setting. On the other hand, Halliday's focus is on the social functions of language, meaning "those contexts which are significant in that we are able to specify some of the meaning potential that is characteristically, and explainably, associated with them" (1977, p. 18-19). And Halliday is interested in how the meaning potential associated with certain contexts sheds light on certain features of the internal organization of language. In this respect Halliday differs from Chomsky in that he believes that "[w]hat the child does with language tends to determine structure" (1977, p. 19). In other words, Halliday believes that the internal form directly reflects the function of the language. This idea contradicts Chomsky's theory that language use reflects knowledge of language--that rules and representations of the mind can be derived from investigating primary linguistic data. Halliday maintains that a close match between structure and function can be brought out by a functional analysis of the system in terms of its meaning potential. In other words, the structures that the child has mastered are a direct reflection of the functions that the language serves for him/her. In this respect, Halliday's theory of language acquisition fits with Piaget's philosophy of contructivism--a child constructs the structure of a language to serve his/her needs, and through the process of building upon abstractions based upon experience, he becomes proficient in his native language. While this process may be going on to an extent, it still does not explain how a child creates novel sentences, and constructs sentences before the need arises. Linguistic Structure Depends on Soclal Function More specifically, as stated above, Halliday describes the process o On Communicative Competence 38 building structure in terms of three main functions--'instrumental,' 'regulatory,' and 'interactional.' Each of these has subordinate components. Figure 1 (below) illustrates the system Nigel has developed for the 'instrumental' Figure 1. Nigel at 19 months: part of the instrumental component. Instrumental = (rising tone) Examples: yes no positive 'res' response __ R :pos (do you . « i ~ want?) ~egative}{basic 'no' R R:neg ) supplementary initiating Q... = more (I want) OorS {~brtad specific -[111Lat food cake -[ 0 :f -{trtakfast general drink _goods 0 pou:dtr toiletry -{ 0 :t toothpaste _[light amenitr:-r water A -:- S.am music (Bartok) S = Otl services . -[(take this) off S assistance S:ass (help me) gtt t!own bread breakfast more bread powder water on Bartok on R:pos R:ncg O:f O:f , Q + O:f O:t A ..,.. S:am A+ S:am = ✓ .,, / - .,,, .,.J -- ., -- .,, ?e no b.11.: b.uka m:, b.u b ga w:,ta 1 :,n tab:, ~:,n yes I no I 1 want I want my I want some I want I want the I want a want it don't some breakfast more bread some water tap record · want it bread powder turned on put on off get down Elements: S:ass S:ass 0 object of desire [food, toiletry] / S service [amenity, assistance] ?.,,,_f, .• :t va dYkao R response [positive, negative) I want I want to Q quantifier my bib getdo\iln A amenity taken off Note: From M.A. K. Halliday (19n). Explorations in the Functions of Language. Copyright 1973 by M. A. K. Halliday. Instrumental = (rising tone) Examples: positive 'res' response __ R :pos (do you . « i ~ want?) ~egative}{basic 'no' R R:neg ) supplementary initiating Q... = more (I want) OorS {~brtad specific -[111Lat food cake -[ 0 :f -{trtakfast general drink _goods 0 pou:dtr toiletry -{ 0 :t toothpaste _[light amenitr:-r water A -:- S.am music (Bartok) S = Otl services . -[(take this) off S assistance S:ass (help me) gtt t!own Instrumental = (rising tone) E l positive 'res' response __ R :pos (do you . « i ~ want?) ~egative}{basic 'no' R R:neg ) supplementary initiating Q... Linguistic Structure Depends on Soclal Function = more (I want) OorS {~brtad specific -[111Lat food cake -[ 0 :f -{trtakfast general drink _goods 0 pou:dtr toiletry -{ 0 :t toothpaste _[light amenitr:-r water A -:- S.am music (Bartok) S = Otl services . -[(take this) off S assistance S:ass (help me) gtt t!own Instrumental = (rising tone) Note: From M.A. K. Halliday (19n). Explorations in the Functions of Language. Copyright 1973 by M. A. K. Halliday. On Communicative Competence 39 function of language which refers to the use of language for the purpose of satisfying needs. In the instrumental component there are five structure­ forming elements: the response element, the object of desire, the service desired, the quantifier, and the amenity. Figure 2. Nigel at 19 months: part of the regulatory component. Regulatory =(rising_ tone, or gesture) E..umples: come overthere A:1en A:loc ., ktm ~uvadca follow come over me there with me oranges-&-lemons P:song - - ' ­ :,..iWelema sing 'oranges and lemons' [accompanied g~:;! . cneutrat• presence - (- urgent A [ U ••• = no:t.• specific A:loc [ovuthue -[down room initiation•· _ P:~~g = (music gesture) [ repetition . R == again sing orangts and lnnons P (London) bridge Jlar draw­[ p :pict train down now overthere now room star train A:loc U + A:loc..., U + A:loc P:pict P:pict dau nau :iuvadu nau .1em da tYafa come (& come over come into draw a draw a sit) down there with me )i>ur room star train with me at once with me at once again. Elements: R A accompany (general, locational] _.,,,, P perform [song, picture] agael U urgency sing that R repetition again by music gesture] Note: From M.A. K. Halliday (19TT). Explorations in the Functions of Language. Copyright 1973 by M. A. K. Halliday. What Halliday proposes is that there is a connection between, for Regulatory =(rising_ tone, or gesture) g~:;! . cneutrat• presence - (- urgent A [ U ••• = no:t.• specific A:loc [ovuthue -[down room initiation•· _ P:~~g = (music gesture) [ repetition . R == again sing orangts and lnnons P (London) bridge Jlar draw­[ p :pict train Regulatory =(rising_ tone, or gesture) g~:;! . cneutrat• presence - (- urgent A [ U ••• = no:t.• specific A:loc [ovuthue -[down room initiation•· _ P:~~g = (music gesture) [ repetition . R == again sing orangts and lnnons P (London) bridge Jlar draw­[ p :pict train g~:;! Linguistic Structure Depends on Soclal Function Regulatory ( i i down now overthere now room star train A:loc U + A:loc..., U + A:loc P:pict P:pict dau nau :iuvadu nau .1em da tYafa come (& come over come into draw a draw a sit) down there with me )i>ur room star train with me at once with me at once Note: From M.A. K. Halliday (19TT). Explorations in the Functions of Language. Copyright 1973 by M. A. K. Halliday. Note: From M.A. K. Halliday (19TT). Explorations in the Functions of Language. g g Copyright 1973 by M. A. K. Halliday. g g Copyright 1973 by M. A. K. Halliday. g g Copyright 1973 by M. A. K. Halliday. What Halliday proposes is that there is a connection between, for On Communicative Competence 40 example, 'the object of desire' and the structure derived from it--the structures of language are expressed in terms of functional elements (and not of classes, such as noun and verb); and they are contextually determined according to the particular function of language. Although Halliday presents early stages of language learning, he claims the same process occurs in later stages, and the emphasis is on the form of the language. He states: I shall suggest, however, that in principle the same is true of the elements of structure of the adult language: that these also have their origin in the social functions of language, though in a way that is less direct and therefore less immediately apparent. Even such a 'purely grammatical' function as 'subject' is derivable from language in use: in fact the notion that there are 'purely grammatical' elements of structure is really self-contradictory. (Halliday, 1977, p. 23) I shall suggest, however, that in principle the same is true of the elements of structure of the adult language: that these also have their origin in the social functions of language, though in a way that is less direct and therefore less immediately apparent. Even such a 'purely grammatical' function as 'subject' is derivable from language in use: in fact the notion that there are 'purely grammatical' elements of structure is really self-contradictory. (Halliday, 1977, p. 23) Figure 2 (above) illustrates the 'regulatory' use of language which is used to control the behavior of others, to manipulate the persons in the environment. Linguistic Structure Depends on Soclal Function It consists of four elements: a demand for company, request for performance, demand for repetition of prior actions, and an urgency command. Halliday points out that there is no negative in the regulatory function at this stage, which means that this is not yet an option in the child's meaning potential. Figure 3 (below) illustrates the 'interactional' component which is the child's use of language to interact with those around him/her--the 'me' and 'you' function of language. It consists of three elements: interaction, need, and search. Halliday does not draw a sharp line among the three main language systems. He says there is a connection between the instrumental and the regulatory function, in that both represent types of demands placed on the addressee. The regulatory and the interactional are also related, in that both On Communicative Competence 41 involve interpersonal relationships. Nevertheless, the functions are distinguishable from one another; "and this is important, because it is through the gradual extension of his meaning potential into new functions that the child's linguistic horizons become enlarged" (Halliday, 1977, p. 25). Figure 3. Nigel at 19 months: part of the interactional component. Figure 3. Nigel at 19 months: part of the interactional component. generalized I: gen Afummy - DaddJ [ A1111a not seeking response personalized = falling tone lnl<"ractional I :pers -{ I seeking response rising tone call ---} \ -[ n~~ N = comt ...____ search [ S••• = where greeting Examples: hullo hullo Mummy Anna Mumm'r Anna where J:gcn.,,, _I:ge1;,... t!buwa tli'uwa hullo! hullo? (greeting) who's [narrow there? tone;+ (call) smile] [wide I :n_ers_ meml Mummy! here you are! l:pers - .,, ana Anna: where are you? come l:pers + N mtmi kem ~fommy. I want you. come l:pcrs + N ina k'm Anna where are you? I want you. Daddy S.::r I :pcrs wn did~ I'm looking for Dadd;­ tone] where iiummy S + l:pcrs Elements: - ✓ wta m'l!m I I want to know where I N S interaction [general, personal] need search Mummy is Note. From M. A. K. Halliday (19n). Explorations in the Functions of Language Copyright 1973 by M. A. K. Halliday. Linguistic Structure Depends on Soclal Function Halliday defines meaning potential as the •unguistic realization of the behaviour potential; 'can mean' is 'can do' when translated into language generalized I: gen Afummy - DaddJ [ A1111a not seeking response personalized = falling tone lnl<"ractional I :pers -{ I seeking response rising tone call ---} \ -[ n~~ N = comt ...____ search [ S••• = where greeting generalized I: gen Afummy - DaddJ [ A1111a not seeking response personalized = falling tone lnl<"ractional I :pers -{ I seeking response rising tone call ---} \ -[ n~~ N = comt ...____ search [ S••• = where greeting lnl<"ractional I Note. From M. A. K. Halliday (19n). Explorations in the Functions o Language Copyright 1973 by M. A. K. Halliday. Halliday defines meaning potential as the •unguistic realization of the behaviour potential; 'can mean' is 'can do' when translated into language. On Communicative Competence 42 The meaning potential is in turn realized in the language system as lexico­ grammatical potential, which is what the speaker 'can say"' (Halliday, 1977, p. 43). Halliday further explains meaning potential as the range of significant variation that is at the disposal of the speaker. The notion is not unlike Dell Hymes 1 notion of 'communicative competence', except that Hymes defines this in terms of 'competence' in the Chomskyan sense of what the speaker knows, whereas we are talking of a potential--what he can do, in the special linguistic sense of what he can mean--and avoiding the additional complication of a distinction between doing and knowing. (1973, p. 46) the range of significant variation that is at the disposal of the speaker. The notion is not unlike Dell Hymes 1 notion of 'communicative competence', except that Hymes defines this in terms of 'competence' in the Chomskyan sense of what the speaker knows, whereas we are talking of a potential--what he can do, in the special linguistic sense of what he can mean--and avoiding the additional complication of a distinction between doing and knowing. (1973, p. 46) We know that Hymes does not define competence in the Chomskyan sense because he does not sever it from ability as Chomsky does. However, Halliday's meaning potential is similar to Hymes' communicative competence because Hymes investigates language from a socio-cultural perspective in the same way that Halliday explores language. Furthermore, Halliday differentiates meaning potential from the Chomskyan notion of competence. Linguistic Structure Depends on Soclal Function Meaning potential is not defined in terms of the mind; rather, it is defined in terms of culture; "not as what the speaker knows, but as what he can do--in the special sense of what he can do linguistically (what he 'can mean', as we have expressed it)" (Halliday, 1977, p. 44). In conclusion, Halliday asserts the following: (A]lthough this connection between the functions of language and the linguistic system is clearest in the case of the language of very young children, it is essentially, I think, a feature of language as a whole. The internal organization of natural language can best be explained in the light of the social functions which language has evolved to serve. Language is as it is because of what it has to do. (1977, p. 26). Essentially, Halliday investigates language from a sociolinguistic perspective. While his theory has its merits, it constitutes only a part of language learning. Although Halliday's complex models of language use On Communicative Competence 43 may play a significant role in how the child manipulates language to serve his/her needs, it still does not explain how the well-formed sentences are generated for the child to internalize, nor does it explain how the child creates novel sentences and makes sense of primary linguistic data. On Communicative Competence 43 may play a significant role in how the child manipulates language to serve his/her needs, it still does not explain how the well-formed sentences are generated for the child to internalize, nor does it explain how the child creates novel sentences and makes sense of primary linguistic data. Ultimately, Halliday admits that the investigation of structure leads to "a fairly abstract grammar (fairly 'deep', in the Chomskyan sense) ..." (1977, p. 87). Nonetheless, Halliday continues to explain language learning exclusively in terms of its functions, despite the fact that he cannot explain the complexity of the structure of language, and how a child masters complex rules of his/her native language. This is where Chomsky's theory of UG seems to fit in, but Halliday refuses to admit this. The following chapter demonstrates the limitations of Halliday's socio-semantic or Hymes' socio­ cultural theory of CC. Alternatives to the Term Communicative Competence What is important to remember, as many linguists agree, is that UG or the mind's mechanism to organize and interpret language is a necessary component for communicative language ability. Chomsky asserts that this innate linguistic knowledge makes it possible for a child to acquire language in a relatively short period of time given the complexity of human language. What linguists suggest and this paper contends is that the "language faculty" converges with language input and produces successful communicative interaction. Without grammatical competence or underlying knowledge of grammar, communication would not take place. This is a theory that, although is not ,directly stated, can be inferred from many linguistic theories. On Communicative Competence 44 Where non-verbal and verbal communicative input and UG converge there is communication, or to use another term proposed by Brown, communicative cognizance takes place (1984, p. 600). By using another term other than communicative competence, Brown solves the confusing problem of using Chomsky's linguistic term of competence which does not mean ability. Others such as Taylor have replaced the term competence with the term proficiency (1980, p. 163). Other linguists define communicative competence in terms of ability, and make it clear from the start that they are not talking about the language instinct or innate knowledge of language as Chomsky defines it. For example, Geis (1995) acknowledges Chomsky's distinction between linguistic competence and linguistic performance. However, Geis maintains that linguistic performance is part of a much more complex competence, "namely speakers' communicative competence" (1995, p. 215). Geis defines CC as is defines CC as (a) a quite general ability to construct communicative plans, including, in particular, conversational plans, in an attempt to achieve one's (normally) nonlinguistic goals and recognize and identify the plans and therefore the goals of others and (b) an ability to produce and to understand plan-relevant messages (including utterances, silences, and other verbal and nonverbal behaviors). (1995, p. 215) Thus, Geis draws the distinction between the knowledge speakers have of their languages and how speakers use their languages. The term CC is then used to refer to "use" and is strictly associated with "ability." Chomsky's grammatical competence is a separate component; although like Chomsky, Geis is interested in how linguistic competence and CC relate. Alternatives to the Term Communicative Competence Geis' theory of CC is that communication or utterance generation is On Communicative Competence 45 pragmatically driven, because the goal in producing utterances is to construct utterances that are not only semantically appropriate, but also contextually appropriate-­ utterances that advance the purposes of the talk exchange in which they occur (Grice 1975) and are consistent in style, politeness, register, and discourse with the context in which they occur. Such utterances may or may not be grammatical in the sense that they are generatable by a conventional grammar of a language. Since the term communicative competence seems to have been born out of a reaction to Chomsky's theory of grammatical competence, and the term "'competence' entered the technical literature in an effort to avoid entanglement with the slew of problems relating to 'knowledge"' (Chomsky, 1980, p. 59) in general, this paper asserts that the term CC should be changed to communicative proficiency (CP) as suggested by Taylor (1988). Thus, Chomsky's linguistic term can be understood as originally intended-­ severed from ability, and the confusion regarding the term communicative competence can be eliminated. Although I will continue to use the term CC because that is how it is discussed in linguistic circles, I believe I have sufficiently illustrated the need to use a different term when discussing theories of communicative language proficiency. By taking this position, I am not making the claim that communicative behavior or pragmatic competence is not underlain by abstract mental knowledge structures as Chomsky proposes for grammatical competence. On the contrary, based on Chomsky's definition of competence, the term CC suggests that we possess underlying knowledge of the rules of language use. However, Hymes does not pursue this investigation. Instead, he discusses CC in terms of social matrices as though language use is merely a On Communicative Competence product of its functions. Thus, the term CP better defines what Hymes investigates--how we learn the social and cultural aspects of language. 46 However, if one wants to gain insight into what is involved in successful communication, then it seems one should look closely at the possibility of underlying rules for pragmatic competence in that same way that Chomsky investigates underlying rules for grammatical competence. Alternatives to the Term Communicative Competence If Hymes uses the term CC to mean all that is involved in communicative language ability, then one needs to consider what abstract rules and representations enter into CC--Chomsky's grammatical competence as well as the possibility of pragmatic competence in the Chomskyan sense. In other words, Hymes' theory of CC should take into consideration the underlying abstract rules that make communication possible in context. The following chapter which provides evidence for Chomsky's grammatical competence opens the door for an investigation of pragmatic competence-­ underlying abstract rules of language use. On Communicative Competence 4 Chapter 3 Psycholinguistic View versus Sociolinguistic View: Chomsky's Defense 47 Psycholinguistic View versus Sociolinguistic View: Chomsky's Defense While Chomsky was looking at the individual and attempting to determine through generative grammar what constitutes universal rules of grammar, Hymes was looking at communication between or among people, and defining language in sociolinguistic terms. In contrast to Chomsky, Hymes proposed that we stop looking at knowledge in the mind of the individual, but think of knowledge as between minds of individuals (Cazden, 1996, p. 7). Chomsky continues to investigate formal models to illuminate abstract properties of the mind/brain, but he never intended the investigation of the universal aspects of language to limit the study of language and all that it encompasses as Hymes claims. As stated earlier, he denies that his theories of abstract rules and representation of the mind/brain have direct application to the teaching of language (Chomsky, 1966, p. 43). While Chomsky successfully demonstrates that there are formal properties of language, he always maintains in all his writings that performance, or 'speech styles' cannot be abstracted from social context. Even Hymes, a sociolinguist, credits Chomsky with a profound linguistic theory of the intrinsic structure of language (Hymes, 1972, p. 273). Still, Hymes attacks Chomsky for revering knowledge and depreciating use. Linguists such as Taylor (1988, p.156) claim Hymes misunderstood Chomsky who was defining competence in terms of the individual's tacit knowledge, knowledge as a state, not knowledge as a process. Hymes looks to antiquity for support for his sociolinguistic views of language acquisition where On Communicative Competence 48 "structure was a means to use, and the grammarian subordinate to the rhetor" (1972, p. 272). Although Hymes, a sociolinguist, sought to transcend the notion of competence in a homogeneous speech community independent of sociological features, he does not preclude the existence of innate linguistic knowledge. However, he does claim that social life affects inner knowledge as well as outward performance. In this regard, Hymes theories are based in empiricist philosophy. Whereas Chomsky differentiates between grammatical competence and performance, Hymes combines knowledge and ability to create his model of communicative competence. In this regard, Hymes has misappropriated the linguistic term competence as defined by Chomsky, since Chomsky severs the term competence from ability (1980, p. 59). Hymes claims Chomsky attacks traditions by using "his own language [as] a basis for theory: intuiting, rather than observing, and rejecting the social world with disdain" (Hymes, 1989, p. 245). Hymes does not equate language acquisition with a universalist view. Psycholinguistic View versus Sociolinguistic View: Chomsky's Defense "Chomsky pursues whatever may provide evidence of the innate. That sets aside much of what users, learners and teachers of language know and do" (Hymes, 1989, p. 248). While Hymes blames Chomsky for disabling linguists from pursuing the righteous path of investigating the ethnography of speaking, I believe Hymes is responsible for confusing the issue by coining the term communicative competence which by Chomsky's definition is a contradiction of terms. For Chomsky, communication encompasses performance and ability, while 'competence• is knowledge of language severed from ability (Chomsky, 1980, p. 59). On Communicative Competence 49 Hymes' allegations (seep. 25) regarding Chomsky's work illustrates why Hymes, not Chomsky, is at least in part responsible for the injustices mentioned earlier related to language learning. To attribute these injustices to Chomsky is to completely misinterpret his work and goals--to determine underlying rules and representations which make language acquisition possible. It does not logically follow that because Chomsky focuses his attention on competence or the formal models of language, that he discounts performance. What Chomsky did do was to shift the focus from behavior or the products of behavior to states of the mind/brain that enter into behavior. Chomsky's concern is knowledge of language: its nature, origin, and use. The three basic questions that he attempts to answer are the following: (i) What constitutes knowledge of language? (i) What constitutes knowledge of language? (ii) How is knowledge of language acquired? (iii) How is knowledge of language put to use? (Chomsky, 1986, p. 3) So while Chomsky's main focus is on competence severed from ability, he is acutely aware of its relationship to use, and is interested in how knowledge of language is put to use. As Spolsky proposed earlier, there is a point at which competence and performance overlap (1989, p. 139). Chomsky proposes how to pursue the answers to the above three basic questions: The answer to the first question is given by a particular generative grammar, a theory concerned with the state of the mind/brain of the person who knows a particular language. The answer to the second is· given by a specification of UG along with an account of the ways in which its principles interact with experience to yield a particular language; UG is a theory of the "initial state" of the language faculty, prior to any linguistic experience. Psycholinguistic View versus Sociolinguistic View: Chomsky's Defense The answer to the third question would be a theory of how the knowledge of language attained enters into the expression of thought and the understanding of presented On Communicative Competence 50 specimens of language, and derivatively, into communication and other special uses of language. (Chomsky, 1986, p. 4) On Communicative Competence 50 specimens of language, and derivatively, into communication and other special uses of language. (Chomsky, 1986, p. 4) 50 Chomsky goes on to say that he is doing nothing more than taking up classical questions that had been set aside for many years. By investigating the 'steady state,· Chomsky demonstrates that there are generalizations about all languages, not just English, which characterize UG, a theory of knowledge, not of behavior. In fact, the UG theory gains its power by being applied to many languages; and since the 1980's many languages have been studied, in particular the Romance languages and Japanese ( Cook & Newson, 1996, p. 3). Chomsky defines UG as "the system of principles, conditions, and rules that are elements or properties of all human languages ... the essence of human language" (Chomsky, 1975, p. 29). Again, Chomsky's theory of competence does not say how a speaker might proceed, nor does it support a method of language teaching. So Hymes' claim that Chomsky is responsible for the list of injustices (see p. 25) with respect to language learning is unfounded. The Chomsky/Piaget debates (below) illuminate the complexities of language learning. These debates demonstrate that language learning extends beyond Halliday's socio-semantic theory of language and Hymes' socio-cultural theory of language. Empiricism versus Natlviam, Piaget/Chomsky Debates Piaget's Theory of Language Learning In the book, Language and Learning, Piaget and Chomsky debate the issue of empiricism versus rationalism with respect to language learning. At issue was whether human linguistic capacities can be considered a product On Communicative Competence 51 of general "constructed" intellectual development (as Piaget contended), or whether they are a highly specialized part of human genetic inheritance--a kind of innate knowledge that has only to unfold (as Chomsky asserts) (Piattelli-Palmarini, 1980, p. xxvii). In the opening chapter by Piaget, "The Psychogenesis of Knowledge and Its Epistemological Significance," Piaget asserts the following: Fifty years of experience have taught us that knowledge does not result from a mere recording of observations without a structuring activity on the part of the subject. Nor do any apriori or innate cognitive structures exist in man; the functioning of intelligence alone is hereditary and creates structures only through an organization of successive actions performed on objects. (Piaget, 1980, p. 23) Piaget does not consider himself a behaviorist or a nativist, but rather he claims to be a constructivist, which he defines as a strong empiricist position regarding the acquisition of knowledge or human understanding. In this respect, Piaget's view is representative of Halliday's theory of language learning--that the character of language has been shaped and determined by what we use it for, and that language is a process of building meaning potential associated with the social functions of language. Piaget explains his anti-behaviorist and non-nativist position as follows: Knowledge proceeds from action or experience similar to the empiricist and behaviorist argument, but the relationship between objects and subjects is not merely "adaptive" or simply a process of association. Rather it is a process which Piaget defines as " the integration of new objects or new situations and events into previous schemes of action which are developed as a result of generalizations made based on experience" (1980, p.164). On Communicative Competence 52 Adaptation does occur but not in an isolated state, because there are logical generalizations constructed based on experience; and new experiences are then integrated into these constructed schemes of action. Piaget maintains that learning is a continual process of construction of schemes and that as we mature we begin to reflect on knowledge gained through the constructed schemes of action. Empiricism versus Natlviam, Piaget/Chomsky Debates He states: "As a rule, all reflecting on a new plane leads to and necessitates a reorganization, and it is this reconstruction, productive of new concepts, that we call "reflection"" (1980, pp. 27-28). According to Piaget, we progress from constructive generalizations based on sensorimotor assimilation to reflective abstraction. Piaget explains this process further: Piaget explains this process further: [R]eflecting on a higher plane of an element taken from a lower level (for example, the interiorization of an action into a conceptualized representation) constitutes an establishment of correspondences, which is itself already a new concept, and this then opens the way to other possible correspondences, which represents a new "opening." The element transferred into the new level is then constituted from those that were already there or those that are going to be added, which is now the work of the "reflection" and no longer of the "reflecting" ( although initially elicited by the latter). (1980, p. 27) In other words, we pass from sensorimotor assimilation (assimilating objects to schemes) to representational assimilation (assimilating objects to each other) through the formation of the semiotic function (evocation of objects not presently perceived). The semiotic function commences when sensorimotor signifiers (cues or signals) are differentiated from what is thereby signified and when signifiers can correspond to a multiplicity of things signified (Piaget, 1980, p. 29). In addition to vocal and learned language, semiotic functions include deferred imitations, symbolic play, On Communicative Competence 53 mental image which is an interiorized imitation, and sign language. In so far as the semiotic function is concerned, Piaget's theory of language learning diverges from Halliday's theory, because Halliday refers to Malinowski's theory that language learning is a direct result of experience with objects that are present. Of course, as stated before, we know that language acquisition is not the result of interaction with the environment alone. There is a biological aspect to language acquisition, and although Piaget and Chomsky disagree on just what that is, they do agree that the biological endowment possesses an innate component. Piaget argues against Chomsky's hypothesis of an "innate fixed nucleus" or the transformational aspects of Chomsky's doctrine. Piaget maintains that at every level of learning a mechanism called autoregulation plays a role. Empiricism versus Natlviam, Piaget/Chomsky Debates Autoregulation is the process by which we construct schemes of action via sensorimotor assimilation, and pass from action to representation due to the semiotic function--differentiation of sensorimotor signifiers from those signifiers that remain as a result of constructive generalizations. While these reflective abstractions are not at the time dependent on sensory input, they were initially produced by sensorimotor intelligence. So that the initial state for Piaget consists of the hereditary mechanism of autoregulation, common to biological and mental processes (1980, pp. 30-31). Piaget maintains that a non-innate fixed nucleus is the "result of the constructions of sensorimotor intelligence, which is prior to language and results from those joint organic and behavioral autoregulations that determine this epigenesis" (1980, p. 31 ). He admits that autoregulation is in part innate, but more in terms of functioning than in terms of structure (1980, On Communicative Competence 54 p. 381). This is similar to Halliday's theory of language learning--that the structure of language is clearly related to the type of function which the language is being made to serve (1977, p. 21). Chomsky's Reply to Piaget In the chapter by Chomsky, "On Cognitive Structures and Their Development: A Reply to Piaget/ Chomsky argues against Piaget's two basic tenets: that innate structures are "biologically inexplicable," and that a fixed non-innate structure can be explained as the 'necessary' result of constructions of sensorimotor intelligence (1980, p. 35). Although Chomsky agrees that the evolutionary development is no doubt, "biologically unexplained," he does not believe that it is "biologically inexplicable." Chomsky argues that there are no substantive proposals involving 'construction of sensorimotor intelligence' that offer any hope of accounting for the phenomenon of language. He draws an analogy between cognitive structures and physical structures of the human body: The expectation that constructions oi sensorimotor intelligence determines the character of a mental organ such as language seems to me hardly more plausible than a proposal that the fundamental properties of the eye or the visual cortex or the heart develop on this basis. (Chomsky, 1980, p. 37) By investigating the 'steady state' attained by puberty, Chomsky claims we can construct a hypothesis as to the grammar internally represented; and we could gain further insight into the growth of language by investigating the intermediate states--the sequences of states that progress from the genetically determined 1initial state' (So) to the •steady state' (Ss). Empiricism versus Natlviam, Piaget/Chomsky Debates By looking at the steady state of a language Chomsky postulates abstract mental On Communicative Competence 55 processing of a ·nontrivial' sort which explains how a child can comprehend and construct well-formed sentences from degenerative information. In other words, primary linguistic data does not provide enough information to explain how we comprehend and construct well-formed sentences. Given this poverty of stimulus, Chomsky proposes innate knowledge of language--rules and representations that provide the basis from which we derive well-formed, comprehensible sentences. Hypothesis 1 is a "structure independent rule" and H2 is a "structure On Communicative Competence dependent rule" {Chomsky, 1980, p.39). Chomsky explains further: Thus, H1 requires analysis of the declarative into just a sequence of words, whereas H2 requires an analysis into successive words and also abstract phrases such as "noun phrase." The phrases are abstract in that their boundaries and labeling are not in general physically marked in any way; rather they are mental constructions. (1980, p. 39). Thus, H1 requires analysis of the declarative into just a sequence of words, whereas H2 requires an analysis into successive words and also abstract phrases such as "noun phrase." The phrases are abstract in that their boundaries and labeling are not in general physically marked in any way; rather they are mental constructions. (1980, p. 39). To illustrate the fact that the structure independent rule, H1, is invalid, yntactic Structures Are Bound by Principles and Parameters Syntactic Structures Are Bound by Principles and Parameters Chomsky presents examples of later states of language acquisition that are not determined by experience, that is, elements of language that are known but for which there appear to be no relevant evidence that allows for the formation of well-formed sentences. In other words, experience with language in a linguistic environment does not provide the stimulus to produce well-formed sentences. Chomsky considers the process of the formation of simple yes-or-no questions. He gives the following examples: (1) The man is here.--ls the man here? (2) The man will leave. -Will the man leave? (2) The man will leave. -Will the man leave? Chomsky puts forth two hypothesis to account for this infinite class of pairs: H1: process the declarative from the beginning to end (left to right), word by word, until reaching the first occurrence of the words is, will, etc.; transpose this occurrence to the beginning (left), forming the associated interrogative H2: same as H1, but select the first occurrence of is, will, etc., following the first noun phrase of the declarative. (1980, p.39) 56 Chomsky gives the following data: (2) The man who is here is tall.--ls the man who is here tall? The man who is tall will leave.--Will the man who is tall leave? These data are predicted by H2 and refute H1, which would predict rather the interrogatives (3): (2) The man who is here is tall.--ls the man who is here tall? The man who is tall will leave.--Will the man who is tall leave? These data are predicted by H2 and refute H1, which would predict rather the interrogatives (3): g ( ) (3) Is the man who here is tall? Is the man who tall will leave? (1980, p. 39). Chomsky explains the preference for H2 over H1, even when no child is taught the relevant facts, by postulating the existence of innate mental representations of rules--rules that are structure dependent. "The child need not consider H 1, it is ruled out by properties of his initial mental state, So" (Chomsky, 1980, p. 40). Chomsky likens language learning to other genetically determined physical properties of the human body. In other words, the different organs of the body are predetermined to function in a certain way, and the mind/brain is no different in that it is genetically predetermined to acquire language. The initial state which possesses rules of grammar provide the resource or foundation for the acquisition of language. This theory explains why children are never heard to make errors like those in example (3). Chomsky explains further: [T]hough as in the case of many other genetically determined properties, (for example, the onset of puberty, the termination of growth) the appearance of this mental characteristic may be delayed many years after birth, and may be conditional on the triggering effect On Communicative Competence 58 Phonology Is Governed by Abstract Universal Rules 58 Even at the level of sound structure, there is evidence that abstract representations are formed and manipulated by a restrictive schematism that specifies the choice of relevant phonetic properties. In other words, in the same way that universal rules govern syntax as illustrated above, universal rules also govern phonology. A careful investigation of sound reveals general principles of organization governing phonological rules. For example, it has been observed that certain phonological rules operate in a cycle. This principle is called the principle of cyclic application (Chomsky, 1972, p. 45). Some simple effects of this principle are illustrated below: a. relaxation, emendation, elasticity, connectivity b. illustration, demonstration, devastation, anecdotal (Chomsky, 1972, p. 44). The unitalicized vowels are reduced to [a] in b, but they retain their original quality in a. Example a differs from bin that the former are derived from underlying forms--relax, emend, elastic, connective, that contain primary stress on the unitalicized vowel. Those in b do not have the same property; and what Chomsky contends is that vowel reduction is contingent upon lack of stress. He accounts for the distinction between forms a and b by employing the principle of cyciic application. In the case of a on the first, innermost cycle, stress will be assigned by general abstract rules to the unitalicized vowels. On the next cycle, stress is shifted, but the abstract stress (the stress on the underlying form) assigned in the first cycle is sufficient to protect the vowel from reduction. In the case of b the underlying forms do not contain a primary stress In the case of b the underlying forms do not contain a primary stress On Communicative Competence 59 on the unitalicized vowel--illustrate, demonstrate, devastate, anecdote. Therefore, the lack of stress in the first cycle does not protect the vowel from reduction. "Thus, it is the abstract underlying representation that determines the phonetic form, a primary role being played by the abstract stress that is virtually eliminated in the phonetic form" (Chomsky, 1972, p. 44-45). Since it is difficult to explain how a language learner might derive this principle by "induction" from the data presented to him, Chomsky concludes that "the principle of cyclic application of phonological rules is an innate organizing principle of universal grammar that is used in determining the character of linguistic experience and in constructing a grammar that constitutes the acquired knowledge of language" (1972, p. 45). 59 Chomsky Refutes Behaviorism and Constructivism Through a close investigation of language in its 'steady state' (Ss). not only does Chomsky demonstrate that language is not learned through an inductive process, as the behaviorists attempt to show, but he also undermines Piaget's theory of constructivism. According to Piaget, who considers himself an anti-nativist and anti-behaviorist, cognitive development is a constructive exchange with the environment. In other words, cognitive development is the construction of the new in the mind/brain, and through assimilation, some structure without is turned into some structure within. Once assimilated, the new structure becomes part of the subject's repertoire, and then can enter as a component into more complex constructions which then build upon themselves, so that the subject can create constructions of constructions within the mind/brain. For Piaget the initial state consists of the 60 On Communicative Competence 60 On Communicative Competence 60 hereditary mechanism of autoregulation, the process by which we construct emes of action via sensorimotor intelligence. Piaget explains further: [CJognitive autoregulation makes use of the general systems of organic regulation such as are found at every genetic, morphogenetic, physiological, and nervous level, and forthwith adapts them to their new situation (new, that is, by relation to the preceding levels, but still present in every animal series).This situation constitutes the exchanges with environment that form the basis of behavior. ... [T]he operational structures of the intelligence are transformation systems of a kind which maintains the system as an invariant totality. This same definition could be applied to the living organism itself, since its two basic properties are that it serves as the field for multiple interactions {=transformations), though at the same time leaving unchanged both the overall form (=conservation) and even a certain number of invariant relationships. (1971, p. 34) However, based on his investigation of the steady state (Ss),Chomsky argues that the process of language development cannot be fully explained by Piaget's theory of constructivism. Chomsky's principles and parameters theory, first known as Government/Binding (GB) theory (1981 ), makes the claim that language knowledge consists of principles universal to all languages and parameters that vary from one language to another. These principles reveal the existence of a fixed nucleus or innate knowledge of language which when exposed to primary linguistic data organizes the input so that it is phonologically, syntactically, and semantically comprehensible. Chomsky Refutes Behaviorism and Constructivism Piaget admits that there is something innate as far as functioning {but not structure) is concerned: "no one has ever been able to make an intelligent man out of an idiot'' {Piaget, 1980, p. 168). However, Chomsky argues that if there are elements of innateness involved in the structure of language, then one must postulate innate structures; you cannot have it both ways. On Communicative Competence Fodor Argues Against Constructivism 61 In a continuation of the rebuttal to Piaget's theory of constructivism, Fodor argues "On the Impossibility of Acquiring 'More Powerful' Structures" (Fodor, 1980, p. 142). While Piaget is in favor of some kind of compromise between innatism and constructivism, Chomsky and Fodor are not. In fact, Fodor considers himself a radical innatist. As stated earlier Piaget admits that some structures are innate, but these innate structures are not highly specific linguistic rules as Chomsky proposes in his theory of UG. Instead, according to Piaget, these rules are the sophisticated outcome of the interactions among simpler primitives. The resulting rules are constructed using the subject's innate repertoire of computational abilities of the mind interacting among themselves and with the environment. Furthermore, Piaget claims that richer or stronger concepts can be constructed from impoverished or weaker concepts. Fodor explains why this inductive learning theory does not fully explain the origin of concepts. Most learning theories are based on experiments which demonstrate how a subject maps certain attributes or characteristics onto an object. For example, the subject, through trial and error, associates the word miv with a red square. This exemplifies the theory of inductive learning, but Fodor argues that this says nothing about the origin of concepts. "What it doesn't tell you is where the hypothesis (and the concepts that they deploy) come from" (Fodor, 1980, p. 145). Furthermore, Fodor argues that Piaget's theory of constructing richer structures from weaker structures is also flawed, because if you define learning as hypothesis formation and confirmation, it is never possible to learn a ric~er On Communicative Competence 62 logic based on a weaker logic. Fodor explains that in order to get from stage 1 to stage 2 by a process of hypothesis formation and confirmation {the only learning theory we have) we would have to learn the truth conditions upon which the hypothesis in stage 1 is based. And to learn these truth conditions we need to formulate a hypothesis using the conceptual apparatus available at stage 1. However, such a hypothesis cannot be formulated because the conceptual apparatus available at stage 1 does not provide the means to formulate a hypothesis which could then theoretically bridge the gap between stage 1 and stage 2. In summary, what the above discussion illustrates is that language learning is different from general learning in that it is not learned inductively. On Communicative Competence Fodor Argues Against Constructivism In order to explain language acquisition, one must include Chomsky's theory of principles and parameters, or UG, because grammatical competence, a component of communication, develops despite the poverty of stimulus. Thus, in the process of acquiring communicative skills we induce from universal rules of grammar how to structure and organize language. Several questions remain: Is pragmatic competence like grammatical competence, in that some part of communicative language ability is underlain by abstract knowledge? Are there universal rules of language use as suggested by Chomsky (1980) and Grice (1975)? Do we possess knowledge of pragmatics like we possess grammatical knowledge? In other words, do certain parts of the brain govern pragmatics like specific modules of the mind govern grammar (i.e. Broca's and Wernicke's aphasia)? These are some of the questions I will attempt to answer in the ensuing chapters. In Chapter 41 review Savignon's {1983) theory of CC and Bachman's On Communicative Competence 63 (1990) theory of Communicative Language Ability. Both linguists describe an integrative theory of CC. Like Chomsky, they make a distinction between competence and performance, but they combine these two entities into one theory. Savignon refers to her theory as a theory of CC; whereas, Bachman avoids the terminological confusion by developing a theory of C i i L Abili (CLA) Integrative Theories of Communicative Competence Both Savignon (1983) and Bachman (1990) develop models of CC that include grammatical competence as one of the integral components of CC. They also theorize that we possess pragmatic competence--underlying knowledge of language use. By theorizing that we possess pragmatic competence, Bachman and Savignon suggest that language skills are subserved by competence--tacit knowledge structures severed from ability. This knowledge of language use enters into performance in the same way that knowledge of grammar enters into performance. Just as there are organizing principles and parameters for grammar, there are organizing principles and parameters for pragmatics. Furthermore, Bachman and Savignon suggest that there is a competence of communication--CC. Communicative Language Ability (CLA). Included in Bachman's theory, and suggested by Savignon, is a theory of pragmatic competence. They theorize that pragmatic competence may be like grammatical competence in that there are basic underlying rules for pragmatics. Through exploring the theories of Savignon (1983) and Bachman (1990) this paper reaches a more complete picture of what it means to achieve communicative competence, or communicative proficiency (Taylor, 1988). It is the contention of this thesis that these theories move beyond Hymes' theory of CC, a socially constituted linguistic theory. Savignon (1983) and Bachman (1990) provide a more comprehensive theory of CC, by integrating Chomsky's theory of grammatical competence into their theory of CC or CLA (Bachman, 1990). In addition, they suggest that we do possess pragmatic competence in the Chomskyan sense. On Communicative Competence 64 Chapter 4 64 Savignon's Theory and Model of CC As early as 1972 Savignon followed her intuition and developed a method of teaching that addressed the needs of communication-a continuous process of expression, interpretation, and negotiation between or among people (1972, p. 8). In her book, Communicative Competence: An Experiment in Foreign Language Teaching (1972), Savignon compares three groups of students' ability to use French in four different situations-­ discussion of a school related topic, information getting, reporting about personal life, and description of the characteristics of an actor. After 18 weeks of institutional teaching, those students who received training in communicative skills performed at a superior level compared with the other On Communicative Competence 65 two groups who did not --a culture group that discussed French culture and attended cultural events, and a control group that attended the language laboratory for two 30-minute sessions a week. These two 30-minute sessions in the language laboratory were in addition to four 50-minute lessons using an instructional program based on the audiolingual method of teaching language which all three groups attended. The experimental group received a 50-minute supplementary session of communicative acts in place of the two 30-minute sessions in the language laboratory for the control group. The results revealed that all groups performed the same (there were no significant differences) on the CEEB tests of listening and reading. However, the experimental communicative group scored higher on communication skills. These findings suggest that there is a difference between linguistic competence an the one hand, and communicative competence on the other hand. Savignon differentiates linguistic competence from communicative competence in practice--through her study in foreign language teaching. In addition, her theory carefully integrates grammatical competence with the other competencies. At the same time, she acknowledges underlying grammatical competence in the strict Chomskyan sense as the basis for language performance or language acquisition. Chomsky defines grammatical competence as the cognitive state that encompasses all those aspects of form and meaning and their relation, including underlying structures that enter into that relation, which are properly assigned to the specific subsystem of the human mind that relates representations of form and meaning. A bit misleadingly perhaps, I will continue to call this · subsystem "the language faculty." (1980, p. 59) Savignon differentiates linguistic competence from communicative competence in practice--through her study in foreign language teaching. In addition, her theory carefully integrates grammatical competence with the other competencies. (1980, p. 30). anale and Swain define strategic competence as the following: Canale and Swain define strategic competence as the following: verbal and non-verbal communication strategies that may be called into action to compensate for breakdowns in communication due to performance variables or to insufficient competence. Such strategies will be of two main types: those that relate primarily to grammatical competence (e.g. how to paraphrase grammatical forms that one has not mastered or cannot recall momentarily) and those that relate more to sociolinguistic competence (e.g. various role-playing strategies, how to address strangers when unsure of their social status). (1980, p. 30-31) The only difference between Canale and Swain's model and Savignon's model is that Savignon proposes a separate component for discourse competence. Later, in 1983, Canale distinguishes sociolinguistic competence from discourse competence (Canale, 1983, p. 9-10). Thus, by discussing Savignon's theory of CC which reconfigures Canale and Swain's theory of CC, I will have outlined the contents and boundaries of each of their components, and will not discuss Canale and Swain's theory separately. On Communicative Competence On Communicative Competence 67 (1980, p. 30). Savignon's Theory and Model of CC At the same time, she acknowledges underlying grammatical competence in the strict Chomskyan sense as the basis for language performance or language acquisition. Chomsky defines grammatical competence as the cognitive state that encompasses all those aspects of form and meaning and their relation, including underlying structures that enter into that relation, which are properly assigned to the specific subsystem of the human mind that relates representations of form and meaning. A bit misleadingly perhaps, I will continue to call this · subsystem "the language faculty." (1980, p. 59) On Communicative Competence 66 The language faculty is understood to be a particular component of the human mind; and UG, a theory of linguistic structure that aims to discover the principles and parameters of attainable human languages, may be regarded as a characterization of the genetically determined language faculty (Chomsky, 1986, p. 3). Thus, knowledge of language plays a critical role in language performance; although as Chomsky explains, and Savignon agrees, a speaker/hearer's implicit knowledge of language is not evident from his performance because errors occur in real situations due to "memory limitations, distractions, shifts of attention and interest ..." (Chomsky, 1965, p. 3). Since Savignon's focus is L2 acquisition, her model (1983) is based on Canale and Swain's (1980) theoretical framework for curriculum design and evaluation in L2 programs. Canale and Swain's theoretical framework for communicative competence minimally includes three main components: grammatical competence, sociolinguistic competence, and strategic competence (1980, p.28). Grammatical competence includes knowledge of lexical items and of rules of morphology, syntax, sentence-grammar semantics, and phonology (1980, p. 29). They refer to Chomsky for a definition of grammatical competence. Sociolinguistic competence consists of two sets of rules: socio­ cultural rules of use and rules of discourse. Sociocultural rules specify the ways in which utterances are produced appropriately within a given context. Rules of discourse are defined in terms of cohesion (i.e. grammatical links) and coherence (i.e. appropriate combination of communicative functions) Savignon's Grammatical Competence Savignon (1983) defines grammatical competence as "linguistic competence in the restricted sense of the term as it has been used by Chomsky ... and most other linguists. It is that part of language performance with which we are most familiar, that is, the grammatical well-formedness that has provided the focus of L2 studies for centuries" (Savignon, 1983, p. 36). Savignon explains in more detail: Savignon explains in more detail: Grammatical competence is mastery of the linguistic code, the ability to recognize the lexical, morphological, syntactic, and phonological features to form words and sentences. Grammatical competence is not linked to any single theory of grammar, nor does it assume the ability to make explicit the rules of usage. A person demonstrates grammatical competence by using a rule, not by stating a rule" (1983, p. 37). 68 On Communicative Competence 68 Savignon's definition of grammatical competence fits with Chomsky's theory that grammar is a system of rules underlying the creative aspects of language. For Chomsky grammatical competence constitutes knowledge of tanguage--the mental structures and representations of the mind/brain with respect to language. The term competence is used because of the problems relating to knowledge; Chomsky wants to sever competence from the meaning of 'ability' (1980, p. 59). Grammatical competence is concerned . with those aspects of form and meaning that are determined by the "language faculty." Universal Grammar provides the basis for developing the rules of grammar of a particular language and all of this knowledge is tacit or implicit. As Savignon states above, grammatical competence is demonstrated by using the rules, not by stating the rules of a language of which there are hundreds. While Savignon's theory of grammatical competence is used as the basis for L2 teaching, Chomsky is skeptical that the theoretical underpinnings of grammatical competence would support a method of language teaching (Chomsky, 1966, p. 43). Nonetheless, Chomsky believes it is important to study the processes by which transition takes place from the 'initial state' of the language faculty prior to any linguistic experience to the 'final state,' that is, language acquisition (Chomsky, 1980, p. 202). Savlgnon's Sociolinguistic Competence The second component of CC, according to Savignon, is sociolinguistic competence which is an interdisciplinary field of study that relates to the socio-cultural rules of language use. Savignon defines it as the On Communicative Competence 69 following: Sociolinguistic competence requires an understanding of the social context in which language is used: the roles of the participants, the information they share. and the function of the interaction. Only in full context of this kind can judgments be made on the appropriateness of a particular utterance in the terms elaborated by Hymes. (19n, p. 37) Sociolinguistic competence requires an understanding of the social context in which language is used: the roles of the participants, the information they share. and the function of the interaction. Only in full context of this kind can judgments be made on the appropriateness of a particular utterance in the terms elaborated by Hymes. (19n, p. 37) Savignon admits that we are far from formulating a satisfactory description of socio-cultural rules of appropriateness. However, one of the goals of intercultural analysis is to make explicit the rules of a culture in order to help nonnatives adapt to unfamiliar cultures. Savignon explains sociolinguistic competence further: Judgments of appropriateness involve more than knowing what to say in a situation and how to say it. They also involve knowing when to remain silent. Or, in fact, when to appear incompetent.. Women of my generation may remember being cautioned by their mothers not to talk up too much in class, not to "show up" the boys, and counseled to "act dumb» on occasion so as to give the men in their lives a feeling of superiority. The appearance of incompetence in this instance was considered appropriate, that is, a sign of sociolinguistic competence. (1983, p. 37). Another example of appropriate behavior is when L2 speakers deliberately maintain a formal register, where in the same situation an informal register might be appropriate for a native speaker. These L2 speakers are playing the role of "foreigner," which they believe has been assigned to them by a native speaker. Thus, there are specific roles to play in a given social situation, and knowing the social rules provides the speaker/ listener with the tools to use language correctly--according to convention, and effectively so that s/he may accomplish his/her goals. Savlgnon's Sociolinguistic Competence Both native and non-native speakers need to know and understand cultural rules of behavior in order to adapt or assimilate into society. In general, the purpose of On Communicative Competence achieving sociolinguistic competence is to promote successful communication. 70 Savignon's Discourse Competence Discourse competence, the third component of CC as defined by Savignon, is also an interdisciplinary inquiry like sociolinguistic competence. "The theory and analysis of discourse bring together many disciplines--for example, linguistics, literary criticism, psychology, sociology, philosophy, anthropology, print, and broadcast media" (Savignon, 1983, p. 38). Discourse competence is concerned with the connections of sentences or utterances used to form a meaningful whole. Organizational patterns of discourse differ depending on the nature of the text and the content in which it appears. Morgan (1981) refers to the various structures underlying discourse as discourse grammar. As stated above, Canale and Swain discuss rules of discourse under the heading of sociolinguistic competence; although they distinguish rules of discourse from socio-cultural rules or rules of appropriateness (1980, p. 30). However, as mentioned above, in 1983 Canale made a further distinction between sociolinguistic competence (socio-cultural rules) and discourse competence (cohesion and coherence). Canale and Swain (1980) refer to Halliday and Hasan (1976) and Widdowson (1978) for a discussion of rules of discourse. These rules are defined in terms of the cohesion (grammatical links) and coherence (appropriate combinations of communicative functions) of groups of utterances (1980, p. 30). Halliday and Hasan (1976) identify and explain cohesive devices used to create a meaningful sentence. Formal cohesive On Communicative Competence 71 devices used to connect language include: pronouns, conjunctions, synonyms, ellipsis, comparisons, and parallel structures. These devices will be discussed in detail in Bachman's (1990) model of CC, since he also relies on Halliday and Hasan (1976) for a definition of what Bachman refers to as 'textual competence.' Connections or structural links between sentences are often not explicit--there may be no overt expression of a link between one proposition and another. However, a listener infers meaning based on knowledge of the world as well as familiarity with a particular context. The following examples by Savignon illustrate the role of inference in the interpretation of discourse: 1) Chico suddenly turned and ran because he saw a policeman coming down the street. 2) Chico saw a policeman coming down the street. Suddenly he turned and ran (1983 p 39) 1) Chico suddenly turned and ran because he saw a policeman coming down the street. 2) Chico saw a policeman coming down the street. Suddenly he turned and ran. (1983, p. 39) In sentence 1) the relationship between the first independent clause and the second dependent clause is explicit. Savignon's Discourse Competence The use of the conjunction 'because', a cohesive device, makes it clear why Chico ran. However, in sentence 2) we need to infer why Chico , an because there is no cohesive device used to make the obvious unambiguous connection, and grammatical knowledge will not provide this information. Our original interpretation might be the same as sentence 1), but with more contextual information, this could be invalidated. To illustrate, Savignon gives the following example of how the text might continue: In sentence 1) the relationship between the first independent clause and the second dependent clause is explicit. The use of the conjunction 'because', a cohesive device, makes it clear why Chico ran. However, in sentence 2) we need to infer why Chico , an because there is no cohesive device used to make the obvious unambiguous connection, and grammatical knowledge will not provide this information. Our original interpretation might be the same as sentence 1), but with more contextual information, this could be invalidated. To illustrate, Savignon gives the following example of how the text might continue: 3) Chico saw the policeman coming down the street. Suddenly he turned and ran. The 5th street bus had just passed him by and he was going to be late for school again. There was no time to ask about Pedro. (1983, p. 39) On Communicative Competence 72 Hence, discourse competence is the ability to grasp the meaning of the text beyond the sentence-level structure. As Savignon's above example illustrates, a single sentence becomes meaningful only in context, the ref ore creating a 'global' meaning. Text coherence or a global meaning can either be created through cohesive devices such as in sentence 1), or by giving more related information in discourse, as in example 3) above. Furthermore, discourse competence is the ability to interpret text connected by implicit or explicit means in order to form a meaningful whole, and to be able to construct a meaningful whole through cohesion or coherence so that others may comprehend the global meaning. Successful communication "is dependent on the knowledge shared by the writer/speaker and the reader/hearer--knowledge of the real world, knowledge of discourse structure and knowledge of the social setting" (Savignon, 1983, p. 40). Savignon's Strategic Competence Strategic competence, the fourth component of CC, is analogous to survival strategies with respect to communication. In other words, survival strategies are the coping and strategic skills used to successfully communicate, and to sustain communication. Savignon defines strategic competence further: There is no such person as an ideal speaker/hearer, who knows the language perfectly and uses it appropriately in all social interactions . . . . The Strategies that one uses to compensate for imperfect knowledge of rules--or limiting factors in their application such as fatigue, distraction, and inattention- -may be characterized as strategic competence.... The strategies we use to sustain communication include paraphrasing, circumlocution, repetition, hesitation, avoidance, and guessing, as well as shifts in register and style. (1983, p. 40-41) On Communicative Competence 73 Thus, strategic competence is the ability of a native or nonnative speaker/hearer to rephrase, repeat, emphasize, clarify, avoid unknown words, topics or situations, and modify the message when a breakdown in communication occurs. This adaptation in L 1 and L2 requires the speaker/hearer to ·empathize' with others. Horwitz and Horwitz describe empathy as the power of imaginatively experiencing others' experiences, and they believe it is a necessary component for communicative competence: 73 Given a 'complete' repertoire of all the appropriate linguistic and sociolinguistic skills, a person without empathy would still be unable to define from a mutual perspective that of the other person (as well as his own) what the particular interpersonal context was and what kind of language it required. (1977, p. 110) In addition to these four linguistic components of CC, there are paralinguistic features of communication to take into consideration. Paralinguistic features include gestures, distance, posture, and facial expressions which accompany words and facilitate communication. According to Savignon, just as there is a linguistic code, there is also a gesture code--there are sociolinguistic rules which govern gesture as well as speech (1983, p. 43). For example, an appropriate gesture may replace a word as a coping strategy during a moment of silence, and may prevent a communication breakdown. These paralinguistic features such as gestures used during speech communication can serve the same purpose as linguistic features in the framework of CC. Thus, different paralinguistic features may fall within one of the four main components depending on the function they serve. On Communicative Competence 7 4 Savignon's Model of Communicative Competence 7 4 The question now remains: How do these four linguistic components interact to produce CC in a particular setting or context? Figure 4 (below) shows Savignon's theoretical model of CC. What this diagram proposes is that even without any grammatical competence, sociolinguistic competence and strategic competence interact to afford a measure of CC. In other words, successful communication can occur without the use of words through gestures, facial expressions, etc., provided there is a willing partner. Savignon includes strategic competence as a component of CC at all levels because coping strategies are always in play, and "regardless of experience or level of proficiency one never knows all of a language" (1983, p. 46). She states that the proportions drawn have no empirical basis, but minimally illustrate that CC is greater than linguistic or grammatical competence, and that one does not move from one competence to the other. "Rather, an increase in one component interacts with the other components to produce a corresponding increase in overall communicative competence" (Savignon, 1983, p. 45). The question now remains: How do these four linguistic components interact to produce CC in a particular setting or context? Figure 4 (below) shows Savignon's theoretical model of CC. What this diagram proposes is that even without any grammatical competence, sociolinguistic competence and strategic competence interact to afford a measure of CC. In other words, successful communication can occur without the use of words through gestures, facial expressions, etc., provided there is a willing partner. Savignon includes strategic competence as a component of CC at all levels because coping strategies are always in play, and "regardless of experience or level of proficiency one never knows all of a language" (1983, p. 46). It is true that grammatical competence would increase with respect to grammatical knowledge of a particular language as the tacit knowledge of rules governing a particular language became part of one's repertoire in the process of language acquisition. However, since Savignon defines grammatical competence in the strict Chomskyan sense it also must begin at the point of the pyramid, like strategic competence, at least in so far as she conceives CC. Universal grammar constitutes "'the initial state' of the language faculty prior to any linguistic experience" (Chomsky, 1986, p. 4). 75 On Communicative Competence 7 Figure 4. Savignon*s Components of Communicative Competence. On Communicative Competence 75 Figure 4. Savignon*s Components of Communicative Competence. Note. On Communicative Competence 7 4 Savignon's Model of Communicative Competence From Savignon, S. J. (1983). Communicative Competence: Theory and Classroom Practice Copyright 1983 by Addison Wesley Publishing Company, Inc. Figure 4. Savignon*s Components of Communicative Competence. Note. From Savignon, S. J. (1983). Communicative Competence: Theory and Classroom Practice Copyright 1983 by Addison Wesley Publishing Company, Inc. My interpretation of the above model is that we as social animals are biologically endowed with a predisposition to communicate, and therefore, we possess innate mechanisms which are designed to use strategies and conform to sociolinguistic rules. This knowledge--strategic and sociolinguistic competence, is not the same as Chomsky's language facuity characterized by UG; however, we possess a general knowledge for communication. Chomsky (1988) and Piaget (1980) both discuss general innate learning mechanisms which are engaged during communication. On the other hand, because of the way the model is drawn with strategic competence and sociolinguistic competence beginning at the point of the pyramid, it seems possible that Savignon is suggesting that we have knowledge of underlying rules of pragmatics-pragmatic competence in the On Communicative Competence 76 Chomskyan sense. Bachman (1990) theorizes that we possess pragmatic competence in the Chomskyan sense of grammatical competence. Under the heading of Language Competence, which Bachman defines as knowledge of language, he includes organizational competence (Chomsky's grammatical competence and textual competence) and pragmatic competence (illocutionary competence and sociolinguistic competence). Below is a review of Bachman's theory. Bachman's Theory and Model of Communicative Language Ability Bachman (1990) includes all of the same components in his theory of CC, but his theoretical models are drawn differently with many more delineations of the four basic components discussed above from Savignon (1983) and Canale and Swain (1980). Instead of using the term communicative competence, Bachman coins his own term--communicative language ability (CLA). In this respect, he avoids the confusion caused by using the term CC, since Chomsky entered the term competence as a technical linguistic term severed from ability. Bachman's framework of CLA (Figure 5) includes three main components: language competence, strategic competence, and psychophysiological mechanisms. Bachman describes CLA in a way that provides a broad basis for the development and use of language tests and language testing research. He extends the framework of earlier models of CC by Hymes (1972), who focuses on L1, Savignon {1983), Canale and Swain {1980), and Canale (1983), all of whom focus on L2. Therefore, Bachman's models are applicable to L 1 and L2. Therefore, Bachman's models are applicable to L 1 and L2. On Communicative Competence 77 Language competence consists of specific knowledge of language used in communication. Strategic competence refers to the language user's appropriate implementation of his knowledge of language in a particular setting. "Psychophysiological mechanisms refer to the neurological and psychological processes involved in the actual execution of language as a physical phenomenon (sound, light)" (Bachman, 1990, p. 84). Below is Bachman's diagram of the interaction of these three basic components of CLA with the language user's knowledge of the world within a particular context. The diagram illustrates how knowledge of the world and knowledge of language provide the tools for strategic competence--the ability to use linguistic knowledge and knowledge of the world to negotiate meaning so that successful communication takes place between speaker/hearer, and writer/reader. What the rest of the diagram illustrates is that neurological and psychological mechanisms interact with strategic competence and context of situation. In other words, how we employ our brains with respect to language is impacted by our level of strategic competence as well as the context in which the communication takes place. Certain psychophysiological mechanisms are engaged when we are placed in a specific linguistic context; and certain psychophysiological mechanisms are employed with respect to our level of strategic competence. Bachman's Theory and Model of Communicative Language Ability On the other hand, our level of strategic competence is impacted by the condition of our psychophysiological mechanisms, and by the context of situation. All three components work together and are affected by one another. Ultimately, they are governed by knowledge structures and It is the interaction of the three main components--language It is the interaction of the three main components--language 78 On Communicative Competence Figure 5. Bachman's Components of Communicative Language Ability On Communicative Competence Figure 5. Bachman's Components of Communicative Language Ability Note. From Bachman, L. F. (1990). Fundamental Considerations in Language Testing. Copyright 1990 by Oxford University Press. language competence. p Figure 5. Bachman's Components of Communicative Language Ability gure 5. Bachman's Components of Communicative Language Ability Note. From Bachman, L. F. (1990). Fundamental Considerations in Language Testing. Copyright 1990 by Oxford University Press competence, strategic competence, and psychophysiological mechanisms, with context of situation and knowledge of the world that defines Bachman's Communicative Language Ability. with context of situation and knowledge of the world that defines Bachman's Communicative Language Ability. Similarly, Savignon states "communicative competence is context specific. Communication takes place in an infinite variety of situations, and success in a particular role depends on one's understanding of the context and on prior experience of a similar kind. It requires making appropriate choices of register and style in terms of the situation and the other participantsn (1983, p. 8). Thus, both Savignon and Bachman agree that communication is like a weaving of all the different competencies and our On Communicative Competence 79 choice of 'design' (style) and 'color' (register) depends on our knowledge of the world and the context of our situation. Defining Bachman's Language Competence For Bachman, language competence includes some of the components found in Canale and Swain's (1980) and Savignon's (1983) models of CC. Language competence is divided into two types: organizational competence and pragmatic competence, and each of these is broken down into its constituent parts. Below is a tree diagram which extends earlier models of CC. Bachman explains that the tree diagram is intended as a visual metaphor, not a theoretical model. The relationships among the components of language competence as shown in the diagram are not in reality independent of each other. Rather, they interact with each other, and it is the interaction among the various competencies and the language use context that characterizes CLA. Canale and Swain ( 1980) and Savignon (1983) make the same point, that it is how these components interact that characterizes CC. With respect to Bachman's model of language competence, Bachman and Palmer (1983) developed a battery of tests that included grammatical competence (morphology and syntax), pragmatic competence (vocabulary, cohesion, and organization) and sociolinguistic competence (sensitivity to register, naturalness, and cultural references). The results of their study revealed empirical evidence that grammatical and pragmatic competence were closely related; whereas, the components of sociolinguistic competence were distinct. Bachman's model of language competence is based on the empirical findings of this study. Pragmatic On Communicative Competence Figure 6: Bachman's Components of Language Competence On Communicative Competence Figure 6: Bachman's Components of Language Competence 80 On Communicative Competen Figure 6: Bachman's Components of Language Competence LANGUAGE COMPETENCE ,,/ PRAGMATIC COMPETENCE ORGANIZATIONAL COMPETENCE /"~ //~, ILLOCUTIONARY SOCIOLINGUISTIC GRAMMATICAL TEXTUAL COMPETENCE COMPETENCE ~\ ·!\·. COMPETENCE COMPETENCE //: / I . /;1\ Ii~ /\ ///I\ // / \ \., Ideal Marip Hell. mag Sensit Sensll Sc'iSII Cu!!ural Cohes Rhet Voc. Maritt. S)rt ~. Org. Functs.Funcls. Functs. Functs to Dial lo Reg. lo Nd!. Rcfs.8 or Varoely Figs or Speei:,h Note. From L. F. Bachman (1990). Fundamental Considerations in Language Testing. Copyright 1990 by Oxford University Press. LANGUAGE COMPETENCE ,,/ PRAGMATIC COMPETENCE ORGANIZATIONAL COMPETENCE /"~ //~, ILLOCUTIONARY SOCIOLINGUISTIC GRAMMATICAL TEXTUAL COMPETENCE COMPETENCE ~\ ·!\·. COMPETENCE COMPETENCE //: / I . /;1\ Ii~ /\ ///I\ // / \ \., Ideal Marip Hell. mag Sensit Sensll Sc'iSII Cu!!ural Cohes Rhet Voc. Maritt. S)rt ~. Org. Functs.Funcls. Functs. Functs to Dial lo Reg. lo Nd!. Rcfs.8 or Varoely Figs or Speei:,h LANGUAGE COMPETENCE ,,/ ORGANIZATIONAL COMPETENCE /" PRAGMATIC COMPETENCE SOCIOLINGUISTIC COMPETENCE / 1\ Note. From L. F. Bachman (1990). Fundamental Considerations in Language Testing. Copyright 1990 by Oxford University Press. competence is redefined to include not only Bachman and Palmer's (1983) sociolinguistic competence, but also illocutionary competence--those abilities related to the functions that are performed through language use. Although Bachman and Palmer's (1983) study revealed that grammatical and discourse competence are related, they felt that there is strong theoretical justification for hypothesizing a distinction between organization--grammar, vocabulary, and cohesion--and organizational strategies of coherence (p. 462). Bachman's model of language competence is based on the empirical findings that vocabulary, morphology, syntax, cohesion, and organization are related; and these components fall under the heading of organizational competence. Pragmatic competence is then redefined to include On Communicative Competence 81 sociolinguistic competence and illocutionary competence (see Figure 6). "Language competencies can thus be classified into two types: organizational competence and pragmatic competence" (Bachman, 1990, p. 86.) In this regard, Bachman extends the definition of competence to include pragmatic competence as suggested by Chomsky (1980}. Bachman {1990} describes how these competencies interact under the heading of strategic competence. Thus, while strategic competence is theoretically conceived as part of CLA, Bachman developed a separate model which he adapts from Faerch and Kasper (1983) (see p. 91 ). As one can see, Bachman's theory of CC is not only based on empirical study, but also expands on the framework of Canale and Swain (1980) and Savignon's (1983) model of CC. On Communicative Competen Figure 6: Bachman's Components of Language Competence Remember that Savignon also drew an empirical distinction between grammatical competence and communicative skills based on her study in foreign language teaching. Bachman's Grammatical Competence It On Communicative Competence 83 should be noted that if (as here) it is meant to refer to the knowledge of how the language system is realized as use in social contexts, then it includes competence in the more restricted Chomskyan sense of the term. (1978, p.163) 83 Bachman agrees with Widdowson that knowledge of language or grammatical competence governs " the choice of words to express specific significations, their forms, their arrangement in utterances to express propositions and their physical realization, either as sounds or written symbols" (1990, p. 87). Similarly, Canale and Swain define grammatical competence as those competencies which provide the "knowledge of how to determine and express accurately the literal meaning of utterances" (1980, p. 30). Bachman's Grammatical Competence Bachman's grammatical competence is based on the competencies described by Widdowson (1978). These consist of the same competencies described by Canale and Swain (1980) with an additional competency~­ graphology or written symbols. In Widdowson's chapter on "Usage and Use" he states that "we are generally required to use our knowledge of the language system in order to achieve some kind of communicative purpose" (1978, p. 3). In other words, the abstract system of the language or tacit knowledge of language is generally not manifested out of context. While we possess the tacit On Communicative Competence 82 knowledge of grammatical rules, and we as students study grammatical rules, there are hundreds of rules that are too complex to teach, and yet are employed in instances of language use. Widdowson makes a distinction between 'usage' and 'use' similar to De Saussure's langue (a system of psychological signs) and parole (social side of speech) (1959, pp. 14-15). Chomsky makes a similar distinction between competence and performance. Like Chomsky, De Saussure believes that the langue can be studied separately; "indeed, the science of language is possible only if the other elements are excluded. Whereas speech is heterogeneous, language [langue]. as defined is homogeneous. It is a system of signs in which the only essential thing is the union of meanings and sound-images, and in which both parts of the sign are psychological" (1959, p. 15). 82 For Widdowson, production of a grammatical sentence out of context is referred to as 'usage'--making use of grammatical knowledge or what Chomsky calls grammatical competence in a non-communicative manner. "The notion of competence has to do with a language user's knowledge of abstract linguistic rules. This knowledge has to be put into effect as behavior; it has to be revealed through performance" (Widdowson, 1978, p. 3). If we employ our knowledge of language or grammatical competence in context, then we are demonstrating 'use,' our ability to use linguistic knowledge for successful communication. Widdowson maintains that there is a natural coincidence of 'usage' and 'use.' Thus, Bachman describes grammatical competence in Widdowson's terms, and Widdowson relies on Chomsky for his definition. Widdowson states: The term 'communicative competence' is now very much in fashion and for this reason alone it is as well to be wary of it: particularly since it does not seem to be used in the same sense by different writers. Bachman's Textual Competence The second component of organizational competence is textual competence, which is equivalent to Savignon's discourse competence. "Textual competence includes the knowledge of the conventions for joining utterances together to form a text, which is essentially a unit of language-­ spoken or written-consisting of two or more utterances in sentences that are structured according to rules of cohesion and rhetorical organization" (Bachman, 1990, p. 88). Text is defined by Halliday and Hasan "as any passage, spoken or written, of whatever length, that does form a unified whole" (1976, p. 1). Bachman defines cohesion in terms of cohesive devices as delineated by Halliday and Hasan (1976). Cohesive devices provide a way to semantically connect parts of a sentence or a number of complete sentences in a text. "Cohesive devices may refer either to upcoming text or Example Sadie is a good dog. She stays in the yard. That was the best dinner ever. She's not as smart as I thought. He told her to run home. She did so. She likes to run. I don't [like to run]. I thought he could read well, but he couldn't. The child ran across the street. The toddler scared me. (Adapted from McCabe, 1998,p.277) Cohesion also includes the proper ordering of old information and new information in discourse. For example, a pronoun (new information) cannot be used unless its reference is known (old information). Rhetorical organization refers to the organizational conventions used in different forms of writing or speaking, such as narrative, expository, descriptive, and comparative. Textual competence also includes conventions of conversation often discussed in terms of Grice's conversational maxims. These maxims include four unwritten rules for efficient speech: 1. The Maxim of Quality: Make your contribution one that is true. 2. The Maxim of Manner: Be brief and orderly. Avoid obscurity and ambiguity. 3. The Maxim of Quantity: Make your contribution as informative as required for the current purposes of the exchange. 4. The Maxim of Relation: Be relevant. (Adapted from Grice, 1975, p. 45) On Communicative Competence 85 (It is important to note that Chomsky (1980) considers Grice's Maxims a part of pragmatic competence, a system of rules and principles that determine how knowledge of language is put to use). Thus far, under the main heading of language competence, Bachman has discussed the necessary organizational competencies of a speaker/hearer. In addition to grammatical competence or innate knowledge of language, all of the above abilities associated with textual competence are essential to achieving the communicative goals of the interlocutors. However. the ability to organize and create a cohesive text is only part of communicative language because communication takes place in context. Knowledge of pragmatics or the conventional rules of language use in context provide the speaker/hearer or writer/reader with the tools to make utterances acceptable. Bachman's Pragmatic Competence Bachman refers to the knowledge of pragmatic conventions for performing acceptable language functions within a specific context as illocutionary competence; and knowledge of sociolinguistic conventions for performing language functions appropriately in a given context is called sociolinguistic competence. Bachman breaks down each of these two components of pragmatic competence into specific abilities. Bachman's lllocutionary Competence Bachman's definition of illocutionary competence is taken from Searle (1969) who developed a theory of speech acts. Searle distinguishes three On Communicative Competence 86 types of speech acts: a) Uttering words (morphemes, sentences)= performing utterance acts. b) Referring and predicating= performing propositional acts. c) Stating, questioning, commanding, promising, etc. = performing illocutionary acts. (1969, p. 24) What Searle maintains is that when uttering a sentence which might be a statement, a question, an exclamation, or an order, the speaker is simultaneously performing three distinct speech acts as listed above. Bachman further defines illocutionary acts in terms of language functions based on Halliday's (1973, 1977) work. Bachman describes four functions of language use as it relates to both the expression of language (speech and writing) and its interpretation (listening and reading). The first and most widely used function in language use is the ideational function-­ expressing meaning in terms of our experience of the real world (Halliday, 1973, p. 27). For example, language is used ideationally to present knowledge in lectures or scholarly articles, and language is used to express feelings either by confiding in a friend or writing in a diary. Manipulative functions are used to effect change and are classified as instrumental, regulatory, or interactional. An example of an instrumental function is to get someone to do something by suggestion, a command or a warning. A regulatory function is used to control the behavior of others by stating rules, laws or norms of behavior (Halliday, 1973, p. 23). The interactional function of a language is the use of interpersonal language to form, maintain or change relationships between people. "Phatic language use, such as greetings, ritual inquiries about health, or comments on the weather, is primarily interactional in function. Its propositional content is On Communicative Competence 87 subordinate to the relationship maintaining function" (Bachman, 1990, p. 93). Imaginative functions of language such as telling jokes, story telling, creating metaphors, as well as attending plays and films enable us to create or enhance our environment for humorous or aesthetic purposes. On Communicative Competence 87 subordinate to the relationship maintaining function" (Bachman, 1990, p. 93). Imaginative functions of language such as telling jokes, story telling, creating metaphors, as well as attending plays and films enable us to create or enhance our environment for humorous or aesthetic purposes. Finally, Bachman emphasizes that these functions are not distinct. Bachman's lllocutionary Competence Like Searle's (1969) theory of speech acts where several speech acts are performed within one utterance, several functions are performed simultaneously within one utterance or written expression. Furthermore, in the majority of cases, utterances seldom stand alone, but are connected and they perform multiple functions. "[l]t is the connections among these functions that provide coherence to discourse" (Bachman, 1990, p. 94). Bachman's Sociollngulstlc Competence Thus, illocutionary competence enables us to use language to express a wide range of functions. The appropriateness of these functions and how they are performed in context are determined according to socio­ cultural and discourse features. Sociolinguistic competence is the sensitivity to the socio-cultural conventions of language use and the ability to respond appropriately to the demands of a particular context. According to Bachman, sociolinguistic competence includes the following abilities: "sensitivity to differences in dialect or variety, to differences in register and to naturalness, and the ability to interpret cultural references and figures of speech" (1990, p. 95). An example of sensitivity to variety or different dialect is that of a Black student who appropriately uses 'Standard American English' in a classroom; On Communicative Competence 88 whereas, outside of the classroom with Black friends, (s)he would use 'Black English Vernacular.' With respect to discourse, there are important choices to make in different contexts because the appropriate use of a dialect or register in a given situation could make the difference between 'acceptability' and 'unacceptability.' For an understanding of the term 'register,' Bachman refers to Halliday, McIntosh, and Strevens (1964) who define register as the variation in language use within a single variety or dialect. Halliday et. al. distinguish differences in register in terms of three aspects of language use in context: ' field of discourse' (subject matter of the language use), 'mode of discourse' (spoken and written), and 'style of discourse' (how participants relate to one another in the language use context) (1964, pp. 90-92). 88 Joos (1967) distinguishes five different levels of style, or register in language use: frozen, formal, consultative, casual, and intimate. When in communication with others in spoken or written discourse the speaker or writer must choose the most appropriate style of language use for the context. An inappropriate choice of style could be interpreted as presumptuous or rude. The third aspect of sociolinguistic competence is sensitivity to naturalness which allows the user to interpret and speak like a native speaker. In other words, a sensitive speaker/hearer will respond in a manner or style that is culturally appropriate. Stilted or unnatural language not only sounds strange but could be misinterpreted. Bachman uses the following examples to illustrate his point: "Compare ... Bachman's Sociollngulstlc Competence 'I wish you wouldn't do that' with 'I would feel better by your not doing that,' or 'I have my doubts' with 'I have several doubts"' (1990, p. 97). On Communicative Competence 89 The final aspect of sociolinguistic competence is the ability to interpret cultural references and figures of speech. Knowledge of referential meaning specific to a culture allows the language user to accurately interpret discourse. For example, a referential expression such as, 'He met his Waterloo' can only be adequately understood if the language user knows what 'Waterloo' symbolizes in American culture. 89 Interpreting figures of speech involves more than knowledge of referential meaning. For example, interpretation of hyperbola such as. "'It's a jungle out there,· requires more than a knowledge of the signification of the words and grammatical structures involved ..." (Bachman, 1990, p. 98). Correct interpretation also involves knowledge of specific meanings and images that are evoked when using figurative speech that is deeply rooted in the culture of a given society or speech community (Bachman, 1990, p. 98). Bachman's Strategic Competence Finally, like Canale and Swain (1980) and Savignon (1983), Bachman recognizes that language is a dynamic process--that the components of language competence interact with one another. The ability to assess relevant information in context, and negotiate meaning on the part of the language user is called 'strategic competence.' According to Bachman, there are two approaches to defining communication strategies: "the 'interactional' definition (Tarone, 1980) and the 'psycholinguistic' definition (Faerch and Kasper, 1984). Tarone defines interactional communication strategies as the "mutual attempt by two interlocutors to agree on a meaning in situations where the requisite meaning structures do not seem to be shared" (1980, p. On Communicative Competence 90 419). On the other hand, Faerch and Kasper believe that this definition is too narrow in scope since it only applies to two interlocutors, and often language use involves only one individual in the case of reading and writing. Tarone does describe another type of strategy called 'production strategy' which she defines as "an attempt to use one's linguistic system efficiently and clearly, with a minimum of effort" (1980, p. 419). Production strategies are like communication strategies in that they are distinct from the language user's language competence, but unlike communicative competence, they lack the interactional focus on the negotiation of meaning. Faerch and Kasper consider Tarone's interactional definition of communicative strategies a subset of the strategies encompassed in the psycholinguistic definition of strategic competence (1984, p. 61 ). Their psycholinguistic definition of strategic competence provides a more comprehensive definition of strategic competence. Both Savignon and Bachman incorporate strategic competence within their framework and view it as a distinct ability separate from language competence. In addition, Bachman as well as Savignon accept the interactional definition of strategic competence in the broader sense as defined by Faerch and Kasper. Similarly, Canale and Swain include strategic competence as a separate component of CC. They describe strategic competence as being . made up of verbal and nonverbal communicative strategies that may be called into action to compensate for breakdowns in communication due to performance variables or to insufficient competence. Such strategies will be of two types: those that relate primarily to grammatical competence (e.g. how to paraphrase grammatical forms that one has not mastered or cannot recall momentarily) and those that relate more to sociolinguistic competence (e.g. Bachman's Strategic Competence various role­ playing strategies, how to address strangers when unsure of their Similarly, Canale and Swain include strategic competence as a separate component of CC. They describe strategic competence as being . made up of verbal and nonverbal communicative strategies that may be called into action to compensate for breakdowns in communication due to performance variables or to insufficient competence. Such strategies will be of two types: those that relate primarily to grammatical competence (e.g. how to paraphrase grammatical forms that one has not mastered or cannot recall momentarily) and those that relate more to sociolinguistic competence (e.g. various role­ playing strategies, how to address strangers when unsure of their 91 On Communicative Competence On Communicative Competence 91 social status). (1980, p. 30-31). social status). (1980, p. 30-31). social status). (1980, p. 30-31). Later Canale extended his definition of strategic competence to include enhancement characteristics of production strategies--mastery of verbal and nonverbal strategies to enhance the rhetorical effect of utterances (1983, p. 339). (1983, p. 339). Figure 7. A Model of Language Use ·---·--- -~ GOAL 1· Interpret or express speech I with specific 1 !unction. modality. , and co_nt_en_t___J -------.i _____.,,_ ·•7 ' SITUATIONAL:~...; PLANNING PROCESS ~-- LANGUAGE ASSESSMENTJ '. Retrieve items , ICOMPETENCE - ; from language t 1 competence Organizational competence --- . ---' - ... --· ·- . ---·1 Pragmatic PLAN I Competence Composed of items, , the realization of I which is expected l to lead to a I communicative goal , .... ________ .,, ____J '· l EXECUTION PSYCHOPHYSIOLOGICAL A neurological and MECHANISMS physiological process UTTERANCE Express or interpret language Note. From L F. Bachman {1990). Fundamental Considerations in Language Testing. Copyright 1990 by Oxford University Press. Figure 7. A Model of Language Use PSYCHOPHYSIOLOGICAL MECHANISMS UTTERANCE Express or interpret language Note. From L F. Bachman {1990). Fundamental Considerations in Language Testing. Copyright 1990 by Oxford University Press. On Communicative Competence 92 Faerch and Kasper's psycholinguistic model of speech production includes a planning phase and an execution phase. The planning phase is described as an interaction of three components--communicative goals, communicative resources available to the individual. and the assessment of the communicative situation. "The execution phase of Faerch and Kasper's model consists of 'neurological and physiological processes· that implement the plan, resulting in language use" (Bachman, 1990, p. 100). However. Bachman's Strategic Competence Faerch and Kasper's model is limited to the use of communication strategies in interlanguage communication where language abilities are deficient, and Bachman views strategic competence to be an important part of all communicative language use, not just compensatory language use. 92 Furthermore. Bachman claims that the above definitions of strategic competence are not adequate because they do not describe the mechanisms by which strategic competence operates. He extends Faerch and Kasper's formulation by adding a separate assessment component; whereas Faerch and Kasper include assessment within the planning phase. Bachman includes three components in his model of strategic competence: assessment, planning, and execution. Figure 7 (above) is a model of how these three components interact with other components of CLA to produce language utterances. Bachman defines the assessment component similar t F h d K ' l i h to Faerch and Kasper's planning phase: The assessment component enables us to (1) identify the information­ including the language variety, or dialect - that is needed for realizing a particular communicative goal in a given context; (2) determine what language competencies (native language, second or foreign language) are at our disposal for most effectively bringing that information to bear in achieving the communicative goal; (3) ascertain the abilities and knowledge that are shared by our interlocutor; and· ( 4) following the communication attempt, evaluate the extent to which the 93 As the model above illustrates, during the planning process of Bachman's model, the interlocutor retrieves relevant items from language compc:tence and formulates a plan to achieve the communicative goals either by using L 1, Li (interlanguage), or L2 (second or foreign language). Like Faerch and Kasper, Bachman defines the planning process as a dynamic interchange between context and discourse: The interpretation of discourse, in other words, requires the ability to utilize available language competencies to assess the context for relevant information and to then match this information to information in the discourse. (1990, p. 102). Finally, Bachman's execution component, similar to Faerch and Kasper's execution phase, draws on the psychophysiological mechanisms (neurological and physiological processes) to implement the plan in the style and register appropriate to the common goal, and context of situation. Summary Figure 7 (above) illustrates Bachman's perception of the interactional process of language use, similar to Canale and Swain and Savignon's models of CC. All of the above linguists include grammatical competence in the strict Chomskyan sense as part of their theoretical model of CC. In other words, Chomsky's term grammatical competence (innate knowledge of language and emergent knowledge of language) is included as one of the components of communicative competence. Although Chomsky defines competence as knowledge of the rules of grammar independent of use, these On Communicative Competence 94 rules enter into communication. According to Chomsky, the language faculty possesses specific properties, structures, and organization in its initial state; and UG, which characterizes the language f acuity, is accessible upon exposure to primary linguistic data. In addition, knowledge of the tacit rules of a particular language are also involved in the communicative process. As Chomsky states in Rules and Representations, it is the goal of the investigator to determine the nature of the competence system, and to show how this abstract system of rules of grammar is put to use (1980, p. 203). In Bachman's theory of language competence, he includes pragmatic competence, knowledge of language use that subserves performance or communication. There are neurolinguistic studies that support Bachman's theory, which I will discuss below. In the following chapter I present my own integrated model and explanation of communicative proficiency. It is similar to Savignon and Bachman's models of CC, in that it includes Chomsky's grammatical competence. However, I place more importance on the organizational properties of UG, a part of grammatical competence. In addition, as suggested by Chomsky (1980) UG includes knowledge of universal rules of language use--Grice's Conversational Maxims (1975), as well as universal rules of grammar. Thus, UG provides o~anizing principles and parameters for the development of language in context. The rules and constraints of UG and knowledge of grammar and pragmatics provide the basis for successful language acquisition. On Communicative Competence Chapter 5 On Communicative Competence Chapter 5 95 Deductive Model of Communicative Proficiency Based on the inferential evidence of innate knowledge of language provided by Chomsky, I propose a deductive model of communicative proficiency. Chomsky's theory of UG basically states that in order to create and understand novel sentences we rely on our internal system of rules and representations; language learning is a deductive process whereby we deduce from innate and tacit knowledge of grammar and phonology how to create and comprehend language. Furthermore, Chomsky states that "[c]reativity is predicated on a system of rules and forms, in part determined by intrinsic human capacities. Without such constraints. we have arbitrary and random behavior, not creative acts" (Chomsky, 1975, p. 133). Figure 8 (below) illustrates that our innate system of language knowledge prevents chaos, or blocks out chaos that would occur without the harmonizing system of the mind/brain. On the basis of suggestions by Grice (1975), Chomsky (1980), and Brown and Levinson (1987), I am extending the theory of UG to include universal rules of pragmatics--Grice's Conversational Maxims. These include truthfulness, relevance, brevity, and informativeness. Brown and Levinson (1987) suggest universal rules of politeness that deviate from Grice's Maxims depending on the context. However, Yu (1999) and Wierzbicka (1990) claim that Brown and Levinson's theory is flawed, because it is based on the theory that all cultures are individually oriented. Yu and Wierzbicka point out that Chinese, Japanese, and Polish cultures are not individually-oriented. Rather, they are community-oriented, and that changes how they view politeness (see pp. 13- On Communicative Competence 96 14). These differences could possibly be seen in the same light as Chomsky's Principles and Parameters Theory. Grice's Maxims would constitute the basic principles of pragmatic competence, but there would be parameters like the head parameter for grammar. These pragmatic parameters could only vary in one of a few ways depending on the culture. For example, in making a request in individually oriented cultures, one would use indirect speech. However, in a community oriented culture, one would use direct speech. Yu (1999) determined that when making requests in China, a community oriented culture, it is polite to use the direct form. Wierzbicka (1991) determined the same with respect to Polish culture (seep. 14). 96 The deductive model (below) illustrates that language learning is governed by both the environment and the innate properties of the mind/brain. Knowledge of language and UG intersect with primary linguistic data and produces or creates CP. Deductive Model of Communicative Proficiency Without UG, in its extended definition, providing the overall organization of language, we would have "arbitrary and random behavior, not creative acts" (Chomsky, 1975, p.133). In other words, the theory of UG provides a schema to which any particular language must conform, and leads to language acquisition through its interaction with the cultural and functional aspects of language as outlined by Hymes (1996) and Halliday (1977), respectively. UG or innate knowledge of language provides the foundation for language acquisition, and allows language learning to proceed in an orderly and unchaotic manner. UG affects all the components of communicative proficiency by providing the underlying rules for organized development of language in context. On Communicative Competence 97 Like Savignon and Bachman, I maintain a distinction between competence and performance. Recent studies in neurolinguistics show Figure 8. Deductive Model of Communicative Proficiency. Ethnography of Speaking Strategic Competence Sociolinguistic Competence Discourse Competence Pragmatic Competence Grammatical Competence On Communicative Competence 97 Like Savignon and Bachman, I maintain a distinction between competence and performance. Recent studies in neurolinguistics show Figure 8. Deductive Model of Communicative Proficiency. 97 Ethnography of Speaking Strategic Competence Sociolinguistic Competence Discourse Competence Pragmatic Competence Grammatical Competence Note: Mary Lou Emerson (1999). Model of Communicative Proficiency. Copyright 1999 by Mary Lou Emerson. Note: Mary Lou Emerson (1999). Model of Communicative Proficiency. Copyright 1999 by Mary Lou Emerson. Note: Mary Lou Emerson (1999). Model of Communicative Proficiency. Copyright 1999 by Mary Lou Emerson. On Communicative Competence 98 evidence that "(n]ot only is (context-independent) sentence grammar theoretically separable from other aspects of sentence interpretation in normal language use, but it has in fact been shown to be clearly separated neurofunctionally" (Paradis, 1998, p. 4). Neurofunctionally focal left hemisphere (LH) damage causes context-independent sentence grammar deficits, and right hemisphere (RH) damage causes deficits in context­ dependent interpretations, non-literal interpretations and affect-related aspects of language processing (Paradis, 1998, p. 4). For example, pragmatics of reference is preserved in patients with Broca's and Wernicke's aphasia, despite syndrome specific problems in retrieving content words such as nouns, verbs, and adjectives, or closed-class grammatical elements such as the, and, and from (Paradis, 1998, p. 7). 98 Bloom and Obler (1998) reported that patients with right-brain-damage produced ambiguous and poorly structured discourse that contained less information, fewer concepts, and more irrelevant remarks than normal control subjects. Chantraine, Joanette and Ska (1998) found that in general RHO patients in conversation displayed deficits such as irrelevant comments, digressions, and inferential problems. On the other hand, the subjects' grammar remained intact. Dronkers, Ludy, and Redfern (1998) observed that aphasic patients who were not able to utter a word demonstrated nearly normal pragmatic competence in communication, exhibiting skills such as maintaining normal proxemics, turn-taking, paralinguistics such as gesturing, facial expressions, and eye gaze. From the results of their study with aphasics with left hemisphere damage, they concluded that pragmatics functions independent On Communicative Competence 99 of grammatical competence; although Paradis (1998), Chomsky (1980) and this paper argues that pragmatic competence is not completely independent of grammatical competence. 99 Affecting Pragmatic Competence Paradis (1998) asserts that neither the right side nor the left side of the brain is sufficient for the processing of language--both are necessary. Recent studies reveal that deficits in the left hemisphere (LH) affect pragmatics. For example, Avent, Wertz and Auther (1998) observed that individuals with aphasia generally display more intact pragmatic skills than they do in the more formal skills such as syntax. Nonetheless, pragmatic deficits are still present in individuals with aphasia caused by left hemisphere damage. Prutting and Kirchner (1987) observed that aphasic subjects showed a mean of 82 % appropriate pragmatic performance. Avent et. al. concluded the same as Prutting and Kirchner--that aphasic people communicated better than they talked (1998, p. 217). In general, their sample of aphasic people displayed better pragmatic performance in conversation than language ability on a standardized test for aphasics. As with Prutting and Kirchner, Avent et. al. noted deficits in pragmatic competence. Pragmatic behaviors that were most impaired were specificity/accuracy, cohesion, and intelligibility. Avent et. al. associated these deficient aspects of pragmatic behavior with semantic, syntactic, and phonological measures of linguistic competence measured by the standardized test, the PICA. Their results indicate pragmatic performance during conversation and performance on a standardized aphasia test are significantly related in a sample of aphasic people (left hemisphere damaged patients). · On Communicative Competence 100 Thus, performance on one measure predicts performance on another. However, the strength of the relationship correlation range from +.57 to+.68 is not overwhelming. (Avent et al., 1998, p. 219) The above studies demonstrate what Chomsky (1986) and Bachman (1990) theorize, that knowledge of grammar affects pragmatic performance. This exemplifies how knowledge of language or in this case how lack of knowledge of cohesion is put to use--deficits occur in pragmatic behavior. On Communicative Competence 100 Thus, performance on one measure predicts performance on another. However, the strength of the relationship correlation range from +.57 to+.68 is not overwhelming. (Avent et al., 1998, p. 219) The above studies demonstrate what Chomsky (1986) and Bachman This exemplifies how knowledge of language or in this case how lack of knowledge of cohesion is put to use--deficits occur in pragmatic behavior. Discourse studies address the pragmatic aspects of communication, as well as linguistic aspects, because they are conducted in a social setting where linguistics, paralinguistics, and nonverbal skills are observable. Affecting Pragmatic Competence Discourse studies address the pragmatic aspects of communication, as well as linguistic aspects, because they are conducted in a social setting where linguistics, paralinguistics, and nonverbal skills are observable. Utalowska, North, and Macaluso-Haynes studied a group of 1 Oaphasics with left hemisphere (LH) damage and 1Onormals in their production of narrative and procedural discourse--telling stories, producing summaries, giving morals to the stories and producing procedures such as brushing teeth. In general the results of the study showed that mildly aphasic individuals produced well-structured narratives and procedural discourse; however, language of the aphasics was reduced in complexity and amount. Aphasics have difficulty in producing summaries and giving morals for the stories. Content and clarity of discourse were rated lower than those produced by the normals. More specifically, Content and clarity of discourse were rated lower than those produced by the normals. More specifically, Sentence level analysis of the summaries showed that aphasics produced much simpler language than the normals in terms of the amount of embedding and percentage of dependent clauses, and nonfinite clauses. The aphasics used more pronouns as compared to nouns than normals. They also used more deictic expressions such as up there , and generalized verbs such as get . (Utalowska, et. al., 1981, p. 358) In narratives, it was the amount of evaluation or elaboration which was primarily reduced in aphasics. Evaluation (explaining the point of the In narratives, it was the amount of evaluation or elaboration which was i il d d i h i E l ti ( l i i th i t f th In narratives, it was the amount of evaluation or elaboration whic primarily reduced in aphasics. Evaluation (explaining the point of the On Communicative Competence 101 narrative) involves use of some complex syntactic devices such as comparatives, negatives, and modals. The authors concluded that this reduction of evaluation in narratives was related either to its function (less important information) or its form (more complex language), or a combination of both (Utalowska, et al., 1981, p. 366). 101 With respect to summaries, the aphasics' inability to produce morals was not related to a lack of linguistic resources, but rather was associated with a well documented abstract attitude in aphasics. As stated above, content and clarity were rated lower for aphasics than for normals. Affecting Pragmatic Competence The authors posited that the quality of content might correspond to the notion of coherence; whereas, the clarity of language might depend on utilization of devices producing linguistic cohesion (Utalowska, et. al., 1981, p. 367). Although Savignon (1983), Canale and Swain (1980), and Halliday and Hasan (1976) consider cohesion and coherence a part of discourse competence which falls under the heading of pragmatics, Utalowska et al. are classifying them as part of linguistic competence. In a study in 1983 Bachman and Palmer defined pragmatic competence as vocabulary, cohesion, and organization; however, the results of the study revealed that these elements were closely related to grammatical competence (morphology and syntax) (see pp. 79-80). Hence, Bachman (1990) created the term organizational competence to include grammatical competence and textual competence. Pragmatic competence was then redefined to include illocutionary competence and sociolinguistic competence. Note that Paradis (1998) also found that patients with Broca's and Wernicke's aphasia had problems retrieving closed-class words such as the, and, and from, On Communicative Competence 102 which include cohesive devices (seep. 98). Thus, in so far as cohesion is defined as grammatical links, they fall under the heading of grammatical competence, or according to Bachman, organizational competence. Although Bachman and Palmer (1983) did not find a separation of the components of grammar and those of discourse in their study, they believe there is strong theoretical justification for making a distinction between organization in terms of grammar, vocabulary, and cohesion, and the organizational strategies of coherence (p. 462). Thus, the above studies show evidence that grammatical competence does provide a framework for the production of comprehensible language, and that grammatical competence plays a significant role in pragmatic competence--how successfully language is used. The above studies also suggest that pragmatic competence is like grammatical competence. In the same way that the left hemisphere is dominant for grammar, the right hemisphere forms the foundation for pragmatics. The following is a summary of some of the studies that advance the theory that the right hemisphere specializes in extralinguistic aspects of communication, like the left hemisphere specializes in linguistic aspects of language. Pragmatics Is Dominated by the Right Hemisphere Extralinguistic aspects of communication which come under the label of pragmatics include features of speech, such as intonation, pitch, and voice quality. These features are referred to as prosody, and disordered prosody as dysprosody or aprosody (Code, 1987, p. 88). Variations in prosody--pitch intonation, rhythm and word stress communicate affective information such On Communicative Competence 103 as mood. Prosodic cues also differentiate grammatical and semantic contrasts such as whether a sentence is a question or a statement, or what a sentence means depending on the placement of stress. For example, the sentence, 'She is hot,' has two different meanings depending on whether or not 'hot' is stressed or 'She' is stressed. 103 Boss (1996) claims the right hemisphere mediates prosody, attitude, emotion, and gestures. ''These right hemisphere aspects of communication are collectively known as pragmatics" (Boss, 1996, p. 81). Recent research examines the special role of the right hemisphere in processing visuospatial, musical, and emotional aspects of communication. Based on their studies on right hemisphere damaged patients, Wapner, Hamby, and Gardner (1981) propose a theory that the right hemisphere is responsible for processing context-dependent linguistic entities; whereas, the left hemisphere subserves context-independent componential aspects of linguistics. The following discussion supports their theory. Kertesz (1983) reports evidence that visuospatial deficits such as poor map reading, neglect of the left side of a design or a word, and spatial disorientation are caused by right hemisphere post-Rolandic lesions, and in some cases more anterior portions of the right hemisphere are substantially involved. Code (1987) reports evidence that skilled musicians depend on left hemisphere processing, and untrained musicians rely on right hemisphere processing. In a dichotic study, Bever and Chiarello (1974) found a right ear advantage in trained musicians and a left ear advantage in non-musicians in a melody recognition task. They concluded that the trained musicians were On Communicative Competence 104 concerned with the separate componential elements of the melodies--the grammar of music; whereas the non-musicians used a more holistic approach, not being trained in reading, writing, and transcribing music. Related to music are studies on prosody (pitch, intonation, and rhythm) and the right hemisphere. Like music, speech sounds possess acoustical properties. "All speech sounds are composed of complex sound waves; that is, they contain many different frequencies simultaneously, like a musical chord that contains many notes" (Yeni-Komshian, 1998, p. 118). Pragmatics Is Dominated by the Right Hemisphere "It is now generally accepted that the right hemisphere is involved in the processing of prosody" (Code, 1987, p. 94). Results of several studies on prosody are similar to the studies in music. Van Lancker and Fromkin (1973) observed a right ear advantage where tone is used phonologically to distinguish and contrast individual words in tone languages such as Chinese and Thai. Zurif (197 4) reported similar findings--that the right ear is superior at linguistics; however, the left ear is better at comprehending non-linguistic intonational speech. Zurif (1974) states the following: [W]hen the subjects' task was a linguistic one, that is, when they had to make use of fundamental frequencies to distinguish voiced from voiceless syllables, they generated a significant right ear advantage. In contrast, when the subjects had to identify the emotional tones of utterances, and presumably, had to process long-term variations in fundamental frequency to do so, they produced a significant left ear superiority... Thus, when the acoustic parameters of intonational contour or emotional tone must be processed or matched independently of their linguistic medium, they become tied to the right hemisphere. In contrast, when these same parameters are used in the service of linguistic decisions, they are focused upon and utilized by the language mechanisms of the left hemisphere. (p. 395) Zurif theorizes that intonation by itself is probably processed by acoustic analyzers in the right hemisphere, and thereby enhances the left On Communicative Competence 105 hemisphere's ability to carry out linguistic decisions; thus, language is a collaborative process between the mind and the environment as well as within the mind. Certainly the environment interacts with the mind to produce language; however, pragmatic competence may be like grammatical competence in that there are specific modules that determine how we interact with the environment--a language faculty for pragmatics. 105 Based on the studies on right hemisphere damage and prosody, Ross ( 1981) developed a theory of right hemisphere specialization for prosody which mirrors the classical left hemisphere model for language. He suggests that prosody is functionally and structurally represented in the right hemisphere, in the same way as formal linguistic aspects of language are represented in the left hemisphere. Pragmatic Competence Mirrors Grammatical Competence Pragmatic Competence Mirrors Grammatical Competence Ross (1981) defined different kinds of aprosodias (a general lack of prosody), and associated them with specific right hemisphere damage. He further theorized the these aprosodias are analogous to the aphasias resulting from damage to homogeneous areas of the left hemisphere. Because the eight known categories of aphasia--motor, sensory, conduction, global, transcortical motor, transcortical sensory, anomic, and mixed transcortical--can be classified by observations concerning (1) spontaneous speech (fluency versus nonfluency); (2) repetition ability; (3) comprehension of spoken language; and (4) visual language skills (naming and reading), patients with focal right hemisphere damage might be examined in a similar manner by evaluating (1) spontaneous prosody and emotional gesturing; (2) the ability to repeat sentences with prosodic-affective variation; (3) the ability to comprehend the prosodic-affective components of language; and (4) the ability to comprehend emotional gesturing. (Ross, 1983, p. 498) On Communicative Competence 106 For example, Ross (1981) discovered that injury to the right frontal and anterior-inferior parietal lobe produced motor aprosodia--a flat monotone speech and loss of spontaneous gesturing. This is analogous to lesions in the left hemisphere known to cause Broca (motor) aphasia. Sensory aprosodia, characterized by an impaired ability to understand the emotion and/or mood being conveyed auditorily or visually by gestures and facial expressions, was associated with damage to the right posterior-superior temporal and inferior-posterior parietal lobes. "This distribution is consistent with left hemisphere lesions known to cause Wernicke (sensory) aphasia" (Ross, 1983, p. 502). Ross (1983) admits that the number of cases are limited; however, he did observe six different aprosodias--motor, sensory, global, transcortical motor, transcortical sensory, and mixed transcortical. The lesions in the right hemisphere associated with these syndromes correspond functionally and anatomically to lesions in the left hemisphere associated with aphasias of propositional language. Visuospatial, musical, prosodic and emotional functions are closely tied together, and the right hemisphere has been shown to underlie the processes of these affective components of language. Ross theorizes a language faculty for pragmatics similar to Chomsky's language faculty for grammar. Ross' (1981) finding that sensory aprosodia is caused by damage to the right hemisphere lends support to other studies that propose the right hemisphere is superior in processing emotion. Schwartz, Davidson, and Maer (1975) recorded the lateral eye movements (LEM) of normal subjects while they were answering emotionally On Communicative Competence 10 charged and neutral questions. Pragmatic Competence Mirrors Grammatical Competence Significantly more leftward LEMs (right hemisphere) were observed while answering emotional than neutral questions. 107 Borod, Koff, and Caron (1983) examined normals in facial asymmetry during eight posed emotional expressions. They selected four that were communicative--greeting (mild smiling), clowning (silly--to amuse a child), flirtation ("come hither"), and disapproval (scolding), and four others that were more reactive--confusion (perplexed), disgust (apprehending a "bad smell"), horror (terrified), grief (crying). The results of their study revealed that the left side of the face was more involved during posed emotional expressions. Borod et al. (1983) concluded that facial expression is primarily related to emotion, and is mediated by the right hemisphere. The significance of these results is that facial expression, an affective part of language, facilitates linguistic communication. Results of Wapner, Hamby and Gardner's (1981) study with 16 right hemisphere brain-damaged patients revealed that right hemisphere patients had no problem using appropriate phonology and syntax when retelling a story; however, they were unable to use their own words; they could not infer a moral for a story; and their mode of delivery was flat. Furthermore, the right hemisphere patients embellished stories three times as often as left hemisphere patients and normals (Wapner et al., 1981, p. 23) Wapner found that those patients with large anterior lesions of the right hemisphere most frequently embellished the stories. In general, right hemisphere patients had difficulty separating out the actual plot from their own invention. All groups were successful in ordering temporal elements of a story. Wapner et al. On Communicative Competence 108 determined that right hemisphere patients suffered from deficits in the processing of ideational and conceptual factors rather than in terms of linguistic ones. They concluded: "While the left hemisphere might appreciate some of Groucho's puns, the right hemisphere might be entertained by the antics of Harpo, only the two hemispheres can appreciate a whole Marx Brother's routine" (Wapner et al., 1981, p. 32). 108 Gardner, Brownell, Wapner, and Michelow (1983) conducted a similar study and corroborated the results of Wapner et al.'s study. Overall, the right hemisphere patients exhibited great difficulty in handling complex ideational materials--paraphrasing and interpreting stories, appreciating humor, inferring the moral of a story, assessing the appropriateness of various facts and situations, and comprehending emotional forms of information. Gardner et al. Pragmatic Competence Mirrors Grammatical Competence conclude the following: To be sure, when memory is sufficiently acute, or cues sufficiently abundant, subjects may well mention all the major points. But their inability to negotiate noncanonical elements, their frequent confabulations and injections of personal details, all suggest that the basic scaffolding of the story has not been apprehended. Without an organizing principle, the patients are consigned to undirected rambling, unable to judge which details matter, and what overarching points they yield. (1983, p.187) Although Gardner et al. (1983) depict the left hemisphere as a context­ free machine, and the right hemisphere as context-dependent machine, they agree with other neuropsychologists that "no functions are housed solely in a particular region of the brain, that all important human capacities have wide cortical representation" (1983, p. 188-189). This is a position that is suggested by Savignon (1983), and theorized by Bachman (1990)--that language is a dynamic process not only between the mind/brain and the 109 On Communicative Competence environment, but also between the hemispheres of the brain. Bara, Tirassa, and Zettin (1997), who investigate the possibility of underlying knowledge for pragmatics, assert that Chomsky's distinction between competence and performance is a fundamental of cognitive science. "Ideally, a cognitive theory should describe both the competence and the performance of the system under analysis" (Bara et al., 1997, p. 9). Bara et al. (1997) maintain that the study of performance impairments after brain damage helps to constrain the underlying competence theory. Given the above neuropsychological evidence for certain pragmatic skills, this paper argues that pragmatics is subserved by domain-specific knowledge structures. Therefore, pragmatic competence is like grammatical competence in that it is also underlain by competence--knowledge of language use. Language Is an Interactive Process Between Knowledge Structures and the Environment Of course, language learning cannot be purely innate because we don't all speak the same language; we speak the language that we hear. Linguistic data or input is required for language acquisition, and input determines output to a certain extent. However, as Chomsky and neurolinguists have shown, innate properties of the mind/brain provide the principles and parameters of all languages, and make it possible for a five year old, who has not yet developed formal operations, to master his/her native language. The top diamond of the above model (see Figure 8) includes UG in its On Communicative Competence 11 O extended definition, and knowledge of language--grammatical knowledge of syntax and phonology, and pragmatic knowledge of prosody, emotion and gestures. Ross (1981, 1983) theorizes that the right hemisphere is functionally and anatomically like the left hemisphere in that there are localized areas of the brain that govern pragmatics. Thus, the top diamond represents our biological endowment--UG (universal rules of grammar and pragmatics--Grice's Maxims), as well as knowledge of grammar (syntax and phonology), and knowledge of pragmatics (prosody, emotion and visuospatial elements of language--gestures). 11 O The brain possesses innate properties that subserve linguistics and extralinguistics or pragmatics. Without a healthy brain, deficits in communication result, depending on the location of the injury. Comprehension and production of emotional messages through facial expressions and prosody is dependent upon an intact right hemisphere. On the other hand, comprehension and production of grammatical speech is dependent on an intact left hemisphere. However, in order to achieve communicative proficiency, both hemispheres must be intact. Language is an interactive process between the organizational properties of the left and right hemispheres. Achieving communicative competence is not simply a matter of the mind/orain interacting with the environment as explained by Hymes (1972) and Halliday (1977). Language acquisition is a product of two healthy hemispheres working together, as well the mind/brain interacting with the environment. In addition to innate knowledge of language and UG, mind/orain is included in the top diamond, because there are other innate learning On Communicative Competence 111 mechanisms besides the language faculty that interact with the environment to produce CP. These are distinct from Chomsky's UG, and would include what Bachman (1990) refers to as the psychophysiological mechanisms of the mind/brain. Language Is an Interactive Process Between Knowledge Structures and the Environment In the case of the study of language Chomsky states the following: Primary linguistic data (the environment) include Hymes' (1996) ethnographic components of language, and Halliday's (1977) functional aspects of language. While Hymes and Halliday's theories have their merits, the cultural and functional aspects of language explain only part of the process of language acquisition. The innate properties of mind, which include UG in its extended definition, and knowledge of language must interact with the environment to produce CP which is comprised of five main components--grammatical competence, pragmatic competence, sociolinguistic competence, discourse competence, and strategic competence. Grammatical competence and pragmatic competence form the base of the pyramid because they are the foundation of communicative competence. They provide the organizing principles that allow language acquisition to take place in context. UG, which indudes Chomsky's universal rules of grammar and Grice's Conversational Maxims, provides the underlying principles and parameters for the orderly processing of primary linguistic data and production of language in context. Unlike Bachman, who On Communicative Competence 112 includes Grice's Maxims under textual competence, I consider these maxims universal rules of pragmatics. In addition, cohesion (grammatical links) would be subsumed under grammatical competence; and rhetorical organization and strategies of coherence would come under the heading of discourse competence. Concluslon In the way that Hymes (1971) defines CC--tacit knowledge of language and ability for use, he does not address competence in the same sense as Chomsky (1980) defines it--underlying knowledge of language severed from ability. While Chomsky maintains the distinction between competence and performance, knowledge of language enters into performance. In fact, it allows language acquisition and language use to take place successfully. How does this reflect on the term CC? Do we possess underlying knowledge of communication that allows communication to successfully take place? Based on the neurolinguistic studies presented above, I believe there is evidence that we possess CC in the Chomskyan sense. My conclusion that there are universal rules of pragmatics is not based on any evidence. Rather it is based on theories of Chomsky (1980)1 Grice (1975), and Brown and Levinson (1987). On the other hand, neuropsychological studies reveal evidence that we possess knowledge of prosody--intonation, pitch, and rhythm (Code, 1987). Included in pragmatic competence would be knowledge of the suprasegmentals--pitch, tone, stress and rhythm. Based on Ross' (1981) On Communicative Competence 113 studies we possess specific modules of the mind that allow us to produce and comprehend prosody of language. How these elements of pragmatics overlap with grammar to facilitate communication constitutes pragmatic competence. Communicative behaviors such as hugging, kissing, and crying would not be included in the theory of pragmatic competence. Only those aspects of language use that directly overlap with grammar would be included in pragmatic competence. Therefore, CC is knowledge of prosody, the musical components of language that express emotion in conjunction with the linguistic aspects of language. Thus, pragmatic competence and grammatical competence work together to produce CP--all that is involved in communication. 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https://openalex.org/W3165600736
https://link.springer.com/content/pdf/10.1007/s12024-021-00379-9.pdf
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The handling of SARS-CoV-2 associated deaths - infectivity of the body
Forensic science, medicine and pathology
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Abstract The body of a deceased with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is considered infectious. In this study, we present the results of infectivity testing of the body and testing of mortuary staff for SARS- CoV-2. We performed real-time quantitative polymerase chain reaction (RT-qPCR) for SARS-CoV-2 on 33 decedents with ante mortem confirmed SARS-CoV-2 infection. Swabs of the body surface from five different body regions and from the body bag or coffin were examined. A subset of the swabs was brought into cell culture. In addition, screening of 25 Institute of Legal Medicine (ILM) personnel for ongoing or past SARS-CoV-2 infection was performed at two different time points during the pandemic. Swabs from all locations of the body surface and the body environment were negative in cases of negative post mortem nasopharyngeal testing (n=9). When the post mortem nasopharyngeal swab tested positive (n=24), between 0 and 5 of the body surface swabs were also positive, primarily the perioral region. In six of the cases, the body bag also yielded a positive result. The longest postmortem interval with positive SARS-CoV-2 RT-qPCR at the body surface was nine days. In no case viable SARS-CoV-2 was found on the skin of the bodies or the body bags. One employee (autopsy technician) had possible occupational infection with SARS-CoV-2; all other employees were tested negative for SARS-CoV-2 RNA or antibody twice. Our data indicate that with adequate management of general safety precautions, transmission of SARS-CoV-2 through autopsies and handling of bodies is unlikely. Keywords  Coronavirus · SARS-CoV-2 · COVID-19 · Autopsy · Infectivity · External Examination Ann Sophie Schröder1 · Carolin Edler1 · Benjamin Ondruschka1 · Klaus Püschel1 · Julia Schädler1 · Axel Heinemann1 · Fabian Heinrich1 · Marc Lütgehetmann2 · Susanne Pfefferle2 · Martin Aepfelbacher2 · Antonia Fitzek1 · Jan‑Peter Sperhake1 Ann Sophie Schröder1 · Carolin Edler1 · Benjamin Ondruschka1 · Klaus Püschel1 · Julia Schädler1 · Axel Heinemann1 · Fabian Heinrich1 · Marc Lütgehetmann2 · Susanne Pfefferle2 · Martin Aepfelbacher2 · Antonia Fitzek1 · Jan‑Peter Sperhake1 Accepted: 7 April 2021 © The Author(s) 2021 / Published online: 2 June 2021 Accepted: 7 April 2021 © The Author(s) 2021 / Published online: 2 June 2021 2 Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20251 Hamburg, Germany 1 Institute of Legal Medicine, University Medical Center Hamburg-Eppendorf, Butenfeld 34, 22529 Hamburg, Germany * Ann Sophie Schröder as.schroeder@uke.de ORIGINAL ARTICLE ORIGINAL ARTICLE https://doi.org/10.1007/s12024-021-00379-9 Forensic Science, Medicine and Pathology (2021) 17:411–418 https://doi.org/10.1007/s12024-021-00379-9 Forensic Science, Medicine and Pathology (2021) 17:411–418 Introduction infection-causing) virus can also be found in cadavers [1–6]. The predominant routes of transmission of SARS-CoV-2 via droplet infection and aerosols are unlikely to be important in non-invasive handling of a deceased person (e.g. external necropsy, coffining). Consideration should be given to smear and contact infections from the body fluids that may be pre- sent in the mouth, on the skin, and surrounding of the body. The body of a deceased person with ante mortem confirmed infection with severe acute respiratory syndrome coronavi- rus 2 (SARS-CoV-2) is considered contagious. It has been shown that PCR-based postmortem detection of SARS- CoV-2 RNA is possible and that viable (and thus potentially However, autopsies and ablutions of SARS-CoV-2 decedents may produce infectious droplets or aerosols. To date, we are not aware of any safely confirmed occu- pational SARS-CoV-2 infections of autopsy teams during autopsies of SARS-CoV-2 positive decedents [7–10]. Since the beginning of the pandemic, the potential infectivity of the body has led to widespread adoption of a restrictive approach to deaths associated with coronavirus disease  2019 (COVID-19), avoiding any contact with the body. In the interim, there have been several recommendations. Dijkhuizen et al. provide an overview of various recommendations in the management of SARS-CoV-2 associated deaths [11]. Ann Sophie Schröder and Carolin Edler share first authorship Antonia Fitzek and Jan-Peter Sperhake share last authorship * Ann Sophie Schröder as.schroeder@uke.de 1 Institute of Legal Medicine, University Medical Center Hamburg-Eppendorf, Butenfeld 34, 22529 Hamburg, Germany 2 Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20251 Hamburg, Germany Ann Sophie Schröder and Carolin Edler share first authorship Antonia Fitzek and Jan-Peter Sperhake share last authorship Ann Sophie Schröder and Carolin Edler share first authorship Antonia Fitzek and Jan-Peter Sperhake share last authorship * Ann Sophie Schröder as.schroeder@uke.de (0121 3456789) 3 412 Forensic Science, Medicine and Pathology (2021) 17:411–418 However, there are no legally binding rules for the manage- ment of SARS-CoV-2 associated deaths. In this study, we present the results of our COVID-19 cadaveric infection investigations and a staff test for SARS- CoV-2. The aim was to investigate the relevance and effec- tiveness of infection control measures in the management of SARS-CoV-2-associated deaths in Hamburg, Germany. Introduction Despite initial recommendations released in the spring of 2020 to avoid autopsies due to the risk of infection, numer- ous autopsies have been performed at the Institute of Legal Medicine in Hamburg (ILM) of the University Medical Center Hamburg-Eppendorf (UKE) since the first COVID- 19 deaths occurred in Hamburg. Many confirmed SARS- CoV-2 infected decedents, as well as suspected cases, have been investigated [12–14]. Handling of known COVID-19 deaths was performed with extended personal protective equipment, including category 2 or 3 filtering face pieces (FFP2, FFP3), disposable gloves, and protective gowns. For autopsies, additional eye protection, head covering, and pro- tective footwear were worn by all personnel. Autopsies were performed in a separate autopsy room equipped with a table exhaust device, low-pressure ventilation, and an air exchange circulation system with a minimum of 10 air changes per hour. The cranial cavity was opened using an oscillating saw with suction, or a hand saw. In deceased who tested positive for SARS-CoV-2 incidentally, often no masks, or only a simple mouthguard (as opposed to FFP2- or FFP3- Masks) was worn during the external examination. Fig. 1   Location of the swaps taken from the skin of the body Results The results of body and body environment testing for SARS- CoV-2 are shown in Table 1. The mean age of the cohort of 33 decedents was 79 years (range 55–99 years). Seventeen patients died during hospitalization, 16 in the outpatient setting. On average, swabs were collected 3.5 days post- mortem (range 0–17 days). Overall, 24 deceased (72.7%) tested RT-qPCR positive for SARS-CoV-2 in the postmor- tem nasopharyngeal swab (highlighted in orange in Table 1), indicating SARS-CoV-2 infection at the time of death, and nine tested negative (highlighted in blue in Table 1). Con- sistently, in all cases with RT-qPCR-negative nasopharyn- geal swabs, all other swabs from the body surface and cadav- eric environment were also negative. In cases with positive postmortem nasopharyngeal swab, most frequently perio- ral swabs were confirmed positive (21/24; 87.5%). In six of these cases, the body bag tested positive (at least > 1000 copies/mL). SARS-CoV-2 RNA loads in swabs from the immediate cadaveric environment were found to correlate significantly with nasopharyngeal viral loads, as shown by simple linear regression (region 2 - perioral: p < 0.0001, region 3 - hands: p = 0.0005, region 4 - wrist: p = 0.0005, region 5- shoulder/hip: p = 0.002). However, the viral RNA loads detected on the body bags did not significantly cor- relate with the nasopharyngeal SARS-CoV-2 RNA loads of each body (site 1: p = 0.10). The longest PMI with a positive test result from the body surface was 9 days in two cases, while this PMI was examined in three individuals. One of the deceased also tested positive at regions 2 to 5, and the other case was weakly positive at the perioral region. For virus viability testing, 500 µl of each swab medium (modified Amies medium) was used for virus adsorption onto a well of a 24-well plate containing ~ 80% conflu- ent Vero cells (ATCC CCL-81) for 1 h. Cells were then washed once with phosphate-buffered saline (PBS) and fresh DMEM (Dulbecco’s Modified Eagle’s Medium con- taining 3% fetal calf serum, 1% penicillin–streptomycin, 1% L-glutamine [200 mM], 1% sodium pyruvate, and 1% non- essential amino acids [all from Gibco/Thermo Fisher Sci- entific, Waltham, MA, USA]) was added. After incubation at 37 °C for 72 h, virus growth was analyzed as previously described [19]. The following data were collected on our cohort: Age, place of death (outpatient vs. Methods Between October 27, 2020 and November 15, 2020, the swabs of regions 1 to 5 were collected twice from 15 of the deceased patients. RT-qPCR for SARS-CoV-2 RNA was performed in one swab and the second swab was stored at -80°C. When RT-qPCR results were positive, cell culture experiments of the second swab were set up in a biosafety level 3 (BSL-3) laboratory in 30 specimens out of 11 cases. Between October 27, 2020 and November 15, 2020, the swabs of regions 1 to 5 were collected twice from 15 of the deceased patients. RT-qPCR for SARS-CoV-2 RNA was performed in one swab and the second swab was stored at -80°C. When RT-qPCR results were positive, cell culture experiments of the second swab were set up in a biosafety level 3 (BSL-3) laboratory in 30 specimens out of 11 cases. For virus viability testing, 500 µl of each swab medium (modified Amies medium) was used for virus adsorption onto a well of a 24-well plate containing ~ 80% conflu- ent Vero cells (ATCC CCL-81) for 1 h. Cells were then washed once with phosphate-buffered saline (PBS) and fresh DMEM (Dulbecco’s Modified Eagle’s Medium con- taining 3% fetal calf serum, 1% penicillin–streptomycin, 1% L-glutamine [200 mM], 1% sodium pyruvate, and 1% non- essential amino acids [all from Gibco/Thermo Fisher Sci- entific, Waltham, MA, USA]) was added. After incubation at 37 °C for 72 h, virus growth was analyzed as previously described [19]. Methods Between June 2, 2020, and November 15, 2020, 33 dece- dents who were tested positive for SARS-CoV-2 ante mortem and who were clinically considered to have died of COVID-19 were examined post mortem at the ILM. A nasopharyngeal swab and swabs from the following regions were collected prior to autopsy or other invasive procedures or cleaning: 1 - body bag or coffin, 2 - perioral, 3 - hands (dorsal and palmar), 4 - wrists, and 5 - shoulder and hip regions (com- bined) (Fig. 1). If the deceased was wrapped in two body bags, the outer body bag was examined. If there was no body bag but a coffin, the latter was examined externally. Gloves were changed after each swabbing. To avoid further contamination, all contact with the body and surfaces was avoided. For the external surfaces (region 1 to 5), the 1 3 Fig. 1   Location of the swaps taken from the skin of the body 1 3 Forensic Science, Medicine and Pathology (2021) 17:411–418 413 swab (COPAN eSwab™, Brescia, Italy) was moistened in the liquid transport medium of the swab tube before col- lection. Swabs were collected with circular movements and gentle pressure for 15 s. The swabs were examined as part of routine diagnostic procedures at the Institute of Medical Microbiology, Virology and Hygiene (IMV) of the UKE. Detection and quantification of viral ribonucleic acid (RNA) was performed by quantitative real-time polymerase chain reaction (RT-qPCR) as previously described [15–18]. 5%. Therefore, a second assay was performed targeting the viral spike protein (Anti-SARS-CoV-2 (S1/S2), Liaison XL, DiaSorin, Saluggia, Italy). A second round was per- formed six months later. These analyses were performed at the IMV of the UKE. The following data were col- lected from the employees: Sex, age, position, and symp- toms of general illness in the last seven days before the tests. The data were analyzed descriptively. 5%. Therefore, a second assay was performed targeting the viral spike protein (Anti-SARS-CoV-2 (S1/S2), Liaison XL, DiaSorin, Saluggia, Italy). A second round was per- formed six months later. These analyses were performed at the IMV of the UKE. The following data were col- lected from the employees: Sex, age, position, and symp- toms of general illness in the last seven days before the tests. The data were analyzed descriptively. Results inpatient), postmor- tem interval (PMI) between death and swab collection. SARS-CoV-2 RNA loads of the swabs were categorized as follows: Negative (-), low positive (+ : < 1000 copies/ml), positive (+ + : 1000–100,000 copies/ml), and high positive (+ + + > 100,000 copies/ml). Data were analyzed descrip- tively and statistical analysis was performed using Graph- Pad Prism software version 9.0.0 (GraphPad Software, CA, U.S.A.). After 3½ months of daily work with SARS-CoV- 2 positive decedents at ILM and several crematoria, including external examinations and 163 conventional autopsies, 25 ILM personnel were tested for active or past SARS-CoV-2 infection. RT-qPCR was performed from a throat swab in universal transport medium as pre- viously described [15]. SARS-CoV-2 antibodies (IgG/ IgM/IgA) directed against the viral nucleocapsid were tested using the Cobas e411 system (Roche Diagnostics, Manheim, Germany) according to the manufacturer’s rec- ommendations. To confirm positive serologic reactions, we used an orthogonal testing strategy as recommended by the Centers for Disease Control and Prevention (USA) for regions with SARS-CoV-2 prevalence below Viable virus could not be detected in any of the 30 specimens examined in cell culture with viral RNA loads of < 1000 up to > 100,000 (Table 1). The results of screening employees for active or past SARS-CoV-2 infection are shown in Table 2. After 3½ months of the ongoing pandemic, no SARS-CoV-2 infec- tion or SARS-CoV-2 antibodies were detected in any of the ILM employees. Results Six months later, RT-qPCR testing for SARS-CoV-2 infection was still negative in all 25 employees tested, but IgM antibodies to SARS-CoV-2 were detected in 1 3 Forensic Science, Medicine and Pathology (2021) 17:411–418 414 Table 1   SARS-CoV-2 presence and copy numbers on the body and the body environment Case Age [years] Place of death PMI [days] Naso- pharyngeal Body bag Perioral Hands Wrists Shoulder & hips 1 94 Hospital 4 - - - - - - 2 99 Hospital 9 ++ - + - - - 3 84 Outpatient 1 - - - - - - 4 62 Hospital 2 - - - - - - 5 78 Hospital 9 - - - - - - 6 55 Hospital 17 - - - - - - 7 62 Hospital 4 ++ - - - - - 8 93 Hospital 5 - - - - - - 9 89 Hospital 0 +++ - - - - - 10 68 Hospital 3 ++ - - - - - 11 76 Hospital 1 - - - - - - 12 81 Hospital 3 +++ +++ + + + ++ 13 85 Hospital 5 +++ - + + + + 14 84 Outpatient 3 +++ - ++ + + + 15 59 Outpatient 3 ++ - ++ - + - 16 87 Outpatient 0 - - - - - - 17 71 Hospital 6 +++ - ++ - - - 18 80 Outpatient 9 ++ - ++ ++ ++ ++ 19 67 Outpatient 1 +++ ++ +++* ++* ++* ++* 20 98 Outpatient 3 +++ - ++* ++* ++* ++ 21 79 Outpatient 5 +++ +++* ++* ++* ++* ++* 22 75 Outpatient 2 +++ ++* +++* + +* ++* 23 79 Outpatient 1 +++ -# ++* +* ++* - 24 76 Hospital 2 ++ - + - - - 25 94 Hospital 5 +++ - +++* + ++ ++* 26 83 Hospital 3 +++ -# ++ ++ + - 27 80 Outpatient 2 ++ ++ ++* ++ - ++* 28 74 Outpatient 1 ++ - ++* - - - 29 82 Outpatient 2 +++ - + - - - 30 92 Outpatient 3 - - - - - - 31 60 Hospital 0 +++ - ++* + - + 32 81 Outpatient 2 +++ +++ ++* - + ++ 33 86 Outpatient 1 +++ - ++* ++* ++* ++* SARS-CoV-2 RNA copies: +++: > 1000000 copies/ml, ++: 1000- 1000000 copies/ml, +: < 1000 copies/ml, -: negative #Coffin not Body bag *Specimen with inoculation of a cell culture for pathogen cultivation Table 1   SARS-CoV-2 presence and copy numbers on the body and the body environment 1 3 Forensic Science, Medicine and Pathology (2021) 17:411–418 415 Table 2   Testing of the employees for SARS-CoV-2 Case Position Age [years] Sex Disease symptoms* SARS-CoV-2 qPCR SARS-CoV-2 antibodies 1 Autopsy assistent 25 f - - - 2 Autopsy assistent 26 f - - - 3 Autopsy assistent 20 f y - - 4 Autopsy assistent 21 f y - - 5 Autopsy assistent 39 f - - y 6 Autopsy assistent 27 f y - - 7 Autopsy assistent 36 f - - - 8 Autopsy assistent 51 m - - - 9 Physician 40 f y - - 10 Physician 51 m y - - 11 Physician 34 f - - - 12 Physician 35 f - - - 13 Physician 28 f - - - 14 Physician 42 f - - - 15 Physician 29 f - - 16 Physician 33 m - - 17 Physician 34 f - - 18 Physician 36 f - - 19 Physician 33 m - - - 20 Physician 38 f y - - 21 Physician 68 m - - - 22 Physician 47 f - - 23 Physician 51 m - - - 24 Student 29 f - - - 25 Student 21 f y - - *Diseases symptoms: Any symptom of illness in the last 7 days before the first test. Results The following symptoms of were reported: headache, diarrhe Table 2   Testing of the employees for SARS-CoV-2 one employee (an autopsy technician). However, it remains unclear whether transmission occurred in a private or occu- pational context. surfaces are wood (coffin), plastic (body bag), textiles, and skin. In addition, there may be a particular risk of trans- mission through infectious body fluids. Under laboratory conditions, viable viruses have been detected in three stud- ies on wood between 24 h and 7 days after contamination, on plastic between 3 and 7 days, and on textiles or clothing between 24 h and 4 days [20–22]. The study by Liu et al. consistently showed longer survival times of the virus on all surfaces compared to the other two studies. One reason for this could be the higher amount of virus used for in vitro contamination. In practice, shorter viral survival times are more likely due to UV radiation, temperature fluctuations, as well as varying humidity and a lower amount of virus. In a study by Harbourt et al. the stability of SARS-CoV-2 was tested on the skin of pigs, and of clothing placed on the pigs [23]. The virus was found to be stable on skin at 4 °C for the duration of the experiment of 14 days and on clothing for at least 96 h. This is similar to the temperature of refrigerated Discussion SARS-CoV-2 infections are usually transmitted by drop- let infection and aerosols. However, transmission of the virus via surfaces is possible in principle. The stability of coronaviruses in the environment depends on temperature, humidity, surface properties, virus quantity, and the virus strain. Specifically for SARS-CoV-2, Chin et al. showed high stability at temperatures of 4 °C, with more rapid inactiva- tion observed at increasing temperatures (inactivation in the medium at 37 °C after two days, after five minutes at 70 °C); high stability was also shown at pH values between 3 and 10 and high humidity [20]. When handling bodies, the relevant 1 3 1 Forensic Science, Medicine and Pathology (2021) 17:411–418 416 religions, are not performed. Baj et al. provide recommenda- tions on precautions around burial [27]. ILM staff follow the guidelines of the Robert Koch Institute, Germany’s central scientific institution in the field of biomedicine, which recom- mend FFP2 masks, eye and face protection, body protection including clean, long-sleeved, liquid-tight or impervious pro- tective clothing, double-layered gloves, and appropriate work shoes [28]. However, in our daily routine, the recommended protective measures (e.g. FFP2) were not always fully applied while handling bodies that were incidentally known to be infected only after arrival at the ILM. bodies, indicating a possible risk of infection emanating from the human body. At higher temperatures, inactivation occurred more rapidly - on skin after 96 h at 22 °C and 8 h at 37 °C [23]. Although conditions may be different in vivo, viral stability on skin underscores the need for continuous hand hygiene to minimize the possibility of viral transmis- sion in daily routine. In our study, in all cases with a negative postmortem nasopharyngeal swab, all other swabs from the body and body bag were also negative. In deceased cases with a posi- tive postmortem nasopharyngeal swab, between 0–5 swabs from the body surface and cadaveric environment tested positive in RT-qPCR, most commonly in the perioral area. Nevertheless, we did not find viable virus on the skin of deceased COVID-19 patients or on surfaces, including body bags. Moreover, viable virus was not detected in any of the samples from bodies that had a short PMI (1 day) either. All bodies transported to the ILM were consecutively screened for SARS-CoV-2 upon arrival. ’Normal’ means of protection in the morgue are body protection and gloves. Discussion This way of handling a body applies to the usual daily work of morticians and crematory workers. In our institute, since the beginning of the pandemic, there has been a suspicion that the risk of infection of COVID-19 deceased may have been over- estimated. If protective measures are complied with, the risk of transmission should be minimal at best. Deceased persons with potentially aerogenously transmissible infectious diseases (e.g. tuberculosis) are autopsied daily by pathologists worldwide, taking safety precautions into account. Nevertheless, infec- tions may occur in the autopsy room due to aerosol-forming measures. Ultimately, however, apart from the autopsy itself, the handling of a deceased person’s body, e.g. during transport, careful washing, or burial, is likely to be relatively harmless ("a body does not cough"). Consistent with the distribution of RT-qPCR-positive test results in the nasopharyngeal and perioral areas, possible contamination via the nasopharyngeal-perioral route is one explanation for the SARS-CoV-2 detection on the other sur- faces. The absence of viable virus in cell culture suggests that no infectivity emanated from the body surface, although SARS-CoV-2 RNA was detected by RT-qPCR. Neverthe- less, infection via contaminated skin or other surfaces cannot be completely excluded. There were limitations to this study. Firstly, samples were only collected from five body regions. However, these regions are the most exposed regions when handling a deceased person in daily practice and all other body regions are usually touched less frequently while examining, mov- ing, or transporting a deceased. Secondly, the sam- ple size was rather small and the cell culture experiments were only performed on a subset of 11 cases or 30 samples, respectively. In international comparison the ILM in Hamburg has autopsied a relatively large number of COVID-19 cases [12]. The ILM staff all tested negative for SARS-CoV-2 after 3½ months, even after exposure to numerous COVID-19 dece- dents. After 9½ months, one autopsy assistant tested positive for IgM antibody. The mode of transmission remains unclear. However, this occurred at a time when the disease was gener- ally very prevalent in Germany. We believe that autopsies in COVID-19-related deaths are an indispensable tool for obtaining scientific data that can contribute critically to a deeper understanding of the disease [10, 24]. Nevertheless, there was considerable reluctance to perform autopsies, particularly at the beginning of the pandemic, which continues to some extent [25, 26]. References 1. Barton LM, Duval EJ, Stroberg E, Ghosh S, Mukhopadhyay S. COVID-19 autopsies, Oklahoma, USA. Am J Clin Pathol. 2020;153:725–33.f Our data shows, that with reasonable management of gen- eral safety precautions, SARS-CoV-2 transmission risk from handling bodies, including external examinations and autopsies, is very low. 2. Braun F, Lütgehetmann M, Pfefferle S, Wong MN, Carsten A, et al. SARS-CoV-2 renal tropism associates with acute kidney injury. Lancet. 2020;396:597–8. 3. Heinrich F, Meißner K, Langenwalder F, Püschel K, Nörz D, et al. Postmortem stability of SARS-CoV-2 in nasopharyngeal mucosa. Emerg Infect Dis. 2020. https://​doi.​org/​10.​3201/​eid27​01.​203112. Discussion This was partly because the risk of infection posed by bodies was difficult to assess. Concerns exist not only among physi- cians who perform autopsies, but also technical personnel and physicians who only perform external post mortems. In contrast to these concerns, however, there are also reports indicating that autopsy personnel are not actually exposed to any particular risk when protective measures are applied [7, 9]. In our cohort, we found no evidence of an increased risk of transmission of the infection to funeral personnel and health care professionals who have to handle SARS-CoV- 2-infected deceased persons. The greatest risk likely comes from unprotected contact with relatives and the unavailabil- ity of infection control measures. However, further research is needed, particularly in the often overlooked funeral indus- try, to analyze, for example, the prevalence of SARS-CoV-2 antibodies among workers in this field and adherence to the use of protective measures in daily practice. It must be acknowledged that the department’s own risk assessment was a rather optimistic estimate for which no evidence was available at the onset of the pandemic. In particular, little was known about the potential infectivity of surfaces and instruments. However, the present results underline the cor- rectness of this assessment. Concerns about the handling of the potentially infectious body also relate to burial practices. Open casket burials are not permitted in some cases. Ritual washing and embalming of the deceased, which are common in various cultures and 3 3 417 Forensic Science, Medicine and Pathology (2021) 17:411–418 Key Points 4. Lindner D, Fitzek A, Bräuninger H, Aleshcheva G, Edler C, et al. Association of cardiac Infection with SARS-CoV-2 in confirmed COVID-19 autopsy cases. JAMA Cardiol. 2020. https://​doi.​org/​ 10.​1001/​jamac​ardio.​2020.​3551. 1. No viable virus was found on the skin of deceased COVID-19 patients nor surfaces, including body bags. 5. Puelles VG, Lütgehetmann M, Lindenmeyer MT, Sperhake JP, Wong MN, et al. Multiorgan and renal tropism of SARS-CoV-2. N Engl J Med. 2020;383:590–2. 2. If usual safety precautions (personal protective equip- ment and appropriate autopsy room) are respected, there is no concern about performing full autopsies. 6. Wichmann D, Sperhake J-P, Lütgehetmann M, Steurer S, Edler C, et al. Autopsy findings and venous thromboembolism in patients with COVID-19. A prospective cohort study. Ann Intern Med. 2020;173:268–77. 3. Open-casket farewells may also be permitted, although unprotected touching of the body should be avoided. 7. Davis GG, Williamson AK. Risk of COVID-19 transmission dur- ing autopsy. Arch Pathol Lab Med. 2020;144:1445a–1445. 4. The infection risk from transporting an infected body in a body bag is low. 8. European Center for Disease Prevention and Control (ECDC). Considerations related to the safe handling of bodies of deceased persons with suspected or confirmed COVID-19. 2020. https://​ www.​ecdc.​europa.​eu/​sites/​defau​lt/​files/​docum​ents/​COVID-​19-​ safe-​handl​ing-​of-​bodies-​or-​perso​ns-​dying-​from-​COVID​19.​pdf. 5. Washing and embalming procedures can be performed by trained personnel if safety precautions analogous to autopsies are taken. 9. Han B, Bhall F, da Silva LF, Van der Heide RS, Love GL, et al. Coronavirus disease 2019 autopsies and personal protective equip- ment. Arch Pathol Lab Med. 2020;144:1295a–1295. 10. Sperhake J-P. Autopsies of COVID-19 deceased? Absolutely! Leg Med (Tokyo). 2020. https://​doi.​org/​10.​1016/j.​legal​med.​2020.​ 101769 Funding  Open Access funding enabled and organized by Projekt DEAL. The Institute of Legal Medicine was supported by a grant from the Authorities for Social Welfare, Hamburg, Germany and the Federal Ministry for Education and Research (BMBF), Germany and report on grants from the Authorities for Social Welfare, Hamburg, Germany. AF was supported by a grant from the ExStra-Fonds of the University of Hamburg, funded by the BMBF. 11. Dijkhuizen LGM, Gelderma HT, Duijst WLJM. Review: The safe handling of a corpse (suspected) with COVID-19. J Forensic Leg Med. 2020. https://​doi.​org/​10.​1016/j.​jflm.​2020.​101999. 12. Edler C, Schröder AS, Aepfelbacher M, Fitzek A, Heinemann A, et al. Dying with SARS-CoV-2 infection—an autopsy study of the first consecutive 80 cases in Hamburg. Germany Int J Leg Med. 2020;134:1275–84. Declarations 13. Fitzek A, Sperhake J, Edler C, Schröder AS, Heinemann A, et al. Evidence for systematic autopsies in COVID-19 positive deceased. Case report of the first German investigated COVID-19 death. Rechtsmedizin. 2020;184–9. Ethical approval  This study was approved by the ethics commission of the Hamburg Medical Association (PV7311). 14. Heinrich F, Sperhake J-P, Heinemann A, Mushumba H, Lennartz M, et al. Germany`s first COVID-19 deceased: a 59-year-old man presenting with diffuse alveolar damage due to SARS-CoV-2 infection. Virchows Arch. 2020;477:335–9. Open Access  This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. 15. Nörz D, Fischer N, Schultze A, Kluge S, Mayer-Runge U, et al. Clinical evaluation of a SARS-CoV-2 RT-PCR assay on a fully automated system for rapid on-demand testing in the hospital set- ting. J Clin Virol. 2020. https://​doi.​org/​10.​1016/j.​jcv.​2020.​104390. 16. Nörz D, Frontzek A, Eigner U, Oestereich L, Wichmann D, et al. Pushing beyond specifications: Evaluation of linearity and clinical performance of the cobas 6800/8800 SARS-CoV-2 RT-PCR assay for reliable quantification in blood and other materials outside recommendations. J Clin Virol. 2020. https://​doi.​org/​10.​1016/j.​ jcv.​2020.​104650. 17. Loeffelholz MJ, Alland D, Butler-Wu SM, Pandey U, Perno CF, et al. Multicenter evaluation of the Cepheid Xpert Xpress SARS- CoV-2 Test. J Clin Microbiol. 2020;58:e00926-e1020. 1 3 Forensic Science, Medicine and Pathology (2021) 17:411–418 418 18. Pfefferle S, Reucher S, Nörz D, Lütgehetmann M. Evaluation of a quantitative RT-PCR assay for the detection of the emerging coronavirus SARS-CoV-2 using a high throughput system. Euro Surveill. 2020. https://​doi.​org/​10.​2807/​1560-​7917.​ES.​2020.​25.9.​ 20001​52. 25. Sapino A, Facchetti F, Bonoldi E, Gianatti A, Barbareschi M, et al. The autopsy debate during the COVID-19 emergency: the Italian experience. Virchows Arch. 2020;476:821–3. 26. Teresiński G, Jurek T. Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Declarations Recommendations of the Polish Society of Forensic Medicine and Criminology and National Consultant for Forensic Medicine with regard to performing forensic post- mortem examinations in case of confirmed COVID-19 disease and suspected SARS CoV-2 infections. Arch Med Sadowej Kryminol. 2019;69:147–57. 19. Pfefferle S, Huang J, Nörz D, Indenbirken D, Lütgehetmann M, et al. Complete genome sequence of a SARS-CoV-2 strain isolated in Northern Germany. Microbiol Resour Announc. 2020;9:e00520-e620. 27. Baj J, Ciesielka M, Buszewicz G, Maciejewski R, Budzyńska B, et al. COVID-19 in the autopsy room–requirements, safety, recommendations and pathological findings. Forensic Sci Med Pathol. 2020. https://​doi.​org/​10.​1007/​s12024-​020-​00341-1. 20. Chin AWH, Chu JTS, Perera MRA, Hui KPY, Yen H-LY, et al. Stability of SARS-CoV-2 in different environmental conditions. Lancet Microbe. 2020; https://​doi.​org/​10.​1016/​S2666-​5247(20)​ 30003-3 21. Liu Y, Li T, Deng, Y Liu S, Zhang D, et al. Stability of SARS- CoV-2 on environmental surfaces and in human excreta. J Hosp Infect. 2021;107:105–7. 28. Robert Koch Institute. Empfehlungen zum Umgang mit SARS- CoV-2-infizierten Verstorbenen. 2021. https://​www.​rki.​de/​DE/​ Conte​nt/​InfAZ/N/​Neuar​tiges_​Coron​avirus/​Verst​orbene.​html. Accessed 19 Jan 2021. 22. Van Doremalen N, Bushmaker T, Morris DH, Holbrook MG, Gamble A, et al. Aerosol and surface stability of SARS-CoV-2 as compared with SARS-CoV-1. N Engl J Med. 2020. https://​doi.​ org/​10.​1056/​NEJMc​20049​73.i Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 23. Harbourt DE, Haddow AD, Piper AE, Bloomfield H, Kearney BJ, et al. Modeling the stability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on skin, currency, and clothing. PLoS Negl Trop Dis. 2020. https://​doi.​org/​10.​1371/​journ​al.​pntd.​ 00088​31. 24. Pomara C, Li Volti G, Cappello F. COVID-19 deaths: Are we sure it is pneumonia? Please, autopsy, autopsy, autopsy! J Clin Med. 2020;9:1259. 1 3
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Lessons Learned from and Prospects of Using Mobile Laboratories for Epidemiological Surveillance over Plague, Other Particularly Dangerous, Natural-Focal, Zoonotic Infectious Diseases
Problemy osobo opasnyh infekcij
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DOI: 10.21055/0370-1069-2020-4-26-33 И.Н. Шарова1, Т.Ю. Красовская1, Е.В. Казорина1, А.В. Казанцев1, М.В. Проскурякова1, В.Е. Куклев1, С.А. Щербакова1, В.В. Кутырев1, Р.И. Адилов2, Е.В. Булычева2, А.А. Троицкая2, Б.Л. Агапов2, И.С. Акимова3, О.Л. Балган3, Н.А. Чумакова3, В.А. Ткаченко3, Э.А. Глушков3, Е.Н. Рождественский4, Г.Х. Базарова4, А.И. Мищенко4, Г.Б. Мухтургин5 g 5Irkutsk Research Anti-Plague Institute of Siberia and Far East, Irkutsk, Russian Federation g y y Altai Plague Control Station, Gorno-Altaisk, Russian Federation; ОБЗОРЫ ОБЗОРЫ Проблемы особо опасных инфекций. 2020; 4 g 2Astrakhan Plague Control Station, Astrakhan, Russian Federation; Опыт и перспективы использования мобильных лабораторий при проведении эпидемиологического надзора за чумой, другими особо опасными, природно-очаговыми, зоонозными инфекционными болезнями 1ФКУЗ «Российский научно-исследовательский противочумный институт «Микроб», Саратов, Российская Федерация; 2ФКУЗ «Астраханская противочумная станция», Астрахань, Российская Федерация; 3ФКУЗ «Тувинская противочумная станция», Кызыл, Российская Федерация; 4ФКУЗ «Алтайская противочумная станция», Горно-Алтайск, Российская Федерация; 5ФКУЗ «Иркутский научно-исследовательский противочумный институт Сибири и Дальнего Востока», Иркутск, Российская Федерация Представлены материалы по использованию мобильных лабораторий, разработанных в РФ, в рамках четырех направлений: мониторинг территорий с целью выявления циркуляции возбудителей природно-очаговых инфек- ционных болезней; мониторинг территорий в период обострения эпизоотической ситуации; участие в ликвидации вспышек инфекционных болезней; мониторинг территории с целью контроля и прогноза эпидемиологической и эпизоотологической ситуации при подготовке к проведению массовых мероприятий. Рассмотрены тактико- технические характеристики и порядок организации работ мобильной лаборатории мониторинга и диагностики, размещенной на базе автомобиля КамАЗ. Отличительной особенностью лаборатории от имеющихся российских и зарубежных аналогов является наличие необходимых условий для проведения исследований с использованием бактериологического анализа, методов экспресс- и ускоренной диагностики, выполнения полного цикла работ – от подготовительного этапа до деструкции инфицированного материала. Высокотехнологичное оборудование по- зволяет использовать две схемы проведения исследования: первая – бактериологический анализ совместно с по- становкой ПЦР, что обеспечивает высокую достоверность полученных результатов; вторая – проведение на пер- вом этапе ПЦР, а при выявлении генетических маркеров возбудителя – выполнение бактериологического анализа положительных проб с целью выделения культуры возбудителя и ее последующей идентификации. Вторая схема позволит сократить объем бактериологических исследований, внести изменения в тактику эпизоотологического обследования. Испытания мобильной лаборатории показали эффективность ее использования при эпизоотологи- ческом обследовании природных очагов чумы, в том числе трансграничных. Благодаря применению мобильной лаборатории в Горно-Алтайском высокогорном природном очаге чумы выявлен новый эпизоотический участок, расположенный на отдаленной территории, используемый населением в качестве летнего пастбища для выпаса домашних животных. Применение мобильных лабораторий обеспечит усиление лабораторной базы учреждений, осуществляющих мониторинг особо опасных, природно-очаговых и других опасных инфекционных болезней, будет способствовать приближению современных диагностических технологий непосредственно к природному очагу, снижению риска развития эпидемических осложнений по чуме и другим особо опасным инфекциям в трансграничных природных очагах. Ключевые слова: мобильная лаборатория, индикация, бактериологический анализ, ПЦР, ИФА, ИХА, эпиде- миологический надзор, эпизоотологический мониторинг, обеспечение биологической безопасности. рр ру щ р р р , p @ Для цитирования: Шарова И.Н., Красовская Т.Ю., Казорина Е.В., Казанцев А.В., Проскурякова М.В., Куклев В.Е., Щербакова С.А., Кутырев В.В., Адилов Р.И., Булычева Е.В., Троицкая А.А., Агапов Б.Л., Акимова И.С., Балган О.Л., Чумакова Н.А., Ткаченко В.А., Глушков Э.А., Рождественский Е.Н., Базарова Г.Х., Мищенко А.И., Мухтургин Г.Б. Опыт и перспективы использования мобильных лабораторий при проведении эпидемиологического надзора за чумой, другими особо опасными, природно-очаговыми, зоонозными инфекционными болезнями. у @ Для цитирования: Шарова И.Н., Красовская Т.Ю., Казорина Е.В., Казанцев А.В., Проскурякова М.В., Куклев В.Е., Щербакова С.А., Кутырев В.В., Адилов Р.И., Булычева Е.В., Троицкая А.А., Агапов Б.Л., Акимова И.С., Балган О.Л., Чумакова Н.А., Ткаченко В.А., Глушков Э.А., Рождественский Е.Н., Базарова Г.Х., Мищенко А.И., Мухтургин Г.Б. Опыт и перспективы использования мобильных лабораторий при проведении эпидемиологического надзора за чумой, другими особо опасными, природно-очаговыми, зоонозными инфекционными болезнями. Проблемы особо опасных инфекций. 2020; 4:26–33. DOI: 10.21055/0370-1069- 2020-4-26-33 1Russian Research Anti-Plague Institute “Microbe”, Saratov, Russian Federation; 2Astrakhan Plague Control Station, Astrakhan, Russian Federation; 1Russian Research Anti-Plague Institute “Microbe”, Saratov, Russian Federation; 2A t kh Pl C t l St ti A t kh R i F d ti Russian Research Anti Plague Institute Microbe , Saratov, Russian Federation; 2Astrakhan Plague Control Station, Astrakhan, Russian Federation; 3 g , , uva Plague Control Station, Kyzyl, Russian Federation; Опыт и перспективы использования мобильных лабораторий при проведении эпидемиологического надзора за чумой, другими особо опасными, природно-очаговыми, зоонозными инфекционными болезнями Проблемы особо опасных инфекций. 2020; 4:26–33. DOI: 10.21055/0370-1069- 2020-4-26-33 2020 4 26 33 Поступила 13.12.18. Отправлена на доработку 18.01.19. Принята к публ. 11.09.20. Поступила 13.12.18. Отправлена на доработку 18.01.19. Принята к публ. 11.09.20. Corresponding author: Irina N. Sharova, e-mail: rusrapi@microbe.ru. Citation: Sharova I.N., Krasovskaya T.Yu., Kazorina E.V., Kazantsev A.V., Proskuryakova M.V., Kuklev V.E., Shcherbakova S.A., Kutyrev V.V., Adilov R.I., Bulycheva E.V., Troitskaya A.A., Agapov B.L., Akimov I.S., Balgan O.L., Chumakova N.A., Tkachenko V.A., Glushkov E.A., Rozhdestvensky E.N., Bazarova G.Kh., Mishchenko A.I., Mukhturgin G.B. Lessons Learned from and Prospects of Using Mobile Laboratories for Epidemiological Surveillance over Plague, Other Particularly Dangerous, Natural-Focal, Zoonotic Infectious Diseases. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2020; 4:26–33. (In Russian). DOI: 10.21055/0370-1069-2020-4-26-33 Received 13.12.18. Revised 18.01.19. Accepted 11.09.20. I.N. Sharova1, T.Yu. Krasovskaya1, E.V. Kazorina1, A.V. Kazantsev1, M.V. Proskuryakova1, V.E. Kuklev1, S.A. Shcherbakova1, V.V. Kutyrev1, R.I. Adilov2, E.V. Bulycheva2, A.A. Troitskaya2, B.L. Agapov2, I.S. Akimov3, O.L. Balgan3, N.A. Chumakova3, V.A. Tkachenko3, E.A. Glushkov3, E.N. Rozhdestvensky4, G.Kh. Bazarova4, A.I. Mishchenko4, G.B. Mukhturgin5 Lessons Learned from and Prospects of Using Mobile Laboratories for Epidemiological Surveillance over Plague, Other Particularly Dangerous, Natural-Focal, Zoonotic Infectious Diseases 1Russian Research Anti-Plague Institute “Microbe”, Saratov, Russian Federation; 2Astrakhan Plague Control Station, Astrakhan, Russian Federation; 3Tuva Plague Control Station, Kyzyl, Russian Federation; 4Altai Plague Control Station, Gorno-Altaisk, Russian Federation; 5Irkutsk Research Anti-Plague Institute of Siberia and Far East, Irkutsk, Russian Federation 26 Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2020; 4 obo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2020; 4 Reviews Abstract. The review presents the materials on the use of mobile laboratories, developed in the Russian Federation, in the framework of four main areas: monitoring of territories to identify the circulation of pathogens of natural-focal infec- tious diseases; monitoring of territories during the exacerbation of the epizootic situation; participation in the elimination of outbreaks of infectious diseases; monitoring of territories to control and predict the epidemiological and epizootiologic situation in preparation for mass events. The tactical and technical characteristics and the procedure for organizing the operation of the mobile laboratory for monitoring and diagnostics mounted on the platform of the KamAZ chassis are considered. A distinctive feature of the laboratory from the existing Russian and foreign counterparts is the availability of the necessary conditions for conducting research using bacteriological analysis, rapid and accelerated diagnostic methods, and performing a full cycle of works – from the preparatory stage to the destruction of infected material. High- tech equipment allows the realization of two research schemes: the first – bacteriological analysis alongside the PCR, which ensures high reliability of the results; the second – conducting the PCR at the first stage, and when identifying genetic markers of the pathogen – performing bacteriological analysis of positive samples in order to isolate the culture of the pathogen and identify it subsequently. The second scheme will reduce the volume of bacteriological studies; make changes in the tactics of epizootiological survey. Tests of the mobile laboratory have demonstrated the effectiveness of its use in the epizootiological examination of natural plague foci, including cross-border ones. Owing to the use of a mobile laboratory in the Gorno-Altaisk high-mountain natural focus of plague, a new epizootic site was identified, located in a remote area, used by the population as a summer pasture for grazing domestic animals. The use of mobile laboratories will strengthen the laboratory base of institutions that monitor particularly dangerous, natural-focal and other dangerous infectious diseases; will help to bring the advanced diagnostic technologies directly to the natural focus, reduce the risk of epidemic complications due to plague and other particularly dangerous infections in cross-border natural foci. Key words: mobile laboratory, indication, bacteriological analysis, PCR, ELISA, ICA, epidemiological surveillance, epizootiological monitoring, provision of biological safety. Conflict of interest: The authors declare no conflict of interest. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2020; 4 зовалась при эпидемиологическом расследовании вспышки лептоспироза в д. Полянки Октябрьского района городского округа Саранск Республики Мордовия. Был установлен источник инфекции и фактор передачи, реализован комплекс противоэпи- демических и профилактических мероприятий по локализации и ликвидации очага. зовалась при эпидемиологическом расследовании вспышки лептоспироза в д. Полянки Октябрьского района городского округа Саранск Республики Мордовия. Был установлен источник инфекции и фактор передачи, реализован комплекс противоэпи- демических и профилактических мероприятий по локализации и ликвидации очага. у р Второе направление – мониторинг территорий в период обострения эпизоотической ситуации. Для ре- шения этой задачи мобильная лаборатория использо- валась в 2007 г. в высокогорных районах Кабардино- Балкарской Республики на территории Центрально- Кавказского природного очага чумы. В период работы на базе мобильной лаборатории методом ПЦР в двух пробах блох (Citellophilus tesquorum) с очеса горного суслика и из входов нор выявлена ДНК возбудителя чумы и впервые положительный результат ПЦР под- твержден бактериологическим методом в стационар- ной лаборатории Былымского эпидотряда [16]. Еще одно направление использования мобиль- ной лаборатории – мониторинг территории с целью контроля и прогноза эпидемиологической и эпизоо- тологической ситуации при подготовке к проведе- нию массовых мероприятий. В рамках этого направ- ления лаборатория была использована в 2013 г. в период подготовки к проведению XXVII Всемирной летней Универсиады в Казани [18, 19]. Осуществлен мониторинг циркуляции возбудителей туляремии, лептоспироза, ГЛПС, ЛЗН и других инфекционных болезней, актуальных для Республики Татарстан. ной лаборатории Былымского эпидотряда [16]. В Астраханской области в 2014–2015 гг. эффек- тивность использования мобильной лаборатории и методов диагностики, применяемых в лаборатории, подтверждены в ходе эпидемиологического надзора за чумой на территории Прикаспийского песчаного природного очага чумы при возникновении разлитой эпизоотии этой инфекции. При проведении эпизоото- логического мониторинга ПЦР использовали как на этапе индикации при исследовании нативного мате- риала, так и при идентификации выделенных куль- тур возбудителя чумы. Полученные результаты сви- детельствовали о высокой информативности этого метода при работе с полевым материалом и культура- ми возбудителя. Отмечена высокая информативность ПЦР для поиска эпизоотийных участков. Анализ распределения положительных в ПЦР проб по секто- рам давал возможность корректировать направление поиска таких участков, повышал вероятность выде- ления культуры возбудителя. Быстрота получения ответа при использовании ПЦР позволяла координи- ровать работу зоогрупп, осуществляющих эпизоото- логический мониторинг, своевременно и оперативно вносить коррективы в календарно-территориальные планы [17]. Установлена высокая корреляция резуль- татов ИФА, направленного на выявление ФI чумного микроба, с результатами бактериологического анали- за. Подтверждена эффективность и быстрота получе- ния ответа при исследовании методом ИХА суспен- зий органов трупов грызунов. Современные модификации мобильной лабора- тории на базе автошасси, тактико-технические характеристики лабораторий. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2020; 4 Shcherbakova S.A., ORCID: https://orcid.org/0000-0003-1143-4069 Kutyrev V.V., ORCID: https://orcid.org/0000-0003-3788-3452 ведки и индикации на базе автомашины ГАЗ-2705, разработанная ФКУЗ РосНИПЧИ «Микроб» [8]. ведки и индикации на базе автомашины ГАЗ-2705, разработанная ФКУЗ РосНИПЧИ «Микроб» [8]. Важными стратегическими направлениями со- временной Концепции функционирования системы противочумных учреждений Федеральной службы по надзору в сфере защиты прав потребителей и бла- гополучия человека, принятой до 2020 г., остаются совершенствование эпидемиологического надзо- ра за чумой, другими особо опасными, природно- очаговыми, зоонозными инфекционными болезня- ми, а также совершенствование системы оператив- ного реагирования и предупреждения ЧС санитарно- эпидемиологического характера [1–7]. Реализации обоих направлений способствует разработка и вне- дрение в практику противочумных учреждений мо- бильных лабораторий, оснащенных высокотехноло- гичным оборудованием и обеспечивающих проведе- ние диагностических исследований непосредствен- но в очаге инфекции [8–10]. Эффективность исполь- зования мобильных лабораторий не раз доказана при ликвидации вспышек инфекционных болезней, вызванных особо опасными вирусами [11–14]. р р р За время эксплуатации лаборатории с 2007 г. можно выделить четыре основных направления ее использования. Одно из них – мониторинг терри- торий с целью выявления циркуляции возбудителей природно-очаговых инфекционных болезней. До настоящего времени мобильная лаборатория эпидразведки и индикации используется при прове- дении эпизоотологического мониторинга природных очагов опасных инфекционных болезней на терри- тории Саратовской области. Районы области контро- лируются на возможность заноса, распространения и циркуляции возбудителей туляремии, лихорадки Западного Нила, инфекций, передаваемых клещами (иксодового клещевого боррелиоза, клещевого эн- цефалита, гранулозитарного анаплазмоза человека, моноцитарного эрлихиоза человека), геморрагиче- ской лихорадки с почечным синдромом, гриппа птиц и других инфекций. В 2007, 2011, 2015 гг. лаборатория использо- валась с целью мониторинга циркуляции возбуди- теля чумы на территории Прикаспийского Северо- Западного степного природного очага в Республике Калмыкия и Волго-Уральского степного, Волго- Уральского песчаного и Прикаспийского песчаного природных очагов чумы в Астраханской области [15]. В Ставропольском крае в 2007 г. лаборатория была В 2007, 2011, 2015 гг. лаборатория использо- валась с целью мониторинга циркуляции возбуди- теля чумы на территории Прикаспийского Северо- Западного степного природного очага в Республике Калмыкия и Волго-Уральского степного, Волго- Уральского песчаного и Прикаспийского песчаного природных очагов чумы в Астраханской области [15]. В Ставропольском крае в 2007 г. лаборатория была Опыт использования мобильной лаборатории эпидемиологической разведки и индикации. Первой мобильной лабораторией, в которой не только осу- ществлялись исследования с использованием совре- менных методов лабораторной диагностики (ПЦР, ИФА, ИХА, МФА), но и обеспечивался необходимый уровень биологической безопасности работ, стала мобильная лаборатория эпидемиологической раз- 27 Проблемы особо опасных инфекций. 2020; 4 ОБЗОРЫ ОБЗОРЫ задействована для определения мест циркуляции возбудителей КГЛ, ЛЗН, лептоспироза [16]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2020; 4 Опыт конструиро- вания и эксплуатации мобильной лаборатории эпид­ разведки и индикации использован при создании следующего поколения лабораторий на шасси ав- томобиля «Газель» – микробиологической лабора- тории экспресс-диагностики (МЛЭД). Инженерно- техническое оснащение лаборатории обеспечивало повышение уровня биологической безопасности и улучшение эргономических характеристик. у у р р р В октябре 2014 г. вступило в силу распоряжение Правительства Российской Федерации № 1965‑р по оказанию в 2015–2016 гг. государствам-участникам СНГ материально-технической и методической поддержки внедрения положений Международных медико-санитарных правил (2005 г.) в целях укреп­ ления национального потенциала в сфере борьбы с инфекционными болезнями. В рамках реализации данного распоряжения создано 10 лабораторий, которые поставлены в пять стран СНГ (Беларусь, Казахстан, Армению, Кыргызстан, Таджикистан). Еще четыре лаборатории поставлены в Казахстан, Узбекистан, Кыргызстан и Монголию в рамках реализации распоряжения Правительства РФ от 26.05.2017 № 1060-р [20]. Третьим направлением использования мобиль- ной лаборатории является участие в ликвидации вспышек инфекционных болезней. В 2008 г. мо- бильная лаборатория эпидразведки и индикации была использована при проведении эпидемиологи- ческого расследования вспышки сибирской язвы в Янаульском районе Республики Башкортостан, что позволило своевременно осуществить комплекс противоэпидемических и профилактических меро- приятий и ликвидировать вспышку сибирской язвы в предельно короткие сроки. Зимой того же года мобильная лаборатория была задействована при проведении эколого-эпизоотологического обследо- вания лесных массивов, прилегающих к г. Аткарску Саратовской области, в период вспышки ГЛПС в районе. В 2014 г. мобильная лаборатория исполь- р В 2017 г. в соответствии с двухсторонним со- глашением о сотрудничестве ФКУЗ «Российский научно-исследовательский противочумный ин- ститут «Микроб» (РосНИПЧИ «Микроб») с Рес­ публиканским центром карантинных и особо опас- ных инфекций (РЦКиООИ) Министерства здравоох- ранения Кыргызской Республики и в ответ на запрос директора Центра, специалистами сезонного проти- воэпидемического отряда «Манас» совместно со спе- циалистами ФКУЗ РосНИПЧИ «Микроб» проведено эпизоотологическое обследование Манас­ского ме- зоочага Таласского высокогорного природного очага чумы Кыргызской Республики, в ходе которого задей- ствована микробиологическая лаборатория экспресс- диагностики на базе автошасси [21]. Исследования 28 Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2020; 4 Reviews ный блок и технический отсек, изолированный от помещений лаборатории. методами ПЦР и ИФА осуществляли в мобильной лаборатории, бактериологический анализ – в ла- боратории эпидотряда. В 2018 г. лаборатория была использована при проведении эпизоотологического мониторинга Центрального и Южного мезоочагов Аксайского высокогорного очага чумы, долины реки Тарагай, а также в обеспечении эпидемиологическо- го надзора во время проведения III Всемирных игр кочевников в Кыргызской Республике. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2020; 4 р р МЛМД обеспечена холодным и горячим водо- снабжением, электричеством, отоплением, обору- дована системой приточно-вытяжной вентиляции с автоматическим поддержанием разницы давления между помещениями, оснащенной высокоэффектив- ными фильтрами очистки воздуха класса Н14. ф р у Лабораторный блок включает: ф р у Лабораторный блок включает: ф р у Лабораторный блок включает: р у В 2019 г. для проведения эпизоотологического обследования приграничного Хархира-Тургенского природного очага чумы на территории Монголии использована мобильная микробиологическая ла- боратория экспресс-диагностики, подаренная Рос­ сийской Федерацией Национальному центру зооноз­ ных инфекций Министерства здравоохранения Мон­ голии. На базе лаборатории для выявления маркеров Yersinia pestis российско-монгольскими специали- стами исследовано более 200 проб полевого мате- риала [22]. - помещение для снятия личной, надевания ра- бочей и защитной одежды; - душевую; - амбур-шлюз (помещение для снятия защитной одежды); - помещение для микробиологических исследо- ваний; - шлюзовую камеру для приема и передачи ма- териала. Для осуществления диагностических исследо- ваний методами ПЦР, ИФА, МФА, бактериологи- ческого анализа, проведения паразитологических работ, деструкции заразного материала лаборатория оснащена: р В рамках реализации мер по борьбе с панде- мией новой коронавирусной инфекции (COVID-19) использование МЛЭД позволило расширить и укре- пить лабораторную сеть в республиках Казахстан, Узбекистан, Кыргызстан, Таджикистан и обеспечить увеличение объема исследований клинического и биологического материала на наличие возбудителя SARS-CoV-2 методом ПЦР [23, 24]. - боксом микробиологической безопасности БМБ-II «Ламинар-С»-1,5 (БМБ (1,5)); - боксом микробиологической безопасности БМБ-II «Ламинар-С»-1,2 (БМБ (1,2)); - боксом абактериальной воздушной среды для работы с реакционными смесями при проведении ПЦР-диагностики БАВ-ПЦР «Ламинар-С» (ПЦР- бокс); - боксом абактериальной воздушной среды для работы с реакционными смесями при проведении ПЦР-диагностики БАВ-ПЦР «Ламинар-С» (ПЦР- бокс); В соответствии с распоряжением Правительства Российской Федерации от 05.09.2016 № 1864-р в 2017 г. создана следующая модификация мобильной лаборатории – мобильная лаборатория мониторин- га и диагностики (МЛМД). Ее отличительной осо- бенностью от имеющихся российских и зарубежных аналогов является наличие необходимых условий для проведения исследований с использованием бактериологического анализа, методов экспресс- и ускоренной диагностики, выполнения полного цик- ла работ – от подготовительного этапа (розлива пи- тательных сред) до деструкции инфицированного материала [25–27]. ) - 2 лабораторными термостатами; - 3 автомобильными холодильниками с поддер- жанием температур от +3 °С до минус 20 °С; - микроволновой системой обеззараживания ме- дицинских отходов или автоклавом; - оборудованием, позволяющим осуществлять выделение культур, индикацию и идентификацию возбудителей опасных инфекционных болезней; Следует отметить, что инженерно-техническое обеспечение мобильной лаборатории позволяет ис- пользовать для проведения исследований современ- ное высокотехнологичное оборудование, что прибли- жает передовые диагностические технологии к при- родному очагу. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2020; 4 В мобильных лабораториях в автономном режиме методами бактериологическо- го анализа, ИФА и/или ПЦР исследованы 297 проб суспензий органов млекопитающих (сурок, суслик длиннохвостый, даурская пищуха, полуденная и гре- бенщиковая песчанки, серый хомячок), 94 объеди- ненные пробы суспензий эктопаразитов, счесанных с грызунов и собранных из входов нор. С использо- ванием мобильной лаборатории, работающей в авто- номном режиме, в Горно-Алтайском высокогорном природном очаге чумы в августе 2018 г. выявлен но- вый эпизоотический участок Жумалы, расположен- ный на отдаленной, труднодоступной территории, используемый населением района в качестве летнего пастбища для выпаса домашних животных. В 2018 г. мобильная лаборатория мониторинга и диагностики Алтайской противочумной станции использована при проведении совместного эпизоотологического обсле- дования монгольской части (Баян-Ульгийский аймак Монголии) трансграничного Сайлюгемского при- родного очага чумы на территории, расположенной в непосредственной близости от государственной гра- ницы РФ. В ходе обследования на базе лаборатории выделено 47 культур возбудителя чумы [21]. - подготовку проб к исследованию (концентри- рование материала путем центрифугирования, пе- ревод сухих и плотных материалов в жидкую фазу и др.); р ) - индикацию возбудителей инфекционных бо- лезней методами ПЦР, ИФА и МФА; - выявление антител к возбудителям в серологи- ческих реакциях; - выделение культур возбудителей инфекцион- ных болезней бактериальной этиологии; - идентификацию выделенных культур возбуди- телей по сокращенной схеме; р - деструкцию инфицированных объектов; - подготовку дифференциально-диагностиче­ ских питательных сред и сред накопления. Для выполнения каждого раздела регламентиро- ванных работ определены рабочие места. Проведение подготовительных работ, а также работы с необезза- раженным материалом и выделенными культурами микроорганизмов следует осуществлять в БМБ (1,5). Для выделения НК используют БМБ (1,2). После за- вершения всех работ и проведения текущей дезин- фекции боксы можно использовать для розлива пи- тательных сред. Приготовление реакционных смесей для ПЦР выполняют в ПЦР‑боксе. Для учета резуль- татов реакций, проведения паразитологических ис- следований используют соответствующие рабочие места, организованные на лабораторных столах. С целью повышения эффективности и обеспе- чения биологической безопасности работ проведе- ние исследований биологического материала и проб объектов окружающей среды, отобранных в ходе эпизоотологического мониторинга природного оча- га, следует осуществлять в два этапа: первичную подготовку проб, приготовление и посев на пита- тельные среды суспензий эктопаразитов и органов мелких млекопитающих, проб объектов окружаю- щей среды необходимо выполнять до перерыва; ис- следования методом ПЦР, ИФА, МФА, просмотр по- севов на чашках, розлив питательных сред – после проведения текущей дезинфекции, обеззараживания помещения УФ-облучением и перерыва. При проведении испытаний определен ряд важ- ных характеристик мобильной лаборатории и по- рядок организации работ. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2020; 4 Объемно-планировочные решения и размещение оборудования лаборатории обеспечива- ют поточность продвижения ПБА и персонала, со- блюдение требований биологической безопасности при выполнении работ. В лаборатории имеются не- обходимые условия для работы с микроорганизмами I (кроме вирусов) – IV групп патогенности. р [ ] Лаборатория предназначена для осуществле- ния лабораторной диагностики чумы и других опасных инфекционных болезней бактериальной и вирусной этиологии при проведении эпидемиоло- гического надзора за природными очагами инфек- ционных болезней, в том числе трансграничными. Функциональные возможности лаборатории позво- ляют использовать ее для усиления лабораторной базы стационарных лабораторий или как самостоя- тельную единицу при проведении мероприятий по обеспечению санитарно-эпидемиологического бла- гополучия населения в случае обострения эпидеми- ческой ситуации и/или при проведении массовых мероприятий. Испытания мобильной лаборатории монито- ринга и диагностики, порядок организации работ. Функциональные и технические характеристики мобильной лаборатории определили номенклатуру диагностических исследований, которая включает: б б б р р Лаборатория размещена на базе кузова-фургона, установленного на шасси автомобиля КамАЗ, моди- фицированного с учетом особенностей конструкции лаборатории. Внутри кузов разделен на лаборатор- - лабораторные исследования проб биологиче- ского материала и объектов окружающей среды, ото- 29 Проблемы особо опасных инфекций. 2020; 4 ОБЗОРЫ ОБЗОРЫ тор) и зоопаразитолог. бранных в ходе эпизоотологического обследования территории природного очага особо опасных и оча- гов других опасных инфекционных болезней; тор) и зоопаразитолог. тор) и зоопаразитолог. Порядок проведения исследований в мобильной лаборатории предусматривает: - прием, сортировку и регистрацию зоологопа- разитологического или клинического материала; - прием, сортировку и регистрацию зоологопа- разитологического или клинического материала; - лабораторные исследования проб, в том числе клинического материала, при проведении экстрен- ных противоэпидемических мероприятий по выяв- лению, локализации и ликвидации очагов опасных инфекционных болезней бактериальной и вирусной этиологии, в том числе возникших вследствие акти- визации природных очагов инфекций или в период проведения массовых мероприятий. - первичную подготовку зоологопаразитологи- ческого материала к лабораторному исследованию (определение видовой принадлежности млекопи- тающих и эктопаразитов, показателей, характери- зующих генеративное и физиологическое состояние, формирование проб для группового или индивиду- ального исследования, очес и вскрытие грызунов, разбор гнезд и др.); Тестовые испытания трех из пяти мобильных ла- бораторий проведены на базе Алтайской, Тувинской ПЧС, а также Яндыковского противочумного отде- ления и Досангского противоэпидемического отряда Астраханской ПЧС в период эпизоотологического обследования территорий Горно-Алтайского высо- когорного природного очага чумы (участки: окрест- ности озер Каракуль и Зерликоль-Нур, Жумалы, Восточная и Центральная части Курайского хребта), урочищ Кургак, Доргун и Боро-Шивеки Тувинского природного очага чумы, Волго-Уральского песчано- го и Прикаспийского песчаного природных очагов чумы соответственно. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2020; 4 Для эффективного функ- ционирования МЛМД при осуществлении лабора- торной диагностики в рамках профилактических и противоэпидемических мероприятий в природных очагах чумы, сочетанных природных очагах инфек- ционных болезней бактериальной и вирусной этио- логии и обеспечения режима работы в одну смену в штатный состав лаборатории должны входить не менее 4 специалистов лабораторного звена (2 врача, 2 лаборанта или лаборант и медицинский дезинфек- Высокая информативность ПЦР при эпизоото- логическом мониторинге природных очагов инфек- ционных болезней позволяет рассматривать возмож- ность применения двух схем исследования полевого 30 Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2020; 4 Reviews в 2013 г. Сообщение 1. Эпидемиологические риски и основ- ные направления мероприятий по обеспечению санитарно- эпидемиологического благополучия в период подготовки к про- ведению Саммита. Проблемы особо опасных инфекций. 2013; 4:5–10. DOI: 10.21055/0370-1069-2013-4-5-10. в 2013 г. Сообщение 1. Эпидемиологические риски и основ- ные направления мероприятий по обеспечению санитарно- эпидемиологического благополучия в период подготовки к про- ведению Саммита. Проблемы особо опасных инфекций. 2013; 4:5–10. DOI: 10.21055/0370-1069-2013-4-5-10. материала: первая – использование бактериологи- ческого метода совместно с постановкой ПЦР, что обеспечивает высокую достоверность полученных результатов; вторая – проведение на первом этапе ПЦР, а при выявлении генетических маркеров воз- будителя – выполнение бактериологического анали- за положительных проб с целью выделения культу- ры возбудителя и ее последующей идентификации. Использование второй схемы позволит внести изме- нения в тактику эпизоотологического обследования, сократить объем бактериологических исследований, что важно при работе мобильной лаборатории в ав- тономном режиме на отдаленных территориях или в полевых условиях. Перспективность такого подхода показана при проведении эпизоотологического об- следования трансграничных природных очагов чумы Северо-Западной Монголии [22, 28]. 6. Онищенко Г.Г., Кузькин Б.П., Ракитин И.А., Башкетова Н.С., Коржаев Ю.Н., Гречанинова Т.А., Дятлов И.А., Кутырев В.В., Топорков А.В., Карнаухов И.Г., Топорков В.П., Щербакова С А С Об 6. Онищенко Г.Г., Кузькин Б.П., Ракитин И.А., Башкетова Н.С., Коржаев Ю.Н., Гречанинова Т.А., Дятлов И.А., Кутырев В.В., Топорков А.В., Карнаухов И.Г., Топорков В.П., Щербакова С.А., Казакова Е.С., Шарова И.Н. Обеспечение санитарно- эпидемиологического благополучия в период подготовки и проведения саммита «Группы двадцати» в Санкт-Петербурге в 2013 г. Сообщение 2. Организация и приоритетные направления работы в период проведения Саммита. Проблемы особо опасных инфекций. 2013; 4:11–5. DOI: 10.21055/0370-1069-2013-4-11-15. С.А., Казакова Е.С., Шарова И.Н. Обеспечение санитарно- эпидемиологического благополучия в период подготовки и проведения саммита «Группы двадцати» в Санкт-Петербурге в 2013 г. Сообщение 2. Организация и приоритетные направления работы в период проведения Саммита. Проблемы особо опасных инфекций. 2013; 4:11–5. DOI: 10.21055/0370-1069-2013-4-11-15. ф ц ; 7. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2020; 4 Носков А.К., Акимова И.С., Вержуцкий Д.Б., Климов В.Т., Чеснокова М.В., Косилко С.А., Балахонов С.В. Обеспечение эпидемиологического благополучия при проведении региональ- ного мероприятия с международным участием. Эпидемиология и вакцинопрофилактика. 2016; 15(2):34–9. DOI: 10.31631/2073- 3046-2016-15-2-34-39. 8. Кутырев В.В., Топорков А.В., Карнаухов И.Г. Применение мобильных лабораторий для противоэпидемического обеспече- ния населения в условиях чрезвычайных ситуаций. Проблемы особо опасных инфекций. 2007; 1:27–9. ф ц ; 9. Шарова И.Н., Карнаухов И.Г., Казакова Е.С., Щербаков Д.А., Пчелинцева М.В., Чекашов В.Н., Поршаков А.М., Глазков А.Н., Щербакова С.А., Топорков А.В., Кутырев В.В. Разработка мобильной лаборатории индикации для осуществления эпизоо- тологического мониторинга природно-очаговых и других опас- ных инфекционных болезней. Проблемы особо опасных инфек- ций. 2009; 4:45–8. DOI: 10.21055/0370-1069-2009-4(102)-45-48. Таким образом, использование мобильных ла- бораторий обеспечит усиление лабораторной базы учреждений, осуществляющих мониторинг особо опасных, природно-очаговых и других опасных ин- фекционных болезней, будет способствовать прибли- жению современных диагностических технологий непосредственно к природному очагу и совершен- ствованию эпидемиологического надзора за инфек- ционными болезнями в целом, а также реализации научно-практических программ взаимодействия, на- правленных на снижение риска развития эпидемиче- ских осложнений по чуме и другим особо опасным инфекциям в трансграничных природных очагах. ц ; ( ) 10. Parsons A., Matero P., Adams M., Yeh K.B. Examining the utility and readiness of mobile and field transportable labora- tories for biodefence and global health security-related purposes. Global Security: Health, Science and Policy. 2018; 3(1):1–13. DOI: 10.1080/23779497.2018.1480403. 11. 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History of surgery: the evolution of views on the formation of intestinal stoma
Istoriâ mediciny
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history of medicine, history of surgery, coloproctological diseases, intestinal stoma history of medicine, history of surgery, coloproctological diseases, intestinal stoma An intestinal stoma is an anastomosis (link) between the intestines and the skin surface, created by a surgeon (ar­ tificially) or which is a complication of a wound or the natural course of a pathological process (for example, a tumour). When literally translated “stoma” is Greek for “mouth”. Stoma surgery relates to the field of coloproc­ tology, which morphed into a separate area of surgery in the middle of the last century. the development of surgery was haphazard and empirical, and usually boiled down to the accumulation and sharing of isolated facts and personal experiences. Against this backdrop, the evolution of views on the intestinal stoma, in our opinion, went through five stages. Abstract The history of intestinal stoma surgery spans more than one century. Awareness of the possible benefits of creating an intestinal stoma came to the medical community long before the practical possibilities of its safe formation. At the same time, the evolution of the attitude towards the intestinal stoma has passed the stages of understanding the possibility of creating an artificial anus, un­ derstanding the need and expediency of such an intervention through the search for optimal techniques and the type of the stoma itself. Its connotation as the final stage of treatment of various coloproctological diseases has been formed amid the emergence of modern asepsis and antiseptics. And as a result of improving the operational technique of the main stage of the operation, the colo-(ileo-)stoma became only a temporary companion of a significant part of patients. The improvement of care methods has led to a significant improvement in the quality of life, even in life-long stoma patients. i The analysis of the content of works on the description of the use of intestinal stomas in surgery of the abdomen allows us to formulate ideas about the evolution of views on the role and importance of an unnatural anus, in the treatment of diseases and injuries of the intestine. Until the 18th century, professional physicians accumulated isolated personal experience and came to the realization of the possibility of excretion of feces bypassing the rectum. In the 18th century, the fatalism of passive observation of the patient is replaced by active tactics of struggle for their life. In the 19th century, surgeons in Europe are searching for the optimal localization of intestinal stoma and expand the indications for its formation. Since the beginning of the 20th century, there is a variety of kinds of stomas, expanding the range of indications for it. In the second half of the 20th century, the passion for technical aspects of stoma-surgery is replaced by stoma-therapy to improve the quality of life patients with a permanent stoma. History of Medicine, 2019, 6(2): 111–117 DOI: 10.17720/2409-5834.v6.2.2019.07g History of Medicine, 2019, 6(2): 111–117 DOI: 10.17720/2409-5834.v6.2.2019.07g Received:  04 March 2019    Accepted:  06 May 2019     Published online: 15 July 2019 Received:  04 March 2019    Accepted:  06 May 2019     Published online: 15 July 2019 Citation: Garmanova TN, Kazachenko EA, Krylov NN (2019) History of surgery: the evolution of views on the formation of intestinal stoma. History of Medicine 6(2): 111–117. https://doi.org/10.17720/2409-5834.v6.2.2019.07g Tatiana N. Garmanova1, Ekaterina A. Kazachenko1, Nikolay N. Krylov1 1 FSAEI HE I.M. Sechenov First MSMU MOH Russia (Sechenov University) 8 Trubetskaya St., building 2, Moscow 119991, Russia 1 FSAEI HE I.M. Sechenov First MSMU MOH Russia (Sechenov University) 8 Trubetskaya St., building 2, Moscow 119991, Russia Corresponding author: Nikolay N. Krylov (nnkrylov01@yandex.ru) History of surgery: the evolution of views on the formation of intestinal stoma Tatiana N. Garmanova1, Ekaterina A. Kazachenko1, Nikolay N. Krylov1 Second stage: fatalism replaced by active tactics Until the early 18th century, in case of intestinal injury, a wait and see tactic was used with respect to the natural course of a pathological process. The first attempts of ac­ tive intervention in the course of intestinal diseases and injuries - the purposeful formation of an intestinal stoma – relate to this period only. These attempts comprised the pulling out and fixation of the edges of the opening in the intestine on the anterior abdominal wall, which facilitated the discharge of faecal matter. The ideas of passionary physicians gradually came into fruition. More reliable is the account of the first attempt at su­ turing an intestinal defect after injury and interrupting the natural course of events, made in the late 15th century by Hieronymus Brunschwygk (Loria 1948). Still, in the 16th century, the father of military medicine, Ambroise Paré, kept with tradition and recommended to only stick to the elimination of the present eventration without color­rhaphy (Dunphy 1970, Paré 1951). Only Paracelsus suggested creating an artificial path into the intestines af­ ter observing the self-healing of intestinal stomas after abdominal injury. In 1701, Jean Méry, a surgeon at the Hôtel-Dieu hospital in Paris, probably for the first time in the world, was forced to form an “anus contre nature” in a female patient with a strangulated inguinal hernia. He cut out the necrotised portion of the strangulated intes­ tine, and a colic stoma was formed in the pubic region.i In France, in 1776, following his plan rather than re­ lying on the long arm of coincidence, H. Pillore was able to form the first ever colostomy. Until then, attempts had been made to resolve the intestinal obstruction of his pa­ tient by administering a large amount of laxatives and mercury, which turned out to be ineffective. During digi­ tal examination, Pillore detected stricture mimicking rec­ tal cancer and created a cecostoma on the wine merchant, Morel, by sewing the edge of the opening in the caecum to the anterior abdominal wall. The patient died on the 28th day due to complications caused by taking a large amount of mercury. The autopsy showed that the stoma was tightly fixed to the skin (Dinnick 1934). First stage: realization of the possibility of excretion of faeces Surgery evolved from a craft into medical science and became an art after the emergence of anaesthesia (gen­ eral and local), the emergence of topographic anatomy, the emergence of ideas about asepsis and antiseptics, as well as transfusion medicine. Until then, i.e., before the second half of the 19th century and the early 20th century, In the early days of the advent of medical knowledge and skills, people had to rely on folk medicine and pro­ fessional healers. However, for a long time, diseases and injuries of internal organs of the chest and abdo­ armanova, EA Kazachenko, NN Krylov. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0) Garmanova TN, Kazachenko EA, Krylov NN: History of surgery: the evolution of views on the formation of intestinal stoma The names of the first few patients whose treatment comprised the forced creation of an intestinal fistula are known. The first among them was George Deppу, who had sustained an abdominal wound during the Battle of Ramilles on 23 May 1706, after which he had a colostomy formed, which functioned for the remaining 14 years of his life. In 1737, the Queen of Great Britain, Caroline of Brandenburg-Ansbach, the wife of George II, had a spon­ taneous intestinal stoma (“Royal stoma”) after rupturing the membrane of strangulated umbilical hernia. Because the strangulation led not only to obstruction, but bowel gangrene as well, she died three days later. In 1750, Wil­ liam Cheselden operated on 73-year old Margaret White for strangulated umbilical hernia related to incoercible vomiting and cut out 55 cm of the intestine, which was fixed at the level of the hernial orifice. Despite the horri­ ble hygiene conditions, she survived and for a long time cared for the peristomal skin with a towel and a rag (Wu 2012, Cromar 1968 Kingsnorth 2006, Cheselden 1750). men remained terra incognita. In case of a penetrating abdo­minal wound with the opening of a bowel lumen, ancient physicians (Hippocrates, Celsus, Galen) could only rely on the demarcation of the wound from the va­ cant abdo­minal cavity and the natural formation of an intestinal stoma (Graney and Graney 1980), which in rare cases could close on its own. More often than not, until the 19th century inclusively, most penetrating ab­ dominal wounds were considered fatal (Adams 1983). First stage: realization of the possibility of excretion of faeces Celsus is said to have tried to sew up abdominal wall wounds, but usually witnessed a fatal outcome. He, there­ fore, came to the idea that nothing could be done once the small intestines are injured. However, if the large intes­ tine was attached to the abdominal wall at the point of the wound, the patient would survive. Furthermore, he noted that constricted hernia sometimes formed its own outlet through the intestinal stoma (Dinnick 1934). However, Caelius Aurelianus (the 2nd century B.C.) cited Praxagoras (the 4th century B.C.) – the mentor of Herophilos and the cousin of Hippocrates – who argued that, in the absence of the effect of medical treatment of intestinal obstruction, an abdominal incision had to be made in the pubic region, the large intestine had to be opened, its contents removed and the would sewn up (Caelius Aurelianus… 1950, Wu 2012).i Second stage: fatalism replaced by active tactics He sutured the edges of the intestinal wound to the anterior abdominal wall and left the patient until full recovery (Cataldo 1999, Larrey 1823).i colon through an incision in the lumbar region near the edge of the lumbar quadrate muscle and the forma­ tion of an artificial anus at that point was a question­ able solution. However, it was easier to reach the large intestine particularly at that point than in the pelvic area (Callisen 1800). Abdominal gunshot wounds with intestinal injury were associated with an unfavourable outcome. Hopes for the spontaneous formation of a stoma or the Larrey oper­ ation were still considered less feasible. L. J.-B. Baudens performed probably the first laparotomy in the history of surgery for a gunshot wound with suture of the in­ testinal wound during the French conquest of Algeria in 1830. One of the two patients survived (Baudens 1836). Encouraged by this success, with increasing frequency, the surgeon performed digital exploration of wounds during the Crimean War (1853–1856). And upon detec­ ting faeces and gases, he insisted on laparotomy under anaesthesia, arguing that celiotomy would be used more frequently in case of injury to internal organs of the abdo­ men (Baudens 1858).i The first reference to special-purpose faecal collectors was made in 1795. Through an operation, M. Daguesceau formed an intestinal stoma in the pelvic area of a farmer who had sustained injury with damage to the intestines. The farmer was subsequently fitted with a small leather bag for collecting faeces, with which he lived until the age of 81 (Daguesceau 1844). Almost nothing is known about how other patients coped with the sanitation of the stoma and peristomial area.i In the first half of the 19th century, the formation of a colostomy during disease and not intestinal wounds, gradually transformed from one-off interventions into a category of operations familiar to a wider circle of physicians. However, due to the risk of peritonitis, not all surgeons were willing to perform it. In 1839, French surgeon Jean Zùlema Amussat analysed 29 case studies of the formation of a colostomy from 1776 (i.e., descrip­ tions of the first cases of colostomy by H. Pillore), where only four patients survived. In all cases, the operation was performed using abdominal approach (Amussat 1839, р. 204). Shaken by the death of his friend from obstruc­ tive rectal cancer, J.Z. 1 Translated by N.N. Krylov. 2 Stages of J.Z. Amussat’s operation: https://commons.wikimedia. org/wiki/File:Amassat%27s_lumber_colostomy,_stages_in_the_ operation._Wellcome_L0010081.jpg Second stage: fatalism replaced by active tactics In 1710, French surgeon Alexis Littré for the first time described the death of a six-day-old infant with an imper­ forate anus as a result of rectal atresia and suggested the formation of an intestinal stoma as a method of treating this birth defect (Littre 1710). The idea was too revolu­ tionary and it was only in 1757 that German anato­mist and surgeon, Lorenz Heister, returned to the idea of the need and the possibility of creating an intestinal stoma in the event intestinal injury or disease (Tebala 2015, Heister 1743). He argued that, since the edge of the in­ testinal wound can spontaneously fuse with the edge of abdominal wall wound, this tip from nature had to be utilised. Colleagues argued that the exteriorisation of the intestines was extremely unpleasant for the patient. His argument was that it was better to give up one of the con­ veniences of life than life itself (Cataldo 1999). In 1783, A. Dubois unsuccessfully attempted to realise Littré’s idea: his three-day old patient lived just 10 days after the colostomy (Amussat 1839, р. 88). It was only three years later, in 1793, when a newborn with proctatre­ sia was successfully operated on for the first time. C. Duret created a colostomy in the left iliac region of a four-day- old infant, who went on to live with the colostomy for 45 years (until his death). Duret initially used this technique on a deceased 15-year old patient, and then decided to use the experience in clinical practice (Duret 1798). 112 History of Medicine, 2019, 6(2): 111–117 Double-barrel colostomy was formed for the first time in 1797 by P. Fine in Geneva on a 63-year old woman with an obstructive tumour of the rectosigmoid junc­ tion. The patient lived for 3.5 months after the operation. During the autopsy, it turned out that the surgeon had cre­ ated a transverse colostoma instead of an ileostomy as intended (Amussat 1839, р. 109, 114). During the Battle of Abukir in 1799, Dominique Larrey fixed the damaged intestines that were lying freely in the abdominal cavi­ ty of a soldier that had sustained an abdominal gunshot wound. Second stage: fatalism replaced by active tactics Amussat set out to find a way to treat colorectal cancer: “I was well prepared to no longer remain a passive spectator of death by obstruction of the rectum...”1 (Magill 1895, р. 34). p Individual and collective experience in this field was gradually accumulated. Despite the risks, it became clear that colostomy prolongs the life of patients with intesti­ nal obstruction caused by colon cancer, with abnormal development of the rectum, as well as with penetrating abdominal wounds, at a time when the creation of an in­ testinal anastomosis was not yet possible. In cases with cance­rous diseases, the formation of a stoma could cer­ tainly not prolong the life of patients, since no radical surgery was performed and the tumour was not removed from the patient. At this stage of the development of med­ icine, the formation of an intestinal fistula was a forced measure in cases when non-surgery treatment was im­ possible. Furthermore, there were no special techniques for caring for the stoma and the risk of complications was high due to the rudimentary equipment, as a result of which surgeons themselves opted not to perform the operation. Among physicians, attitude towards this tech­ nique was rather negative. One would agree with S.S. Yudin’s opinion of the surgery of that time: “In those old days, the determination of surgeons had to contend with the inflexible will to live of the patients themselves” (Iu­ din 1955, p. 16). In order to eliminate the risk of peritonitis, he elaborated the idea of the extraperitoneal formation of a colostomy via a transverse cut in the lumbar region.2 The change of ap­ proach enabled to expand indications for the formation of a stoma. Although, J.Z. Amussat did not put forward a meth­ od of treating colorectal cancer, he considered the easing of the suffering of patients with acute intestinal obstruction (Amussat 1856) to be his main achievement. In England in 1841, independent of the French sur­ geon’s work, J. Erichsen, based on previously published data, articulated the expanded indications for the forma­ tion of a colostomy: rectal atresia, treatment-resistant constipation, colon obstruction and rectal cancer with severe pain (Erichsen 1841). These indications in parti­ cular were taken into account by T. Billroth, who opera­ Third stage: the search for the optimal localization of intestinal stoma (analysis of alternatives) and expansion of indications for it Fourth stage: variety of kinds of stomas, wide range of indications The first half of the 20th century is associated with the rapid development of new areas of surgery, including colorectal surgery. By that time, results (successes and failures) of the use of the intestinal stoma had been ac­ cumulated, which enabled to develop a new tactic. A non-radical analogue of this operation, but sim­ pler in terms of technical execution, was the formation of an end colostomy in 1884 by O. Madelung from Rostock and M. Polloson from Lyon for treating rec­ tal cancer. It was performed through laparotomy with complete cut-off of the sigmoid colon loop, where the afferent end was pulled into the wound onto the ante­ rior abdominal wall, leaving the lumen open, and the distal end was sewn tightly and sunk into the abdomi­ nal cavity. The creation of such a colostomy marked a new stage in the development of colon surgery, since it enabled to avoid the creation of an intestinal anas­ tomosis after its cutting. It also prevented contents of the intestines from entering the distal segment directly above tumour, and it could be a palliative alternative to emergency surgery for an inextricable tumour (Made­ lung 1884, Polloson 1884). The alternative radical operations for colon can­ cer that had been developed required the formation of a colostomy for temporary or permanent removal of faeces in the postoperative period. The established position was to perform as the first stage, in the most severely stricken, debilitated patients, preliminary in­ testinal decompression with a preventive (discharge) stoma – cecostoma (according to G.F. Zeidler, 1897) or transversosigmostomy (according to H. Schloffer, 1903). This enabled to plan and implement the two- or three-stage tactic for treating tumorous intestinal ob­ struction in the approaching 20th century. The second stage comprised resection of the intestine carrying the tumour, after which the safety intestinal fistula was re­ moved at the third stage. The creation of a temporary intestinal stoma prevented intestinal contents from en­ tering the distal segment of the intestines during the healing of the intestinal anastomosis sutures (Tebala 2015, Cataldo 1999). An alternative to single-barrel colostomy was pro­ posed for the first time in 1885 by Russian surgeon A. Kni from Moscow. He suggested that double-barrel colostomy, which removes faeces, had to be performed, since the spurs separating the two parts of the intestines are enough to prevent faeces from entering the distal efferent limb. Third stage: the search for the optimal localization of intestinal stoma (analysis of alternatives) and expansion of indications for it In 1800, H. Callisen from Copenhagen insisted on the need for translumbar approach when performing co­ lostomy since he considered the transabdominal ap­ proach dangerous due to the high risk of peritonitis. He explained that access to the caecum or descending 113 Garmanova TN, Kazachenko EA, Krylov NN: History of surgery: the evolution of views on the formation of intestinal stoma ted on N.A. Nekrasov on 12 April 1879 for progressive rectal obstruction, caused by stricture mimicking cancer. Colostomy under chloroform anaesthesia (Belogolovy 1878) was conducted in the poet’s apartment (36 Liteyny Prospect, Saint Petersburg) by retroperitoneal approach as required by J.Z. Amussat. The use of such a rod enabled to create an artificial anus which protrudes above the surface of the body, which enabled complete discharge of the contents of the intes­ tines and became the next stage in the evolution of the quest for the most effective and safest intestinal stoma (Allingham 1887).i The first ileostomy was formed nearly 150 years after the first colostomy, in 1879, by German surgeon W.G. Baum. A patient with intestinal obstruction during proximal colon cancer had an ileostomy formed as the first stage of treatment. After 8 weeks, resection of the ileocecal segment with ileocolostomy was performed. Unfortunately, the patient died on the 9th day after the operation due to the failure of anastomosis sutures and peritonitis (Cataldo 1999, Baum 1879). The general atmosphere in those days was vividly described by German surgeon G. Wegner in 1876, who stressed that, like himself, his contemporaries were brought up in fear of God and peritonitis. This fear would loom over surgeons, right until the advent of antibiotics. In 1879, obstructive resection of the colon with a tu­ mour was performed for the first time in Austria: colonic – T. Billroth, and sigmoid – Carl Gussenbauer, with su­ ture of the peripheral end, its sinking into the abdominal cavity and the pulling out of the proximal end onto the abdominal wall in the form of an end colostomy. Due to the absence of an entero-entero anastomosis and the possible risk of failure of its suture, this way of ending the operation enabled to drastically reduce postoperative lethality (Billroth 1879, Magill 1895, Maydl 1888). Fourth stage: variety of kinds of stomas, wide range of indications The widespread use of temporary intestinal stoma fa­ cilitated the accumulation of data on the nature, sever­ ity and frequency of complications which significantly influence mortality rate. D. Patey (1951) and C. Butler (1952) offered to perform abdominoperineal extirpa­ tion of the rectum during cancerous diseases with the formation of a skin-intestinal suture. The introduction of this technology into practice and the extraperitoneal location of the colostomy enabled to lower the risk of such dangerous complications as prolapse and stenosis. In 1952, B. Brooke proposed pulling out the mucous lining of the stump of the ileum and attaching it to the anterior abdominal wall by suture, thereby reducing the probability of the common dilatation of the stenosed “column” of the ileum (Brooke 1952).fi i Successes in the surgery of rectal and colon can­ cer occurred concurrently with the improvement of the technique of creating stomas. C.H. Mayo in 1904 (Mayo and Dixon 1928) and W.Е. Miles in 1908 (Miles 1908) explained the creation of a terminal colostomy when completing abdominoperineal extirpation of the rectum. It enabled to perform the operation as much radically as possible, which in turn reduced the fre­ quency of tumour recurrence and improved the long- term outcome. Thus, colostomy gained its place in the treatment of rectal cancer. J.P. Lockhart-Mummery in 1907 developed a technique for two-stage intermediate resection of the rectum: a loop colostomy was created 10 days prior to the intermediate resection of the rec­ tum (Lockhart-Mummery 1923).i Caring for the stoma was extremely difficult until the 1950s, when R. Turnbull introduced the concept of the rehabilitation of stoma patients (Turnbull and Turnbull 1991). Various modifications of the creation of intesti­ nal stomas were subsequently proposed, although from the second half of the 20th century the basic concept of their use did not change significantly. Technical inno­ vations (particularly the now commonplace mechani­ cal, staple, intestinal suture) enabled to standardise the results of operations performed by surgeons worldwide, reduce the risk of postoperative complications asso­ ciated with both the presence of the stoma and the pos­ sible failure of anastomosis sutures, which undoubtedly considerably improved the overall results, length and quality of life of patients. In 1921 H. Fourth stage: variety of kinds of stomas, wide range of indications The surgeon tested his ideas during ex­ periments on animals (Knie 1885). In 1888, K. Maydl published the description of a new technique of form­ ing a loop stoma in clinical practice. The intestine loop was pulled out of the abdominal cavity and, through the mesentery; a support rod was placed underneath it. Even a goose quill was used emergency situations (Maydl 1888). The idea was also used by H.W. Alling­ ham in 1887. He described the formation of a colostomy using a glass rod which was passed underneath a middle intestine loop, which prevented its retraction into the abdominal cavity until the edge of the intestines were fused with the edge of the wound of the abdominal wall. The colostomy played the same role during J. Miku­ licz’s multi-stage treatment of colon cancer. Not satis­ fied with the results of single-step resection with forma­ tion of a primary anastomosis, he described three-stage treatment of colon cancer in 1903. The first stage com­ prised pulling out part of the intestines with the tumour and creating a colostomy from the proximal end. The pulled loop with the tumour was cut out and the two limbs of the intestines (proximal and distal) were sewn together with the subsequent extended restoration of the continuity of the intestines. J. Mikulicz published material on 16 cases of this operation (one fatal case) in 1903 (Mikulicz 1903). 114 History of Medicine, 2019, 6(2): 111–117 widespread use of this technique. This turned out to be critical in patients with pancolitis, the removal of which required the life-long presence of an ileostomy. How­ ever, after A. Parks proposed the creation of an ileal pouch (“neorectum” from the ileum) with preservation of the anal sphincter, ileostomy evolved into a category of a temporary measure for removal of faeces (Parks et al. 1980). With time, the relative advantages and shortcomings of extraperitoneal resection with colostomy became clear, since the main operation becomes shorter, the likelihood of infectious complications is lower and the probability of the patient recovering quickly is higher. He argued that all this outweighs the inconvenience of a colostomy on the anterior abdominal cavity. The cre­ ation of a preventive stoma enabled him to reduce the frequency of lethal complications associated with sin­ gle-step resection from 50% to 12.5% – based on the results of the first 100 operations (Mikulicz 1937). Fourth stage: variety of kinds of stomas, wide range of indications Hartmann proposed a modification of the radical treatment of pelvic colon cancer, which in­ cludes the resection of the rectosigmoid segment, tight­ ly sewing the distal end, placing the rectal stump below the pelvic peritoneum and creating a terminal colosto­ my from the proximal end. This operation essentially embodied the previous idea put forward by T. Billroth and C. Gussenbauer, albeit in an area more difficult for manoeuvre. This operation was proposed as an alter­ native to abdominoperineal rectum resection. H. Hart­ mann followed the concept of delayed formation of an entero-entero anastomosis with elimination of the sto­ ma. However, he admitted the extremely high technical difficulty of restoring faecal flow. As a result, nearly a hundred years from the description of the operation, Hartmann allows for the formation of the continent co­ lostomy, since later on up to 30–50% of patients do not return to have the stoma closed (Hartmann 1931). Fifth stage: stoma surgery gives rise to stoma therapy The creation of temporary or permanent intestinal sto­ mas became a common operation in the 20th century. However, right up to the 1970–1980s, there were no special tools for caring for stomas. In 1924, a proposal was made to wash the colostomy, which was the only way of looking after stomas. The attachable colosto­ my bag was invented by Danish nurse Elise Sørensen in 1954. Norma Gill, a patient with an ileostomy, who had been operated on by American surgeon Turnbull for ulcerative colitis, became the first professional in providing care to stoma patients in 1958. The first de­ vice for collecting intestinal contents with odour con­ tainment was invented in the 1980s. Stoma patients now had a choice between the standard procedure of The use of indications to ileostomy became wide­ spread in the 20th century. Ileostomy could be tem­ porary during aggravation of ulcerative colitis, and the natural flow of the faecal stream was restored after the onset of remission. The technique was proposed in 1913 by J.Y. Brown (Brown 1913). After the publication of the work of C.C. Miller et al in 1949, with increasing frequency surgeons resorted to total colproctectomy with permanent ileostomy for radical treatment of se­ vere ulcerative colitis (Miller et al. 1949). The lack or temporary bypass of the colon in such patients normally led to complications (uncontrolled dehydration, peristomal dermatitis), which hampered 115 Garmanova TN, Kazachenko EA, Krylov NN: History of surgery: the evolution of views on the formation of intestinal stoma in just 15–25% of rectal cancer patients that have un­ dergone radical surgical treatment. washing the stoma or a tool for collecting intestinal contents (Ca­taldo 1999). Therefore, the history of intestinal stoma surgery spans more than one century. The recognition of the possible benefits of creating an intestinal stoma came to the medical community long before the practical possibilities of its safe formation. At the same time, the evolution of the attitude towards the intestinal stoma has passed the stages of understanding the possibility of creating an artificial anus, understanding the need and expediency of such an intervention through the search for optimal techniques and the type of the stoma itself. Its connotation as the final stage of treatment of var­ ious coloproctological diseases was formed amid the emergence of modern asepsis and antiseptics. References Adams DB (1983) Abdominal Gunshot Wounds in Warfare: A Historical Review. Military Medicine 148 (1): 15–20. https://doi.org/10.1093/ milmed/148.1.15 Callisen H (1800) Chirurgia imperforationis ani. Systema Chirurgiae Hodiernae in Usum Publicum Et Privatum Adornatum. Hafniae. P. 688–689. Cataldo P (1999) Intestinal stomas: 200 years of digging. Diseases of the Colon and Rectum 42 (2):137–142. https://doi.org/10.1007/ BF02237118 Allingham HW (1887) Inguinal colotomy; its advantages over the lum­ bar operation, with special reference to a method for preventing fæces passing below the artificial anus. British Medical Journal 2: 874–878. Cheselden W (1750) The anatomy of the human body. Case report of Margaret White. London. 324 p. Amussat JZ (1839) Mémoire sur la possibilité d’établir un anus artificiel dans la région lombaire sans pénétrer dans le péritoine. Paris. Cromar CDL (1968) The evolution of colostomy. Diseases of the Colon and Rectum 11 (6): 423–446. doi: 10.1007/BF02616774 Amussat JZ (1856) Memoires sur l’enterotomie du gros inestin. G. Baillière Paris. 484 p. Daguesceau M (1844) Artificial anus. The Medical Times and Ga­ zette10: 446 р. Baudens LJ-B (1836) Clinique des plaies d’armes à feu. Paris: Jean-Bap­ tiste Baillière. 610 p. Dinnick T (1934) The origins and evolution of colostomy. British journal of Surgery 22: 142–154. Baudens LJ-B (1858) La Guerre de Crimée. Les campements, les bris, les ambulances, les hôpitaux. Paris: Michel Lévy frères. 412 p. Baudens LJ-B (1858) La Guerre de Crimée. Les campements, les ( ) p les ambulances, les hôpitaux. Paris: Michel Lévy frères. 412 p. Dunphy JE (1970) The cut gut. The American Journal of Surgery 119: 1–8. Baum WG (1879) Resection eines carcinomatosen Dickdarmstuckes. Centralblatt fur Chirugie 6: 169–176. Duret C (1798) Observation sur un enfant ne sans anus. Receuil Pério­ dique 4: 45–50. Erichsen J (1841) On the formation of artificial anus in adults, for the relief of retention of the faeces. London Medical Gazette 2: 223–227. Belogolovy NA (1878) Bolezn Nikolaya Alekseevicha Nekrasova [Dis­ ease of Nikolai Alekseevich Nekrasov]. Otechestvennye zapiski 10: 314–340. (In Russ.) Graney MJ, Graney CM (1980) Colorectal surgery from the antiquity to the modern era. 23: 432–441. Billroth T (1879) Anus praeternaturalis; Enteroteraraphie; Heilung. Arch. J. Klin. Chir. Berl. 18 (9): 582–585. Hartmann PH (1931) Chirurgie du rectum. Masson. 398 р. Brooke BN (1952) The management of an ileostomy including its com­ plications. Lancet 2: 102–104. Heister L (1743) A general system of surgery in three parts. London: W. Innys, C. Davis, J. Clark, R. Fifth stage: stoma surgery gives rise to stoma therapy And as a result of improving the operational technique of the main stage of the operation, the colo-(ileo-)stoma be­ came only a temporary companion of a significant part of patients. The improvement of care methods has led to a significant improvement in the quality of life, even in life-long stoma patients. Attempts were made to create an alternative to colosto­ my bags by creating reservoirs and continent stomas. The technique of creating a “containment” ileostomy was first proposed by N. Kock in 1969 (Kock 1969). Numerous attempts were subsequently made to create the so-called continent stoma. However, most of the attempts were un­ successful. New complications emerged and the degree of containment left a lot to be desired. The proposed tech­ niques did not grain widespread currency. In the early 21st century, with the emergence of sphincter-saving operations, the need for intestinal stomas dropped drastically; operations are increasing­ ly performed with the primary creation of an intestinal anastomosis. However, due to its “low” position, the frequency of failure of its mechanical sutures is rela­ tively high. Hence the creation of a preventive stoma became the cornerstone of success in preventing this complication. Thanks to the development of new treat­ ment techniques, today a permanent stoma is necessary References International abstracts of surgery 87: 21–549. Polloson M (1884) Nouvelle méthode opératoire pour la cure radicale de cancer du rectus. Lyon Medical 17: 28–31. Madelung О (1884) Über eine Modifikation der Colotomie wegen Car­ cinoma recti. Centralblatt Chir 11: 68–69. Tebala GD (2015) History of colorectal surgery. A comprehensive his­ torical review from the ancient Egyptians to the surgical robot. 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Access mode: https://archive.org/details/generalsystemofs1743heis/ Brown JY (1913) The value of complete physiological rest of the large bowel in the treatment of certain ulcerative and obstructive lesions of this organ. Surgery, gynecology and obstetrics 16: 610–613. Iudin SS (1955) Etyudy zheludochnoy khirurgii [Etudes of Gastric Sur­ gery]. Moscow: Medgiz. (In Russ.) this organ. Surgery, gynecology and obstetrics 16: 610–613. Kingsnorth AN (2006) Hernia Management. Fundamentals of Surgical Practice. Ed. A.N. Kingsnorth and A.A. Majid. Cambridge University Press. 265 p. Kingsnorth AN (2006) Hernia Management. Fundamentals of Su Caelius Aurelianus. On acute diseases and on chronic diseases (1950). Edited and translated by Drabkin I.E. Chicago: The University of Chicago Press. P. 403. 116 History of Medicine, 2019, 6(2): 111–117 Knie A (1885) Zur Technik der Kolotomie. Centralblatt Chir 25: 433– 436. Mikulicz von J (1903) Chirurgische Erfahrungen über das Darmcarci­ nom. Arch klin Chir 69: 28–47. Mikulicz von J (1903) Chirurgische Erfahrungen über das Darmcarci­ nom. Arch klin Chir 69: 28–47. Kock NG (1969) Intra-abdominal “reservoir” in patients with permanent ileostomy. Preliminary observations on a procedure resulting in fecal “continence” in five ileostomy patients. The Archives of Surgery 99 (2): 223–231. Mikulicz von J (1937) Surgical experiences with intestinal carcinoma. Medical Classics 2: 210–229. Miles WE (1908) A method of performing abdomino-perineal excision for carcinoma of the rectum and of the terminal portion of the pelvic colon. Lancet 2: 1812–1813. Larrey DJ (1823) Surgical Essays. Sonic observations on wounds of the intestines. Translated from French by John Revere. Baltimore: N. G. Maxwell. 335 p. Miller CC, McGardner C, Ripstein GB (1949) Primary resection of the colon in ulcerative colitis. Canadian Medical Association Journal 60: 584–585. Littre A (1710) Diverses observations anatomiques. Histoire de l’Acadé­ mie Royale de Science. Paris. P. 36–37. Paré A (1951) The apologie and treatise of Ambroise Paré, containing the voyages made into divers places with many of his writings upon Lockhart-Mummery JP (1923) Diseases of the rectum and colon and their surgical treatment. London: Baillière Tindall & Cox. 872 р. surgery. Ed. G. Keynes. London: Falcon Educational Books. 206 р. Parks AG, Nicholls RJ, Belliveau P (1980) Proctocolectomy with ileal res­ ervoir and anal anastomosis. British Journal of Surgery 67: 533–538. Loria FL (1948) Historical aspects of penetrating wounds of the Loria FL (1948) Historical aspects of penetrating wounds of the abdo­ men. International abstracts of surgery 87: 21–549.i men. About the authors Tatiana Nikolaevna Garmanova – Candidate of Medical Sciences, Docent at the Department of Surgery, Faculty of Medicine and Prevention, FSAEI HE I.M. Sechenov First MSMU MOH Russia (Sechenov University), Moscow. Email: tatianagarmanova@gmail.com Ekaterina Aleksandrovna Kazachenko – 4th-year student at the Medical faculty, FSAEI HE I.M. Sechenov First MSMU MOH Russia (Sechenov University), Moscow. Email: ekaterina.k.97@mail.ru Ekaterina Aleksandrovna Kazachenko – 4th-year student at the Medical faculty, FSAEI HE I.M. Sechenov F ( h ) l k k l Aleksandrovna Kazachenko – 4th-year student at the Medical faculty, FSAEI HE I.M. Sechenov First MSMU ussia (Sechenov University), Moscow. Email: ekaterina.k.97@mail.ru Ekaterina Aleksandrovna Kazachenko 4th year student at the Medical faculty, FSAEI HE I.M. Sechenov F MOH Russia (Sechenov University), Moscow. Email: ekaterina.k.97@mail.ru Nikolay Nikolaevich Krylov – Doctor of Medical Sciences, Professor at the Department of Human Studies, Insti­ tute of Social Science, FSAEI HE I.M. Sechenov First MSMU MOH Russia (Sechenov University), Moscow. Email: nnkrylov01@yandex.ru 117 117
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“Монголын тухай БХК(б)Н-ын баримт бичигт” (1920-1932) архивын баримт бичгийн эмхэтгэлийн тухай
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НОМЫНШҮҮМЖ НОМЫНШҮҮМЖ НОМЫНШҮҮМЖ 157 ,О.Батсайхан - ОУСХ-ийн эрдэм шинжилгээний тэргүүлэх ажилтан, доктор(Зс.О), профессор «Монголын тухай БХК(б)Н-ын баримт бичигг» (1920 一 1932) архивын баримт бичгийн эмхтгэлийн тухай О.Батсайхан Ардчилал шинэчлэлийн үрээр ОХУ - аас урьд хаалттай байсан архивын фондуудыг, ялангуяа БХК(б)Н-ын Төв Хорооны Улс төрийн Товчооны маш нууцын онцгой зэрэглэл бүхий тэмдэгтэй «тусгай хавтаст» баримтуудыг нээлттэй болгосны ачаар түүхийн үнэн бодит мэдээлэл, сурвалж агуулсан баримт бичгүүдийг судлаачид судлан илрүүлж, улмаар өргөн олон түмний хүртээл болгох сайхан боломж нээгдсэн билээ, 1992 онд байгуулагдсан Архивын салбарт хамтран ажиллах Монгол - Оросын Засгийн газар хоорондын хамтарсан комиссын ажлын хүрээнд доктор С.Дамдинсүрэн, доктор Шепелев В.Н, нарын хоёр улсын судлаачид сүүлийн жилүүдэд Оросын төрийн нийгэм улс төрийн түүхийн архив (РГАСПИ - Российский государственный архив соииально-политической истории) 一 ын фондуудад хайлт судалгаа хийж, ОК(б)Н, БХК(б)Н -аас Монголын талаар явуулж байсан бодлого, үйл ажидлагаанд холбогдох 1920 - 1952 оны үеийн олон мянган хуудас баримт бичгийг олж ирүүлсэн юм. Үүний үр дүнл «Монголын тухай БХК(б)Н-ын баримт бичигт» гэсэн архивын эх сурвгшж баримт бичгийн эмхтгэлийн 1 дүгээр ботийг (1920-1932) 2002 онд, 2 дугаар ботийг (1933-1952) 2003 онд тус тус хэвлэн нийтлэх, дараагийн он жилүүдайн өөрөөр хэлбэл 1953-1991 оны баримт бичгүүдайг нээлтгэй болгохын хэрээр судлаж илрүүлж үргэлжлүүдэн боть болгож дэс дараатайгаар хэвлүүлэхээр төлөвлөсөн болно. Энэ 2002 онд хэвлэгдэн гарч буй дээрх баримт бичгийн эмхтгэлийн нэгдүгээр боть болбоос Монголын түүхийн нэгэн онцлог үе болох 1920 онд Монгол Ардын Намын төлөөлөгчид ОК(б)Н-ын Төв Хорооны Сибирийн Товчоотой холбоо тогтоосон үеэс 1932 онд БХК(б)Н-ын Төв Хорооны Улс Төрийн Товчооны Монголын комиссыг байгуулж Монголд явуулах бодлогыг үндсээр нь өөрчилж «Шинэ эргэлтийн бодлого» хэмээхийг хэрэгжүүлж эхэлсэн үеийг хамарч байна. - Энэхүү эмхтгэлд ОК(б)Н - БХК(б)Н-ын Төв Хорооны Улс Төрийн Товчоо, Зохион байгуулах Товчоо, Нарийн бичгийн дарга нарын газар зэрэг дээд удирдлагын түвшинд болон ОК(б)Н-ын ,О.Батсайхан - ОУСХ-ийн эрдэм шинжилгээний тэргүүлэх ажилтан, доктор(Зс.О), профессор тсайхан - ОУСХ-ийн эрдэм шинжилгээний тэргүүлэх ажилтан, доктор(Зс.О), ссор 158 OnOHWICCWLWI Сибирийн Товчоо, Алс Дорнодын Товчоо зэрэг бүс нутгийн байгууллагуу/1ын хүрээнд Монголын талаар гаргаж байсан тоггоол, шийдвэр, протокол, тэмдэглэл, удирдамж, зөвдөмжү\щ5 ЗСБНХУ-ын Ардын комиссариатын Зөвлөлиин зарим чухал шийдвэр, хоёр орны удирдагчид, төлөөлөгчдийн янз бүрийн түвшний уулзалт ярианы тэмдэглэл, харилцсан захидал, бичиг цахилгаанууд зэрэг урьд өмнө нийтлэгдээгүй үлэмж хэмжээний чухал баримт, эх сурвалжууд орсон байна. Эмхтгэлийн хэвлэгдэн буи нэгдүгээр ботид нийт 191 баримтыг сонгон оруулсаны дөч шахам хувь нь ОК(б)Н -ын Төв Хорооны Улс Төрийн Товчооны «тусгай хавтаст» багтсан шийдвэрүүд байна. «Монголын тухай БХК(б)Н-ын баримт бичигг» (1920 一 1932) архивын баримт бичгийн эмхтгэлийн тухай - үү ОК(б)Н - БХК(б)Н-ын Төв Хорооны Улс Төрийн Товчоо, Зохион байгуулах Товчоо, Нарийн бичгийн дарга нарын газрын хура/шаанаас ЗХУ болон Монгол улсын цэрэг, yzic төр, эдийн засаг, гадаад харилиааны төдийгуи Монго/ш ажиллуулах боловсон хүчний асуудал, Монголын удирдлагын бүрэттдхүүн, уил ажиллагааны тухай асуудлыг ч авч хэлэлцэж, тодорхой шийдвэр гарган хэрэгжүүлж байсаныг эмхтгэ/щ орсон баримтуудаас харж болно. Монголын талаар Коминтерний бодлогыг боловсруулах, Монгол дахь Коминтерний төлөөлөгчдийн ажиллагааг чиглүүлэн үнэлэлт дүгнэлт өгч, залж, жолоодож байсан тухай ЗСБНХУ-ын дээд уиирдагчдын шийдвэрүүд эл эмхтгэ/щ тодорхой байр зззлж байна. Эмхтгэ/тд Коминтерн болон ГХАК-ын шугамаар Монготщ ажитшах чухал боловчон хүчнийг ОК(б)Н-ын Төв Хорооны Зохион байгуулах Товчоо, Нарийн бичгийн дарга нарын газар, Улс Төрийн Товчоогоор хэрхэн шалгаруулан томилж ажлын удирдамжийг хянан боловсруулж байсан тухай баримт бичгүүд, мөн Улс Төрийн Товчоо Коминтерний Гүйцэтгэх Хороогоор зуучлуулалгүй Монголын амьдралын чухал асуудлуудыг шууд шийдвэрлэж байсаныг харуулсан эх сурвалж бичгүүд орсон байна. Монголын ард түмний үндэсний эрх чөлөө, тусгаар тогтнолын төлөө тэмцэл, үндэсний ардаилсан хувьсгал, Монгол улсын дотоодын хийгээд олон улсын амьдралд тулгарч байсан чухал зангилаа асуухшуудын талаарх Зөвлөлтийн нам, төрийн дээд удир/шагын байр суурь, бодлогыг тусгасан баримт бичгуүдийг багтаасан нь эл эмхтгэлийн бас нэгэн онцлог болжээ. Монголчуудын үндэсний нэгдлийн тухай түухэн ёсоор дэвшигдэж, Монгол үндэстний х^ваагдмал байдлыг гэтлэн давах гэсэн зүй ёсны сонирхлыг илэрхийлсэн үзэл санааг бусад улс үндэстний эсрэг хандсан харгис үзэл мэтээр зэмлэн бурууггах, түүнд панмонголизм гэсэн нэр зүүж, үзэл суртал, улс төрийн хэт туйлширсан утга өгч хэрхэн гажуудуулах болсоныг харуулсан эх сурвалж бичгүүд цөөнгүй орсон нь баримт бичгийн эмхтгэлийн үнэ цэнийг улам өргөж байна. НОМЫНШҮҮМЖ НОМЫНШҮҮМЖ 159 Тус архивын баримт бичгийн эмхтгэлд томоохон байр эзэлж байгаа нэг гол чиглэл бол Монгол улсын эдийн засгийн амьдралыг төлөвшүүлэх, түүний дотор Монгол улсын банк санхүүгийн системийг шинээр үүсгэн байгуулахад онцгой анхаарал тавьж байсан тухай ба Зөвлөлт Монголын худалдаа эдийн эдийн засгийн харилцааг хөгжүүлэх чигдэлээр БХК(б)Н-ын Төв Хорооны болон ЗСБНХУ-ын АКнЗ-ийн гаргаж байсан тогтоол шийдвэрүүд юм. Иэрэг, батлан хамгаалах асуудал, эрүүл мэнд, боловсрол, соёл шинжлэх ухааны асуудлаар БХК(б)Н-ын Төв Хороо, удирдах төв байгууллагууд их анхаарал тавьж, тогтоол шийдвэр гаргаж байсан тухай баримтууд тусдаа бие даасан эмхтгэл болгохоор бэлтгэж байгаа тул энэ талын баримт бичгүудийг оруулаагүй болно. БХК(б)Н Монголын талаарх бүх талыг хамарсан бодлого хэрэгжүүлэхдээ Монголд ажиллах ЗСБНХУ^ын бүрэн эрхт төлөөлөгч, КИГХ-ны төлөөлөгч, худалдааны төлөөлөгч, Монгол банкны ерөнхий хорооны дарга, төрөл бүрийн комисс, экспедицид ажиллаж байсан хумүүсийн бүрэлдхүүнийг хэрхэн хянан бата/тж байсан тухай тэдгээрийн ажлын удирдамж, заавар, тайлан зэрэг баримтууд мөн Монголын өөрийнх нь боловсон хүчнийг бэлтгэхэд анхаарал тавьж байсныг харуулсан баримт бичгүүд эмхтгэлд тодорхой байр эзэлсэн байна. Энэхүү эмхтгэлд орж буй баримт бичгүүд бүхэ/шээ БХК(б)Н, Зөвлөлт төрөөс Монголын талаар явуулсан бодлого, үйл ажиллагаа, хоёр улсын хооронд тогтсон онцгой хэв маягийн харилцааны тухай урьд да/щ хаалттай байсан арвин баялаг мэдээллийн санг нээн өгч судлаач, сэхээтэн, оюутан, уншигч, өргөн олон түмний хүртээл болгож, хоёр улсын харилцааны түүхэн үнэнийг сэргээх, бидний танин мэдэхүйг уламжлал болсон өрөөсгөл ойлголт, элдэв гажуудлаас чөлөөлөх төдийгүй Монгол, Оросын найрсаг харилцаа, хоёр ард түмний өнөөгийн зорьж буй ардчилал, зах зээл, хүмүүнлэгийн харилцааны хөгжилд сургамж болох утгаараа үнэлж баршгүи ач холбогдолтой болжээ. Энэ ботид Зөвлөлт Орос улсаас Монголын талаар хийгээд Дорнодахинд явуулж баисан бодлого, Монгол Зөвлө/гг Ороснн хооронд тогтсон өвөрмөц харилцааны зарим асуудлуудыг тусгасан олон тооны барим бичгүүд анх удаа орж байна. Энэ ботид хамаарсан баримт бичгүүдийг судлаач түүх сонирхогч хэн бүхэнд үзэж таницдах боломж нээснээрээ юуны өмнө 1920 иод оны үеийн Зөвлөлт Орос улсаас Ази дахин, улмаар дэлхий дахинд явуулж байсаны бодаогыг улам тодруулах боломж өгөхийн зэрэшээ Монгол улсын түүх бодит байдлаар бичигдэх нөхцөл бурдэх юм.
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Psychometric properties of the Danish Hospital Anxiety and Depression Scale in patients with cardiac disease: results from the DenHeart survey
Health and quality of life outcomes
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Citation for pulished version (APA): Christensen, A. V., Dixon, J. K., Juel, K., Ekholm, O., Rasmussen, T. B., Borregaard, B., Mols, R. E., Thrysøe, L., Thorup, C. B., & Berg, S. K. (2020). Psychometric properties of the Danish Hospital Anxiety and Depression Scale in patients with cardiac disease: results from the DenHeart survey. Health and Quality of Life Outcomes, 18, Article 9. https://doi.org/10.1186/s12955-019-1264-0 Download date: 24. Oct. 2024 Open Access Open Access Psychometric properties of the Danish Hospital Anxiety and Depression Scale in patients with cardiac disease: results from the DenHeart survey Anne Vinggaard Christensen1* , Jane K. Dixon2, Knud Juel3, Ola Ekholm3, Trine Bernholdt Rasmussen4, Britt Borregaard5, Rikke Elmose Mols6, Lars Thrysøe7, Charlotte Brun Thorup8 and Selina Kikkenborg Berg1,3,9 Abstract Background: Anxiety and depression symptoms are common among cardiac patients. The Hospital Anxiety and Depression Scale (HADS) is frequently used to measure symptoms of anxiety and depression; however, no study on the validity and reliability of the scale in Danish cardiac patients has been done. The aim, therefore, was to evaluate the psychometric properties of HADS in a large sample of Danish patients with the four most common cardiac diagnoses: ischemic heart disease, arrhythmias, heart failure and heart valve disease. Methods: The DenHeart study was designed as a national cross-sectional survey including the HADS, SF-12 and HeartQoL and combined with data from national registers. Psychometric evaluation included analyses of floor and ceiling effects, structural validity using both exploratory and confirmatory factor analysis and hypotheses testing of convergent and divergent validity by relating the HADS scores to the SF-12 and HeartQoL. Internal consistency reliability was evaluated by Cronbach’s alpha, and differential item functioning by gender was examined using ordinal logistic regression. Results: A total of 12,806 patients (response rate 51%) answered the HADS. Exploratory factor analysis supported the original two-factor structure of the HADS, while confirmatory factor analysis supported a three-factor structure consisting of the original depression subscale and two anxiety subscales as suggested in a previous study. There were floor effects on all items and ceiling effect on item 8. The hypotheses regarding convergent validity were confirmed but those regarding divergent validity for HADS-D were not. Internal consistency was good with a Cronbach’s alpha of 0.87 for HADS-A and 0.82 for HADS-D. There were no indications of noticeable differential item functioning by gender for any items. Conclusions: The present study supported the evidence of convergent validity and high internal consistency for both HADS outcomes in a large sample of Danish patients with cardiac disease. There are, however, conflicting results regarding the factor structure of the scale consistent with previous research. Trial registration: ClinicalTrials.gov: NCT01926145. Trial registration: ClinicalTrials.gov: NCT01926145. Keywords: Hospital anxiety and depression scale, Psychometric evaluation, Cardiac patients, Validity, Reliability * Correspondence: anne.vinggaard.christensen@regionh.dk * Correspondence: anne.vinggaard.christensen@regionh.dk p gg @ g 1Department of Cardiology, Rigshospitalet, Copenhagen University Hospital Blegdamsvej 9, 2100 Copenhagen, Denmark Full list of author information is available at the end of the article p gg g 1Department of Cardiology, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, 2100 Copenhagen, Denmark Full list of author information is available at the end of the article University of Southern Denmark Citation for pulished version (APA): Christensen, A. V., Dixon, J. K., Juel, K., Ekholm, O., Rasmussen, T. B., Borregaard, B., Mols, R. E., Thrysøe, L., Thorup, C. B., & Berg, S. K. (2020). Psychometric properties of the Danish Hospital Anxiety and Depression Scale in patients with cardiac disease: results from the DenHeart survey. Health and Quality of Life Outcomes, 18, Article 9. https://doi.org/10.1186/s12955-019-1264-0 Go to publication entry in University of Southern Denmark's Research Portal Terms of use This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: g y y y Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: • You may download this work for personal use only • You may download this work for personal use only. Y t f th di t ib t th t i l it f y y p g • You may freely distribute the URL identifying this open access version If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk (2020) 18:9 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 https://doi.org/10.1186/s12955-019-1264-0 Christensen et al. Health and Quality of Life Outcomes https://doi.org/10.1186/s12955-019-1264-0 Data collection and sample Data was collected as part of the DenHeart study. The design and methods have been described in the pre- published protocol [25]. The DenHeart study was de- signed as a national cross-sectional survey combined with data from national registers at baseline and one year follow-up. Over a period of one year (April 2013– April 2014) all patients discharged or transferred from one of five national heart centers were asked to fill out a questionnaire at hospital discharge. Excluded were pa- tients under the age of 18, patients without a Danish civil registration number, patients who did not under- stand Danish and patients who were unconscious when transferred from a heart center. The Hospital Anxiety and Depression Scale (HADS) was developed for patients with somatic illness admitted to the hospital [5] and is often used as a self-rating scale to screen for anxiety and depression symptoms across a wide range of patient and general populations. The scale includes two subscales, HADS-A and HADS-D measur- ing anxiety and depression symptoms, respectively. The scale is focused on the psychic symptoms of mood disor- ders, leaving out physical symptoms that can be con- fused with physical illness [5]. This is an advantage in cardiac populations where symptoms such as palpita- tions or dizziness might be related to the underlying cardiac disease and not a potential mood disorder. Based on their discharge diagnosis from the Danish National Patient Register [26], patients were divided into diagnostic sub-groups [2]. Included in the current ana- lyses are patients with ischemic heart disease, arrhyth- mias, heart failure and heart valve diseases. Furthermore, co-morbidity characteristics were col- lected from the Danish National Patient Register [26]. The Tu co-morbidity index was calculated including congestive heart failure, cardiogenic shock, arrhythmia, pulmonary oedema, malignancy, diabetes, cerebrovascu- lar disease, acute/chronic renal failure and chronic ob- structive pulmonary disease – all calculated ten years back [27]. HADS has been extensively tested for validity and reliability in English and other language versions, with satisfactory results across different patient populations, e.g. cardiac disease, cancer, psychological illness and in general populations [6–8]. Looking at previous valid- ation studies of HADS in cardiac populations, however, there are differing results regarding the factor structure of the scale, Table 1. Background most common cardiac diagnoses: ischemic heart disease, arrhythmias, heart failure and heart valve diseases. Anxiety and depression symptoms are common among cardiac patients with prevalence rates of up to 30 and 20%, respectively, at hospital discharge and up to three months after hospitalization. This reflects the possible severity of the physical illness on other aspects of health [1, 2]. Previous studies have shown that anxiety and depression symptoms can predict future morbidity and mortality among cardiac patients [3, 4] underlining the importance of identifying these symptoms in order to initiate interventions to reduce them. A prerequisite for this is having a valid instrument to identify the symptoms. © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 2 of 13 Page 2 of 13 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Data collection and sample The originally proposed two-factor structure is confirmed in six studies [9–14], but eight studies find different versions of a three-factor structure to have the best fit depending on the analytic method used [12, 13, 15–20]. By contrast, one study finds a one- factor structure to have the best fit [21]. Information on demographic characteristics were collected from the Civil Registration System [28] and the Danish Education Register [29]. The HADS questionnaire The HADS is a 14 item questionnaire originally devel- oped to measure anxiety and depression symptoms in patients with somatic disease [5]. The instrument offers two subscales, HADS-A and HADS-D, each consisting of seven items and measuring anxiety and depression symptoms, respectively. HADS-A is focused on symp- toms relating to generalized anxiety and HADS-D on symptoms relating to anhedonia, a central aspect of depression [30]. Each item is scored on a scale of 0–3 with each subscale score ranging from 0 to 21. Eight items are reverse scored with higher scores indicating a better response. These are reversed when summing the two subscales. The recommended cut-off values are 8– 10 for possible presence of a mood disorder and ≥11 for probable presence of a mood disorder [5]. It has previously been found that among cardiac patients the minimal clinically important difference on the HADS is 1.7 points [31]. Differential item functioning (DIF) is a form of meas- urement error at item level by which patients from dif- ferent groups with the same level of a construct being measured do not have the same scores. The presence of DIF by gender has been examined for HADS, but the results are not consistent [22–24]. HADS has been translated into Danish and is fre- quently used in clinical research but the psychometric properties of the Danish version have not been evalu- ated. Even though the scale has been found to be valid and reliable in previous studies, this is no assurance of equivalent validity when used in a different language, culture or context. Therefore, the aim of the current study was to evaluate the psychometric properties of the Danish HADS in a large population of patients with the Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 3 of 13 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 3 of 13 Table 1 Previous validations of HADS in patients with cardiac disease Cronbach’s alpha Reference Language Population Analytic methods Number of factors Sub scale content HADS-A HADS-D Correlation between sub scales Ayis et al. 2018 [9] English Stroke (n = 1443) ML PCA CFA (and IRT) 2 Anxiety: 1,3,5,7,9,11,13 Depression: 2,4,6,8,10,12,14 Kaur et al. The HADS questionnaire 2015 [15] Malaysian Coronary artery disease (n = 189) PCA CFA 3 Anxiety: 1,3,5,7,9,11,13 Anhedonia: 2,4,6,14 Psychomotor retardation: 8, 10,12 0.89 0.69 Anhedonia: 0.70 Psychomotor retardation: 0.51 Anhedonia – psychomotor retardation: 0.35 Anhedonia – anxiety: 0.47 Psychomotor retardation – anxiety: 0.39 De Smedt et al. 2013 [10] 22 European countries CABG, PCI, AMI, myocardial ischemia (n = 8745) CFA 2 Anxiety: 1,3,5,7,9,11,13 Depression: 2,4,6,8,10,12,14 0.82 0.74 0.60 Cosco et al. 2012 [21] English Cardiovascular disease (n = 893) MSA 1 1,2,3,4,5,6,7,9,10,11,12,13 Emons et al. 2012 [16] Dutch Cardiac patients (n = 534) MSA EFA CFA 3 Anxiety: 1,3,5,9,13 Depression: 2,4,6,8,10,12 Restlessness: 7,11,14 Depression – restlessness: 0.62 Restlessness – anxiety: 0.68 Depression – anxiety: 0.66 Kendel et al. 2010 [22] German CABG (n = 1271) Rasch (HADS-D only) Depression: 2,4,6,12 Hunt-Shanks et al. 2010 [17] English Cardiac patients (n = 801) CFA 3 Negative affect: 1,5,7,11 Autonomic anxiety: 3,9,13 Depression: 2,4,6,8,10,12,14 Martin et al. 2008 [18] German Chinese English Coronary heart disease (n = 1793) MGCFA 3 Antonomic anxiety: 3,9,13 Negative affectivity: 1, 5,7,11Anhedonic depression: 2,4,6,8,10,12,14 Pais-Ribeiro et al. 2007 [11] Portuguese Mixed patients incl. Coronary heart disease (n = 1322) EFA CFA 2 Anxiety: 1,3,5,7,9,11,13 Depression: 2,4,6,8,10,12,14 0.76 0.81 0.58 Wang et al. 2006 [12] Chinese Coronary heart disease (n = 154) CFA 2 or 3 2: Anxiety: 1,3,5,9,11 Depression: 2,4,6,7,8,10,12,14 3: Antonomic anxiety: 3,9,13 Negative affectivity: 1,5,7,11 Anhedonic depression: 2,4,6, 8,10,12,14 Barth and Martin 2005 [13] German Coronary heart disease (n = 1320) EFA CFA EFA: 2 CFA: 3 EFA: Anxiety: 1 3 5 7 9 11 13 0.82 (between HADS A d Table 1 Previous validations of HADS in patients with cardiac disease Ayis et al. 2018 [9] English Stroke (n = 1443) ML PCA CFA (and IRT) 2 Anxiety: 1,3,5,7,9,11,13 Depression: 2,4,6,8,10,12,14 Kaur et al. 2015 [15] Malaysian Coronary artery disease (n = 189) PCA CFA 3 Anxiety: 1,3,5,7,9,11,13 Anhedonia: 2,4,6,14 Psychomotor retardation: 8, 10,12 0.89 0.69 Anhedonia: 0.70 Psychomotor retardation: 0.51 Anhedonia – psychomotor retardation: 0.35 Anhedonia – anxiety: 0.47 Psychomotor retardation – anxiety: 0.39 De Smedt et al. 2013 [10] 22 European countries CABG, PCI, AMI, myocardial ischemia (n = 8745) CFA 2 Anxiety: 1,3,5,7,9,11,13 Depression: 2,4,6,8,10,12,14 0.82 0.74 0.60 Cosco et al. 2012 [21] English Cardiovascular disease (n = 893) MSA 1 1,2,3,4,5,6,7,9,10,11,12,13 Emons et al. The HADS questionnaire 2012 [16] Dutch Cardiac patients (n = 534) MSA EFA CFA 3 Anxiety: 1,3,5,9,13 Depression: 2,4,6,8,10,12 Restlessness: 7,11,14 Depression – restlessness: 0.62 Restlessness – anxiety: 0.68 Depression – anxiety: 0.66 Kendel et al. 2010 [22] German CABG (n = 1271) Rasch (HADS-D only) Depression: 2,4,6,12 Hunt-Shanks et al. 2010 [17] English Cardiac patients (n = 801) CFA 3 Negative affect: 1,5,7,11 Autonomic anxiety: 3,9,13 Depression: 2,4,6,8,10,12,14 Martin et al. German Coronary heart MGCFA 3 Antonomic anxiety: 0.39 De Smedt et al. 2013 [10] 22 European countries CABG, PCI, AMI, myocardial ischemia (n = 8745) CFA 2 Anxiety: 1,3,5,7,9,11,13 Depression: 2,4,6,8,10,12,14 0.82 0.74 0.60 Cosco et al. 2012 [21] English Cardiovascular disease (n = 893) MSA 1 1,2,3,4,5,6,7,9,10,11,12,13 Emons et al. 2012 [16] Dutch Cardiac patients (n = 534) MSA EFA CFA 3 Anxiety: 1,3,5,9,13 Depression: 2,4,6,8,10,12 Restlessness: 7,11,14 Depression – restlessness: 0.62 Restlessness Depression – restlessness: 0.62 Restlessness – anxiety: 0.68 Depression – anxiety: 0.66 0.66 Kendel et al. 2010 [22] German CABG (n = 1271) Rasch (HADS-D only) Depression: 2,4,6,12 Hunt-Shanks et al. 2010 [17] English Cardiac patients (n = 801) CFA 3 Negative affect: 1,5,7,11 Autonomic anxiety: 3,9,13 Depression: 2,4,6,8,10,12,14 Martin et al. 2008 [18] German Chinese English Coronary heart disease (n = 1793) MGCFA 3 Antonomic anxiety: 3,9,13 Negative affectivity: 1, 5,7,11Anhedonic depression: 2,4,6,8,10,12,14 Pais-Ribeiro et al. 2007 [11] Portuguese Mixed patients incl. Coronary heart disease (n = 1322) EFA CFA 2 Anxiety: 1,3,5,7,9,11,13 Depression: 2,4,6,8,10,12,14 0.76 0.81 0.58 Wang et al. 2006 [12] Chinese Coronary heart disease (n = 154) CFA 2 or 3 2: Anxiety: 1,3,5,9,11 Depression: 2,4,6,7,8,10,12,14 3: Antonomic anxiety: 3,9,13 Negative affectivity: 1,5,7,11 Anhedonic depression: 2,4,6, 8,10,12,14 Barth and Martin 2005 [13] German Coronary heart disease (n = 1320) EFA CFA EFA: 2 CFA: 3 EFA: Anxiety: 1,3,5,7,9,11,13 Depression: 2,4,6,8,10,12,14 CFA: Psychomotor agitation: 1,7, 11 Psychic anxiety: 0.82 (between HADS-A and HADS-D) Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 4 of 13 Table 1 Previous validations of HADS in patients with cardiac disease (Continued) Cronbach’s alpha Reference Language Population Analytic methods Number of factors Sub scale content HADS-A HADS-D Correlation between sub scales 3,5,9,13 Depression: 2,4,6,8,10,12,14 Martin et al. 2004 [52] Chinese Acute coronary syndrome (n = 138) CFA 3 Different models apply 0.79 0.55 Martin et al. Psychometric properties of HADS The following psychometric properties of the HADS were evaluated. Floor and ceiling effects occur if more than 15% of the patients select the lowest or highest possible score on an item. Floor and ceiling effects can be an indication that extreme items are missing in either end of the scale, which can possibly limit its validity [39, 40]. p y y Construct validity is defined as the degree to which an instrument measures what it is intended to measure. It is evaluated by testing hypotheses about an instrument – for example, relationships between parts of an instru- ment, relationships with scores of other instruments or differences between relevant groups [41]. An aspect of construct validity is structural validity, which is the degree to which the sub-scale scores of an instrument are an adequate reflection of the dimensions of the construct to be measured [41]. Structural validity was evaluated using exploratory factor analysis (EFA) and confirmatory factor analyses (CFA). CFA was conducted for the original two-factor structure suggested by Zigmond and Snaith [5], and also for four three-factor models [15, 42–44] and one one-factor model [21] found in previous studies including cardiac patients. The HADS questionnaire 2003 [19] English MI (n = 335) CFA 3 Anhedonia: 2,4,6,8,10,12,14 Psychic anxiety: 3,5,9,13 Psychomotor agitation: 1,7, 11 0.83–0.86 (3 timepoints) 0.76–0.80 (3 timepoints) Roberts et al. 2001 [14] English Female cardiac patients (n = 167) CFA 2 Anxiety: 1,3,5,7,9,11,13 Depression: 2,4,6,8,10,12,14 0.85 0.80 0.60 Martin and Thompson 2000 [20] English MI (n = 194) EFA 3 1: 2,4,6,7,8,10,12,14 2: 3,9,13 3: 1,5,11 0.76 0.72 0.54 ML maximum likelihood; PCF principal component analysis; CFA confirmatory factor analysis; IRT item response theory; MSA Mokken scale analysis; EFA exploratory factor analysis; CABG coronary artery bypass graft; PCI percutaneous coronary intervention; AMI acute myocardial infarction; MGCFA meta group confirmatory factor analysis; MI myocardial infarction Table 1 Previous validations of HADS in patients with cardiac disease (Continued) ML maximum likelihood; PCF principal component analysis; CFA confirmatory factor analysis; IRT item response theory; MSA Mokken scale analysis; EFA exploratory factor analysis; CABG coronary artery bypass graft; PCI percutaneous coronary intervention; AMI acute myocardial infarction; MGCFA meta group confirmatory factor analysis; MI myocardial infarction Cronbach’s alpha of 0.87 for the emotional subscale and 0.91 for the physical one [38]. The Danish version of HADS has been frequently used for research purposes, both in observational studies and randomized controlled trials, as well as for screening purposes in clinical practice [2, 3, 32–36]. Furthermore, two single items on anxiety and depres- sion allowed patients to rate anxiety and depression on a 10-point Likert scale. The translation of the HADS from English into Danish was evaluated by five independent assessors who were fluent in both English and Danish. For each item the equivalence of the translation was evaluated on a scale from 1 to 4, with higher numbers indicating stronger equivalence. The Translation Validity Index (TVI) was calculated as the proportion of assessments rated posi- tively with score of 3 or 4 [37]. Other instruments The Short-Form 12 health survey (SF-12) is a brief, gen- eric measure of health-related quality of life that gener- ates both a physical (PCS) and a mental component score (MCS). Higher scores indicate better health status [16]. The SF-12 has been validated in a population of pa- tients with coronary heart disease from 22 European countries with satisfactory results for construct validity and a Cronbach’s alpha of 0.87 for PCS and 0.84 for MCS, respectively, indicating high internal consistency reliability [10]. HeartQoL is a disease-specific question- naire that measures quality of life in cardiac patients and produces a global score and two subscales: a physical and an emotional scale ranging from 0 to 3 with higher scores indicating better quality of life status [18–20]. The instrument has been validated in a large sample of coronary patients with results confirming both discrim- inative and convergent validity and high reliability with a Construct validity was also examined through hypoth- eses testing by looking at HADS scores in relation to the Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 5 of 13 Page 5 of 13 Page 5 of 13 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 MCS on SF-12, the emotional subscale of HeartQoL and a single item on anxiety and a single item on depression (convergent construct validity), and in relation to the PCS and physical subscale of HeartQoL (divergent con- struct validity). HADS, SF-12 and HeartQoL subscales was examined by stratifying mean scores of MCS, PCS, and HeartQoL emotional and HeartQoL physical by HADS-A and HADS-D scores above and below 8. HADS, SF-12 and HeartQoL subscales was examined by stratifying mean scores of MCS, PCS, and HeartQoL emotional and HeartQoL physical by HADS-A and HADS-D scores above and below 8. Internal consistency was evaluated by calculating Cronbach’s alpha for subscales and also by corrected item-total correlations. We hypothesized high correlations (r > 0.60) between both HADS-A and HADS-D and the MCS score and the HeartQoL emotional score and high correlations be- tween HADS-A and a single item measuring anxiety, and between HADS-D and a single item measuring de- pression. Furthermore, we hypothesized low correlations (r < 0.30) between HADS-A and HADS-D and PCS and HeartQoL physical as these measures were not supposed to be related to the HADS subscales. Demographic and clinical profile Demographic and clinical characteristics are presented as frequencies or means with standard deviations (SD). Item score distributions are presented as means with SD, frequencies for each response category and missing data. Histograms and the Kolmogorov-Smirnov test were used to determine whether item scores deviated from the normal distribution. Out of 25,241 eligible patients, 12,806 had complete re- sponses to the HADS questionnaire giving a response rate of 51%. Demographic and clinical characteristics are presented in Table 2. Item score statistics and translation validity index Exploratory factor analysis was conducted using prin- cipal axis extraction based on eigenvalues greater than 1. Oblimin rotation was applied with a cut-off point of 0.30 as designating loading on a factor. The item score statistics are presented in Table 3. Item 8 showed markedly different scores compared to the rest of the items, with more patients using high response cat- egories, Table 3. There were floor effects on all items and a ceiling effect on item 8, Table 3. Confirmatory analyses were conducted with the weighted least squared means and variance (WLSMV) estimator. A Root Mean Square Error of Approximation (RMSEA) estimate below 0.06 along with Comparative Fit Index (CFI) and Tucker Lewis Index (TLI) estimates above 0.95 indicated a good model fit [46]. Of the 14 items, 12 had an TVI of 100%, and two (items 3 and 11) had TVI of 60% (both of these were a part of HADS-A. The TVI for the total scale was 94%, Additional file 1: Table S1. Other instruments DIF was examined using multivariate ordinal logistic regression with items as the dependent variable and gender and total score (HADS-A or HADS-D depend- ing on the item) as the independent variables. Because the proportional odds assumption was not fulfilled a partial proportional odds model was used. DIF was evaluated by different criteria. Uniform DIF can be con- sidered if the odds ratio (OR) for gender is statistically significantly different from 1 [45]. Interactions between gender and total score were included to evaluate pos- sible non-uniform DIF. A statistically significant inter- action can be an indication of non-uniform DIF [45]. Because of the large sample size and the risk of finding statistically significant results with no or very little clin- ical meaning, DIF was also evaluated by Nagelkerke’s R.2 A difference in R2 of more than 0.03 between models was an indication of noticeable DIF (both uni- form and non-uniform) [45]. Internal consistency reliability is an indicator of the extent to which the items of an instrument are internally correlated and therefore measure the same construct. This can be evaluated by calculating Cronbach’s alpha. A Cronbach’s alpha of between 0.70 and 0.95 is an indi- cation of good internal consistency [40]. DIF is a form of measurement invariance at item level. DIF means that there are items for which patients from different groups with the same level of the con- struct being measured do not have the same scores. This can indicate that the item measures different things in the different groups. DIF can be uniform or non-uniform depending on whether the differences are present for all values of the scale or just for some values of the scale [45]. Only patients with complete responses to the HADS were included in the analyses. Analyses were conducted using SAS version 9.4, IBM SPSS version 25 and Mplus version 7.4. Differential item functioning There were indications of DIF for item 3, 4 and 13 where women were more likely to have high item scores compared to men and for items 11 and 14 where men were more likely to have high item scores compared to women. There were significant interactions between item and subscale for items 1, 2, 5, 7, 8, 9 and 12, which is an indication of non-uniform DIF. However, in ana- lysis using Nagelkerke’s R2 there was no noticeable DIF for any item, Table 7. The CFA indicated that the three-factor structure sug- gested by Friedman et al. [44] showed the best fit for the models tested, Table 5. The diagram from the CFA of the three-factor structure suggested by Friedman et al. [44] is presented in Fig. 1. Internal consistency For HADS-A mean inter-item correlation was 0.50 (range 0.35–0.61) and Cronbach’s alpha was 0.87. The corrected item-total correlations ranged from 0.52 to 0.71. Cronbach’s alpha would not be improved by the deletion of any item. For HADS-D mean inter-item correlation was 0.41 (range 0.24–0.58). Cronbach’s alpha was 0.82. The cor- rected item-total correlations ranged from 0.44 to 0.67. Cronbach’s alpha would not be improved by the deletion of any item. For all HADS items the mean inter-item correlation was 0.40 (range 0.24–0.61). Looking at the three-factor structure, the Cronbach’s alpha for the psychomotor agitation subscale was 0.74 and 0.83 for the psychic anxiety subscale. The HADS-D subscale was unchanged with a Cronbach’s alpha of 0.82. Cronbach’s alpha would not be improved by the deletion of any item. Factor structure Both the EFA and the CFA were conducted on the total population. Extensive previous literature exists that provide suggestions for models to be tested in the CFA. The results from the EFA indicate that the original two- factor structure of the HADS seems to fit in this cardiac population. However, item 7 showed almost the same loading on each subscale, Table 4. The correlation be- tween HADS-A and HADS-D was 0.66. Spearman’s rank-order correlations were used to de- termine convergent and divergent validity as data were not normally distributed. Convergent validity between Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 6 of 13 Page 6 of 13 Table 2 Demographic and clinical characteristics n 12,806 Male, n (%) 8953 (69.9) Age, mean (SD) 65.1 (12.1) Marital status (n,%) Married Divorced Widowed Unmarried 8307 (64.9) 1728 (13.5) 1533 (12.0) 1238 (9.6) Educational level (n,%) Basic school Upper secondary or vocational school Higher education Missing 3903 (30.5) 5595 (43.7) 3018 (23.5) 290 (2.3) Cardiac diagnosis (n,%) Ischemic heart disease Arrhythmias Heart failure Heart valve diseases 6832 (53.3) 4121 (32.2) 917 (7.2) 936 (7.3) Co-morbidity (n,%) Hypertension 4424 (34.6) Ventricular arrhythmia 589 (4.6) Ischemic heart disease 5544 (43.3) Myocardial infarction 2408 (18.8) Diabetes 1257 (9.8) Heart failure 2210 (17.3) Renal disease 426 (3.3) Chronic obstructive pulmonary disease 837 (6.5) Tu comorbidity score (n,%) 0 5271 (41.2) 1 4378 (34.2) 2 2062 (16.1) ≥3 1095 (8.5) HADS-A and the single item on anxiety was 0.68 and be- tween HADS-D and the single item on depression it was 0.59. This confirmed the stated hypotheses about conver- gent validity. However, the two single items were highly correlated (0.76). Correlations between HADS-A and PCS and Heart- QoL physical were 0.25 and 0.35, respectively. Correla- tions between HADS-D and PCS and HeartQoL physical were 0.50 and 0.55, respectively. This did not confirm the hypotheses on divergent validity for HADS-D. Discussion Looking at MCS, PCS, HeartQoL emotional and Heart- QoL physical scores in relation to HADS scores, patients with scores below 8 on both HADS-A or HADS-D had high scores on MCS and HeartQoL emotional. Con- versely, patients with HADS-A and HADS-D scores above 8 have the lowest scores. The same pattern is found in PCS and HeartQoL physical scores, Table 6. In the present study the psychometric properties of the HADS in a large sample of Danish cardiac patients were evaluated. Floor effects were found on all items and ceiling effect on item 8. The original two-factor structure of the scale was confirmed in EFA, but CFA indicated a three- factor structure. The hypotheses proposed were supported for both subscales, providing evidence for convergent validity. However, for HADS-D the hypotheses proposed for divergent validity were not supported. Thus, divergent validity is not indicated. Internal consistency was good for both HADS-A and HADS-D. Correlations between HADS-A and MCS and HeartQoL emotional were 0.67 and 0.75, respectively. Correlations between HADS-D and MCS and HeartQoL emotional were 0.66 and 0.63, respectively. The correlation between Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 7 of 13 Table 3 Item and score statistics Score distribution, n (%) Mean (SD) 0 1 2 3 Missing HADS-A n = 12,806 5.79 (4.19) 1. I feel tense or ‘wound up’* 1.05 (0.83) 3471 (25.8) 6413 (47.6) 2662 (19.8) 745 (5.5) 172 (1.3) 3. I get a sort of frightened feeling as if something awful is about to happen* 1.09 (0.90) 4050 (30.1) 4702 (34.9) 3754 (27.9) 361 (5.7) 196 (1.5) 5. Worrying thoughts go through my mind* 0.90 (0.89) 5189 (38.5) 5027 (37.3) 2244 (16.7) 790 (5.9) 213 (1.6) 7. I can sit at ease and feel relaxed 0.73 (0.73) 5673 (42.1) 5746 (42.7) 1721 (12.8) 156 (1.2) 167 (1.2) 9. I get a sort of frightened feeling like ‘butterflies’ in the stomach 0.62 (0.72) 6596 (50.0) 5354 (39.8) 1009 (7.5) 304 (2.3) 200 (1.5) 11. I feel restless as I have to be on the move* 0.88 (0.81) 4874 (36.2) 5549 (41.2) 2444 (18.2) 413 (3.1) 183 (1.4) 13. I get sudden feelings of panic* 0.52 (0.69) 7691 (57.1) 4355 (32.4) 1035 (7.7) 163 (1.2) 219 (1.6) HADS-D n = 12,806 4.29 (3.65) 2. Discussion I still enjoy the things I used to enjoy 0.72 (0.78) 6080 (41.2) 5332 (39.6) 1428 (10.6) 433 (3.2) 190 (1.4) 4. I can laugh and see the funny side of things 0.37 (0.64) 9403 (69.8) 2991 (22.2) 766 (5.7) 131 (1.0) 172 (1.3) 6. I feel cheerful* 0.51 (0.65) 8309 (61.7) 3358 (24.9) 1417 (10.5) 209 (1.6) 170 (1.3) 8. I feel as if I am slowed down* 1.40 (0.93) 2078 (15.4) 5912 (43.9) 3177 (23.6) 2122 (15.8) 174 (1.3) 10. I have lost interest in my appearance* 0.43 (0.69) 8983 (66.7) 3076 (22.9) 1080 (8.0) 138 (1.0) 186 (1.4) 12. I look forward with enjoyment to things 0.52 (0.74) 8119 (60.3) 3593 (26.7) 1313 (9.8) 225 (1.7) 213 (1.6) 14. I can enjoy a good book or radio or TV program 0.37 (0.69) 9654 (71.7) 2608 (19.4) 705 (5.2) 289 (2.2) 207 (1.5) Each item is scored on a scale of 0–3 with each subscale ranging from 0 to 21. For six items higher scores indicate a worse response. The eight items highlighted with * are reverse scored. These are reversed when summing the subscales Table 3 Item and score statistics 14. I can enjoy a good book or radio or TV program Each item is scored on a scale of 0–3 with each subscale ranging from 0 to 21. For six items higher scores indicate a worse respo with * are reverse scored. These are reversed when summing the subscales of 0–3 with each subscale ranging from 0 to 21. For six items higher scores indicate a worse response. The eight items highlighted se are reversed when summing the subscales The factor analyses indicate that the factor structure of the HADS is not completely clear. The EFA con- firmed the original two-factor structure suggested by Zigmond and Snaith [5], but the CFA showed that the three-factor structure as found by Friedman et al. [44] in a French sample of patients suffering from major depres- sion had the best model fit. The same result was found by Barth and Martin in a German coronary heart disease population [13]. Several other studies have found varia- tions of a three-factor structure to have the best model fit for the HADS as indicated in Table 5. Discussion The differences in factor structure found across studies might be ex- plained by different methodology such as data extraction method, model fit criteria, translation or type of patients included. (item 1, 7, 11), psychic anxiety (item 3, 5, 9, 13) and de- pression (item 2, 4, 6, 8, 10, 12, 14), the division of items from the original HADS-A into two factors can make sense as relating to two different dimensions of anxiety disorder. The items in the psychomotor agitation sub- scale relate to physical feelings of restlessness and agita- tion while the items in the psychic anxiety subscale relate to emotional representation of anxiety with worry- ing and nervous thoughts. Agitation is, however, also a common symptom among patients with depressive dis- orders and can occur as a side effect of antidepressant medication [47]. The interrelatedness between symptoms of anxiety and depression is further evident in the high correla- tions between HADS-A and HADS-D. This did not change when looking at the three-factor structure instead. It has previously been argued that a high When considering the content of the three factors sug- gested by Friedman et al. [44]; psychomotor agitation Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 8 of 13 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 8 of 13 Table 4 Exploratory factor analysis - rotated factor matrixa Table 4 Exploratory factor analysis - rotated factor matrixa Factor 1 2 Item 9. I get a sort of frightened feeling like ‘butterflies’ in the stomach 0.81 Item 3. I get a sort of frightened feeling as if something awful is about to happen 0.80 Item 5. Worrying thoughts go through my mind 0.69 Item 13. I get sudden feelings of panic 0.71 Item 1. I feel tense or ‘wound up’ 0.60 Item 7. I can sit at ease and feel relaxed 0.41 0.36 Item 11. I feel restless as I have to be on the move 0.46 Item 12. I look forward with enjoyment to things 0.79 Item 6. I feel cheerful 0.67 Item 2. I still enjoy the things I used to enjoy 0.72 Item 4. I can laugh and see the funny side of things 0.62 Item 8. I feel as if I am slowed down 0.54 Item 10. I have lost interest in my appearance 0.55 Item 14. I can enjoy a good book or radio or TV program 0.45 Cumulative % of variance explained 45.22 53.99 Eigenvalue 6.33 1.23 aExploratory factor analyses using principal axis extraction based in eigenvalues greater than 1, Oblimin rotation and cut-off point of 0.30 Loadings> 0.40 in bold Table 5 Fit indices for confirmatory factor analyses of factor structures proposed in previous studies RMSEA Models Number of factors Sub scale content RMSEA 90% CI p-value CFI TLI Zigmond and Snaith 1983 [5] 2 HADS-A: 1,3,5,7,9,11,13 HADS-D: 2,4,6,8,10,12,14 0.071 0.069;0.072 < 0.001 0.973 0.968 Dunbar et al. 2000 [43] 3 Negative affect: 1,5,7,11 Autonomic anxiety: 3,9,13 Depression: 2,4,6,8,10,12,14 0.061 0.059;0.062 < 0.001 0.981 0.976 Friedman et al. 2001 [44] 3 Psychomotor agitation: 1,7,11 Psychic anxiety: 3,5,9,13 Depression: 2,4,6,8,10,12,14 0.060 0.058;0.061 < 0.001 0.981 0.977 Caci et al. 2003 [42] 3 Anxiety: 1,3,5,9,13 Depression: 2,4,6,8,10,12 Restlessness: 7,11,14 0.064 0.062;0.065 < 0.001 0.979 0.974 Kaur et al. 2015 [15] 3 Anxiety: 1,3,5,7,9,11,13 Anhedonia: 2,4,6,14 Psychomotor retardation: 8,10,12 0.069 0.068;0.071 < 0.001 0.975 0.969 Cosco et al. 2012 [21] 1 1,2,3,4,5,6,7,9,10,11,12,13 0.111 0.109;0.113 < 0.001 0.945 0.932 Table 5 Fit indices for confirmatory factor analyses of factor structures proposed in previous studies Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Page 9 of 13 Page 9 of 13 (2020) 18:9 Fig. 1 Diagram from the confirmatory factor analysis presenting the model with the best fit. Standardized loadings (SE). Table 4 Exploratory factor analysis - rotated factor matrixa PAn = psychic anxiety; Dep = depression; PAg = psychomotor agitation In the EFA item 7 was found to load almost equally on both factors. This has been found in previous studies as well [13]. Item 7 reads ‘I can sit at ease and feel relaxed’; this may reflect aspects of both anxiety and depression. Eight items in the HADS are reversely scored. This is a recommended method to avoid acquiescence bias which is the tendency for respondents of a survey to agree with statements regardless of their content. How- ever, research suggests that individual differences in response styles can systematically affect the factor structure [49]. The uncertainty of the factor structure of the HADS is not necessarily a reason to discard the instrument, but rather to be clear on the purpose of using the scale. The two-factor structure may prove useful as a simple indication of either anxiety or de- pression. The possible presence of a third factor indi- cates that the scale may provide more refined results regarding different aspects of anxiety, rather than just an indication of generalized anxiety. Because the results regarding factor structure were not clear, the two- factor structure originally proposed was used in the remaining analyses for the paper. There were floor effects on all items, which may indi- cate that the number of extreme response categories is not sufficient. As the HADS was developed to detect in- dications of a mood disorder, which is not present in the majority of the population, even a population with se- vere illness, it is not surprising that there are floor ef- fects. Item 8 also showed a ceiling effect. The item reads ‘I feel as if I am slowed down’. In a population of elderly, severely ill patients just discharged, it is not surprising that this feeling would be prevalent. This item is suscep- tible to influence from either age or disease which is a bias in terms of validity as an indicator of mood. Fig. 1 Diagram from the confirmatory factor analysis presenting the model with the best fit. Standardized loadings (SE). PAn = psychic anxiety; Dep = depression; PAg = psychomotor agitation correlation between anxiety and depression is to be ex- pected, not because of common symptoms but because it is possible that anxiety can lead to depression and that depression can lead to anxiety. Table 4 Exploratory factor analysis - rotated factor matrixa It is also possible that the two disorders result from a common cause. The causality of this relationship cannot, however, be determined from cross-sectional data [48]. y The analyses of DIF indicated that there could be po- tential problems with DIF for several items. Table 4 Exploratory factor analysis - rotated factor matrixa However, Table 6 HADS scores in relation to SF-12 and HeartQoL scores Table 6 HADS scores in relation to SF-12 and HeartQoL scores HADS-A < 8 ≥8 HADS-D n (%) 8211 (64.1) 553 (4.3) < 8 MCS, mean (SD) 53.03 (7.96) 42.96 (8.63) PCS, mean (SD) 44.13 (10.43) 34.11 (9.07) HeartQoL emotional, mean (SD) 2.50 (0.56) 1.56 (0.68) HeartQoL physical, mean (SD) 1.83 (0.83) 1.47 (0.81) n (%) 2147 (16.8) 1895 (14.8) ≥8 MCS, mean (SD) 42.01 (9.16) 34.11 (9.07) PCS, mean (SD) 33.44 (9.93) 35.53 (10.03) HeartQoL emotional, mean (SD) 1.92 (0.70) 1.04 (0.69) HeartQoL physical, mean (SD) 1.02 (0.71) 0.95 (0.70) HADS Hospital Anxiety and Depression Scale; SF-12 = Short Form 12; HADS-A = Hospital Anxiety and Depression Scale – Anxiety; HADS-D = Hospital Anxiety and Depression Scale – Depression; MCS = Mental Component Scale; PCS = Physical Component Scale; SD = Standard deviation The cut-off of 8 is used as an indicator of possible mood disorder Table 6 HADS scores in relation to SF-12 and HeartQoL scores HADS-A < 8 ≥8 HADS-D n (%) 8211 (64.1) 553 (4.3) < 8 MCS, mean (SD) 53.03 (7.96) 42.96 (8.63) PCS, mean (SD) 44.13 (10.43) 34.11 (9.07) HeartQoL emotional, mean (SD) 2.50 (0.56) 1.56 (0.68) HeartQoL physical, mean (SD) 1.83 (0.83) 1.47 (0.81) n (%) 2147 (16.8) 1895 (14.8) ≥8 MCS, mean (SD) 42.01 (9.16) 34.11 (9.07) PCS, mean (SD) 33.44 (9.93) 35.53 (10.03) HeartQoL emotional, mean (SD) 1.92 (0.70) 1.04 (0.69) HeartQoL physical, mean (SD) 1.02 (0.71) 0.95 (0.70) HADS Hospital Anxiety and Depression Scale; SF-12 = Short Form 12; HADS-A = Hospital Anxiety and Depression Scale – Anxiety; HADS-D = Hospital Anxiety and Depression Scale – Depression; MCS = Mental Component Scale; PCS = Physical Component Scale; SD = Standard deviation The cut-off of 8 is used as an indicator of possible mood disorder HADS Hospital Anxiety and Depression Scale; SF-12 = Short Form 12; HADS-A = Hospital Anxiety and Depression Scale – Anxiety; HADS-D = Hospital Anxiety and Depression Scale – Depression; MCS = Mental Component Scale; PCS = Physical Component Scale; SD = Standard deviation The cut-off of 8 is used as an indicator of possible mood disorder HADS Hospital Anxiety and Depression Scale; SF-12 = Short Form 12; HADS-A = Hospital Anxiety and Depression Scale – Anxiety; HADS-D = Hospital Anxiety and Depression Scale – Depression; MCS = Mental Component Scale; PCS = Physical Component Scale; SD = Standard deviation The cut-off of 8 is used as an indicator of possible mood disorder HADS Hospital Anxiety and Depression Scale; SF-12 = Short Form 12; HADS-A = Hospital Anxiety and Depression Scale – Anxiety; HADS-D = Hospital Anxiety and Depression Scale – Depression; MCS = Mental Component Scale; PCS = Physical Component Scale; SD = Standard deviation The cut-off of 8 is used as an indicator of possible mood disorder Christensen et al. Table 4 Exploratory factor analysis - rotated factor matrixa Health and Quality of Life Outcomes (2020) 18:9 Page 10 of 13 Table 7 Differential item functioning tested for gender OR (95% CI)a for item responses 1, 2 and 3 Overall p-value Significant interaction between gender and sub scale Nagelkerke’s R2 Step 1 Nagelkerke’s R2 Step 2 Nagelkerke’s R2 Step 3 DIF R2 b HADS-A Item 1. I feel tense or ‘wound up’ X 0.6712 0.6714 0.6717 0.0005 Item 3. I get a sort of frightened feeling as if something awful is about to happen 1: 0.948 (0.782;1.149) 2: 0.801 (0.719;0.892) 3: 0.916 (0.820;1.023) 0.0007 0.6287 0.6293 0.6295 0.0008 Item 5. Worrying thoughts go through my mind X 0.6924 0.6930 0.6932 0.0008 Item 7. I can sit at ease and feel relaxed X 0.6008 0.6011 0.6018 0.0010 Item 9. I get a sort of frightened feeling like ‘butterflies’ in the stomach X 0.6718 0.6726 0.6730 0.0012 Item 11. I feel restless as I have to be on the move 1:1.670 (1.315;2.122) 2: 1.385 (1.240;1.545) 3: 1.423 (1.289;1.571) <.0001 0.4746 0.4785 0.4788 0.0042 Item 13. I get sudden feelings of panic 1: 0.667 (0.462;0.963) 2: 0.712 (0.600;0.845) 3: 0.781 (0.703;0.868) <.0001 0.6291 0.6307 0.6308 0.0017 HADS-D Item 2. I still enjoy the things I used to enjoy X 0.6107 0.6112 0.6116 0.0009 Item 4. I can laugh and see the funny side of things 1: 0.865 (0.587;1.274) 2: 0.917 (0.765;1.099) 3: 0.805 (0.719;0.902) 0.0025 0.5739 0.5747 0.5747 0.0008 Item 6. I feel cheerful 1: 1.079 (0.750;1.551) 2: 1.089 (0.937;1.266) 3: 1.071 (0.956;1.200) 0.5055 0.6381 0.6381 0.6385 0.0004 Item 8. I feel as if I am slowed down X 0.5660 0.5676 0.5684 0.0024 Item 10. I have lost interest in my appearance 1: 0.813 (0.561;1.177) 2: 1.050 (0.905;1.218) 3: 0.956 (0.866;1.054) 0.3345 0.4235 0.4237 0.4239 0.0003 Item 12. I look forward with enjoyment to things X 0.6136 0.6142 0.6143 0.0007 Item 14. I can enjoy a good book or radio or TV program 1: 2.132 (1.558;2.918) 2: 1.612 (1.361;1.909) 3: 1.431 (1.289;1.587) <.0001 0.3805 0.3853 0.3855 0.0050 a P i l i l dd d l i h i d d i bl d d d b l i d d i bl M f Acknowledgements To the patients who took the time to participate in the survey, the 800 cardiac nurses involved in data collection and the heart centres for prioritizing this study in a busy clinic. And to Sangchoon Jeon, Yale School of Nursing for statistical support. The large sample size in this study is an advantage be- cause of statistical power and because it allows a hetero- geneous sample. There is, however, a risk of finding statistically significant results of minimal clinical import- ance. Therefore, we have not only looked at p-values to determine validity, but rather measures of strength of correlation, internal consistency and Nagelkerke’s R2 for analyses of DIF. Conclusions because of the risk of finding statistically significant re- sults of minimal clinical importance in this large popula- tion, changes in Nagelkerke’s R2 between models were given priority. These indicated no noticeable DIF for any items. The presence of DIF for gender has been explored in previous studies [22–24, 50], but only one study found substantial DIF for item 14, with men being more likely to endorse this item [22]. The findings of this study supported the validity and re- liability of the HADS in a sample of Danish patients with cardiac disease. EFA supported the original two-factor structure of the scale, while CFA supported a three- factor structure consisting of the original depression subscale and two anxiety subscales; psychomotor agita- tion and psychic anxiety. The hypotheses regarding con- vergent validity were confirmed, but those regarding divergent validity were not confirmed for HADS-D. Internal consistency was good with a Cronbach’s alpha of 0.87 for HADS-A and 0.82 for HADS-D. There were no indications of noticeable DIF by gender for any items. When considering the usefulness of the HADS in clin- ical practice it should also be noted that HADS has been shown to predict morbidity and mortality in this patient population and similar patient populations [3, 4, 51]. Funding This work was supported by Helsefonden; the Heart Centre, Rigshospitalet, The Danish Heart Association, the Novo Nordisk Foundation and Familien Hede Nielsens Fond. The funders played no part in planning or conducting the research. HADS-D Page 11 of 13 Page 11 of 13 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Abbreviations CFA: Confirmatory factor analysis; CFI: Comparative Fit Index; DIF: Differential item functioning; EFA: Exploratory factor analysis; HADS: Hospital Anxiety and Depression Scale; MCS: Mental component score; OR: Odds ratio; PCS: Physical component score; RMSEA: Root Mean Square Error of Approximation; SD: Standard deviation; SF-12: Short-Form 12; TLI: Tucker Lewis Index; WLSMV: Weighted least squared means and variance Newer methods for exploring internal consistency exist, e.g. the use of McDonalds omega. However, for consistency with the methods chosen throughout this paper and for comparison with other HADS validation studies we chose to include Cronbach’s alpha. Authors’ contributions SKB conceived the overall idea for the DenHeart study and all authors designed the study. AVC performed the statistical analyses under the supervision of JKD and wrote the first draft of the manuscript. All revised the manuscript critically. All have given their final approval of the version to be published. The response rate was 51%, which is to be expected in a population of severely ill patients on the day of hos- pital discharge. This may raise concerns about represen- tativeness, however, the proportions of patients in the diagnostic sub-groups were similar to that of the entire eligible population, and responders and non-responders were comparable in terms of their demographic and clinical profiles, suggesting a representative sample [2]. We did, however, find a higher mortality rate in non- responders compared to responders [4]. Availability of data and materials Danish legislation on data security prohibits sharing of data. Danish legislation on data security prohibits sharing of data. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12955-019-1264-0. Additional file 1: Table S1. Translation Validity Index (TVI) for the Danish translation of Hospital Anxiety and Depression Scale (HADS) Ethics approval and consent to participate The DenHeart study complies with the Declaration of Helsinki. Danish legislation does not require surveys to be approved by an ethics committee system but rather by the Danish Data Protection Agency (2007-58-0015/30– 0937). The Danish National Board of Health permitted the use of register data. DenHeart is registered at ClinicalTrials.gov (NCT01926145). Patients provided informed consent. In the present study we used a single question on anx- iety and depression to measure convergent validity. However, the two questions were highly correlated. In- cluding more comprehensive instruments to measure anxiety and depression would have been optimal to examine convergent validity. These were, however, not available in the data. Limitations of the study There is no description of the process of how the HADS was translated into Danish from the questionnaire owner, so it is not clear whether the translation has followed the recommended steps to ensure cross- cultural validity [45]. The current analyses are, in fact, the first specific investigation of the psychometric prop- erties of the Danish language version of HADS. For the current study, we evaluated the TVI for each item and the total scale with satisfactory results. Items 3 and 11 (both in HADS-A) received the lowest rating (60%). References 23. Djukanovic I, Carlsson J, Årestedt K. Is the hospital anxiety and depression scale (HADS) a valid measure in a general population 65–80 years old? A psychometric evaluation study Health Qual Life Outcomes. 2017;15:193. 1. Moser DK, Dracup K, Evangelista LS, Zambroski CH, Lennie TA, Chung ML, et al. Comparison of prevalence of symptoms of depression, anxiety, and hostility in elderly patients with heart failure, myocardial infarction, and a coronary artery bypass graft. Heart Lung. 2010;39:378–85. 24. Cameron IM, Crawford JR, Lawton K, Reid IC. Differential item functioning of the HADS and PHQ-9: an investigation of age, gender and educational background in a clinical UK primary care sample. J Affect Disord. 2013;147:262–8. 2. Berg SK, Rasmussen TB, Thrysoee L, Lauberg A, Borregaard B, Christensen AV, et al. DenHeart: differences in physical and mental health across cardiac diagnoses at hospital discharge. J Psychosom Res. 2017;94:1–9. 25. Berg SK, Svanholm J, Lauberg A, Borregaard B, Herning M, Mygind A, et al. Patient-reported outcomes at hospital discharge from heart Centres, a national cross-sectional survey with a register-based follow-up: the DenHeart study protocol. BMJ Open. 2014;4:e004709. 3. Berg SK, Thygesen LC, Svendsen JH, Christensen AV, Zwisler AD. Anxiety predicts mortality in ICD patients: results from the cross-sectional National Copenheart Survey with register follow-up. Pacing Clin Electrophysiol. 2014; 37:1641–50. 26. Lynge E, Sandegaard JL, Rebolj M. The Danish National Patient Register. Scand J Public Health. 2011;39:30–3. 4. Berg SK, Thorup CB, Borregaard B, Christensen AV, Thrysoee L, Rasmussen TB, et al. Patient-reported outcomes are independent predictors of one-year mortality and cardiac events across cardiac diagnoses: findings from the national DenHeart survey. Eur J Prev Cardiol. 2019;26:624–37. 27. Tu JV, Austin PC, Walld R, Roos L, Agras J, McDonald KM. Development and validation of the Ontario acute myocardial infarction mortality prediction rules. J Am Coll Cardiol. 2001;37:992–7. 5. Zigmond AS, Snaith RP. The hospital anxiety and depression scale. Acta Psychiatr Scand. 1983;67:361–70. 28. Pedersen CB. The Danish civil registration system. Scand J Public Health. 2011;39:22–5. 29. Jensen VM, Rasmussen AW. Danish education registers. Scand J Public Health. 2011;39:91–4. 6. Herrmann C. International experiences with the hospital anxiety and depression scale - a review of validation data and clinical results. J Psychosom Res. 1997;42:17–41. 30. Snaith RP. The hospital depression and anxiety scale. Health Qual Life Outcomes. 2003;1:29. 7. Bjelland I, Dahl AA, Haug TT, Neckelmann D. The validity of the hospital anxiety and depression scale. Competing interests h h d l h Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Page 12 of 13 Page 12 of 13 Page 12 of 13 Christensen et al. Health and Quality of Life Outcomes (2020) 18:9 Author details 1 f 16. Emons WHM, Sijtsma K, Pedersen SS. Dimensionality of the hospital anxiety and depression scale (HADS) in cardiac patients. Assessment. 2012;19:337–53. 1Department of Cardiology, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, 2100 Copenhagen, Denmark. 2Yale School of Nursing, Yale University, 400 West Campus Drive, Orange, CT 06477, USA. 3National Institute of Public Health, University of Southern Denmark, Studiestræde 6, 1455 Copenhagen, Denmark. 4Department of Cardiology, Herlev and Gentofte University Hospital, Kildegaardsvej 28, 2900 Hellerup, Denmark. 5Cardiothoracic- and Vascular Department, Odense University Hospital, J.B.- Winslows Vej 4, 5000 Odense, Denmark. 6Department of Cardiology, Aarhus University Hospital, Palle Juul-Jensens Blv. 99, 8200 Aarhus, Denmark. 7Department of Cardiology, Odense University Hospital, University of Southern Denmark, J.B. 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Latent structure of the hospital anxiety and depression scale: a 10-year systematic review. J Psychosom Res. 2012;72:180–4. 32. Hojskov IE, Moons P, Hansen NV, Greve H, Olsen DB, Cour SL, et al. Early physical training and psycho-educational intervention for patients undergoing coronary artery bypass grafting. The SheppHeart randomized 2 x 2 factorial clinical pilot trial. Eur J Cardiovasc Nurs. 2016;15:425–37. 9. Ayis SA, Ayerbe L, Ashworth M, DA Wolfe C. Evaluation of the hospital anxiety and depression scale (HADS) in screening stroke patients for symptoms: item response theory (IRT) analysis. J Affect Disord. 2018;228: 33–40. 33. Sibilitz KL, Berg SK, Thygesen LC, Hansen TB, Kober L, Hassager C, et al. High readmission rate after heart valve surgery: a nationwide cohort study. Int J Cardiol. 2015;189:96–104. 10. De Smedt D, Clays E, Doyle F, Kotseva K, Prugger C, Pajak A, et al. 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Martin CR, Thompson DR, Chan DS. An examination of the psychometric properties of the hospital anxiety and depression scale in Chinese patients with acute coronary syndrome. Psychiatry Res. 2004;129:279–88. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Division Buyout and Refinancing of Event Risk Covenant Bonds: Evidence from the Long-Term Stock Performance Manish Tewari1 1 School of Business & Economics, State University of New York, Brockport, New York, USA Correspondence: Manish Tewari, School of Business & Economics, State University of New York, 350 New Campus Drive, SUNY, Brockport, NY 14420. USA. Tel: 1-585-395-5678. E-mail: mtewari@brockport.edu Accepted: April 27, 2013 Online Published: May 17, 2013 URL: http://dx.doi.org/10.5539/ibr.v6n6p1 Accepted: April 27, 2013 Online P URL: http://dx.doi.org/10.5539/ibr.v6n6p1 Accepted: April 27, 2013 Online P URL: http://dx.doi.org/10.5539/ibr.v6n6p1 Received: March 30, 2013 doi:10.5539/ibr.v6n6p1 Received: March 30, 2013 doi:10.5539/ibr.v6n6p1 International Business Research; Vol. 6, No. 6; 2013 ISSN 1913-9004 E-ISSN 1913-9012 Published by Canadian Center of Science and Education International Business Research; Vol. 6, No. 6; 2013 ISSN 1913-9004 E-ISSN 1913-9012 Published by Canadian Center of Science and Education Abstract The focus of this paper is to assess the long-term common stock performance of the parent firms that underwent divisional buyout (DBO) and had event risk covenant (ERC) bonds outstanding at the announcement of the DBO. The final sample of 46 parent firms exhibit a common characteristic where all the ERC bonds were redeemed (either called above par or put on the firm at par) or restructured at a higher cost to the firm around DBO announcement date due to the presence of ERCs. ERCs are triggered since the parent firms that divest their assets through a DBO reveal future cash flow volatility, which has potential to lower the value of existing bonds. This refunding of the bonds leads to costly refinancing for the parent firms, which has long-term implications. I find significantly negative cumulative abnormal returns at the issue date of the ERC bonds for these firms due to potential managerial entrenchment and foregone transfer of wealth from bondholders to stockholders. Consistent with the finance literature, I find significantly positive cumulative abnormal returns for parent firms at the announcement of the DBO. These positive short-term returns at the announcement do not translate into long-term positive returns. The common stock of these parent firms significantly underperforms the market over the periods three, four, and five years after the DBO date. This dichotomy can be attributed to the security market overreaction to the announcement of DBO. The long-term underperformance can be attributed to the costly refinancing of the ERC bonds. Keywords: event risk covenants, bonds, division buyout, parent firm, long-term performance 1 Introduction Keywords: event risk covenants, bonds, division buyout, parent firm, long-term performance 1. Introduction Hite and Vetsuypens (1989) find that the shareholders of the parent firms, undergoing DBO, experience positive and significant wealth effect at the announcement of the DBO. trigger protective covenants in these bonds. Due to the transfer of wealth from bondholders to stockholders, the lower bond values result into higher returns for the common stockholders. Hite and Vetsuypens (1989) find that the shareholders of the parent firms, undergoing DBO, experience positive and significant wealth effect at the announcement of the DBO. The event risk covenants (ERCs) in bonds are intended to deter negative wealth effect for the bondholders in the event of a major restructuring of the firm including leveraged and divisional buyouts (Cook & Easterwood, 1994). These covenants allow bondholders to put the bonds back at par on the firm undergoing these events. Bondholders can use the presence of ERCs in bonds as a bargaining tool and require the firms to restructure and reissue the existing bonds or call the bonds at more favorable terms to bondholders. The ERCs are also commonly referred to as poison puts or super poison puts in finance literature. The ERCs are known to be effective in achieving their intended purpose. Billet, Jiang, and Lie (2010) finds that the bonds with ERCs perform significantly better than the bonds without the ERC protection during a leveraged buyout. I investigate a sample of 46 parent firms, with non-convertible ERC bonds outstanding, that underwent DBO. I find that all the bonds were either refunded (put back by bondholders or called prematurely) or restructured around the DBO announcement date. This observation is in conformity with the argument by Asquith and Wizman (1990) who state that the bondholders must be appropriately compensated with a premium for the covenant protected bonds that are called due to a buyout since the buyout violates the protective covenants. The ERC protection results in firms refunding the protected bonds at or above premium or restructuring them to the benefit of the bondholders. This refunding leads to a higher refinancing cost for the firm which has long term implications (Chatfield & Moyer, 1986). The prime focus of this paper is to study the long-term market performance of the parent firms, with ERC bonds outstanding at the DBO announcement, over the periods three, four, and five years after the DBO. 1. Introduction Leveraged buyouts (LBOs) have been widely explored in the finance literature. In an LBO the investors buyout the entire publicly traded firm and take it private primarily with debt financing. Inefficient management and operations have been cited as primary reasons for taking a firm private through a leveraged buyout (Ambrose & Winters, 1992). Smith (1990) finds that firm’s operating performance considerably and significantly increased after the LBO. DeAngelo and DeAngelo (1987) and Kaplan (1989) find that the LBO considerably increases the market value of the target firms at the announcement of LBO. In a divisional buyout transaction (DBO hereafter), firm’s mangers and/or investor(s) buyout a division of a publicly traded firm primarily with debt financing and is typically taken private. Subsequently, the division exists as a separate (new) firm separated from the parent firm which stays public. The net effect of a DBO on the capital structure of the new firm is a substantial increase in the level of debt on the firm’s books since the assets of the new firm, including the future cash flows, are used as collateral for the loans obtained in the DBO. The DBOs are motivated by an opportunity to increase the efficient use of assets for the division at the same time improving the effectiveness and efficiency of decision making for the slimmer parent firm (Hite & Vetsuypens, 1989). The long-term performance of parent firms after the DBO has remained unexplored in the literature. Since, the parent firm stays public after the DBO, the firm’s common stock can be observed to evaluate the firm’s long-term performance. Bae and Jo (2002) find that parent firms with high cash flow volatility and high growth are more likely to divest through a DBO. Conversely, parent firms undergoing DBO likely signal volatile future cash flows. Titman and Wessels (1988) and Friend and Lang (1988) find that a firm’s ability to raise capital through debt generally decreases with an increase in the volatility of the firm’s earnings and cash flow due to the increased risk faced by the bondholders. This increase in risk has potential to lowers the value of outstanding bonds and is likely to 1 1 International Business Research Vol. 6, No. 6; 2013 www.ccsenet.org/ibr trigger protective covenants in these bonds. Due to the transfer of wealth from bondholders to stockholders, the lower bond values result into higher returns for the common stockholders. 1. Introduction The objective is to study how the refunding of these bonds affects the long-term returns to the common stockholders. Using Fama and French (1993) model, I find that these firms significantly underperform the market in the long-term after the DBO. This result conflicts with the observation of significant positive cumulative abnormal returns around the announcement of the DBO. This dichotomy can be attributed to the short-term exuberance by the investors (Barberis, Shleifer, & Vishny, 1998). I also find strong evidence of negative cumulative abnormal returns around the issue date of the ERC bonds for these firms. This further confirms the finding by Roth and McDonald (1999) that ERCs in bonds have the potential to induce managerial entrenchment as well as prevent wealth transfer from bondholders to stockholders. These results have important implications for the bond and common stock investors and funds. 2.1 DBO and the Parent Firm According to Hite and Vetsuypens (1989), the benefit to the parent firms undergoing DBO accrues largely from the improved efficiency and lower complexity of the decision-making for the management of the firm. The expected result after the sale of the division for the parent firm is a smaller, efficient, and more manageable firm. The shareholders of the parent firm, undergoing DBO experience positive wealth effect at the announcement of the DBO (Hite & Vetsuypens, 1989). In contrast the bondholders of the parent firms are susceptible to a significant loss of wealth due to the transfer of wealth from bondholders to stockholders. Maxwell and Rao (2003) find negative abnormal returns for the bondholders of the firms undergoing divisional divestiture in the announcement month. A significant proportion of the wealth accrued to the stockholders at the DBO announcement results from the losses incurred by the bondholders. Bae and Jo (2002) find that a parent firm with higher volatility of cash flow is more likely to divest a division through a DBO. Hence, a parent firm undergoing DBO is likely to signal high variability of future cash flows consequently, lowering the market value of existing bonds. The parent firms undergoing DBO are categorized as high growth firms with high cash volatility (Bae & Jo, 2002) in contrast to non-leveraged divestitures such as spin-offs or equity carve-outs where the specific characteristics of the parent firms remain largely unclear. .2 ERC Bonds and Negative Wealth Effect for Common Stockholders 2.5 Long-Term Performance after an Event The long-term stock performance after a special event has been well documented in finance literature. Speiss and Affleck-Graves (1999) evaluate the long-term performance of common stock of the firms after the issue of the straight debt. They find strong evidence of negative long-term returns. Affleck-Graves and Miller (2003) study the long-term performance of the firms after the optimal call of the debt. They find evidence of positive long-term returns. Tewariand Ramanlal (2010) study the long-term performance of firms issuing bonds with both call and put options embedded in the bonds. They find strong evidence of higher returns from issue date to the put date but find an evidence of lower returns after the put date. 2.4 Effect of Refunding of ERC Bonds Due to the DBO 2.4 Effect of Refunding of ERC Bonds Due to the DBO All the ERC bonds in our sample of parent firms are redeemed, put back, or restructured around the announcement of DBO date. This action can be attributed to the presence of ERCs in these bonds. In all the instances where the bonds are redeemed (called), they are redeemed at a premium. Since this type of redemption is not optimal (suboptimal) for the firm, it comes at a greater cost. According to Vu (1986), the firm is willing to pay a premium when the bonds with restrictive covenants are called. This premium is based on the opportunity cost of terms of covenants. For the bond issues where the terms of the bonds are restructured with new terms more beneficial to the investors, the long-term cost to the firm can be substantial. According to Chatfield and Moyer (1986), the inclusion of a put feature on a bond changes the benefits and costs to the issuer. The issuer enjoys an initially lower anticipated interest cost on the capital raised but faces substantial cost due to the risk of early refunding and a higher interest rate the firm is likely to pay on new bonds to replace (refinance) the issue put back on the firm. Once refunded (suboptimal call or put back), the firm can refinance the bonds only at the higher current market rates. This has potential of substantially increasing long-term financing cost to the parent firm. 2.3 ERC Bonds and Positive Wealth Effect for Common Stockholders The LBOs enable wealth transfer from bondholders to stockholders (Warga & Welch, 1993; Asquith & Wizman, 1990; Cook, Easterwood, & Martin, 1992). Presence of ERCs in bonds effectively limits this transfer of wealth, thereby reducing the potential conflict of interest between the stockholders and bondholders (Myers, 1977; Jensen & Meckling, 1976). This reduction in the conflict between the stockholders and bondholders potentially reduces the financing cost to the firm since the bondholders are willing to accept lower coupon rate for the lower risk of transfer of wealth (Cremers, Nair, & Wei, 2007). 2.2 ERC Bonds and Negative Wealth Effect for Common Stockholders 2.2 ERC Bonds and Negative Wealth Effect for Common Stockholders According to Kahan and Klaussner (1993), ERCs in bonds lower the probability of takeover which can entrench the management. Entrenched management is likely to take actions which may not be in the best interest of the stockholders consequently, increasing the agency cost between the stockholders and managers. This agency problem can lower the stockholder wealth (Shleifer & Vishny, 1989; Roth & McDonald, 1999). In addition, the lower probability of takeover effectively reduces the potential of a premium stockholders are likely to earn in the event of a buyout. This premium largely results from the transfer of wealth from bondholders to stockholders. 2 International Business Research Vol. 6, No. 6; 2013 www.ccsenet.org/ibr This foregone wealth transfer can reduce the stockholder wealth. Perumpral, Davidson, and Sen (1999) attribute negative announcement effect on the stock price to the presence of ERCs in bonds. This foregone wealth transfer can reduce the stockholder wealth. Perumpral, Davidson, and Sen (1999) attribute negative announcement effect on the stock price to the presence of ERCs in bonds. 4. Sample Selection The Securities Data Company Platinum database (SDC Platinum) is used to collect the bond data. The Bloomberg bond data is used to gather details of the terms of redemption of these bonds. The initial sample is collected for all the firms with ERC bond issues after 1986. The year 1986 is chosen as the starting year since that is the historical limit on the Bloomberg bond data. The initial sample is further screened for the ERC bond issuing firms that underwent DBO. The initial sample of firm with ERC bond issues are further screened for the following conditions. a) At least one year should have elapsed from the issue date to the DBO announcement date in order to eliminate the bonds issued to finance DBO. b) The cutoff date for the sample parent firms is set as 2007 since the objective is to measure the long-term performance over the period five years after the DBO announcement date. b) The cutoff date for the sample parent firms is set as 2007 since the objective is to measure the long-term performance over the period five years after the DBO announcement date. c) The issuing firm must be listed on a US exchange. d) The stock prices must be available on the Center for Research in Security Prices (CRSP) database. d) The stock prices must be available on the Center for Research in Security Prices (CRSP) database. The firms are further screened on Bloomberg to determine if the ERC protected bond issue was called, put, or restructured and the terms of restructure. The final full sample consists of 46 issues of the parent firms that underwent DBO and had ERC bonds outstanding that were redeemed or restructured around the DBO date. The firms are further screened on Bloomberg to determine if the ERC protected bond issue was called, put, or restructured and the terms of restructure. The final full sample consists of 46 issues of the parent firms that underwent DBO and had ERC bonds outstanding that were redeemed or restructured around the DBO date. 3. Hypotheses Development All the effects of DBO and the refinancing of the redeemed or restructured ERC bonds on the parent firms are only realized in the long-term. This argument helps formulate hypothesis 3. H3: In the long-term, the common stock of the parent firm should underperform after the DBO date 5. Research Methods 5.1 Short-Term Event Study (Announcement Effect) 3. Hypotheses Development Lehn and Poulsen (1991) argue that the ERCs are included in the bonds of the issuers where the risk of the LBO is high. The market’s expectations of the parent firm being involved in an LBO or a DBO is expected to be much higher for the parent firm that actually undergoes DBO later. Hence, the market’s reaction to the announcement of bond issues with ERCs for these firms (much prior to the DBO) is expected to be negative since forgone wealth transfer and managerial entrenchment far outweigh the benefit of lower agency cost associated with ERCs. This logic formulates hypothesis 1. H1: The common stock cumulative abnormal returns around the issue date of ERC bonds for these firms should be negative. Hite and Vetsuypens (1989) find positive abnormal returns for the parent firm at the DBO announcement. They argue that in addition to the premium the parent receives for the divested division, the DBO reduces the complexity of decision making by reducing the diversity of the assets to be managed by the management team. This potentially provides operational benefit to the parent firm. The common stock investors focused on this benefit are likely to bid up the stock price upon the DBO announcement. This argument helps formulate hypothesis 2. H2: The common stock cumulative abnormal returns for the parent firm around the announcement of DBO should be positive. The DBO reveals the riskiness of the future cash flows for the parent firms, which lowers the value of existing debt. The future debt is likely to be issued at a higher cost. ERCs in bonds require parent firms to redeem or restructure these bonds at DBO. The refinancing of these bonds due to the DBO is likely to substantially increase the long-term financing cost to the parent firm. This financing cost is likely to overshadow any operational 3 International Business Research Vol. 6, No. 6; 2013 www.ccsenet.org/ibr benefit from the sale of the division. The short-term exuberance (positive DBO announcement effect) may not transform into the positive long-term returns (Barberis et al., 1998). All the effects of DBO and the refinancing of the redeemed or restructured ERC bonds on the parent firms are only realized in the long-term. This argument helps formulate hypothesis 3. benefit from the sale of the division. The short-term exuberance (positive DBO announcement effect) may not transform into the positive long-term returns (Barberis et al., 1998). 5.1.1 Fama-French Model 6; 2013 International Business Research www.ccsenet.org/ibr equation 3 below: equation 3 below: equation 3 below: ܣܴ௜௧ൌ ܴ௜௧െ ܴ෠௜௧ (3) ܣܴ௜௧ൌ ܴ௜௧െ ܴ෠௜௧ (3) ܣܴ௜௧ൌ ܴ௜௧െ ܴ෠௜௧ (3) Where ܴ௜௧ is the actual return on stock i on day t in the event window. The CAR is calculated using equation 4: Where ܴ௜௧ is the actual return on stock i on day t in the event window. is the actual return on stock i on day t in the event window. alculated using equation 4: CARሺ௧ଶ,௧ଵሻൌ∑ ܣܴ௧ ௧ଶ ௧ଵ (4) (4) CARሺ௧ଶ,௧ଵሻൌ∑ ܣܴ௧ ௧ଶ ௧ଵ Where t1 and t2 represent beginning and the end of the three day trading window, ݐ א ሺݐ1, ݐ2ሻ. 5.1.2 Market Model Where t1 and t2 represent beginning and the end of the three day trading window, ݐ א ሺݐ1, ݐ2ሻ. 5.1.2 Market Model The results for abnormal results are also obtained using the market model in order to check the robustness of results from the F-F model. The market model uses the equation 5: The market model uses the equation 5: q ൫ܴ௜௧െܴ௙௧൯ൌߙ௜൅ߚ௜൫ܴ௠௧െܴ௙௧൯൅ ߝ௜௧ (5) The expected return over the estimation period [-255, -46] is computed using equation 6: ሺܴ෠௜௧െܴ௙௧ሻൌߙො௜൅ߚመ௜ሺܴ௠௧െܴ௙௧ሻ൅ ߝ̂௜௧ (6) (5) ൫ܴ௜௧െܴ௙௧൯ൌߙ௜൅ߚ௜൫ܴ௠௧െܴ௙௧൯൅ ߝ௜௧ (5) ൫ ௜௧ ௙௧൯ ௜ ߚ௜൫ ௠௧ ௙௧൯ ௜௧ ( ) The expected return over the estimation period [-255, -46] is computed using equation 6: ሺܴ෠௜௧െܴ௙௧ሻൌߙො௜൅ߚመ௜ሺܴ௠௧െܴ௙௧ሻ൅ ߝ̂௜௧ (6) The expected return over the estimation period [-255, -46] is computed using equation 6: ሺܴ෠௜௧െܴ௙௧ሻൌߙො௜൅ߚመ௜ሺܴ௠௧െܴ௙௧ሻ൅ ߝ̂௜௧ The expected return over the estimation period [ 255, 46] is computed using equation 6: ሺܴ෠௜௧െܴ௙௧ሻൌߙො௜൅ߚመ௜ሺܴ௠௧െܴ௙௧ሻ൅ ߝ̂௜௧ (6) ሺܴ෠௜௧െܴ௙௧ሻൌߙො௜൅ߚመ௜ሺܴ௠௧െܴ௙௧ሻ൅ ߝ̂௜௧ The abnormal return is computed using equation 3 and the CAR is computed using equation 4. 5.1.3 Event Study at the Issue Date The abnormal return is computed using equation 3 and the CAR is computed using equation 4. 5 1 3 E t St d t th I D t The abnormal return is computed using equation 3 and the CAR is computed using equation 4. This study uses issue date rather than the announcement date for the bond sample to measure abnormal returns around the issue date. According to Harvey, Lins, and Roper (2004), the announcement date for a typical bond issue almost always occurs either on the issue date or after the issue date. 5.1.1 Fama-French Model The focus of this study is to examine the determinants of cumulative abnormal return on the common stock of the firms over the event period. The abnormal returns are estimated using the Fama and French (1993) three-factor model (F-F hereafter) with a pre-event estimation window of [–255,–46] trading days prior to the event date. The F-F model is represented by the equation 1 below: ൫ܴ௜௧െܴ௙௧൯ൌߙ௜൅ߚ௜൫ܴ௠௧െܴ௙௧൯൅ݏ௜ܵܯܤ௧൅݄௜ܪܯܮ௧൅ ߝ௜௧ (1) Where, ܴ௙௧ = the risk free rate. ܴ௜௧ = the daily return of the stock i on day t. ܴ௜௧ = the daily return of the stock i on day t. ܴ௜௧ = the daily return of the stock i on day t. ܴ௠௧ = the return on the CRSP value-weighted index on day t. ܴ௠௧ = the return on the CRSP value-weighted index on day t. ܴ௠௧ = the return on the CRSP value-weighted index on day t. ܵܯܤ௧ = the return on the mimicking portfolio for size (Small minus Big) on day t. ܵܯܤ௧ = the return on the mimicking portfolio for size (Small minus Big) on day t. ܪܯܮ௧ = the return on the mimicking portfolio for book-to-market (High minus Low). The values and details of the construction of the variablesܴ௠௧, ܴ௙௧, ܵܯܤ௧, and ܪܯܮ௧ are obtained from the Kenneth French website. Equation 1 defines the relationship between the stock returns and the market during the pre-event estimation period [-255, -46]. Regression analysis using equation 1 is used to predict the expected daily returns for a given stock on a given day using CRSP value-weighted index on that day. The expected return ( ܴ෠௜௧ ) for a stock ion day t is represented by equation 2 below: ሺܴ෠௜௧െܴ௙௧ሻൌߙො௜൅ߚመ௜ሺܴ௠௧െܴ௙௧ሻ൅ݏ̂௜ܵܯܤ௧൅݄෠௜ܪܯܮ௧൅ ߝ̂௜௧ (2) (2) The event study captures the effect of some unexpected new information on the firms’ stock prices. The abnormal return is calculated for each day by subtracting the firm’s expected stock return (i.e. the return you would expect given its beta or association with the F-F value weighted overall stock market return over the 255 days prior to the event) from the actual observed return (MacKinlay, 1997). For each firm the daily abnormal return (ܣܴ௜௧) and the cumulative abnormal return (CAR) are calculated in the event window for examining the extent to which the stocks responds to the event. The ܣܴ௜௧ is calculated by 4 4 International Business Research Vol. 6, No. 6; 2013 Vol. 6, No. 5.1.1 Fama-French Model Even in the cases where the announcement is made prior to the issue date, market is unsure whether the issue will actually take place. In addition, the details of the contract parameters of the bonds are not revealed until the issue is registered. Therefore, the event window (0, +3) is primarily used to fully capture the reaction of the market after the details of the debt issue are fully digested by the market. Results for the windows (0, +1) and (0, +2) are also provided to observe the momentum of abnormal returns. 5.1.4 Event Study at DBO Announcement The abnormal returns are measured around the DBO announcement date with expectations that the market is likely to react very sharply to the DBO news. A DBO announcement is likely to produce highest abnormal returns over the (0, +1) window, which is the primary window of focus in this study. The results for the other windows (-1, +2) and (0, +3) are also provided. The (-1, +2) is chosen in order to capture the abnormal returns just in case the DBO news was leaked a trading day prior to the announcement date. The (0, +2) window is used to test whether the momentum of abnormal returns continued for a trading day longer. 5.2 Long-Term Event Study The long-term study to examine the long-run performance of the common stock of the 46 parent firms that underwent DBO also uses F-F regression method. The fourth factor commonly known as momentum factor (Brav, Geczy, &Gompers, 2000) is included to account for the presence of any momentum in the return. Since the objective is to measure the long-term monthly returns after the DBO announcement date, the common stock returns are examined over the periods three, four, and five years after the DBO announcement date. The four factor model used to determine the long run performance is represented by equation 7: ൫ܴ௣௧െܴ௙௧൯ൌߙ൅ߚ൫ܴ௠௧െܴ௙௧൯൅ݏ ሺܵܯܤ௧ሻ൅݄ ሺܪܯܮ௧ሻ൅ ݑ ሺܷܯܦ௧ሻ൅ ߝ௧ actor model used to determine the long run performance is represented by equation 7: ൫ܴ௣௧െܴ௙௧൯ൌߙ൅ߚ൫ܴ௠௧െܴ௙௧൯൅ݏ ሺܵܯܤ௧ሻ൅݄ ሺܪܯܮ௧ሻ൅ ݑ ሺܷܯܦ௧ሻ൅ ߝ௧ (7) or model used to determine the long run performance is represented by equation 7: ൫ܴ௣௧െܴ௙௧൯ൌߙ൅ߚ൫ܴ௠௧െܴ௙௧൯൅ݏ ሺܵܯܤ௧ሻ൅݄ ሺܪܯܮ௧ሻ൅ ݑ ሺܷܯܦ௧ሻ൅ ߝ௧ (7) Where, Where, ܴ௣௧= the return on the portfolio of sample firms in month t. It is calculated using the calendar time average method where each month’s returns represent the monthly average for each stock’s return that falls in that month (Mitchell & Stafford, 2000). The portfolio of firms is formed for each month that falls in the event period and monthly portfolio returns are calculated beginning from the month when the DBO announcement is made and continues over the event period. ܴ௣௧= the return on the portfolio of sample firms in month t. It is calculated using the calendar time average method where each month’s returns represent the monthly average for each stock’s return that falls in that month (Mitchell & Stafford, 2000). The portfolio of firms is formed for each month that falls in the event period and monthly portfolio returns are calculated beginning from the month when the DBO announcement is made and continues over the event period. ܷܯܦ௧ = the momentum factor. The explanation of variables ܴ௠௧, ܴ௙௧, ܵܯܤ௧, and ܪܯܮ௧ is provided in section 5.1.1. The value and the details of construction of ܷܯܦ௧ are obtained from the Kenneth French website. The primary focus in the long-term studies is on the intercept term α,which captures the abnormal returns per 5 International Business Research Vol. 6, No. 6; 2013 www.ccsenet.org/ibr month. The monthly average returns (ܴ௣௧ሻ are calculated by constructing value-weighted (VW) portfolio. The portfolio is constructed by taking weighted average of the returns of all the firms that fall in a particular month, weighted by the firm value (market capitalization) (Fama & French, 1993). The results are presented for the ordinary least squares (OLS) regression method. The results are also presented for the weighted least squares (WLS) regression method to check the robustness of the results. 6. Results Table 1 depicts the CAR over the windows (0, +3), (0, +2), and (0, +1) around the ERC bonds issue date. These results are provided to test the hypothesis 1. The results for F-F model as well as the market model are presented for the equally weighted/value weighted CRSP index. The focus is on the results over the event window (0, +3) as discussed earlier in section 5.1.3. For the F-F equally weighted model the CAR is -1.26% and statistically significant at 5%. For the F-F value weighted model the CAR is -1.29% and is statistically significant at 5%. The CAR for the equally weighted and the value weighted market model is also negative and statistically significant at 5%. The CAR for (0, +2) and (0, +1) is also negative and statistically significant for both equally weighted and value weighted F-F and the market model. These results confirm our hypothesis 1. Table 1. Mean cumulative abnormal returns (CAR) around the ERC bonds issue date Method Event Window (Days) Mean Cumulative Abnormal Return Portfolio Time-Series t-statistic Percent Negative Generalized Sign Z Fama-French/Equally Weighted (0,+1) -0.80% -1.62* 63 -1.29* (0,+2) -1.10% -1.81** 58 -1.86** (0,+3) -1.26% -1.81** 61 -2.06** Fama-French/Value Weighted (0,+1) -0.77% -1.55* 59 -0.83 (0,+2) -1.12% -1.84** 68 -2.01** (0,+3) -1.29% -1.84** 65 -1.71** Market Model/Equally Weighted (0,+1) -0.86% -1.67** 66 -1.55* (0,+2) -1.25% -2.00** 68 -1.88** (0,+3) -1.43% -1.97** 68 -1.88** Market Model/Value Weighted (0,+1) -0.78% -1.51* 66 -1.69** (0,+2) -1.03% -1.64** 61 -1.04 (0,+3) -1.25% -1.71** 63 -1.36* Description: The results show average cumulative abnormal returns for the event windows (0,+1), (0,+2), and (0,+3). (+1), (+2), and (+3)correspond to the first, second, and third trading day after the issue date respectively. Abnormal returns are estimated using returns from 255 days prior to the issue date and ending 46 days before the issue date. The significance of the difference between the proportion of positive/negative returns in the event period and the fraction of positive/negative returns in the estimation period is measured by Generalized Sign Z test. ***, **, and * indicate significance at the 1%, 5%, and 10% level, respectively. Table 1. Mean cumulative abnormal returns (CAR) around the ERC bonds issue date Description: The results show average cumulative abnormal returns for the event windows (0,+1), (0,+2), and (0,+3). (+1), (+2), and (+3)correspond to the first, second, and third trading day after the issue date respectively. 6. Results Abnormal returns are estimated using returns from 255 days prior to the issue date and ending 46 days before the issue date. The significance of the difference between the proportion of positive/negative returns in the event period and the fraction of positive/negative returns in the estimation period is measured by Generalized Sign Z test. ***, **, and * indicate significance at the 1%, 5%, and 10% level, respectively. Table 2 depicts the CAR over the windows (-1, +2), (0, +1), and (0, +2) around the DBO announcement date. These results are provided to test the hypothesis 2. The results for F-F model as well as the market model are presented for the equally weighted/value weighted CRSP index. The focus is on the results over the event window (0, +1) as discussed earlier in section 5.1.4. For the F-F equally weighted model the CAR is 4.66% and statistically significant at 1%. For the F-F value weighted model the CAR is 4.78% and is statistically significant at 1%. The CAR for the equally weighted and the value weighted market model is also positive and statistically significant at 1%. The CAR for (-1, +2) and (0, +2) is also positive and statistically significant at 1% for both equally weighted and value weighted, F-F and the market models. These results confirm our hypothesis 2. International Business Research Vol. 6, No. 6; 2013 www.ccsenet.org/ibr Table 2. Mean cumulative abnormal returns around the DBO announcement date Method Event Window (Days) Mean Cumulative Abnormal Return Portfolio Time-Series t-statistic Percent Positive Generalized Sign Z Fama-French/Equally Weighted (-1,+2) 4.77% 5.245*** 68 2.418*** (0,+1) 4.66% 7.246*** 70 2.747*** (0,+2) 4.61% 5.844*** 65 2.088** Fama-French/Value Weighted (-1,+2) 4.96% 5.436*** 68 2.418*** (0,+1) 4.78% 7.411*** 76 3.405*** (0,+2) 4.75% 6.013*** 65 2.088** Market Model/Equally Weighted (-1,+2) 4.07% 4.317*** 59 1.667** (0,+1) 4.21% 6.322*** 65 2.327*** (0,+2) 4.03% 4.931*** 62 1.997** Market Model/Value Weighted (-1,+2) 4.41% 4.671*** 65 1.455* (0,+1) 4.41% 6.603*** 62 2.113** (0,+2) 4.37% 5.353*** 61 1.784** Description: The results show average cumulative abnormal returns for the event windows (-1,+2), (0,+1), and (0,+2). The days in the windows correspond to a day prior (-1) to the DBO announcement date (0) and the first (+1), and the second (+2) trading day after the DBO announcement date. Abnormal returns are estimated using returns from 255 days prior to the DBO announcement date and ending 46 days before the DBO announcement date. 6. Results The significance of the difference between the proportion of positive/negative returns in the event period and the fraction of positive/negative returns in the estimation period is measured by Generalized Sign Z test. ***, **, and * indicate significance at the 1%, 5%, and 10% level, respectively. Table 2. Mean cumulative abnormal returns around the DBO announcement date windows correspond to a day prior (-1) to the DBO announcement date (0) and the first (+1), and the second (+2) trading day after the DBO announcement date. Abnormal returns are estimated using returns from 255 days prior to the DBO announcement date and ending 46 days before the DBO announcement date. The significance of the difference between the proportion of positive/negative returns in the event period and the fraction of positive/negative returns in the estimation period is measured by Generalized Sign Z test. ***, **, and * indicate significance at the 1%, 5%, and 10% level, respectively. Table 3 provides the results for the calendar time average F-F model (with momentum factor) for the sample of 46 firms. These results are provided to test the hypothesis 3. The panels A, B, and C in table 3 provide the OLS and WLS results for the VW portfolios over the periods three, four, and five years after the DBO announcement date. The focus in the long-term type event studies is on the intercept estimate α, which measures the abnormal monthly return. The α for the event period from issue date to three years after the issue date (Panel A) shows negative monthly return of -0.83%/-0.75%for OLS/WLS methods, statistically significant at 5%. These monthly returns compound to a significant loss of -26%/ -24% over the entire three year period. The α for the event period from issue date to four years after the issue date (Panel B) shows negative monthly return of -0.62%/-0.58%for OLS/WLS methods, statistically significant at 10% and 5% respectively. These monthly returns compound to a significant loss of -26%/ -24% over the entire four year period. The α for the event period from issue date to five years after the issue date (Panel C) shows negative monthly return of -0.51%/-0.60%for OLS/WLS methods, statistically significant at 10% and 5% respectively. These monthly returns compound to significant loss of -26%/ -30% over the entire five year period. Description: OLS corresponds to Ordinary Least Squares regression. WLS corresponds to Weighted Least Squares regression. Value weighted returns are used for both OLS and WLS methods where the returns are weighted by the market capitalization of the firm at the DBO announcement date. White’s method (White, 1980) is used to calculate t-statistics. *** indicates significance at 1% level, ** indicates significance at 5% level, and * indicates significance at 10% level. 6. Results All the results provide strong evidence that these firms significantly underperform three, four, and five years after the DBO date. These results confirm hypothesis 3. Table 3. Long-term performance after the DBO announcement date Event Period α β s h u AdjR² Panel A: 3 Year Returns OLS Regression -0.83 1.11 0.49 0.50 -0.16 39.90% (-2.03)** (11.93)*** (3.90)*** (3.05)*** (-2.63)*** WLS Regression -0.75 1.04 0.53 0.50 -0.21 36.76% (-1.96)** (11.36)*** (5.00)*** (4.02)*** (-3.02)*** Panel B: 4 Year Returns OLS Regression -0.62 1.13 0.50 0.41 -0.08 42.38% (-1.59)* (12.69)*** (4.06)*** (2.58)*** (-2.69)*** WLS Regression -0.58 1.04 0.45 0.35 -0.28 41.09% (-1.67)** (12.41)*** (4.70)*** (3.04)*** (-2.48)*** Panel C: 5 Year Returns OLS Regression -0.51 1.15 0.45 0.43 -0.18 49.03% (-1.47)* (13.46)*** (3.70)*** (2.80)** (-3.22)*** WLS Regression -0.6 1.07 0.43 0.44 -0.09 46.49% (-1.91)** (14.17)*** (5.05)*** (4.30)*** (-3.11)*** Description: OLS corresponds to Ordinary Least Squares regression. WLS corresponds to Weighted Least Squares regression. Value weighted returns are used for both OLS and WLS methods where the returns are weighted by the market capitalization of the firm at the DBO announcement date. White’s method (White, 1980) is used to calculate t-statistics. *** indicates significance at 1% level, ** indicates significance at 5% level, and * indicates significance at 10% level. Table 3. Long-term performance after the DBO announcement date Description: OLS corresponds to Ordinary Least Squares regression. WLS corresponds to Weighted Least Squares regression. Value weighted returns are used for both OLS and WLS methods where the returns are weighted by the market capitalization of the firm at the DBO announcement date. White’s method (White, 1980) is used to calculate t-statistics. *** indicates significance at 1% level, ** indicates significance at 5% level, and * indicates significance at 10% level. 7 Vol. 6, No. 6; 2013 International Business Research www.ccsenet.org/ibr 7. Conclusion The protective provisions, such as ERCs in bonds, intended to provide protection to the bondholders from significant firm restructuring events such as leveraged buyout (LBO) and divisional buyout (DBO) typically, require the issuing (parent) firms to redeem (make suboptimal call or put back by investors) or restructure these bonds and reissue with terms favorable to the investors and unfavorable to the firms. Refinancing of redeemed issues places long-term cost burden on the parent firm (Chatfield & Moyer, 1986). Although, DBO is expected to provide operational benefits to the parent firm (Hite & Vetsuypens, 1989), the parent firms with ERC bonds outstanding are likely to face enormous long-term refinancing cost of these bonds. In analyzing the long-term performance of a sample of 46 parent firms with DBO that had ERC bonds outstanding at the DBO, I find that the refinancing cost of ERC protected bonds overshadows any operational benefits of the DBO. These firms significantly underperform the market in the long-term over the periods three, four, and five years after the DBO. I also find strong evidence of short-term positive announcement effect at the announcement of the DBO for these parent firms. The short-term positive abnormal returns at the DBO announcement do not transform into long-term positive returns. This dichotomy can be attributed to short-term overreaction by the common stock investors (Barberis et al., 1998). I also find significantly negative cumulative abnormal returns at the issue date of the ERC bonds by the parent firms. This confirms the observation by (Perumpral et al., 1999; Billet et al., 2010) that the presence of ERCs in bonds lowers the stock price due to managerial entrenchment and potential loss of restructure premium for the stockholders. These results have significant implications for the bond investors and common stock investors in these types of firms. References Affleck-Graves, J., & Miller, R. E. 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A string of marine shell beads from the Neolithic site of Vršnik (Tarinci, Ovče pole), and other marine shell ornaments in the Neolithic of North Macedonia
Anthropozoologica
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art. 56 (4) — Published on 12 March 2021 www.anthropozoologica.com Directeur de la publication / Publication director : Bruno David Président du Muséum national d’Histoire naturelle Rédactrice en chef / Editor-in-chief: Joséphine Lesur Rédactrice en chef / Editor-in-chief: Joséphine Lesur Rédactrice / Editor: Christine Lefèvre Responsable des actualités scientifiques / Responsible for scientific news: Rémi Berthon Responsable des actualités scientifiques / Responsible for scientific news: Rémi Berthon Assistante de rédaction / Assistant editor: Emmanuelle Rocklin (anthropo@mnhn.fr) Assistante de rédaction / Assistant editor: Emmanuelle Rocklin (anthropo@mnhn.fr) Mise en page / Page layout: Emmanuelle Rocklin, Inist-CNRS Mise en page / Page layout: Emmanuelle Rocklin, Inist-CNRS Louis Chaix (Muséum d’Histoire naturelle, Genève, Suisse) Jean-Pierre Digard (CNRS, Ivry-sur-Seine, France) Allowen Evin (Muséum national d’Histoire naturelle, Paris, France) Bernard Faye (Cirad, Montpellier, France) Carole Ferret (Laboratoire d’Anthropologie Sociale, Paris, France) Giacomo Giacobini (Università di Torino, Turin, Italie) Lionel Gourichon (Université de Nice, Nice, France) Véronique Laroulandie (CNRS, Université de Bordeaux 1, France) Stavros Lazaris (Orient & Méditerranée, Collège de France – CNRS – Sorbonne Université, Paris, France) Nicolas Lescureux (Centre d’Écologie fonctionnelle et évolutive, Montpellier, France) Marco Masseti (University of Florence, Italy) Georges Métailié (Muséum national d’Histoire naturelle, Paris, France) Diego Moreno (Università di Genova, Gènes, Italie) François Moutou (Boulogne-Billancourt, France) Marcel Otte (Université de Liège, Liège, Belgique) Joris Peters (Universität München, Munich, Allemagne) François Poplin (Muséum national d’Histoire naturelle, Paris, France) Jean Trinquier (École Normale Supérieure, Paris, France) Baudouin Van Den Abeele (Université Catholique de Louvain, Louvain, Belgique) Christophe Vendries (Université de Rennes 2, Rennes, France) Denis Vialou (Muséum national d’Histoire naturelle, Paris, France) Jean-Denis Vigne (Muséum national d’Histoire naturelle, Paris, France) Allowen Evin (Muséum national d’Histoire naturelle, Paris, France) Allowen Evin (Muséum national d’Histoire naturelle, Paris, France Bernard Faye (Cirad, Montpellier, France) Carole Ferret (Laboratoire d’Anthropologie Sociale, Pari ( p g Giacomo Giacobini (Università di Torino, Turin, Italie) ( , , ) Lionel Gourichon (Université de Nice, Nice, France) Lionel Gourichon (Université de Nice, Nice, France) Véronique Laroulandie (CNRS, Université de Bordeaux 1, France) Stavros Lazaris (Orient & Méditerranée, Collège de France – CNRS – Sorbonne Université, Paris, France) Nicolas Lescureux (Centre d’Écologie fonctionnelle et évolutive, Montpellier, France) Marco Masseti (University of Florence, Italy) G Mét ilié (M é ti l d’Hi t i t ll P i F ) ( ) Véronique Laroulandie (CNRS, Université de Bordeaux 1, France) q ( Stavros Lazaris (Orient & Méditerranée, Collège de France – CN Nicolas Lescureux (Centre d’Écologie fonctionnelle et évolutive, M g Marco Masseti (University of Florence, Italy) ( y y) Georges Métailié (Muséum national d’Histoire naturelle, François Moutou (Boulogne-Billancourt, France) anthropozoologica A string of marine shell beads from the Neolithic site of Vršnik (Tarinci, Ovče pole), and other marine shell ornaments in the Neolithic of North Macedonia Vesna DIMITRIJEVIĆ, Goce NAUMOV, Ljubo FIDANOSKI & Sofija STEFANOVIĆ Vesna DIMITRIJEVIĆ, Goce NAUMOV, Ljubo FIDANOSKI & Sofija STEFANOVIĆ 56 (4) — Published on 12 March 2021 w.anthropozoologica.com Vesna DIMITRIJEVIĆ, Goce NAUMOV, Ljubo FIDANOSKI & Sofija STEFANOVIĆ art. 56 (4) — Published on 12 March 2021 www.anthropozoologica.com Directeur de la publication / Publication director : Bruno David Président du Muséum national d’Histoire naturelle Directeur de la publication / Publication director : Bruno David Président du Muséum national d’Histoire naturelle Couverture / Cover : Couverture / Cover : Le vaisseau anthropomorphe de Vršnik contenant des perles. Crédits photo : Jugoslav Pendić / The anthropomorphic vessel from Vršnik with beads inside. Photo credits: Jugoslav Pendić. Le vaisseau anthropomorphe de Vršnik contenant des perles. Crédits photo : Jugoslav Pendić / The anthropomorphic vessel from Vršn credits: Jugoslav Pendić. Anthropozoologica est indexé dans / Anthropozoologica is indexed in: – Social Sciences Citation Index – Arts & Humanities Citation Index – Current Contents - Social & Behavioral Sciences – Current Contents - Arts & Humanities – Zoological Record – BIOSIS Previews – Initial list de l’European Science Foundation (ESF) – Norwegian Social Science Data Services (NSD) – Research Bible Anthropozoologica est distribué en version électronique par / Anthropozoologica is distributed electronically by: – BioOne® (http://www.bioone.org) – Social Sciences Citation Index – Arts & Humanities Citation Index – Current Contents - Social & Behavioral Sciences – Current Contents - Arts & Humanities – Initial list de l’European Science Foundation (ESF) – Norwegian Social Science Data Services (NSD) Anthropozoologica est distribué en version électronique par / Anthropozoologica is distributed electronically by: – BioOne® (http://www bioone org) Anthropozoologica est une revue en flux continu publiée par les Publications scientifiques du Muséum, Paris, avec le soutien du CNRS. Anthropozoologica is a fast track journal published by the Museum Science Press, Paris, with the support of the CNRS. Les Publications scientifiques du Muséum publient aussi / The Museum Science Press also publish: Adansonia, Zoosystema, Geodiversitas, European Journal of Taxonomy, Naturae, Cryptogamie sous-sections Algologie, Bryologie, Mycologie, Comptes Rendus Palevol. Diffusion – Publications scientifiques Muséum national d’Histoire naturelle CP 41 – 57 rue Cuvier F-75231 Paris cedex 05 (France) Tél. : 33 (0)1 40 79 48 05 / Fax : 33 (0)1 40 79 38 40 diff.pub@mnhn.fr / https://sciencepress.mnhn.fr Diffusion – Publications scientifiques Muséum national d’Histoire naturelle ff.pub@mnhn.fr / https://sciencepress.mnhn.fr © Publications scientifiques du Muséum national d’Histoire naturelle, Paris, 2021 ISSN (imprimé / print) : 0761-3032 / ISSN (électronique / electronic) : 2107-08817 © Publications scientifiques du Muséum national d’Histoire naturelle, Paris, 2021 ISSN (imprimé / print) : 0761-3032 / ISSN (électronique / electronic) : 2107-08817 KEY WORDS Personal ornaments, Spondylus, Antalis, scaphopods. Sofija STEFANOVIĆ Sofija STEFANOVIĆ Biosense Institute, University of Novi Sad, Dr Zorana Đinđića 1, S-21000 Novi Sad (Serbia) and Laboratory for Bioarchaeology, Department of Archaeology, Faculty of Philosophy, University of Belgrade, Čika Ljubina 18-20, S-11000 Belgrade (Serbia) Submitted on 24 September 2019 | Accepted on 30 September 2020 | Published on 12 March 2021 Submitted on 24 September 2019 | Accepted on 30 September 2020 | Published on 12 March 2021 Dimitrijević V., Naumov G., Fidanoski L. & Stefanović S. 2021. — A string of marine shell beads from the Neolithic site of Vršnik (Tarinci, Ovče pole), and other marine shell ornaments in the Neolithic of North Macedonia. Anthropozoologica 56 (4): 57-70. https://doi.org/10.5252/anthropozoologica2021v56a4. http://anthropozoologica.com/56/4 A string of marine shell beads from the Neolithic site of Vršnik (Tarinci, Ovče pole), and other marine shell ornaments in the Neolithic of North Macedonia Vesna DIMITRIJEVIĆ Laboratory for Bioarchaeology, Department of Archaeology, Faculty of Philosophy, University of Belgrade, Čika Ljubina 18-20, S-11000 Belgrade (Serbia), and Biosense Institute, University of Novi Sad, Dr Zorana Đinđića 1, S-21000 Novi Sad (Serbia) vdimitri@f.bg.ac.rs Goce NAUMOV Museum of North Macedonia, Josif Mihailović 7, NM-1000 Skopje (North Macedonia) Ljubo FIDANOSKI Museum of the City of Skopje Ss Cyril & Methodius, NM-1000 Skopje (North Macedonia) Sofija STEFANOVIĆ Biosense Institute, University of Novi Sad, Dr Zorana Đinđića 1, S-21000 Novi Sad (Serbia) and Laboratory for Bioarchaeology, Department of Archaeology, Faculty of Philosophy, University of Belgrade, Čika Ljubina 18-20, S-11000 Belgrade (Serbia) bmitted on 24 September 2019 | Accepted on 30 September 2020 | Published on 12 March 2021 1. In this paper, the term annulet is used for circlets of continuous and homoge­ nous material of any size, bangle for annulet with a diameter that would allow wearing it on the arm, wrist or ankle, while the term ring is reserved for smaller circlets, suitable for wearing on fingers (after Nikolaidou 2003). MOTS CLÉS Ornements personnels, Spondylus, Antalis, scaphopodes. INTRODUCTION However, these maps usually lack the pre­ cision of shell items distribution in a diachronic perspective, since they usually refer to the Neolithic in a broad sense or Neolithic and Eneolithic/Chalcolithic periods (but see Rigaud The man’s affinity for personal ornamentation is universal and known since the distant past and from all continents (Cordwell 1979; Bar-Yosef Mayer 2005, 2008; Balme & Morse 2006; Zilhão 2007; Kuhn & Stiner 2007; Álvarez-Fernández & Carvajal-Contretas 2010; Szabó et al. 2014). The manner in which people were decorating themselves in prehistory is known mainly on the basis of ornaments made from durable materials. Among those, the most widely distributed and most frequently used in ornament production are items manufactured from shells of marine organisms. In addition to insights into fashion, aesthetic and cultural affinities in prehistory, as well as the social status of individuals, when reliable contextual information is available on shell items as personal belongings, ornaments made of marine shells also tell us about networks of communication and exchange between different prehistoric populations, and distances covered by these networks (Dimitrijević & Tripković 2006; Vanhaeren & d’Errico 2006; Álvarez-Fernández 2011; Rigaud 2011; Micheli 2012; Chapman & Gaydarska 2015). p g j p The fact that our data are incomplete and sometimes mislead­ ing in showing false blank spots on distribution maps, is also manifested by the region of the Republic of North Macedonia. A single site in this region, Amzabegovo, was included in the maps of Spondylus distribution in the Neolithic of Europe (e.g., Müller 1997). In this paper, we are going to show that the distribution of marine shell ornaments in North Macedonia is much greater. We will demonstrate that these ornaments vary in ornament type and raw material – shell species used in their production, especially in the case of the assemblage from the site of Govrlevo. In addition, the collection of shell ornaments from the Neolithic of North Macedonia includes some excep­ tional finds such as beads, probably forming a single string, found in an anthropomorphic vessel at the site of Vršnik in Ovče Pole (Fig. 1), almost forgotten since their brief mention in the original excavation report (Garašanin & Garašanin 1961). g p y Distribution maps of items manufactured from marine shells found at prehistoric sites testify to this. The best known is the distribution of personal ornaments made of Spondylus shell (Willms 1985; Müller 1997: fig. RÉSUMÉ Un collier de perles de coquillages marins provenant du site néolithique de Vršnik (Tarinci, Ovče pole), et d’autres ornements de coquillages marins du Néolithique de la Macédoine du Nord. q g q L’étude des ornements faits de coquillages marins a une importance remarquable pour la compréhen­ sion des sociétés préhistoriques. Ils nous renseignent sur la mode, les affinités esthétiques et culturelles des individus et des groupes sociaux, ainsi que sur les anciens réseaux de communication et d’échange. Le nombre d’objets en coquillages marins connus de la période néolithique de la Macédoine du Nord est relativement faible. Bien que peu nombreux, ils varient en termes de type d’ornement, avec des perles, des bracelets et des pendentifs représentés, et de type de coquillage utilisé comme matière pre­ mière, car ils sont constitués de coquilles de bivalves, de gastéropodes et de scaphopodes. La découverte de 157 perles de coquillages, vraisemblablement issues d’un seul fil, est particulièrement importante. Elles ont été découvertes en 1958 dans un vaisseau anthropomorphe sur le site de Vršnik à Ovče pole. C’est la reconnaissance de cette découverte, et le fait qu’elle était à l’origine mal décrite, puis presque complètement oubliée, qui ont lancé cette étude. La majorité des perles sont tubulaires et constituées de coquilles de deux mollusques ayant une morphologie très différente (bivalves et scaphopodes), mais elles sont étonnamment similaires en taille, forme et couleur. En outre, la collection comprenait des perles tubulaires en pierre blanche, une perle discoïde à coquille unique et trois escargots perforés. Cette trouvaille, ainsi que d’autres provenant de la région de Macédoine du Nord, nous permet de mieux comprendre la distribution des objets en coquillages marins en Europe continentale au Néolithique. De plus, elle ajoute à la visibilité des articles de scaphopodes partagés dans les réseaux d’échange, qui pourraient être sous-estimés en raison des difficultés de leur reconnaissance. INTRODUCTION 2011; Chapman & Gaydarska 2015), if not prehistory as a whole. In addition, maps are mainly too general when the type of ornament is in question (with some exceptions, like Rigaud 2011: annex 2), and also often lack shell taxonomic identification. For example, shell bangles 1, one of the most popular ornament types in the Neolithic, are often auto­ matically ascribed to Spondylus, while frequently, the shell of another bivalve, Glycymeris, was also used in their production (Dimitrijević & Tripković 2006). Also, scaphopod shells seem to be uncommon in the greater part of Europe in various pe­ riods of prehistory, but this may be the consequence of their poor recognition (Dimitrijević 2014; Tripković et al. 2016).h The man’s affinity for personal ornamentation is universal and known since the distant past and from all continents (Cordwell 1979; Bar-Yosef Mayer 2005, 2008; Balme & Morse 2006; Zilhão 2007; Kuhn & Stiner 2007; Álvarez-Fernández & Carvajal-Contretas 2010; Szabó et al. 2014). The manner in which people were decorating themselves in prehistory is known mainly on the basis of ornaments made from durable materials. Among those, the most widely distributed and most frequently used in ornament production are items manufactured from shells of marine organisms. In addition to insights into fashion, aesthetic and cultural affinities in prehistory, as well as the social status of individuals, when reliable contextual information is available on shell items as personal belongings, ornaments made of marine shells also tell us about networks of communication and exchange between different prehistoric populations, and distances covered by these networks (Dimitrijević & Tripković 2006; Vanhaeren & d’Errico 2006; Álvarez-Fernández 2011; Rigaud 2011; Micheli 2012; Chapman & Gaydarska 2015). Distribution maps of items manufactured from marine shells found at prehistoric sites testify to this. The best known is the distribution of personal ornaments made of Spondylus shell (Willms 1985; Müller 1997: fig. 1; Schuster 2002: map 1; Borrello & Micheli 2004: fig. 3; Dimitrijević & Tripković 2006: fig. 10; Séfériadés 2009; Rigaud 2011: figs 159, 160; Siklósi & Csengeri 2011: fig. 1). Ornaments made of Spondylus get the most attention, due to their relative frequency and shell impressibility. ABSTRACTh The study of ornaments made of marine shells has remarkable importance for understanding prehis­ toric societies. They tell us about fashion, aesthetic and cultural affinities of the individuals and social groups, as well as ancient networks of communication and exchange. The number of marine shell items known from the Neolithic period of North Macedonia is relatively low. Albeit few, they vary in orna­ ment type, with beads, bangles and pendants represented, and the kind of shell used as raw material, as they are made of shells of bivalves, gastropods, and scaphopods. Of special importance is a find of 157 shell beads, presumably from a single string, discovered in 1958 in an anthropomorphic vessel at the site of Vršnik in Ovče pole. It was the recognition of this find, and the fact that it was originally poorly described, and later almost completely forgotten, that initiated this study. The majority of beads are tubular and made of shells of two mollusks with very different shell morphology (bivalves and scapho­ pods), yet they are strikingly similar in size, shape, and color. In addition, the collection included white stone tubular beads, a single shell discoid bead, and three perforated snails. This find, as well as others from the region of North Macedonia, enhance our understanding of marine shell items distribution in continental Europe in the Neolithic period. Also, it adds to the visibility of scaphopod items share in exchange networks, which might be underestimated because of the difficulties in their recognition. 57 ANTHROPOZOOLOGICA • 2021 • 56 (4) © Publications scientifiques du Muséum national d’Histoire naturelle, Paris. Dimitrijević V. et al. Dimitrijević V. et al. BACKGROUND – THE NEOLITHIC OF NORTH MACEDONIA Apart from these social features, the pottery also had a sig­ nificant role in symbolic processes and therefore the painted patterns comprised of semiotic components, but some had anthropomorphic features, with the vessel from Vršnik be­ ing one of few (Naumov 2008, 2015). In terms of symbolic objects, Neolithic societies modeled a number of anthropo­ morphic and zoomorphic figurines, but also house models and stamps with human and animal representations (Sanev 2006; Chausidis 2010; Naumov 2010). Regarding rituals, intramural burials were also practiced within the settlements with an apparent preference for infants, children and women buried next to or below the dwellings (Naumov 2014). The beginning of the Neolithic in North Macedonia has been often discussed and some major perspectives are presented. The first agricultural societies in the region appeared at the end of the 7th millennium BC as indicated by radiocarbon dates from several sites, such as Amzabegovo (Gimbutas 1976a; Naumov 2016). Although some dates suggest earlier occupation in the middle of the 7th millennium, most evidence at the moment points to 6100 BC as the initial stage of occupation of this site. The Middle Neolithic starts approximately at 5800 BC while its transition to Late Neolithic is dated to 5300-5200 BC (Gimbutas 1976a; Sanev 1995). g A number of axes indicate forestation and provision of trees for constructing the buildings that were mainly rectangular, made of wattle and daub. Daub structures such as ovens, granaries, and bins used for storing and processing of cere­ als, as well as those for bread production, were often installed within buildings (Stojanova Kanzurova 2008; Tolevski 2009; Fidanoski 2012; Naumov 2013). A large number of seeds found in the vicinity of these structures, as well as a number of flint tools utilized as sickles for agriculture, indicate socie­ ties with a distinct focus on cereal-based diet (Hopf 1961; Renfrew 1976; Beneš et al. 2018; Naumov et al. 2018). In terms of diet, the consumption of cattle, sheep, goat and pig meat is confirmed, as well as the use of various vegetables and fruits in the daily cuisine (Bökönyi 1976; Ivkovska 2009; Naumov et al. 2018). Most likely the Neolithic started with the full set of the so-called ‘Neolithic package’, as no pre-pottery levels are de­ termined so far (Garašanin 1979; Sanev 1994). INTRODUCTION 1; Schuster 2002: map 1; Borrello & Micheli 2004: fig. 3; Dimitrijević & Tripković 2006: fig. 10; Séfériadés 2009; Rigaud 2011: figs 159, 160; Siklósi & Csengeri 2011: fig. 1). Ornaments made of Spondylus get the most attention, due to their relative frequency and shell impressibility. However, these maps usually lack the pre­ cision of shell items distribution in a diachronic perspective, since they usually refer to the Neolithic in a broad sense or Neolithic and Eneolithic/Chalcolithic periods (but see Rigaud 58 ANTHROPOZOOLOGICA • 2021 • 56 (4) Marine shell ornaments in the Neolithic of North Macedonia 50 km N Govrlevo Amzabegovo Vršnik Mogila NORTH MACEDONIA Fig. 1. — The map of North Macedonia with Neolithic sites mentioned in the text. Fig. 1. — The map of North Macedonia with Neolithic sites mentioned in the text. BACKGROUND – THE NEOLITHIC OF NORTH MACEDONIA Thus, since the Early Neolithic, the first agricultural societies produced pottery, figurines, lithic and stone tools, herded domestic animals and cultivated plants. The pottery mostly consists of white-painted vessels with fine fabric in the diversity of shapes, but also coarse pottery mainly used for cooking or as storage containers (Fidanoski 2009). Painted pottery was not only an aesthetical and practical device but also an indicator of the identity of the societies that employed it (Naumov 2015). Consequently, the communities inhabiting a particular region (a valley) had a distinct design of pottery patterns different from those in the other regions. Thus the so- called Amzabegovo – Vršnik and Velušina – Porodin cultural groups were determined, although there is a visual difference of patterns even within them, and particularly within the Amzabegovo – Vršnik group (Garašanin 1979; Sanev 1994). In general, the research of the Neolithic in North Macedonia as an extensive process is still far from sufficient, but current multidisciplinary projects provide new data which will signifi­ cantly help to understand better the first agricultural societies in the region. Nevertheless, the excavations, cabinet research 59 ANTHROPOZOOLOGICA • 2021 • 56 (4) Dimitrijević V. et al. 16 fragmented bangles, one annulet with a possible outer diameter of 3 cm (presumably a ring), one fragmented orna­ ment pierced at one end, and one small bivalve perforated at the hinge (Gimbutas 1976b: figs 203.10-13; 218.2, 3, 5, 7; 215.1-7; pl. 27, 28). These items originate from the phase Anza I, correlated with Early Neolithic (four Spondylus beads, one ring), Anza II, Middle Neolithic (seven bangles), and Anza IV, Late Neolithic (three Spondylus beads, six tubular beads of a fragile white shell, nine bangles, one fragmented ornament pierced at one end, and one small bivalve perfo­ rated at the hinge). Table 1. — Shell ornaments found at the Neolithic sites in North Macedonia. Abbreviations: EN, Early Neolithic; MN, Middle Neolithic; LN, Late Neolithic. to or less than one-third of the diameter (after Nikolaidou 2003). 3. A pendant or an amulet is an item perforated at one end, which cannot rotate when suspended (after Nikolaidou 2003). 2. A bead is defined as an object perforated along its major rotational axis (after Nikolaidou 2003). A tubular bead is characterized by the length equal to or more than three times their diameter, and a discoid bead has the length equal BACKGROUND – THE NEOLITHIC OF NORTH MACEDONIA Site Amzabegovo Govrlevo Vršnik Tumba Mogila Chronology EN MN LN EN EN/MN MN EN/MN Ornament type Bangle – 7 9 – 5 3 – Ring 1 – – – – – – Pendant – – 1 1 – – – Perforated ornament – – – – 1 – – Spondylus bead 4 3 – – – 29 – Scaphopod bead – 6 – – – 98 – Gastropod bead – – – – – 3 – Fragmented ornament – – 1 – – – 1 Table 1. — Shell ornaments found at the Neolithic sites in North Macedonia. Abbreviations: EN, Early Neolithic; MN, Middle Neolithic; LN, Late Neolithic. A fragmented shell bangle was published from the site of Govrlevo (Fidanoski 2012: 57, fig. 80). For the purpose of this paper, we analyzed all shell items from this site. The as­ semblage is rather diverse with respect to the ornament types present and the variability of shell raw material used. There are five fragments of bangles made of a shell of the bivalve genus Glycymeris, which is possible to conclude on the basis of morphological traces preserved: a triangular area with tent- shaped grooves (Fig. 2A), multiple transversal hinge teeth diverging outwards (Fig. 2A-C; G), a trace of an adductor muscle scar (Fig. 2E) and traces of marginal crenulations on the inner side of the valve (Fig. 2F), showing at the same time from which part of the valve the fragments come from (cf. Dimitrijević & Tripković 2006). and laboratory analyses performed so far indicate dynamic farming communities that were interacting with the natural environment and involved themselves in complex social and symbolic processes. The shell ornaments in general, and the beads deposited within the anthropomorphic vessel from Vršnik in particular, confirm such vibrant engagement of people in aesthetics and ritual practices. Three of these five bangle fragments from Govrlevo (Fig. 2E- G) show dark gray – blackish color, created under the influ­ ence of heating. One of them (Fig. 2F) shows a nice uniform grayish color which seemingly arose under reducing condi­ tions. It is possible that the bangles were intentionally heated to change their natural whitish color to dark grey or black (cf. annulets at the Late Neolithic settlement of Dimini [Chapman & Gaydarska 2007], or shells of Tritia neritea (Linnaeus, 1758) in the Lower Mesolithic of Franchthi cave [Perlès & Vanhaeren 2010]).h MARINE SHELL ORNAMENTS IN THE NEOLITHIC OF NORTH MACEDONIA Apart from the exceptional find of beads in an anthropomor­ phic vessel at the site of Vršnik, marine shell ornaments are reported from the Neolithic sites of Amzabegovo in Ovče pole, Govrlevo in Skopje valley, and Tumba Mogila in Pelagonia (Gimbutas 1976b; Simoska et al. 1979: fig. 1; Fidanoski 2009, 2012) (Table 1). In the present section, we shall give an overview of shell items from the collections of the city museums of Štip and Skopje which were available for study (Govrlevo and Vršnik), as well as present the data from the literature for those which were not accessible (Amzabegovo and Tumba Mogila).h There is one complete valve of Cerastoderma glaucum (Bruguière, 1789) (Fig. 2H-J), perforated by scraping off the most elevated part of the umbo. On the inner side of the valve, there are distinct ridges that diminish towards the middle of the valve (Fig. 2I). This feature enables differentia­ tion from its close relative, Cerastoderma edule (Linnaeus, 1758), previously known as Cardium edule. Although the perforation suggests that the item was used as a pendant 3, there are no usage traces on the perforation rims which would be expected if the item was worn on a cord. The usage of this kind of shell in the Neolithic of North Macedonia is also indicated by imprints on ceramic vessel fragments, presumably made by “Cardium” valve margins found at the Late Neolithic site of Mogila – Senokos (Temelkoski & Mitkoski 2008: 59). The studied shell assemblage from Govrlevo comprises the complete shell material collected in the course of the excava­ tion campaigns 1982-2010 and curated in the City Museum of Skopje. From the site of Vršnik, only the material presented at the exhibition in the History Museum in Štip was acces­ sible and studied. Magnifying lenses with a magnification × 20 were used for the observation of the details of morphology and manufacture of ornaments, and for measuring a digital caliper with the pre­ cision of 0.1 mm. A more detailed analysis of the Vršnik string was conducted on the basis of high-resolution photographs. MARINE SHELL ORNAMENTS IN THE NEOLITHIC OF NORTH MACEDONIA Bangles, beads and other types of pierced shell ornaments were recorded in Amzabegovo: seven Spondylus tubular and discoid beads 2, six tubular beads made of a fragile white shell, Magnifying lenses with a magnification × 20 were used for the observation of the details of morphology and manufacture of ornaments, and for measuring a digital caliper with the pre­ cision of 0.1 mm. A more detailed analysis of the Vršnik string was conducted on the basis of high-resolution photographs. Another perforated valve fragment (Fig. 2K-M) originates from a shell similar in size to C. glaucum or edule, but of different appearance, as it is shiny, like the mother of pearl, unlike the opaque, chalky Cerastoderma valve. There are Bangles, beads and other types of pierced shell ornaments were recorded in Amzabegovo: seven Spondylus tubular and discoid beads 2, six tubular beads made of a fragile white shell, 60 ANTHROPOZOOLOGICA • 2021 • 56 (4) Marine shell ornaments in the Neolithic of North Macedonia h l i l i d bl i All f h b l f h i f G l f h Fig. 2. — Marine shell ornaments from Govrlevo. A, one third of the diameter fragment of a shell bangle (inv. no 15132), made from a Glycymeris right valve, w preserved triangular area with tent-shaped grooves and several hinge teeth diverging outwards on both sides of the triangular area; B, the opposite side of the sam fragment showing the triangular umbonal cavity, i.e. the inner side of the hinge plate; C, one third of the diameter fragment of a shell bangle (inv. no 16212) ma from a Glycymeris valve, with preserved anterior margin of the valve with a portion of the plate with hinge teeth; D, the same fragment showing the uniform ban depth; E, a fragment of a Glycymeris bangle (inv. no 16853) with preserved portion of an aductor muscle scar; F, a fragment of a Glycymeris bangle (inv. no 162 with preserved marginal crenulation on the inner side of the valve; G, a fragment of a Glycymeris bangle (inv. no 16302) with preserved hinge teeth; H, left valve Cerastoderma glaucum (Bruguière, 1789) (inv. no 16574), outer side; I, the same specimen, inner side of the valve; J, the same specimen, the umbonal area w a perforation made by scraping; K, perforated middle portion of the bivalve (inv. MARINE SHELL ORNAMENTS IN THE NEOLITHIC OF NORTH MACEDONIA no 16301), outer side; L, the same specimen, inner side; M, the same specime lateral side, to show the depth of the item and the roundness of the edges. Scale bar: 2 cm. Photos credits: Vesna Dimitrijević (A-G; I-K; M); Ljubo Fidanoski (H, A E I B F J C G K D H L M orphological traits preserved to enable taxonomic fication It is the middle part of the valve and the All of the bangles from the site of Govrlevo are from the texts defined as Early Middle Neolithic as well as the shell — Marine shell ornaments from Govrlevo. A, one third of the diameter fragment of a shell bangle (inv. no 15132), made from a Glycymeris right valv ed triangular area with tent-shaped grooves and several hinge teeth diverging outwards on both sides of the triangular area; B, the opposite side of the nt showing the triangular umbonal cavity, i.e. the inner side of the hinge plate; C, one third of the diameter fragment of a shell bangle (inv. no 16212) Glycymeris valve, with preserved anterior margin of the valve with a portion of the plate with hinge teeth; D, the same fragment showing the uniform E, a fragment of a Glycymeris bangle (inv. no 16853) with preserved portion of an aductor muscle scar; F, a fragment of a Glycymeris bangle (inv. no  served marginal crenulation on the inner side of the valve; G, a fragment of a Glycymeris bangle (inv. no 16302) with preserved hinge teeth; H, left v derma glaucum (Bruguière, 1789) (inv. no 16574), outer side; I, the same specimen, inner side of the valve; J, the same specimen, the umbonal are ation made by scraping; K, perforated middle portion of the bivalve (inv. no 16301), outer side; L, the same specimen, inner side; M, the same spe de, to show the depth of the item and the roundness of the edges. Scale bar: 2 cm. Photos credits: Vesna Dimitrijević (A-G; I-K; M); Ljubo Fidanoski A E I B F J C G K D H L M A E B C D A A B C D G I J H H J H I J K L M Fig. 2. — Marine shell ornaments from Govrlevo. A, one third of the diameter fragment of a shell bangle (inv. MARINE SHELL ORNAMENTS IN THE NEOLITHIC OF NORTH MACEDONIA no 15132), made from a Glycymeris right valve, with preserved triangular area with tent-shaped grooves and several hinge teeth diverging outwards on both sides of the triangular area; B, the opposite side of the same fragment showing the triangular umbonal cavity, i.e. the inner side of the hinge plate; C, one third of the diameter fragment of a shell bangle (inv. no 16212) made from a Glycymeris valve, with preserved anterior margin of the valve with a portion of the plate with hinge teeth; D, the same fragment showing the uniform bangle depth; E, a fragment of a Glycymeris bangle (inv. no 16853) with preserved portion of an aductor muscle scar; F, a fragment of a Glycymeris bangle (inv. no 16214) with preserved marginal crenulation on the inner side of the valve; G, a fragment of a Glycymeris bangle (inv. no 16302) with preserved hinge teeth; H, left valve of Cerastoderma glaucum (Bruguière, 1789) (inv. no 16574), outer side; I, the same specimen, inner side of the valve; J, the same specimen, the umbonal area with a perforation made by scraping; K, perforated middle portion of the bivalve (inv. no 16301), outer side; L, the same specimen, inner side; M, the same specimen, lateral side, to show the depth of the item and the roundness of the edges. Scale bar: 2 cm. Photos credits: Vesna Dimitrijević (A-G; I-K; M); Ljubo Fidanoski (H, L). All of the bangles from the site of Govrlevo are from the con­ texts defined as Early-Middle Neolithic, as well as the shell item perforated in its middle, while C. glaucum pendant and the fossil come from the more precisely defined, Early Neolithic settings. no morphological traits preserved to enable taxonomic identification. It is the middle part of the valve and the perforation is positioned centrally. The rim of the item is well rounded. 61 ANTHROPOZOOLOGICA • 2021 • 56 (4) Dimitrijević V. et al. Fig. 3. — Marine shell ornaments from Vršnik. A, more than half of the diameter fragment of a shell bangle (inv. no C/110) made from a Glycymeris valve, with a preserved part of the triangular umbonal cavity and the hinge plate; B, the same specimen, opposite side, showing the marginal crenulation of the inner side of the valve; C, one third of the diameter fragment of a shell bangle (inv. MARINE SHELL ORNAMENTS IN THE NEOLITHIC OF NORTH MACEDONIA no 0/509) made from a Glycymeris valve; D, the same specimen, showing the marginal crenulation of the inner valve along its whole length; E, outer side of a fragment of a shell bangle made from a Spondylus valve (inv.no B&112) with a perforation; F, the same specimen, lateral view with a remnant of an adductor muscle scar; G, a Spondylus bead (inv. no 0/7), lateral view; H, the same specimen, front view. Scale bar: 2 cm. Photos credits: Jugoslav Pendić. A B C D E F G H A B C D B A D D G F H H G Fig. 3. — Marine shell ornaments from Vršnik. A, more than half of the diameter fragment of a shell bangle (inv. no C/110) made from a Glycymeris valve, with a preserved part of the triangular umbonal cavity and the hinge plate; B, the same specimen, opposite side, showing the marginal crenulation of the inner side of the valve; C, one third of the diameter fragment of a shell bangle (inv. no 0/509) made from a Glycymeris valve; D, the same specimen, showing the marginal crenulation of the inner valve along its whole length; E, outer side of a fragment of a shell bangle made from a Spondylus valve (inv.no B&112) with a perforation; F, the same specimen, lateral view with a remnant of an adductor muscle scar; G, a Spondylus bead (inv. no 0/7), lateral view; H, the same specimen, front view. Scale bar: 2 cm. Photos credits: Jugoslav Pendić. Both Glycymeris bangles were cut out along the margins of valves. One of them had more than half of the valve di­ ameter preserved. A remnant of the triangular hollow below the hinge is visible on one end, while the opposite end shows the marginal ornamentation (Fig. 3A, B). Another fragment bears remnants of the ornamentation from the inner margin of the valve on its whole length (Fig. 3C, D). At the site of Vršnik, apart from the beads found in the anthro­ pomorphic vessel which will be described in the next section, three fragmented bangles, and one separate bead were also discovered (Fig. 3). They are previously unpublished and are currently exhib­ ited at the Museum of Štip. The bead and one of these fragmented bangles are made from a Spondylus sp. shell (Fig. 3E-H), and two other bangles from Glycymeris sp. (Fig. 3A-D). MARINE SHELL ORNAMENTS IN THE NEOLITHIC OF NORTH MACEDONIA It has been suggested that Vršnik – Amzabegovo II and III share features and are widely contemporaneous with the Early and Middle Neolithic Starčevo in the Central Balkans, and Vršnik – Amzabegovo IV with the beginning of the Late Neolithic Vinča – Tordoš A (Garašanin 1979; Sanev 1995).h A single fragmented item, probably a pendant made from an unknown mollusk shell, is published from Tumba – Mogila, a tell site in Pelagonia (Simoska et al. 1979: 17; Fidanoski 2009). At Amzabegovo and Govrlevo, there were also bangles made from a material other than shell: fragments of one stone and one clay bangle from Govrlevo (Fig. 4A, B), and nine clay bangles from Amzabegovo (Gimbutas 1976b: 250-252; fig. 216). This is not an unusual find since at a large number of sites where shell bangles were found, they were accompanied with bangles of stone, bone or clay. Usually, they are much fewer than shell bangles, and are often interpreted as their imitations (cf. Dimitrijević & Tripković 2006: 246, fig. 9).i The anthropomorphic vessel has been discovered in block 1, in a layer ascribed to Vršnik II (Garašanin & Garašanin 1961). The vessel is in the form of the lower part of a fe­ male body, decorated with rows of incisions (Fig. 5). It is a unique find – there are vessels in the Neolithic of Balkans and Anatolia with similar traits, but none that we could describe as similar in both presenting the lower part of a female body, and in the manner of shaping and ornamentation of the bowl (Naumov 2008). A find from Govrlevo points to an occurrence of fossil collection, presumably in the surroundings (Fig. 4C). It is a small portion of the original form, presumably a cast of the end of a shell of an uncoiled ammonite. As it has natural spi­ ral furrows, it could have been used as a pendant, or simply collected as a curiosity. “A string of 157 clam and snail beads and one small red stone bead” (Garašanin & Garašanin 1961: 24, figs 33; 34) was found in the vessel. While in the reference paper by Garašanin & Garašanin a single sentence was dedicated to the description of the find in the vessel, and one small photo of rather low quality, it appears that it was later completely forgotten, and not further mentioned in subsequent literature. MARINE SHELL ORNAMENTS IN THE NEOLITHIC OF NORTH MACEDONIA 62 62 ANTHROPOZOOLOGICA • 2021 • 56 (4) Marine shell ornaments in the Neolithic of North Macedonia A B C Fig. 4. — Bangles made from non-shell materials from Govrlevo. A, fragment of a clay bangle; B, fragment of a marble bangle; C, fragmented fossil – the cast of an uncoiled ammonite. Scale bar: 1 cm. Photos credits: Vesna Dimitrijević (A, C); Ljubo Fidanoski (B). Fig. 5. — The anthropomorphic vessel from Vršnik. A, the vessel with beads inside to show their mutual relationship; B, drawing of the vessel by Sead Čerkez, in Benac 1979: pl. XIV, 1a, b. Scale bar: 2 cm. Photo credits: Jugoslav Pendić. A B A B C B Fig. 4. — Bangles made from non-shell materials from Govrlevo. A, fragment of a clay bangle; B, fragment of a marble bangle; C, fragmented fossil – the cast of an uncoiled ammonite. Scale bar: 1 cm. Photos credits: Vesna Dimitrijević (A, C); Ljubo Fidanoski (B). Fig. 5. — The anthropomorphic vessel from Vršnik. A, the vessel with beads inside to show their mutual relationship; B, drawing of the vessel by Sead Čerkez, in Benac 1979: pl. XIV, 1a, b. Scale bar: 2 cm. Photo credits: Jugoslav Pendić. Spondylus bangle bears a pit at the inner side, which is a remnant of the hollow below the valve hinge. Its color is milky white, while the outer side is coated with carbonates. It is perforated on one end. The perforation was performed in two steps, from the outer, and from the inner side (Fig. 3E, F). port was published shortly afterward (Garašanin & Garašanin 1961). Four horizons of habitation structures were distinguished (Vršnik I-IV), which were subsequently correlated with the sequence at the site of Amzabegovo, also known as Anzabegovo, and interpreted by Garašanin (1979) as a distinct cultural group (Amzabegovo – Vršnik I-IV), with Amzabegovo – Vršnik I identified as the Early, Amzabegovo – Vršnik II and III as Middle, and Amzabegovo – Vršnik IV as the Late Neolithic. The phasing was based primarily on the pottery features. MARINE SHELL ORNAMENTS IN THE NEOLITHIC OF NORTH MACEDONIA The anthropomorphic vessel was later discussed and illustrated, but the fact that the beads were found in it was omitted (e.g., Garašanin 1979). Given that the original description was scant, mentioning only that the beads were made from “snails and The context of the discovery of Vršnik beads The context of the discovery of Vršnik beads Systematic excavations at the site of Vršnik began in 1958. The site is also known as Vršnik – Tarinci, designating a more precise location. Remains of dwellings built in wattle and daub technique were recovered, as well as pits and ovens, a large quantity of pottery, a rather modest amount of stone and bone tools, and few anthropomorphic figurines. An excavation re­ 63 ANTHROPOZOOLOGICA • 2021 • 56 (4) Dimitrijević V. et al. C B H D B C D F G H E A Fig. 6. — A, Beads from the Vršnik anthropomorphic vessel in a string, as displayed on the exhibition in the Štip museum. The rectangles designate the magnified details of the string; B, detail of the string, showing tubular beads – ovoid (the first on the top, and two on the bottom) and cylindrical (the second from the top); C, a detail of the string with a discoid bead; D, a detail of the string with cylindrical beads with characteristic sinusoidal ends originating from the lines of growth stagnation in scaphopod shells; E, Antalis vulgaris (da Costa, 1778), the line of growth stagnation is in the middle of the shell; F, G, Tritia neritea (Linnaeus, 1758) beads; H, a detail of the string with two biconical stone/mineral beads between a cylindrical (first on the right) and an ovoid bead. Scale bars: A, 2 cm; B, 1 cm; E, 5 mm; F-H, 1 cm. Photos credits: Jugoslav Pendić (A-D, F-H); Raymond Huet (E; http://www.animalbase.uni-goettingen.de/zooweb/servlet/AnimalBase/home/ picture?id=9128, last consultation on 22 February 2021). B C D C B H D A C B D E E F G H F G G F H G Fig. 6. — A, Beads from the Vršnik anthropomorphic vessel in a string, as displayed on the exhibition in the Štip museum. The context of the discovery of Vršnik beads The rectangles designate the magnified details of the string; B, detail of the string, showing tubular beads – ovoid (the first on the top, and two on the bottom) and cylindrical (the second from the top); C, a detail of the string with a discoid bead; D, a detail of the string with cylindrical beads with characteristic sinusoidal ends originating from the lines of growth stagnation in scaphopod shells; E, Antalis vulgaris (da Costa, 1778), the line of growth stagnation is in the middle of the shell; F, G, Tritia neritea (Linnaeus, 1758) beads; H, a detail of the string with two biconical stone/mineral beads between a cylindrical (first on the right) and an ovoid bead. Scale bars: A, 2 cm; B, 1 cm; E, 5 mm; F-H, 1 cm. Photos credits: Jugoslav Pendić (A-D, F-H); Raymond Huet (E; http://www.animalbase.uni-goettingen.de/zooweb/servlet/AnimalBase/home/ picture?id=9128, last consultation on 22 February 2021). clams”, and the fact that the report (Garašanin & Garašanin 1961) was published in a local journal, it is not surprising that this find is not represented on the maps of shell items in European prehistory. the beads are translucent, thus most probably made from a contemporary shell, as fossil shells regularly loose transpar­ ency and become opaque through the process of fossilization (cf. Dimitrijević & Tripković 2006). As usual in beads, their manufacture largely destroys the morphological traits of the original shell, making the taxonomic identification difficult. However, in some beads, the alterna­ tion of transparent and opaque lines is observable, identical to the appearance of growth lines in bivalves (Fig. 6B, C, G). The width of these lines is uneven, and they are extending at variable angles in relation to bead axes, similarly to growth lines in sections observable in items made from Spondylus shell. Bearing in mind that Spondylus gaederopus Linnaeus, 1758 is one of the most common shell raw material in pre­ history of Europe and the Middle East (Borrello & Micheli 2004; Dimitrijević & Tripković 2006), and that the produc­ tion of massive beads of this size demanded massive valves, hardly occurring in any other Spondylus species, this leads to the assumption that precisely S. gaederopus was used for the manufacture of these beads. Bead descriptionh The beads put together on a string, are currently on dis­ play within the permanent exhibition at the Institute for Protection of Cultural Monuments and Museum – Štip (Fig. 6A). We counted 152 pieces, five less than mentioned in the original excavation report (Garašanin & Garašanin 1961). Three beads are Tritia neritea snails perforated on the last whorl (Fig. 6F, G). Remaining 149 show regular circular cross-section. The “small red stone bead” was not found in the string at the exhibition. g At first glance, all beads except the perforated snails look like they were made from the same material. Their color is whitish. A single one is discoid (Fig. 6D). Two types of shell tubular beads are present, cylindrical and ovoid (subtypes B21-1 and B.2 in bead typology after Bonnardin 2009; from Rigaud 2011: fig. 6) (Fig. 6B). Their differences can be seen in bead profiles. In the cylindrical subtype, the bead diameter is uniform along the length, whereas ovoid beads have the largest diameter in the middle, slightly and gradually taper­ ing toward the ends. When observed under a light source, The alternation of growth lines, i.e. the internal shell struc­ ture, is regularly visible in ovoid beads. Ovoid beads are also characterized by regular circular ends standing at the right angle in relation to bead axes. On the contrary, no growth lines are visible in cylindrical beads, their surfaces are smooth, 64 ANTHROPOZOOLOGICA • 2021 • 56 (4) Marine shell ornaments in the Neolithic of North Macedonia mm 18 16 14 12 10 8 6 4 2 0 145 133 121 109 97 85 73 61 49 37 25 14 1 length width inner diameter Number of specimens Fig. 8. — The length, width and inner diameter of Vršnik beads. Bead type Raw material snail perforated gastropod shell stone/mineral biconical cilindrical ovoid Schematic presentation bivalve shell discoid cf. Spondylus gaederopus Tritia neritea bivalve shell cf. Spondylus gaederopus bivalve shell cf. Antalis vulgaris at the last whorl Fig. 7. — The typology of beads from the Vršnik string. Fig. 8. — The length, width and inner diameter of Vršnik beads. we leave the question of species attribution of the shell mate­ rial of cylindrical Vršnik beads open. To make matters even more complex, a certain number of tubular white beads differ from both types described above (Fig. 6H). They were made of homogenous semitranspar­ ent material. Bead descriptionh This “sinusoidal” appearance of ends is characteristic for beads originating from the shells of scaphopods. Namely, in scaphopod shells, usually a single or several growth lines are found, most often in the form of sinusoidal curves (Fig. 6E). Scaphopod shells break most easily along these growth lines. Accordingly, scaphopod beads, no matter whether the shells were collected already broken on the beach, or obtained by breaking shell by pressure, very often have this kind of si­ nusoidal ends (Dimitrijević et al. 2010; Dimitrijević 2014; Tripković et al. 2016). The surface of cylindrical beads is smooth – it is without longitudinal ribs that characterize the shell of most of the scaphopods. Based on this fact, we could speculate that cylindrical beads might originate from the anterior (wider) end of Antalis vulgaris (da Costa, 1778), given that the shell of this species is ornamented with longi­ tudinal ribs in the posterior end, but soon disappearing, thus the anterior end is smooth. Antalis vulgaris is one of c. ten scaphopod species living in the Mediterranean and the larg­ est among them, with maximal length of 60 mm (Ozturk 2011; Martínez-Ortí & Cádiz 2012; Kurzawska et al. 2013: appendix A and references therein). The size of the scaphopod shell used in the production of Vršnik beads had to be quite massive, and maybe even larger then maximal size recorded in recent animals. However, in their study of Natufian scapho­ pod ornaments, Kurzawska et al. (2013) claim that some of the Antalis vulgaris shells found at archaeological sites seem to be thicker and larger than recent specimens, which also might be true for the European Neolithic sites. In any case, Accordingly, Vršnik beads are made from shells of three very different marine mollusks – gastropod coiled snails, Tritia neritea, bivalves, most probably Spondylus gaederopus, scaphopods, possibly Antalis vulgaris, and some sort of whit­ ish semitransparent stone. They belong to five different bead types – perforated snails, discoid, ovoid, cylindrical and bi­ conical (Fig. 7). Bead descriptionh They do not have growth lines like the beads made from bivalves, and they differ from scaphopod beads by very regular straight ends. In addition, they are slightly but clearly biconical, thus unlikely made from a scaphopod shell tapering toward one end. Probably, these beads were not made from mollusk shell, but from some sort of whitish, soft, semitransparent stone or mineral. Their number is smaller compared to ovoid beads made from bivalves and cylindrical beads originating from scaphopods. Fig. 7. — The typology of beads from the Vršnik string. while their ends are oblique or curved in a sinusoidal line (Fig. 6D). This “sinusoidal” appearance of ends is characteristic for beads originating from the shells of scaphopods. Namely, in scaphopod shells, usually a single or several growth lines are found, most often in the form of sinusoidal curves (Fig. 6E). Scaphopod shells break most easily along these growth lines. Accordingly, scaphopod beads, no matter whether the shells were collected already broken on the beach, or obtained by breaking shell by pressure, very often have this kind of si­ nusoidal ends (Dimitrijević et al. 2010; Dimitrijević 2014; Tripković et al. 2016). The surface of cylindrical beads is smooth – it is without longitudinal ribs that characterize the shell of most of the scaphopods. Based on this fact, we could speculate that cylindrical beads might originate from the anterior (wider) end of Antalis vulgaris (da Costa, 1778), given that the shell of this species is ornamented with longi­ tudinal ribs in the posterior end, but soon disappearing, thus the anterior end is smooth. Antalis vulgaris is one of c. ten scaphopod species living in the Mediterranean and the larg­ est among them, with maximal length of 60 mm (Ozturk 2011; Martínez-Ortí & Cádiz 2012; Kurzawska et al. 2013: appendix A and references therein). The size of the scaphopod shell used in the production of Vršnik beads had to be quite massive, and maybe even larger then maximal size recorded in recent animals. However, in their study of Natufian scapho­ pod ornaments, Kurzawska et al. (2013) claim that some of the Antalis vulgaris shells found at archaeological sites seem to be thicker and larger than recent specimens, which also might be true for the European Neolithic sites. In any case, while their ends are oblique or curved in a sinusoidal line (Fig. 6D). Bead dimensions and stringingf Tubular beads from shell do not differ significantly, especially if their width and the inner diameter is considered (Fig. 8). Their average length is 10 mm, the average width in the middle is 5.7 mm, and the average depth of the bead wall is 2.8 mm. The length of the beads ranges from maximal 15.9 mm to minimal 4.3 mm, the latter observed in a single discoid bead. In that range, the length is very variable. The width of the beads shows less variation, from 4.3 to 8 mm, and once again the width of a single discoid bead is exceptional, being the largest. The inner diameter is uniform, with an average of c. 3 mm. This uniformity of the inner diameter suggests that all beads most likely originate from the same string. Certainly, this hypothesis is further supported by the fact that all beads were found on a pile and in a vessel, therefore, if not literally strung on a single thread, obviously deposited as a whole. Of course, particular beads could have also been strung on two or more threads, whether in the moment of their deposition in the vessel or during their previous usage. 65 ANTHROPOZOOLOGICA • 2021 • 56 (4) Dimitrijević V. et al. 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 Scaphopoda Bivalvia Gastropoda stone 16 14 12 10 8 6 4 Width (mm) Length (mm) Fig. 9. — The length/width ratio of Vršnik beads. we take a look at figurines from Pelagonia, we may observe that some of them bear representations of ornaments. Judging by their size and shape, it might be assumed that Spondylus items (such as pendants, necklaces or belts) were depicted (Todorova & Vajsov 1993: fig. 170), speaking in the favor of Spondylus appreciation, and probably its actual presence in the region.i g Individual shell finds are mostly attributed to one of the four established Neolithic phases in the region, whereas the chronological attribution of a few specimens is only noted as the Neolithic (Table 1). From the Early Neolithic (Amzabegovo – Vršnik I), four Spondylus beads and one ring originate from the site Amzabegovo, and one perforated Cerastoderma glaucum was found at Govrlevo. Five Glycymeris bangles and one perforated bivalve from Govrlevo and fragment­ ed ornament from Tumba Mogila were attributed to the Early or Middle Neolithic. Bead dimensions and stringingf Seven Spondylus and Glycymeris bangles, three Spondylus and six scaphopod beads from Amzabegovo are dated to the Middle Neolithic (Amzabegovo – Vršnik II), as well as one Spondylus and two Glycymeris bangles. A find of beads in an anthropomorphic vessel at this site is also related to the Middle Neolithic. From the Late Neolithic (Amzabegovo – Vršnik IV), there are nine bangles, one frag­ mented ornament pierced on one end, and one small bivalve perforated at the hinge from Amzabegovo. Fig. 9. — The length/width ratio of Vršnik beads. But, in any case, they were ultimately combined as a whole, which is important for our further discussion. p If we consider the length/breadth proportion (Fig. 9), we observe that the discoid bead is separate, while all the others show a positive correlation. Stone beads tend to be smaller than average, while bivalve beads tend to be larger than av­ erage. Scaphopod beads are most numerous and of all sizes. Consequently, it should be noted that the chronological resolution for this study is fairly low. The Neolithic period is too complex and long, and the shell collection available for study does not allow for a discussion on the diachronic distribution of ornaments, i.e. such questions as: when did the discussed types of ornaments first appear in the region, were there local differences, and how did their value change through time? Instead, we shall point to the appearance of the same or similar types of ornaments in adjacent regions, the Mediterranean and continental parts of the Balkan Peninsula, in order to connect the finds from North Macedonia with regions which yielded much more data on this type of ornaments.h MARINE SHELL ORNAMENTS FROM THE NEOLITHIC PERIOD IN NORTH MACEDONIA AND COMPARATIVE FINDINGS FROM ADJACENT AREAS Having in mind that the number of Neolithic settlements discovered in North Macedonia is quite large, with some of them excavated extensively, the number of discovered shell ornaments is rather small. However, it should be borne in mind that the probability of omitting potential finds of shell ornaments would have been very high, for multiple reasons. In the first place, small and fragile shell items are often over­ looked if sieving and flotation are not performed, which was largely the case in the excavation strategies of these sites. Even in cases when they were found and curated, mostly shell items were not identified and published, but rather, according to common archaeological practice, they were often lumped to­ gether with stone or bone tools or remained unselected from animal bones. Besides the shell ornaments described, there were no other finds of marine shells which would indicate that marine mollusks were used in the diet. Therefore, shells were probably perceived as artefacts and prestige materials, opposite to the shells of mollusks collected for the subsistence (cf. Dupont 2019). They were most probably imported as fin­ ished products obtained by exchange with coastal populations. The best-represented ornament class are bangles. As in Greece (Infantidis 2019: xxx), they occur in North Macedonia from the Middle Neolithic and are made from Spondylus and Glycymeris, more precisely only from Glycymeris at Govrlevo. The Amzabegovo collection contains 16 bangles. Although they were originally all identified as Spondylus (Gimbutas 1976b), it is very likely that at least one of them is made from Glycymeris. Namely, in the published drawing (Gimbutas 1976b: fig. 215.6), a fragment of a hinge is shown, with a series of hinge teeth which are concordant with Glycymerys hinge teeth. On the other hand, such teeth are non-existent in Spondylus, which is characterized by two large cardinal teeth and two sockets. Bangles are found throughout the Neolithic Europe (Rigaud 2011: fig. 160; Chapman & Gaydarska 2015). At many sites in Greece (Infantidis 2019) and Balkans (Dimitrijević & Tripković 2006) they represent the dominant class of orna­ ments. They are often accompanied with bangles made from clay or stone, supposedly imitations of shell bangles, which were also found at Amzabegovo and Govrlevo. g Somewhat larger assemblages are only known from Amzabegovo in Ovče Pole and Govrlevo in Skopje Valley. MARINE SHELL ORNAMENTS FROM THE NEOLITHIC PERIOD IN NORTH MACEDONIA AND COMPARATIVE FINDINGS FROM ADJACENT AREAS On the other hand, beads from Vršnik, although presumed to be A. vulgaris, were made from mas­ sive and obviously much larger shells. They were made from shell segments where the narrowing of the shell characteris­ tic for scaphopods is not easily observed. One can imagine that this type of bead is not easily identified as a scaphopod bead, and even easily taken for granted as Spondylus bead, as our description in the above section has shown. In any case, we failed to find analogies for the Vršnik type of scaphopod beads in literature. An item made from the middle part of the valve of an unidentified bivalve from Govrlevo is also a single find of its kind in North Macedonia. It was also most likely an element of a string. However, it was probably suspended differently, since the perforation is positioned in the middle of the item, therefore the bead would not rest comfortably against the body of a wearer, as beads or pendants with a balanced perforation do. One possibility is that it was placed on the separate end of a string with the knot, but even more appealing would be the hypothesis that it represented one of the elements of a string for producing noise, as documented in ethnographic examples (Kolotourou 2007; Rainio & Mannermaa 2014). A similar example could have been pieced which were also perforated in the middle, but made of a different material (marble and C. glaucum valve), found in association at Dispilio (Infantidis 2019: fig. 4.62). i g When scaphopods used as beads are concerned, they were discovered at Amzabegovo and Govrlevo. Whole shells of scaphopods or segments of variable length were used as beads in the wider area of Europe and were found at a relatively large number of sites, especially in Greece (Karali 1999; Nikolaidou 2003; Infantidis 2019). In Neolithic Greece, they are most often identified as Dentalium sp., although probably in the sense of Scaphopoda indet., rather than actually referring to the genus Dentalium. They were also found in the Western Mediterranean and Central Europe (Rigaud 2011: 251, 263), where they were mostly identi­ fied as Antalis sp. or Antalis vulgaris. The distance from the Mediterranean coast probably influenced their quantity, therefore, they were rarely found in the hinterlands. Along the Black Sea coast, scaphopods identified as Antalis sp. MARINE SHELL ORNAMENTS FROM THE NEOLITHIC PERIOD IN NORTH MACEDONIA AND COMPARATIVE FINDINGS FROM ADJACENT AREAS From the vast valley of Pelagonia, where many Neolithic tumba (tell) sites are found, there is a single shell find from Tumba Mogila (Simoska et al. 1979; Fidanoski 2009). However, if 66 ANTHROPOZOOLOGICA • 2021 • 56 (4) Marine shell ornaments in the Neolithic of North Macedonia Marine shell ornaments in the Neolithic of North Macedonia A perforated valve made from Cerastoderma glaucum from Govrlevo is a single find of this kind in North Macedonia. In Neolithic Greece, shells of this bivalve species are amongst the most commonly used jewelry raw material (Infantidis 2019: 153). There are a few examples of a direct analogy to the Govrlevo specimen if the location of the perforation and the way it was produced are considered. At the Neolithic site of Dispilio, a necklace was found, consisting of seven valves of this species perforated in the exactly same manner as the Govrlevo specimen – by rubbing the clam beak against a rough surface (Infantidis 2019: fig. 4.43). A possible necklace made from Cerastoderma glaucum is also mentioned from Nea Nikomedia (Infantidis 2019: 218). In some instances, scaphopod beads were found in piles, thus probably originally forming necklaces or strings. To mention just a few, six scaphopod beads and one marble pendant were found in Stavroupoli Thessaloniki (Infantidis 2019), and a group of 22 scaphopod beads in Sitagroi Drama (Nikolaidou 2003: 346, 347; pl. 9.13). Analogous to Vršnik, beads de­ posited in ceramic vessels were found in a vase at Gyalos of Spata, Aticca (Infantidis 2019: 76). Also, the jewelry was placed in a ceramic vessel in the Early Neolithic hoard from Galabnik (Western Bulgaria), with more than 10000 beads made of scaphopods, stone, and bone, along with the annulets manufactured from Spondylus and nephrite (Chapman 2000). Scaphopod beads, jointly with Spondylus beads and perfo­ rated plates of a belt, were also found at the Late Neolithic site of Čepin in Croatia, in this case, deposited in a large Spondylus valve (Tripković et al. 2016). The scaphopods were identified as Antalis vulgaris, due to the preservation of ante­ rior openings and a larger portion of the shell body in most of the specimens. These were tiny shells, less than 1.5 cm long and very thin. Scaphopod beads from Amzabegovo are very similar, and it is possible that they originate from shells of the same species. MARINE SHELL ORNAMENTS FROM THE NEOLITHIC PERIOD IN NORTH MACEDONIA AND COMPARATIVE FINDINGS FROM ADJACENT AREAS occur in large numbers starting with the fifth millennium, probably due to the maritime trade with the Aegean (Ivanova 2012). Ornaments deposited in graves, especially at the necropolis of Durankulak and Varna (Avramova 2002; Todorova 2002), offered particular insights regarding the manner they were assembled and worn. Apart from contemporary, fossil shells were also used, especially in the middle Danube area and in the Carpathian Basin (Todorova 1995; Sztancs & Beldiman 2010; Dimitrijević et al. 2010) sometimes continuously during a considerable period of time (Dimitrijević 2014). In Italy, fossil scaphopods were used in the region of the river Po basin, while contemporary shells were recorded in Liguria and Campaign (Micheli 2004, 2012). Acknowledgements Borrello M. A. & Micheli R. 2004. — Spondylus gaederopus, gioiello dell’ Europa preistorica. Preistoria Alpina 40 (Suppl 1): 71-82. C J F A h l P l Pl We would like to express our gratitude to colleagues and staff of the Institute for Protection of Cultural Monuments and Museum – Štip and the Museum of the City of Skopje, for permission to work on shell items from their collec­ tions. We thank Ivana Živaljević for her help in English editing, Predrag Filipović for the artwork in illustrations (Figs. 2-5; 7), as well as the authors of the photographs used in illustrations. We deeply respect invested efforts and qual­ ity of publication aspired by editors and Publishing service of the journal Anthropozoologica. Finally, we appreciate valu­ able and regardful comments received from the reviewers, Catherine Dupont and Louis Chaix.This paper is a result of the Project “BIRTH: Births, mothers and babies: prehistoric fertility in the Balkans between 10 000-5000 BC”, funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant Agreement no 640557). Chapman J. 2000. — Fragmentation in Archaeology: People, Places and Broken Objects in the Prehistory of Southeastern Europe. Rout­ ledge, London, New York, 296 p. Chapman J. & Gaydarska B. 2007. — Parts and Wholes. Frag­ mentation in Prehistoric Context. Oxbow Books, Oxford, 233 p. https://doi.org/10.5334/pia.312 Chapman J. & Gaydarska B. 2015. — Spondylus gaederopus/Gly­ cymeris exchange networks in the European Neolithic and Chal­ colithic, in Fowler J., Harding J. & Hofmann D. (eds), The Oxford Handbook of Neolithic Europe. https://doi.org/10.1093/ oxfordhb/9780199545841.013.014i Chausidis N. 2010. — Neolithic ceramic figurines in the shape of a woman – house from the Republic of Macedonia, in Gheo­ rghiu D. & Cyphers A. (eds), Anthropomorphic and zoomor­ phic miniature figures in Eurasia, Africa and Meso-America: morphology, materiality, technology, function and context. BAR International Series 2138: 25-35.h Cordwell J. M. 1979. — The very human arts of transformation, in Cordwell J. M. & Schwarz R. A. (eds), The Fabrics of Culture. The Anthropology of Clothing and Adornment. Mouton Publishers, The Hague: 47-75. https://doi.org/10.1515/9783111631523.47h h Dimitrijević V. 2014. — The provenance and use of fossil scapho­ pod’s shells at the Late Neolithic/Eneolithic site Vinča – Belo Brdo, in Szabó K., Dupont C., Dimitrijević V., Gastélum Gómez L. G. & Serrand N. (eds), Archaeomalacology: shells in the archaeological record. CONCLUSION Albeit small, the collection of marine shell ornaments from North Macedonia is nevertheless quite diverse in terms of or­ nament types and raw material – i.e. the shell species used in their production. Bangles made from Spondylus and Glycymeris valves were found at most sites. In addition, beads made from Tritia neritea, Spondylus and scaphopod shells occur, as well as a ring and perforated ornaments made from Cerastoderma glaucum in one case, and from unknown bivalve species in three more cases. In the anthropomorphic vessel at the site of Vršnik, more than 150 beads made from shells of gastropods, bivalves, scaphopods, and some sort of whitish semitransparent stone were found. Judging by the size of beads, especially their uniform inner diameter, along with the fact that they were found deposited in a pile in a vessel, it can be assumed that they probably formed a single string. When considering body decorations in the archaeological record, obviously we only have a partial insight into their original appearance, and it is often difficult to reconstruct the manner in which they were worn. This is due to the fact that as a rule, we are missing perishable materials or their traces, by which the preserved ornaments of durable materials were assembled. Moreover, 67 ANTHROPOZOOLOGICA • 2021 • 56 (4) Dimitrijević V. et al. Bar-Yosef Mayer D. E. 2008. — Invertebrate analysis, in Pears­ all D. (ed.), Encyclopedia of Archaeology. Academic Press, New York: 1560-1565. we usually find elements of composite ornaments separated. A string from Vršnik is a rare exception, as it offers an insight into aesthetic preferences and manufacturing skills that we rarely have the opportunity to discover in the archaeological record. In this case, the skill was manifested by combining materials from very different raw material with a single com­ mon feature – their white color, into elements masterfully modified into a similar shape. Benac A. (ed.) 1979. — Praistorija jugoslavenskih zemalja. II: Neolit­ sko doba. Svjetlost, Sarajevo, 705 p. Beneš J., Naumov G., Majerovičová T., Budilová K., Živaljević I., Dimitrijević V., Bumerl J., Komárková V., Kovárník J., Vychronová M. & Stefanović S. 2018. — Onsite bioarchaeological knowledge of the Neolithic settlements in the Balkans: the case of Vrbjanska Čuka, a tell-site in Pelagonia, Republic of Macedonia. Interdisciplinaria Archaeologica Natural Sciences in Archaeology 9 (1): 121-145. Acknowledgements Proceedings of the 11th ICAZ Interna­ tional Conference – Archaeomalacology working group, 23-28 August 2010, France. BAR International Series 2666: 33-41. https://doi.org/10.13140/2.1.4388.7368 zenodo.1260412 Bökönyi S. 1976. — The vertebrate fauna from Anza, in Gimbu­ tas M. (ed.), Anza, Neolithic Macedonia, as reflected by Excavation at Anza, Southeast Yugoslavia. Institute of Archaeology, University of California, Los Angeles: 313-363. Bonnardin S. 2009. — La parure funéraire au Néolithique ancient dans les Bassins parisien et rhénan. Rubané, Hinkelstein et Vil­ leneuve-Saint-Germain. Société préhistorique française (coll. Mémoire de la SPF ; 49), Paris, 322 p. https://doi.org/10.1016/j. anthro.2010.03.003 CONCLUSION https://doi.org/10.5281/ i Our data also shows that what looked like almost empty space on the maps of the distribution of Spondylus/marine shell items in Neolithic Europe, no longer appears to have been excluded from prehistoric exchange networks. 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Analysis of shared heritability in common disorders of the brain
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Analysis of Shared Heritability in Common Disorders of the Brain 2) Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA 3) Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA 4) Department of Mathematics, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA 21 5) Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA 22 6) Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK 2 6) Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK 3 23 7) Cardiff University, Medical Research Council Center for Neuropsychiatric Genetics & Genomics, Institute of Psychology, Medicine & Clinical Neuroscience, Cardiff, Wales, 24 UK 25 ) Cardiff University, Medical Research Council Center for Neuropsychiatric Genetics & Genomics, Institute of Psychology, Medicine & Clinical Neuro UK 8) Center for Human Genetic Research, Department of Neurology, Massachusetts General Hospital, Boston, MA, USA 9) Psychiatric and Neurodevelopmental Genetics Unit (PNGU), Center for Human Genetics Research, Massachusetts General Hospital, Boston, M 10) Institute for Stroke and Dementia Research, Klinikum der Universität München, Ludwig-Maximilians-University, Munich, Germany 28 11) Department of Neurology, Brigham & Women’s Hospital, Harvard Medical School, Boston, MA 29 2) Charite Universitatsmedizin Berlin, Berlin, Germany 29 12) Charite Universitatsmedizin Berlin, Berlin, Germany 30 12) Charite Universitatsmedizin Berlin, Berlin, Germany 30 13) Department of Computer Science, New Jersey Institute of Technology, New Jersey, USA 31 31 14) Kings College London, Institute of Psychiatry, Psychology & Neuroscience, Social Genetics & Developmental Psychiatry Center, MRC, London, England 32 31 14) Kings College London, Institute of Psychiatry, Psychology & Neuroscience, Social Genetics & Developmental Psychiatry Center, MRC, London, England 32 15) NIHR Bi d R h C t f M t l H lth S th L d & M d l NHS T t & I tit t P hi t L d E l d 3 14) Kings College London, Institute of Psychiatry, Psychology & Neuroscience, Social Genetics & Developmental Psychiatry Center, MRC, London, England 32 HR, Biomed Research Center for Mental Health, South London & Maudsley 33 16) Departments of Psychiatry and Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. 34 34 17) Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint 49 32) Department of Genetics and Psychiatry, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA 50 33) Neuropsychiatric Genetics Research Group, Department of Psychiatry, Trinity College Dublin, Ireland 51 Analysis of Shared Heritability in Common Disorders of the Brain 1 V Anttila*1,1,2,3, B Bulik-Sullivan1,3, H Finucane4,5, R Walters1,2,3, J Bras6, L Duncan1,3, V Escott- 2 Price7, G Falcone8, P Gormley9, R Malik10, N Patsopoulos3,11, S Ripke1,2,3,12, Z Wei13, D Yu2,9, 3 PH Lee2,9, P Turley1,3, IGAP consortium, IHGC consortium, ILAE Consortium on Complex 4 Epilepsies, IMSGC consortium, IPDGC consortium, METASTROKE and Intracerebral 5 Hemorrhage Studies of the International Stroke Genetics Consortium, Attention-Deficit 6 Hyperactivity Disorder Working Group of the Psychiatric Genomics Consortium, Autism 7 Spectrum Disorders Working Group of The Psychiatric Genomics Consortium, Bipolar 8 Disorders Working Group of the Psychiatric Genomics Consortium, Eating Disorders Working 9 Group of the Psychiatric Genomics Consortium, Major Depressive Disorder Working Group of 10 the Psychiatric Genomics Consortium, Tourette Syndrome and Obsessive Compulsive Disorder 11 Working Group of the Psychiatric Genomics Consortium, Schizophrenia Working Group of the 12 Psychiatric Genomics Consortium, G Breen14,15, C Churchhouse1,2,3, C Bulik16,17, M Daly1,2,3, M 13 Dichgans10,18, SV Faraone19, R Guerreiro20, P Holmans7, K Kendler21, B Koeleman22, CA 14 Mathews23, AL Price3,5, JM Scharf2,3,8,9,24,25, P Sklar26, J Williams7, N Wood20,27,28, C 15 Cotsapas3,29, A Palotie1,2,3,9,30,31, JW Smoller2,9, P Sullivan16,32, J Rosand3,8,25, A Corvin†*2,33, 16 BM Neale †*3,1,2,3, on behalf of the Brainstorm consortium 17 Analysis of Shared Heritability in Common Disorders of the Brain 1 V Anttila*1,1,2,3, B Bulik-Sullivan1,3, H Finucane4,5, R Walters1,2,3, J Bras6, L Duncan1,3, V Escott- 2 Price7, G Falcone8, P Gormley9, R Malik10, N Patsopoulos3,11, S Ripke1,2,3,12, Z Wei13, D Yu2,9, 3 PH Lee2,9, P Turley1,3, IGAP consortium, IHGC consortium, ILAE Consortium on Complex 4 Epilepsies, IMSGC consortium, IPDGC consortium, METASTROKE and Intracerebral 5 Hemorrhage Studies of the International Stroke Genetics Consortium, Attention-Deficit 6 Hyperactivity Disorder Working Group of the Psychiatric Genomics Consortium, Autism 7 Spectrum Disorders Working Group of The Psychiatric Genomics Consortium, Bipolar 8 Disorders Working Group of the Psychiatric Genomics Consortium, Eating Disorders Working 9 Group of the Psychiatric Genomics Consortium, Major Depressive Disorder Working Group of 10 the Psychiatric Genomics Consortium, Tourette Syndrome and Obsessive Compulsive Disorder 11 Working Group of the Psychiatric Genomics Consortium, Schizophrenia Working Group of the 12 Psychiatric Genomics Consortium, G Breen14,15, C Churchhouse1,2,3, C Bulik16,17, M Daly1,2,3, M 13 Dichgans10,18, SV Faraone19, R Guerreiro20, P Holmans7, K Kendler21, B Koeleman22, CA 14 Mathews23, AL Price3,5, JM Scharf2,3,8,9,24,25, P Sklar26, J Williams7, N Wood20,27,28, C 15 Cotsapas3,29, A Palotie1,2,3,9,30,31, JW Smoller2,9, P Sullivan16,32, J Rosand3,8,25, A Corvin†*2,33, 16 BM Neale †*3,1,2,3, on behalf of the Brainstorm consortium 17 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint 1 Analysis of Shared Heritability in Common Disorders of the Brain 1 V Anttila*1,1,2,3, B Bulik-Sullivan1,3, H Finucane4,5, R Walters1,2,3, J Bras6, L Duncan1,3, V Escott 2 Price7, G Falcone8, P Gormley9, R Malik10, N Patsopoulos3,11, S Ripke1,2,3,12, Z Wei13, D Yu2, 3 PH Lee2,9, P Turley1,3, IGAP consortium, IHGC consortium, ILAE Consortium on Comple 4 Epilepsies, IMSGC consortium, IPDGC consortium, METASTROKE and Intracerebra 5 Hemorrhage Studies of the International Stroke Genetics Consortium, Attention-Defic 6 Hyperactivity Disorder Working Group of the Psychiatric Genomics Consortium, Autism 7 Spectrum Disorders Working Group of The Psychiatric Genomics Consortium, Bipola 8 Disorders Working Group of the Psychiatric Genomics Consortium, Eating Disorders Workin 9 Group of the Psychiatric Genomics Consortium, Major Depressive Disorder Working Group o 10 the Psychiatric Genomics Consortium, Tourette Syndrome and Obsessive Compulsive Disorde 11 Working Group of the Psychiatric Genomics Consortium, Schizophrenia Working Group of th 12 Psychiatric Genomics Consortium, G Breen14,15, C Churchhouse1,2,3, C Bulik16,17, M Daly1,2,3, M 13 Dichgans10,18, SV Faraone19, R Guerreiro20, P Holmans7, K Kendler21, B Koeleman22, CA 14 Mathews23, AL Price3,5, JM Scharf2,3,8,9,24,25, P Sklar26, J Williams7, N Wood20,27,28, C 15 Cotsapas3,29, A Palotie1,2,3,9,30,31, JW Smoller2,9, P Sullivan16,32, J Rosand3,8,25, A Corvin†*2,3 16 BM Neale †*3,1,2,3, on behalf of the Brainstorm consortium 17 1) Analytic and Translational Genetics Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA 18 2) Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA 19 3) Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA 20 4) Department of Mathematics, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA 21 5) Department of Epidemiology, Harvard T.H. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint Chan School of Public Health, Boston, Massachusetts, USA 22 6) Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK 23 7) Cardiff University, Medical Research Council Center for Neuropsychiatric Genetics & Genomics, Institute of Psychology, Medicine & Clinical Neuroscience, Cardiff, Wales, 24 UK 25 8) Center for Human Genetic Research, Department of Neurology, Massachusetts General Hospital, Boston, MA, USA 26 9) Psychiatric and Neurodevelopmental Genetics Unit (PNGU), Center for Human Genetics Research, Massachusetts General Hospital, Boston, MA, USA 27 10) Institute for Stroke and Dementia Research, Klinikum der Universität München, Ludwig-Maximilians-University, Munich, Germany 28 11) Department of Neurology, Brigham & Women’s Hospital, Harvard Medical School, Boston, MA 29 12) Charite Universitatsmedizin Berlin, Berlin, Germany 30 13) Department of Computer Science, New Jersey Institute of Technology, New Jersey, USA 31 14) Kings College London, Institute of Psychiatry, Psychology & Neuroscience, Social Genetics & Developmental Psychiatry Center, MRC, London, England 32 15) NIHR, Biomed Research Center for Mental Health, South London & Maudsley NHS Trust & Institute Psychiatry, London, England 33 16) Departments of Psychiatry and Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. 34 17) Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden. 35 18) Munich Cluster for Systems Neurology (SyNergy), Munich, Germany 36 19 Departments of Psychiatry and of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY, USA 37 20) Department of Molecular Neuroscience, Institute of Neurology, University College London, London, UK 38 21) Department of Psychiatry, Virginia Commonwealth University, Richmond, VA, USA 39 22) Division Biomedical Genetics, University Medical Center Utrecht, Utrecht, the Netherlands 40 23) Department of Psychiatry and UF Genetics Institute, University of Florida: Gainesville, Florida, USA 41 24) Division of Cognitive and Behavioral Neurology, Brigham and Women's Hospital, Boston, MA, USA 42 25) Department of Neurology, Massachusetts General Hospital, Boston, MA, USA 43 26) Icahn School of Medicine at Mount Sinai, New York, New York, USA 44 27) Sobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, University College London, London, UK 45 28) Institute of Genetics, University College London, London, UK 46 29) Department of Neurology, Yale School of Medicine, New Haven, CT, USA 47 30) Institute for Molecular Medicine Finland (FIMM), Helsinki, Finland 48 31) Department of Neurology, Massachusetts General Hospital, Boston, MA, USA. Analysis of Shared Heritability in Common Disorders of the Brain A division between neurology and psychiatry developed, with the 75 more directly observable phenomena (such as the presence of emboli protein tangles or unusual 76 52 † These authors jointly supervised this work 53 *1 verneri.anttila@gmail.com 54 *2 acorvin@tcd.ie 55 *3 bneale@broadinstitute.org 56 57 One Sentence Summary: Comprehensive heritability analysis of brain phenotypes demonstrates a 58 clear role for common genetic variation across neurological and psychiatric disorders and 59 behavioral-cognitive traits, with substantial overlaps in genetic risk. 60 Abstract: Disorders of the brain exhibit considerable epidemiological comorbidity and frequently share 61 symptoms, provoking debate about the extent of their etiologic overlap. We quantified the genetic sharing 62 of 25 brain disorders based on summary statistics from genome-wide association studies of 215,683 63 patients and 657,164 controls, and their relationship to 17 phenotypes from 1,191,588 individuals. 64 Psychiatric disorders show substantial sharing of common variant risk, while neurological disorders 65 appear more distinct from one another. We observe limited evidence of sharing between neurological and 66 psychiatric disorders, but do identify robust sharing between disorders and several cognitive measures, as 67 well as disorders and personality types. We also performed extensive simulations to explore how power, 68 diagnostic misclassification and phenotypic heterogeneity affect genetic correlations. These results 69 One Sentence Summary: Comprehensive heritability analysis of brain phenotypes demonstrates a 58 clear role for common genetic variation across neurological and psychiatric disorders and 59 behavioral-cognitive traits, with substantial overlaps in genetic risk. 60 Abstract: Disorders of the brain exhibit considerable epidemiological comorbidity and frequently share 61 symptoms, provoking debate about the extent of their etiologic overlap. We quantified the genetic sharing 62 of 25 brain disorders based on summary statistics from genome-wide association studies of 215,683 63 patients and 657,164 controls, and their relationship to 17 phenotypes from 1,191,588 individuals. 64 Psychiatric disorders show substantial sharing of common variant risk, while neurological disorders 65 appear more distinct from one another. We observe limited evidence of sharing between neurological and 66 psychiatric disorders, but do identify robust sharing between disorders and several cognitive measures, as 67 well as disorders and personality types. We also performed extensive simulations to explore how power, 68 diagnostic misclassification and phenotypic heterogeneity affect genetic correlations. These results 69 highlight the importance of common genetic variation as a source of risk for brain disorders and the value 70 of heritability-based methods in understanding their etiology. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint Analysis of Shared Heritability in Common Disorders of the Brain 35 18) Munich Cluster for Systems Neurology (SyNergy), Munich, Germany 35 18) Munich Cluster for Systems Neurology (SyNergy), Munich, Germany 36 ) Munich Cluster for Systems Neurology (SyNergy), Munich, Germany 36 19 Departments of Psychiatry and of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY, USA 37 19 Departments of Psychiatry and of Neuroscience and Physiology, SUNY U 37 20) Department of Molecular Neuroscience, Institute of Neurology, University College London, London, UK 38 38 21) Department of Psychiatry, Virginia Commonwealth University, Richmond, VA, USA 39 40 23) Department of Psychiatry and UF Genetics Institute, University of Florida: Gainesville, Florida, USA 41 41 24) Division of Cognitive and Behavioral Neurology, Brigham and Women's Hospital, Boston, MA, USA 42 25) Department of Neurology, Massachusetts General Hospital, Boston, MA, USA 42 25) Department of Neurology, Massachusetts General Hospital, Boston, MA, USA 43 43 26) Icahn School of Medicine at Mount Sinai, New York, New York, USA 44 43 26) Icahn School of Medicine at Mount Sinai, New York, New York, USA 44 ) Icahn School of Medicine at Mount Sinai, New York, New York, USA 44 27) Sobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, University College London, London, UK 45 ) Sobell Department of Motor Neuroscience and Movement Disorders, Instit 45 28) Institute of Genetics, University College London, London, UK 46 28) Institute of Genetics, University College London, London, UK 46 46 29) Department of Neurology, Yale School of Medicine, New Haven, CT, USA 47 47 30) Institute for Molecular Medicine Finland (FIMM), Helsinki, Finland 48 ) Institute for Molecular Medicine Finland (FIMM), Helsinki, Finland 48 31) Department of Neurology, Massachusetts General Hospital, Boston, MA, USA. 49 49 32) Department of Genetics and Psychiatry, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA 50 50 33) Neuropsychiatric Genetics Research Group, Department of Psychiatry, Trinity College Dublin, Ireland 51 1 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Analysis of Shared Heritability in Common Disorders of the Brain Epidemiological and twin studies 82 have explored patterns of phenotypic overlaps(5-7), and substantial comorbidity has been 83 reported for many pairs of disorders, including bipolar disorder-migraine(8), stroke-major 84 depressive disorder(MDD)(9), epilepsy-autism spectrum disorders (ASD) and epilepsy-attention 85 deficit hyperactivity disorder (ADHD)(10, 11). Furthermore, neurological and psychiatric 86 research has shown that mutations in the same ion channel genes confer pleiotropic risk for 87 multiple distinct brain phenotypes(12-14). Recently, genome-wide association studies (GWAS) 88 have demonstrated that individual common risk variants show overlap across traditional 89 diagnostic boundaries (15, 16), and that disorders like schizophrenia, MDD and bipolar disorder 90 can have strong genetic correlations(17). 91 52 † These authors jointly supervised this work 53 *1 verneri.anttila@gmail.com 54 *2 acorvin@tcd.ie 55 *3 bneale@broadinstitute.org 56 57 One Sentence Summary: Comprehensive heritability analysis of brain phenotypes demonstrates a 58 clear role for common genetic variation across neurological and psychiatric disorders and 59 behavioral-cognitive traits, with substantial overlaps in genetic risk. 60 Abstract: Disorders of the brain exhibit considerable epidemiological comorbidity and frequently share 61 symptoms, provoking debate about the extent of their etiologic overlap. We quantified the genetic sharing 62 of 25 brain disorders based on summary statistics from genome-wide association studies of 215,683 63 patients and 657,164 controls, and their relationship to 17 phenotypes from 1,191,588 individuals. 64 Psychiatric disorders show substantial sharing of common variant risk, while neurological disorders 65 appear more distinct from one another. We observe limited evidence of sharing between neurological and 66 psychiatric disorders, but do identify robust sharing between disorders and several cognitive measures, as 67 well as disorders and personality types. We also performed extensive simulations to explore how power, 68 diagnostic misclassification and phenotypic heterogeneity affect genetic correlations. These results 69 highlight the importance of common genetic variation as a source of risk for brain disorders and the value 70 of heritability-based methods in understanding their etiology. 71 The classification of brain disorders has evolved over the past century, reflecting the 72 medical and scientific communities’ best assessments of the presumed root causes of clinical 73 phenomena such as behavioral change, loss of motor function, spontaneous movements or 74 alterations of consciousness. Analysis of Shared Heritability in Common Disorders of the Brain It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint 52 † These authors jointly supervised this work 53 *1 verneri.anttila@gmail.com 54 *2 acorvin@tcd.ie 55 *3 bneale@broadinstitute.org 56 57 One Sentence Summary: Comprehensive heritability analysis of brain phenotypes demonstrates a 58 clear role for common genetic variation across neurological and psychiatric disorders and 59 behavioral-cognitive traits, with substantial overlaps in genetic risk. 60 Abstract: Disorders of the brain exhibit considerable epidemiological comorbidity and frequently share 61 symptoms, provoking debate about the extent of their etiologic overlap. We quantified the genetic sharing 62 of 25 brain disorders based on summary statistics from genome-wide association studies of 215,683 63 patients and 657,164 controls, and their relationship to 17 phenotypes from 1,191,588 individuals. 64 Psychiatric disorders show substantial sharing of common variant risk, while neurological disorders 65 appear more distinct from one another. We observe limited evidence of sharing between neurological and 66 psychiatric disorders, but do identify robust sharing between disorders and several cognitive measures, as 67 well as disorders and personality types. We also performed extensive simulations to explore how power, 68 diagnostic misclassification and phenotypic heterogeneity affect genetic correlations. These results 69 highlight the importance of common genetic variation as a source of risk for brain disorders and the value 70 of heritability-based methods in understanding their etiology. 71 The classification of brain disorders has evolved over the past century, reflecting the 72 medical and scientific communities’ best assessments of the presumed root causes of clinical 73 phenomena such as behavioral change, loss of motor function, spontaneous movements or 74 alterations of consciousness. A division between neurology and psychiatry developed, with the 75 more directly observable phenomena (such as the presence of emboli, protein tangles, or unusual 76 electrical activity patterns) generally defining the neurological disorders(1). Applying modern 77 methods to understand the genetic underpinnings and categorical distinctions between brain 78 disorders may be helpful in informing next steps in the search for the biological pathways 79 underlying their pathophysiology(2, 3). 80 In general, brain disorders (here excepting those caused by trauma, infection or cancer) 81 show substantial heritability from twin and family studies (4). Analysis of Shared Heritability in Common Disorders of the Brain In addition to locus discovery, the degree of 95 distinctiveness (23) across a wide set of neurological and psychiatric phenotypes can now be 96 evaluated with the introduction of novel heritability-based methods(24) and sufficiently large 97 sample sizes. These analyses can shed light on the nature of these diagnostic boundaries and 98 explore the extent of shared common variant genetic influences. 99 100 Study design 101 We formed the Brainstorm consortium, a collaboration among GWAS meta-analysis 102 consortia of 25 disorders (see Data sources), to perform the first comprehensive heritability and 103 correlation analysis of brain disorders. We included all common brain disorders for which we 104 could identify a GWAS meta-analysis consortium of sufficient size for heritability analysis that 105 was willing to participate. The total study sample consists of 215,683 cases of different brain 106 disorders and 657,164 controls (Table 1), and provides coverage of a majority of ICD-10 blocks 107 covering mental and behavioral disorders and diseases of the central nervous system. Also 108 included are 1,191,588 samples for 13 “behavioral-cognitive” phenotypes (n=744,486) chosen 109 for being traditionally viewed as brain-related, and four “additional” phenotypes (n=447,102) 110 selected to represent known, well-delineated etiological processes (e.g. immune disorders 111 [Crohn’s disease] and vascular disease [coronary artery disease]; Table 2) or anthropomorphic 112 measures (height and BMI). 113 GWAS summary statistics for the 42 disorders and phenotypes were centralized and 114 underwent uniform quality control and processing(25). Where necessary, we generated 115 European-only meta-analyses for each disorder to avoid potential biases arising from ancestry 116 differences, as many of the brain disorder datasets included sample sets from diverse ancestries. 117 Clinically relevant subtypes from three disorders (epilepsy, migraine and ischemic stroke) were 118 also included; in these cases, the analyzed datasets are subsets of the top-level dataset, as shown 119 in Table 1. 120 We have recently developed a novel heritability estimation method, linkage 121 disequilibrium score regression (LDSC)(24), which was used to calculate heritability estimates 122 and correlations, as well as to estimate their statistical significance from block jack-knife-based 123 standard errors. Heritability for binary disorders and phenotypes was transformed to the liability- 124 scale. We further performed a weighted-least squares regression analysis to evaluate whether 125 GWAS have also demonstrated that common genetic variation substantially contributes 92 to the heritability of brain disorders. Analysis of Shared Heritability in Common Disorders of the Brain In addition to locus discovery, the degree of 95 distinctiveness (23) across a wide set of neurological and psychiatric phenotypes can now be 96 evaluated with the introduction of novel heritability-based methods(24) and sufficiently large 97 sample sizes. These analyses can shed light on the nature of these diagnostic boundaries and 98 explore the extent of shared common variant genetic influences. 99 100 Study design 101 We formed the Brainstorm consortium, a collaboration among GWAS meta-analysis 102 consortia of 25 disorders (see Data sources), to perform the first comprehensive heritability and 103 correlation analysis of brain disorders. We included all common brain disorders for which we 104 could identify a GWAS meta-analysis consortium of sufficient size for heritability analysis that 105 was willing to participate. The total study sample consists of 215,683 cases of different brain 106 disorders and 657,164 controls (Table 1), and provides coverage of a majority of ICD-10 blocks 107 covering mental and behavioral disorders and diseases of the central nervous system. Also 108 included are 1,191,588 samples for 13 “behavioral-cognitive” phenotypes (n=744,486) chosen 109 for being traditionally viewed as brain-related, and four “additional” phenotypes (n=447,102) 110 selected to represent known, well-delineated etiological processes (e.g. immune disorders 111 [Crohn’s disease] and vascular disease [coronary artery disease]; Table 2) or anthropomorphic 112 measures (height and BMI). 113 GWAS summary statistics for the 42 disorders and phenotypes were centralized and 114 underwent uniform quality control and processing(25). Where necessary, we generated 115 GWAS have also demonstrated that common genetic variation substantially contributes 92 to the heritability of brain disorders. In most cases, this occurs via many common variants, each 93 of small effect, with examples in Alzheimer’s disease(18), bipolar disorder(19), migraine(20), 94 Parkinson’s disease(21), and schizophrenia(22). In addition to locus discovery, the degree of 95 distinctiveness (23) across a wide set of neurological and psychiatric phenotypes can now be 96 evaluated with the introduction of novel heritability-based methods(24) and sufficiently large 97 sample sizes. These analyses can shed light on the nature of these diagnostic boundaries and 98 explore the extent of shared common variant genetic influences. 99 We formed the Brainstorm consortium, a collaboration among GWAS meta-analysis 102 consortia of 25 disorders (see Data sources), to perform the first comprehensive heritability and 103 correlation analysis of brain disorders. Analysis of Shared Heritability in Common Disorders of the Brain In most cases, this occurs via many common variants, each 93 of small effect, with examples in Alzheimer’s disease(18), bipolar disorder(19), migraine(20), 94 Parkinson’s disease(21), and schizophrenia(22). In addition to locus discovery, the degree of 95 distinctiveness (23) across a wide set of neurological and psychiatric phenotypes can now be 96 evaluated with the introduction of novel heritability-based methods(24) and sufficiently large 97 sample sizes. These analyses can shed light on the nature of these diagnostic boundaries and 98 explore the extent of shared common variant genetic influences. 99 100 Study design 101 We formed the Brainstorm consortium, a collaboration among GWAS meta-analysis 102 consortia of 25 disorders (see Data sources), to perform the first comprehensive heritability and 103 correlation analysis of brain disorders. We included all common brain disorders for which we 104 could identify a GWAS meta-analysis consortium of sufficient size for heritability analysis that 105 was willing to participate. The total study sample consists of 215,683 cases of different brain 106 disorders and 657,164 controls (Table 1), and provides coverage of a majority of ICD-10 blocks 107 covering mental and behavioral disorders and diseases of the central nervous system. Also 108 included are 1,191,588 samples for 13 “behavioral-cognitive” phenotypes (n=744,486) chosen 109 for being traditionally viewed as brain-related, and four “additional” phenotypes (n=447,102) 110 selected to represent known, well-delineated etiological processes (e.g. immune disorders 111 [Crohn’s disease] and vascular disease [coronary artery disease]; Table 2) or anthropomorphic 112 measures (height and BMI). 113 GWAS summary statistics for the 42 disorders and phenotypes were centralized and 114 underwent uniform quality control and processing(25). Where necessary, we generated 115 European-only meta-analyses for each disorder to avoid potential biases arising from ancestry 116 differences, as many of the brain disorder datasets included sample sets from diverse ancestries. 117 Clinically relevant subtypes from three disorders (epilepsy, migraine and ischemic stroke) were 118 also included; in these cases, the analyzed datasets are subsets of the top-level dataset, as shown 119 in Table 1. 120 We have recently developed a novel heritability estimation method, linkage 121 GWAS have also demonstrated that common genetic variation substantially contributes 92 to the heritability of brain disorders. In most cases, this occurs via many common variants, each 93 of small effect, with examples in Alzheimer’s disease(18), bipolar disorder(19), migraine(20), 94 Parkinson’s disease(21), and schizophrenia(22). Analysis of Shared Heritability in Common Disorders of the Brain 71 The classification of brain disorders has evolved over the past century, reflecting the 72 medical and scientific communities’ best assessments of the presumed root causes of clinical 73 phenomena such as behavioral change, loss of motor function, spontaneous movements or 74 alterations of consciousness. A division between neurology and psychiatry developed, with the 75 more directly observable phenomena (such as the presence of emboli, protein tangles, or unusual 76 electrical activity patterns) generally defining the neurological disorders(1). Applying modern 77 methods to understand the genetic underpinnings and categorical distinctions between brain 78 disorders may be helpful in informing next steps in the search for the biological pathways 79 underlying their pathophysiology(2, 3). 80 In general, brain disorders (here excepting those caused by trauma, infection or cancer) 81 show substantial heritability from twin and family studies (4). Epidemiological and twin studies 82 have explored patterns of phenotypic overlaps(5-7), and substantial comorbidity has been 83 reported for many pairs of disorders, including bipolar disorder-migraine(8), stroke-major 84 depressive disorder(MDD)(9), epilepsy-autism spectrum disorders (ASD) and epilepsy-attention 85 deficit hyperactivity disorder (ADHD)(10, 11). Furthermore, neurological and psychiatric 86 research has shown that mutations in the same ion channel genes confer pleiotropic risk for 87 multiple distinct brain phenotypes(12-14). Recently, genome-wide association studies (GWAS) 88 have demonstrated that individual common risk variants show overlap across traditional 89 diagnostic boundaries (15, 16), and that disorders like schizophrenia, MDD and bipolar disorder 90 can have strong genetic correlations(17). 91 2 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint GWAS have also demonstrated that common genetic variation substantially contributes 92 to the heritability of brain disorders. In most cases, this occurs via many common variants, each 93 of small effect, with examples in Alzheimer’s disease(18), bipolar disorder(19), migraine(20), 94 Parkinson’s disease(21), and schizophrenia(22). Heritability and correlations among brain disorders We observed a similar range of heritability estimates among the disorders and the 135 behavioral-cognitive phenotypes (Fig. S1A-B and Table S1, S2), roughly in line with previously 136 reported estimates obtained from smaller datasets (see Table S3 and Supplementary Text). Three 137 ischemic stroke subtypes (cardioembolic, large-vessel disease and small-vessel disease) as well 138 as the “agreeableness” personality measure from NEO Five-Factor Inventory(27) had insufficient 139 evidence of additive heritability for robust analysis and thus were excluded from further 140 analysis(25). We did not observe a correlation between heritability estimates and factors relating 141 to study makeup (Table S4; Fig. S1C-F). Since some of the results interpretation depends on lack 142 of observed correlation, we explored the behavior of observed correlation vs power (Fig. S2A), 143 standard errors (Fig. S2B) and the individual results (Fig. S2C and D) to identify where we can 144 be reasonably robust in claiming lack of correlation with current datasets. 145 In expanding on the number of pairwise comparisons in brain disorders, we observed 146 widespread sharing across psychiatric disorders (Fig. 1 and S3) beyond those previously reported 147 (17), but not among neurological disorders. Among the psychiatric disorders, schizophrenia 148 showed significant genetic correlation with most of the psychiatric disorders, while MDD was 149 positively (though not necessarily significantly) correlated with every other disorder tested. 150 Further, schizophrenia, bipolar disorder, anxiety disorders, MDD and ADHD each showed a high 151 degree of correlation to the four others (average rg=0.40; Table S5). Anorexia nervosa, 152 obsessive-compulsive disorder (OCD) and schizophrenia also demonstrated significant sharing 153 amongst themselves. On the other hand, the common variant risk of both ASD and Tourette 154 Syndrome (TS) appear to be somewhat distinct from other psychiatric disorders, although with 155 significant correlation between TS, OCD and MDD, as well as between ASD and schizophrenia. 156 Post-traumatic stress disorder (PTSD) alone showed no significant correlation with any of the 157 other psychiatric phenotypes (though some correlation to ADHD and MDD was observed, Fig. 158 1). The modest power of the ASD, PTSD and TS meta-analyses, however, limits the strength of 159 this conclusion (Fig. S2C). 160 Neurological disorders revealed greater specificity, and a more limited extent of genetic 161 correlation than the psychiatric disorders (Fig. 2 and S4, Table S5). Parkinson’s disease, 162 Alzheimer’s disease, generalized epilepsy and multiple sclerosis showed little to no correlation 163 with any other brain disorders. Analysis of Shared Heritability in Common Disorders of the Brain It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint specific regulatory partitions of the genome, using the ten top-level tissue-type and 53 functional 129 partitions from Finucane et al. (26). Finally, simulated phenotype data was generated under 130 several different scenarios by permuting the 120,267 genotyped individuals from the UK 131 Biobank (25) to both evaluate power and aid in interpreting the results (see Supplementary Text). 132 133 Heritability and correlations among brain disorders 134 We observed a similar range of heritability estimates among the disorders and the 135 behavioral-cognitive phenotypes (Fig. S1A-B and Table S1, S2), roughly in line with previously 136 reported estimates obtained from smaller datasets (see Table S3 and Supplementary Text). Three 137 ischemic stroke subtypes (cardioembolic, large-vessel disease and small-vessel disease) as well 138 as the “agreeableness” personality measure from NEO Five-Factor Inventory(27) had insufficient 139 evidence of additive heritability for robust analysis and thus were excluded from further 140 analysis(25). We did not observe a correlation between heritability estimates and factors relating 141 to study makeup (Table S4; Fig. S1C-F). Since some of the results interpretation depends on lack 142 of observed correlation, we explored the behavior of observed correlation vs power (Fig. S2A), 143 standard errors (Fig. S2B) and the individual results (Fig. S2C and D) to identify where we can 144 be reasonably robust in claiming lack of correlation with current datasets. 145 specific regulatory partitions of the genome, using the ten top-level tissue-type and 53 functional 129 partitions from Finucane et al. (26). Finally, simulated phenotype data was generated under 130 several different scenarios by permuting the 120,267 genotyped individuals from the UK 131 Biobank (25) to both evaluate power and aid in interpreting the results (see Supplementary Text). 132 133 Analysis of Shared Heritability in Common Disorders of the Brain We included all common brain disorders for which we 104 could identify a GWAS meta-analysis consortium of sufficient size for heritability analysis that 105 was willing to participate. The total study sample consists of 215,683 cases of different brain 106 disorders and 657,164 controls (Table 1), and provides coverage of a majority of ICD-10 blocks 107 covering mental and behavioral disorders and diseases of the central nervous system. Also 108 included are 1,191,588 samples for 13 “behavioral-cognitive” phenotypes (n=744,486) chosen 109 for being traditionally viewed as brain-related, and four “additional” phenotypes (n=447,102) 110 selected to represent known, well-delineated etiological processes (e.g. immune disorders 111 [Crohn’s disease] and vascular disease [coronary artery disease]; Table 2) or anthropomorphic 112 measures (height and BMI). 113 GWAS summary statistics for the 42 disorders and phenotypes were centralized and 114 underwent uniform quality control and processing(25). Where necessary, we generated 115 European-only meta-analyses for each disorder to avoid potential biases arising from ancestry 116 differences, as many of the brain disorder datasets included sample sets from diverse ancestries. 117 Clinically relevant subtypes from three disorders (epilepsy, migraine and ischemic stroke) were 118 also included; in these cases, the analyzed datasets are subsets of the top-level dataset, as shown 119 in Table 1. 120 We have recently developed a novel heritability estimation method, linkage 121 disequilibrium score regression (LDSC)(24), which was used to calculate heritability estimates 122 and correlations, as well as to estimate their statistical significance from block jack-knife-based 123 standard errors. Heritability for binary disorders and phenotypes was transformed to the liability- 124 scale. We further performed a weighted-least squares regression analysis to evaluate whether 125 differences relating to study makeup (such as sample size) were correlated with the magnitude of 126 the correlation estimates. We also performed a heritability partitioning analysis using stratified 127 LD score regression to examine whether the observed heritability was enriched in any tissue- 128 3 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Heritability and correlations among brain disorders 186 Among the personality measures, significant positive correlations were observed f 187 neuroticism (anorexia nervosa, anxiety disorders, migraine, migraine without aura, MDD, OC 188 schizophrenia and Tourette Syndrome; Fig. S6A), depressive symptoms (ADHD, anxie 189 disorder, bipolar disorder, MDD, and schizophrenia) and subjective well-being (anxiety disorde 190 bipolar disorder, MDD, as well as replicating the previously reported correlation betwe 191 neuroticism with both MDD and schizophrenia(29)). For smoking-related measures, the on 192 significant genetic correlations were to never/ever smoked (MDD: rg=0.33, p=3.10 x 10-11 a 193 ADHD: rg=0.37, p=3.15 x 10-6). 194 Among the additional phenotypes, the two diseases chosen as examples of disorders wi 195 well-defined etiologies had different results: Crohn’s disease, representing immunologic 196 pathophysiology, showed no correlation with any of the study phenotypes, while the phenoty 197 representing vascular pathophysiology (coronary artery disease) showed significant correlati 198 t MDD ( 0 19 8 71 10-5) ll th t t k l t d h t ( 0 69 2 47 199 none were significant, reflecting the relatively modest power of the current focal epilepsy meta- 166 analysis (Fig. S2C). However, the modest heritability and the broad pattern of sharing observed 167 for focal epilepsy may be consistent with considerable heterogeneity and potentially even 168 diagnostic misclassification across a range of neurological conditions. 169 In the cross-category correlation analysis, the overall pattern is consistent with limited 170 sharing across the included neurological and psychiatric disorders (Fig. 3; average rg=0.03). The 171 only significant cross-category correlations were with migraine, suggesting it may share some of 172 its genetic architecture with psychiatric disorders; migraine-ADHD (rg=0.26, p=8.81 x 10-8), 173 migraine-TS (rg=0.19, p=1.80 x 10-5), and migraine-MDD (rg=0.32, p=1.42 x 10-22 for all 174 migraine, rg=0.23, p=5.23 x 10-5 for migraine without aura, rg=0.28, p=1.00 x 10-4 for migraine 175 with aura). 176 We observed several significant genetic correlations between the behavioral-cognitive or 177 additional phenotypes and brain disorders (Fig. 4, Table S6). Results for cognitive traits were 178 dichotomous among psychiatric phenotypes (Fig. S5A), with ADHD, anxiety disorders, MDD 179 and Tourette Syndrome showing negative correlations to the cognitive measures, while anorexia 180 nervosa, ASD, bipolar disorder and OCD showed positive correlations. Schizophrenia showed 181 more mixed results, with significantly negative correlation to intelligence but positive correlation 182 to years of education. Among neurological phenotypes (Fig. Heritability and correlations among brain disorders Focal epilepsy showed the highest degree of genetic correlation 164 among the neurological disorders (average rg =0.46, excluding other epilepsy datasets), though 165 4 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint none were significant, reflecting the relatively modest power of the current focal epilepsy met 166 analysis (Fig. S2C). However, the modest heritability and the broad pattern of sharing observ 167 for focal epilepsy may be consistent with considerable heterogeneity and potentially ev 168 diagnostic misclassification across a range of neurological conditions. 169 In the cross-category correlation analysis, the overall pattern is consistent with limit 170 sharing across the included neurological and psychiatric disorders (Fig. 3; average rg=0.03). T 171 only significant cross-category correlations were with migraine, suggesting it may share some 172 its genetic architecture with psychiatric disorders; migraine-ADHD (rg=0.26, p=8.81 x 10- 173 migraine-TS (rg=0.19, p=1.80 x 10-5), and migraine-MDD (rg=0.32, p=1.42 x 10-22 for 174 migraine, rg=0.23, p=5.23 x 10-5 for migraine without aura, rg=0.28, p=1.00 x 10-4 for migrai 175 with aura). 176 We observed several significant genetic correlations between the behavioral-cognitive 177 additional phenotypes and brain disorders (Fig. 4, Table S6). Results for cognitive traits we 178 dichotomous among psychiatric phenotypes (Fig. S5A), with ADHD, anxiety disorders, MD 179 and Tourette Syndrome showing negative correlations to the cognitive measures, while anorex 180 nervosa, ASD, bipolar disorder and OCD showed positive correlations. Schizophrenia show 181 more mixed results, with significantly negative correlation to intelligence but positive correlati 182 to years of education. Among neurological phenotypes (Fig. S5B), the correlations were a 183 either negative or null, with Alzheimer’s disease, epilepsy, ICH, ischemic stroke, early-ons 184 stroke and migraine showing significantly negative correlations. Correlations with bipol 185 disorder(24), Alzheimer’s disease and schizophrenia have been previously reported(28)). Heritability and correlations among brain disorders S5B), the correlations were all 183 either negative or null, with Alzheimer’s disease, epilepsy, ICH, ischemic stroke, early-onset 184 stroke and migraine showing significantly negative correlations. Correlations with bipolar 185 disorder(24), Alzheimer’s disease and schizophrenia have been previously reported(28)). 186 Among the personality measures, significant positive correlations were observed for 187 neuroticism (anorexia nervosa, anxiety disorders, migraine, migraine without aura, MDD, OCD, 188 schizophrenia and Tourette Syndrome; Fig. S6A), depressive symptoms (ADHD, anxiety 189 disorder, bipolar disorder, MDD, and schizophrenia) and subjective well-being (anxiety disorder, 190 bipolar disorder, MDD, as well as replicating the previously reported correlation between 191 neuroticism with both MDD and schizophrenia(29)). For smoking-related measures, the only 192 significant genetic correlations were to never/ever smoked (MDD: rg=0.33, p=3.10 x 10-11 and 193 ADHD: rg=0.37, p=3.15 x 10-6). 194 Among the additional phenotypes, the two diseases chosen as examples of disorders with 195 well-defined etiologies had different results: Crohn’s disease, representing immunological 196 pathophysiology, showed no correlation with any of the study phenotypes, while the phenotype 197 representing vascular pathophysiology (coronary artery disease) showed significant correlation 198 to MDD (rg=0.19, p=8.71 x 10-5) as well as the two stroke-related phenotypes (rg=0.69, p=2.47 x 199 10-6 to ischemic stroke and rg=0.86, p=2.26 x 10-5 for early-onset stroke), suggesting shared 200 genetic effects across these phenotype. Significant correlations were also observed for BMI, 201 which was positively correlated with ADHD and MDD, and negatively correlated with anorexia 202 nervosa (as previously reported with a different dataset(24)) and schizophrenia. 203 5 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint Our enrichment analysis (Fig. Heritability and correlations among brain disorders S7, Table S7 and S8) demonstrated novel significant 204 heritability enrichments between central nervous system (CNS) and generalized epilepsy, MDD, 205 TS, college attainment, intelligence, neuroticism, never/ever smoked); depressive symptoms and 206 adrenal/pancreatic cells and tissues, as well as between immune system cells and multiple 207 sclerosis. We also note with interest that the psychiatric disorders with large numbers of 208 identified GWAS loci (bipolar disorder, MDD and schizophrenia) and the only cross-correlated 209 neurological disorder with the same (migraine) all show enrichment to conserved regions, while 210 the other neurological disorders with similar numbers of loci (MS and Alzheimer’s and 211 Parkinson’s diseases) do not (Fig. S7A, B). Significant enrichment to conserved regions was also 212 observed to neuroticism, intelligence and college attainment and to H3K9ac peaks for BMI. We 213 also replicate the previously reported (CNS) enrichment for schizophrenia, bipolar disorder and 214 years of education (here in a larger dataset compared to the original report, but with considerable 215 sample overlap), and observe the previously reported enrichments for BMI (CNS), years of 216 education (CNS), height (connective tissues and bone, cardiovascular system and other) and 217 Crohn’s disease (hematopoietic cells) from the same datasets (Fig. S7C, D) (26). 218 219 Discussion 220 By integrating and analyzing the current genome-wide association summary statistic data 221 from consortia of 25 brain disorders, we find that psychiatric disorders broadly share a 222 considerable portion of their common variant genetic risk, especially across schizophrenia, 223 MDD, bipolar disorder, anxiety disorder and ADHD, while neurological disorders are more 224 genetically distinct. Across categories, psychiatric and neurologic disorders share relatively little 225 of their common genetic risk, suggesting that multiple different and largely independently 226 regulated etiological pathways may give rise to similar clinical manifestations (e.g., psychosis, 227 which manifests in both schizophrenia(30) and Alzheimer’s disease(31)). Except for migraine, 228 which appears to share some genetic architecture with psychiatric disorders, the existing clinical 229 delineation between neurology and psychiatry is recapitulated at the level of common variant 230 risk for the studied disorders. 231 Given that the broad and continuous nature of psychiatric disorder spectra in particular 232 has been clinically recognized for a long time(32-34) and that patients can, in small numbers, 233 progress from one diagnosis to another(35), we evaluated to what extent diagnostic 234 misclassification could explain the observed correlations. Heritability and correlations among brain disorders Genetic correlation could arise if, for 235 example, substantial numbers of patients progress through multiple diagnoses over their lifetime, 236 or if some specific diagnostic boundaries between phenotype pairs are particularly porous to 237 Our enrichment analysis (Fig. S7, Table S7 and S8) demonstrated novel significant 204 heritability enrichments between central nervous system (CNS) and generalized epilepsy, MDD, 205 TS, college attainment, intelligence, neuroticism, never/ever smoked); depressive symptoms and 206 adrenal/pancreatic cells and tissues, as well as between immune system cells and multiple 207 sclerosis. We also note with interest that the psychiatric disorders with large numbers of 208 identified GWAS loci (bipolar disorder, MDD and schizophrenia) and the only cross-correlated 209 neurological disorder with the same (migraine) all show enrichment to conserved regions, while 210 the other neurological disorders with similar numbers of loci (MS and Alzheimer’s and 211 Parkinson’s diseases) do not (Fig. S7A, B). Significant enrichment to conserved regions was also 212 observed to neuroticism, intelligence and college attainment and to H3K9ac peaks for BMI. We 213 also replicate the previously reported (CNS) enrichment for schizophrenia, bipolar disorder and 214 years of education (here in a larger dataset compared to the original report, but with considerable 215 sample overlap), and observe the previously reported enrichments for BMI (CNS), years of 216 education (CNS), height (connective tissues and bone, cardiovascular system and other) and 217 Crohn’s disease (hematopoietic cells) from the same datasets (Fig. S7C, D) (26). 218 Discussion 220 We sought to confirm 242 and expand upon these estimates by performing large-scale simulations and calculating the 243 resulting correlations across a variety of scenarios (Fig. S8, S9, Table S9 and Supplementary 244 Text). First, we established that the observed heritability of the simulated misclassified traits 245 behaves as expected (Fig. S8A), and that the effects on observed correlation (Fig. S8B and S8C) 246 are in line with the estimates from family data(36). We further explored the effect of 247 misclassification on observed rg given the correlation observed in real data. Reasonably low 248 levels of misclassification or changes to the exact level of heritability appear unlikely to induce 249 substantial changes in the estimated genetic correlation, though a lower observed heritability 250 caused by substantial misclassification (Fig. S8A) will decrease the power to estimate the genetic 251 overlap, as observed in the power analysis (Fig. S10). Further, such evidence of genetic overlap 252 is unlikely to appear in the absence of underlying genetic correlation (Table S10), as it is 253 apparent that a very high degree of misclassification (up to 79%) would be required to produce 254 the observed correlations in the absence of any true genetic correlation. Therefore, the observed 255 correlations suggest true sharing of a substantial fraction of the common variant genetic 256 architecture among psychiatric disorders as well as between behavioral-cognitive measures and 257 brain disorders. 258 The high degree of genetic correlation among the psychiatric disorders adds further 259 evidence that current clinical diagnostics do not reflect the underlying genetic etiology of these 260 disorders, and that genetic risk factors for psychiatric disorders do not respect clinical diagnostic 261 boundaries. This suggests an interconnected nature for their genetic etiology, in contrast to 262 neurological disorders, and underscores the need to refine psychiatric diagnostics. This study 263 may provide important ‘scaffolding’ to support a new research framework for investigating 264 mental disorders, incorporating many levels of information to understand basic dimensions of 265 brain function, such as through the National Institute of Mental Health’s RDoC initiative. 266 The observed positive genetic correlations are consistent with a few different scenarios. 267 For example, rg may reflect the existence of some portion of common genetic risk factors 268 conferring equal risks to multiple disorders where other distinct additional factors contribute to 269 the eventual clinical presentation. Discussion 220 By integrating and analyzing the current genome-wide association summary statistic data 221 from consortia of 25 brain disorders, we find that psychiatric disorders broadly share a 222 considerable portion of their common variant genetic risk, especially across schizophrenia, 223 MDD, bipolar disorder, anxiety disorder and ADHD, while neurological disorders are more 224 genetically distinct. Across categories, psychiatric and neurologic disorders share relatively little 225 of their common genetic risk, suggesting that multiple different and largely independently 226 regulated etiological pathways may give rise to similar clinical manifestations (e.g., psychosis, 227 which manifests in both schizophrenia(30) and Alzheimer’s disease(31)). Except for migraine, 228 which appears to share some genetic architecture with psychiatric disorders, the existing clinical 229 delineation between neurology and psychiatry is recapitulated at the level of common variant 230 risk for the studied disorders. 231 Given that the broad and continuous nature of psychiatric disorder spectra in particular 232 has been clinically recognized for a long time(32-34) and that patients can, in small numbers, 233 progress from one diagnosis to another(35), we evaluated to what extent diagnostic 234 misclassification could explain the observed correlations. Genetic correlation could arise if, for 235 example, substantial numbers of patients progress through multiple diagnoses over their lifetime, 236 or if some specific diagnostic boundaries between phenotype pairs are particularly porous to 237 misclassification; while it would be unlikely to observe large-scale misclassification of migraine 238 as schizophrenia, for example, there may be more substantial misclassification between other 239 pairs, consistent with the clinical controversies in classification. Previous work(36) suggests that 240 substantial misclassification (on the order of 15-30%, depending on whether it is uni- or 241 6 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint bidirectional) is required to introduce high levels of genetic correlation. Discussion 220 The presence of significant genetic correlation may also reflect 270 the phenotypic overlap between any two disorders; for example, the sharing between 271 schizophrenia and ADHD might reflect underlying difficulties in executive functioning, which 272 are well-established in both disorders(37). Similarly, the sharing between anorexia nervosa, OCD 273 and schizophrenia may reflect a shared mechanism underlying cognitive biases that extend from 274 overvalued ideas to delusions. Another scenario is that a heritable intermediate trait confers risk 275 to multiple outcomes, thereby giving rise to the genetic correlation, as the genetic influences on 276 this trait will be shared for both outcomes (e.g., obesity as a risk factor for both type 2 diabetes 277 and coronary artery disease), or that even the majority of common genetic effects are shared 278 between a pair of traits, but each individual effect may confer different degrees of risk and lead 279 to different aggregate genetic risk profiles. While a combination of these is likely, it will become 280 7 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint increasingly feasible to evaluate these overlaps at the locus level as more genome-wide 281 significant loci are identified in the future. 282 The low correlations observed across neurological disorders suggest that the current 283 classification reflects relatively specific genetic etiologies, although the limited sample size for 284 some of these disorders and lack of inclusion of disorders conceived as “circuit-based” in the 285 literature, such as restless legs syndrome, sleep disorders and possibly essential tremor, 286 constrains the generalizability of this conclusion. Generally, this analysis recapitulates the 287 current understanding of the relatively distinct primary etiology underlying these disorders; 288 degenerative disorders (such as Alzheimer’s and Parkinson’s diseases) would not be expected a 289 priori to share their polygenic risk profiles with a neuro-immunological disorder (like multiple 290 sclerosis) or neurovascular disorder (like ischemic stroke). Discussion 220 It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint psychiatric disorders, ASD and TS showed a similar absence of correlation with other disorders, 320 although this could reflect small sample sizes. 321 Analysis of the Big Five personality measures suggest that the current sample sizes for 322 personality data are beginning to be sufficiently large for correlation testing; neuroticism, which 323 has by far the largest sample size, shows several significant correlations. Most significant of 324 these was to MDD (rg=0.737, p=5.04 x 10-96), providing further evidence for the link between 325 these phenotypes, reported previously with polygenic risk scores(50) and twin studies(51, 52); 326 others included schizophrenia, anxiety disorders, migraine, migraine without aura, and OCD 327 (Table S6). Further, the observation of strong correlation between MDD and anxiety disorders 328 together with their remarkably strong and similar patterns of correlation between each of these 329 disorders and the dimensional measures of depressive symptoms, subjective well-being, and 330 neuroticism suggests that they all tag a fundamentally similar underlying etiology. The novel 331 significant correlation between coronary artery disease and MDD supports the long-standing 332 epidemiological observation of a link between MDD and CAD(53), while the observed 333 correlation between ADHD and smoking initiation (rg=0.374, p=3.15 x 10-6) is consistent with 334 the epidemiological evidence of overlap(54) and findings from twin studies(55), supporting the 335 existing hypothesis that impulsivity inherent in ADHD may drive smoking initiation and 336 potentially dependence (though other explanations, such as reward system dysfunction would fit 337 as well). 338 For the neurological disorders, five (Alzheimer’s disease, intracerebral hemorrhage, 339 ischemic and early-onset stroke, and migraine) showed significant negative genetic correlation to 340 the cognitive measures, while a further two (epilepsy and focal epilepsy) showed moderate 341 negative genetic correlation (Fig. S5). For Alzheimer’s disease, poor cognitive performance in 342 early life has been linked to increased risk for developing the disorder in later life(56), but to our 343 knowledge no such connection has been reported for the other phenotypes. Discussion 220 Similarly, we see limited evidence for 291 the reported co-morbidity between migraine with aura and ischemic stroke(38) (rg=0.29, 292 p=0.099); however, the standard errors of this comparison are too high to draw strong 293 conclusions. At the disorder subtype level, migraine with and without aura (rg=0.48, p=1.79 x 10- 294 5) shows substantial genetic correlation, while focal and generalized epilepsy (rg=0.16, p=0.388) 295 show much less. 296 The few significant correlations across neurology and psychiatry, namely between 297 migraine and ADHD, MDD and TS, suggest modest shared etiological overlap across the 298 neurology/psychiatry distinction. The co-morbidity of migraine with MDD, Tourette Syndrome 299 and ADHD has been previously reported in epidemiological studies (39-42), while in contrast, 300 the previously reported co-morbidity between migraine and bipolar disorder seen in 301 epidemiological studies (43) was not reflected in our estimate of genetic correlation (rg=-0.03, 302 p=0.406). 303 Several phenotypes show only very low-level correlations with any of the other disorders 304 and phenotypes studied here, despite large sample size and robust evidence for heritability, 305 suggesting their common variant genetic risk may largely be unique. Alzheimer’s disease, 306 Parkinson’s disease, and multiple sclerosis show extremely limited sharing with the other 307 phenotypes and with each other. Neuroinflammation has been implicated in the pathophysiology 308 of each of these conditions(44-46), as it has for migraine(47) and many psychiatric conditions, 309 including schizophrenia(48), but no considerable shared heritability was observed with either of 310 those conditions nor with Crohn’s disease, nor did we observe enrichment for immune-related 311 tissues in the functional partitioning (Fig. S7) as we did for Crohn’s disease. While this 312 observation does not preclude shared neuroinflammatory mechanisms in these disorders, it does 313 suggest that on a large scale, common variant genetic influences on these inflammatory 314 mechanisms are not shared between these disorders. Further, we only observed significant 315 enrichment of heritability for immunological cells and tissues in multiple sclerosis, showing that 316 inflammation-specific regulatory marks in the genome do not show overall enrichment for 317 common variant risk for either Alzheimer’s or Parkinson’s diseases (though this does not 318 preclude the effects of specific, non-polygenic neuroinflammatory mechanisms(49)). Among 319 8 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Discussion 220 ADHD, anxiety 344 disorders and MDD show a significant negative correlation to cognitive and education attainment 345 measures, while the remaining five of the eight psychiatric disorders (anorexia nervosa, ASD, 346 bipolar disorder, OCD, and schizophrenia) showed significant positive genetic correlation with 347 one or more cognitive measures. These results strongly suggest the existence of a link between 348 cognitive performance already in early life and the genetic risk for both psychiatric and 349 neurological brain disorders. The basis of the genetic correlations between education, cognition 350 and brain disorders may have a variety of root causes including indexing performance 351 differences based on behavioral dysregulation (e.g., ADHD relating to attentional problems 352 during cognitive tests) or may reflect ascertainment biases in certain disorders conditional on 353 impaired cognition (e.g., individuals with lower cognitive reserve being more rapidly identified 354 for Alzheimer’s disease). 355 BMI shows significant positive genetic correlation to ADHD, consistent with a meta- 356 analysis linking ADHD to obesity(57), and negative genetic correlation with anorexia nervosa, 357 OCD and schizophrenia. These results are consistent with the evidence for enrichment of BMI 358 9 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint heritability in CNS tissues(26) and that many reported signals suggest neuronal involvement(58); 359 this may also provide a partial genetic explanation for lower BMI in anorexia nervosa patients 360 even after recovery(59). Given that no strong correlations were observed between BMI and any 361 of the neurological phenotypes, it is possible to hypothesize that BMI’s brain-specific genetic 362 architecture is more closely related to behavioral phenotypes. Ischemic stroke and BMI show 363 surprisingly little genetic correlation in this analysis (rg=0.07, p=0.26), suggesting that although 364 BMI is a strong risk factor for stroke(60), there is little evidence for shared common genetic 365 effects. Author Information Correspondence and requests for materials should be addressed to V.A. 390 (verneri.anttila@gmail.com), A.C. (acorvin@tcd.ie) or B.M.N. (bneale@broadinstitute.org). 391 References: 394 1. J. B. Martin, The integration of neurology, psychiatry, and neuroscience in the 21st century. Am J 395 Psychiatry 159, 695 (May, 2002). 396 2. J. W. Smoller, Disorders and borders: psychiatric genetics and nosology. Am J Med Genet B 397 Neuropsychiatr Genet 162B, 559 (Oct, 2013). 398 3. T. R. Insel, P. S. 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Discussion 220 It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint Discussion 220 These analyses also suggest that the reported reduced rates of cardiovascular disease in 366 individuals with histories of anorexia nervosa (61, 62) are due to BMI-related effects; with the 367 limited evidence of genetic correlation of anorexia nervosa with intracerebral hemorrhage, 368 ischemic stroke, early-onset stroke and coronary artery disease, these results suggest that any 369 lower cardiovascular mortality is more likely due to direct BMI-related effects rather than 370 shared common genetic risk variants. 371 It is broadly apparent from the results presented here that the current clinical boundaries 372 for the studied psychiatric phenotypes do not reflect distinct underlying pathogenic processes 373 based on the genetic evidence, while in contrast, the studied neurological disorders show much 374 greater genetic specificity. Although it is important to emphasize that while some disorders are 375 under-represented here (e.g. personality disorders in psychiatry and circuit-based disorders [such 376 as restless leg syndrome] in neurology), these results clearly demonstrate the limited evidence for 377 widespread common genetic risk sharing between psychiatric and neurological disorders, while 378 providing strong evidence for links between them and behavioral-cognitive measures. We 379 highlight the need for some degree of restructuring of psychiatric nosology and that genetically 380 informed analyses may provide a good basis for such activities, consistent with the historical 381 knowledge from twin and family-based results. Further elucidation of individual disorders and 382 their genetic overlap, especially as distinct loci map onto a subset of disorders and etiological 383 processes, may form the basis for either defining new clinical phenotypes or support a move to a 384 more continuous view of psychiatric phenotypes. Further study is needed to evaluate whether 385 overlapping genetic contributions to psychiatric pathology may influence optimal treatment 386 choices. Ultimately, such developments give hope to reducing diagnostic heterogeneity and 387 eventually improving the diagnostics and treatment of psychiatric disorders. 388 10 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . 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It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint 67. D. Woo et al., Meta-analysis of genome-wide association studies identifies 1q22 as a 537 susceptibility locus for intracerebral hemorrhage. References: 394 Schunkert et al., Large-scale association analysis identifies 13 new susceptibility loci for 558 coronary artery disease. Nat Genet 43, 333 (Apr, 2011). 559 77. H. Schunkert et al., Large-scale association analysis identifies 13 new susceptibility loci for 558 coronary artery disease. Nat Genet 43, 333 (Apr, 2011). 559 562 14 14 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint Acknowledgments The authors wish to acknowledge Rosy Hoskins, Jaana Wessman and Joanna Martin for thei 563 cromulent comments on the manuscript, Matthew Whittall for inspiration, and the patients and participants of th 564 respective consortia. For study-specific acknowledgments, see Supplementary Materials. GWAS summary statistic 565 used in the paper are available either directly from, or via application submitted in, the web addresses listed below 566 Data on coronary artery disease has been contributed by CARDIoGRAMplusC4D investigators and have been 567 downloaded from www.CARDIOGRAMPLUSC4D.ORG. matSpD is available a 568 neurogenetics.qimrberghofer.edu.au/matSpD/. This research has been conducted using the UK Biobank Resourc 569 (application #18597). References: 394 570 571 572 Data sources 573 Disorder or phenotype – Consortium or dataset identifier – web address: 574 Psychiatric disorders 575 ADHD – PGC-ADD2 - http://www.med.unc.edu/pgc/results-and-downloads 576 Anorexia nervosa(63) – PGC-ED - http://www.med.unc.edu/pgc/results-and-downloads 577 Anxiety disorder(64) – ANGST - http://www.med.unc.edu/pgc/results-and-downloads 578 Autism spectrum disorders(65) – PGC-AUT - http://www.med.unc.edu/pgc/results-and-downloads 579 Bipolar disorder – PGC-BIP2 - http://www.med.unc.edu/pgc/results-and-downloads (soon) 580 Major depressive disorder – PGC-MDD2 - http://www.med.unc.edu/pgc/results-and-downloads (soon) 581 OCD – PGC-OCDTS - http://www.med.unc.edu/pgc/results-and-downloads 582 PTSD – PGC-PTSD - http://www.med.unc.edu/pgc/results-and-downloads 583 Schizophrenia(22) – PGC-SCZ2 – http://www.med.unc.edu/pgc/results-and-downloads 584 Tourette Syndrome – TSAIGC – http://www.med.unc.edu/pgc/results-and-downloads 585 586 Neurological disorders 587 Alzheimer's disease(18) – IGAP - http://www.pasteur-lille.fr/en/recherche/u744/igap 588 Epilepsy and subtypes, focal and generalized(66) – ILAE – http://www.epigad.org/page/show/gwas_index 589 Intracerebral hemorrhage(67) – ISGC - http://www.strokegenetics.com/ 590 Ischemic stroke and subtypes (cardioembolic, early-onset, small-vessel and large-vessel)(68) – METASTROKE 591 dataset of the ISGC – http://www.strokegenetics.com/ 592 Migraine and subtypes, migraine with and without aura – IHGC – www.headachegenetics.org 593 Multiple sclerosis(69) – IMSGC - http://eaglep.case.edu/imsgc_web 594 Parkinson's disease(21) – IPDGC – www.pdgene.org 595 596 Behavioral-cognitive phenotypes 597 College attainment, years of education(70) – SSGAC – http://www.thessgac.org/data 598 Childhood cognitive performance(71) – SSGAC – http://www.thessgac.org/data 599 Extraversion, agreeableness, conscientiousness and openness (27) – GPC – http://www.tweelingenregister.org/GPC/ 600 IQ(72) – CTG - http://ctg.cncr.nl/software/summary_statistics 601 Neuroticism, depressive symptoms and subjective well-being (73) – SSGAC - http://www.thessgac.org/data 602 Never/ever smoked, cigarettes per day(74) - TAG - http://www.med.unc.edu/pgc/results-and-downloads 603 604 Additional phenotypes 605 BMI(58) – GIANT – https://www.broadinstitute.org/collaboration/giant 606 Height(75) – GIANT – https://www.broadinstitute.org/collaboration/giant 607 Crohn’s disease(76) – IIBDGC - http://www.ibdgenetics.org/downloads.html 608 Coronary artery disease(77) – Cardiogram – http://www.cardiogramplusc4d.org/downloads/ 609 Acknowledgments The authors wish to acknowledge Rosy Hoskins, Jaana Wessman and Joanna Martin for their 563 cromulent comments on the manuscript, Matthew Whittall for inspiration, and the patients and participants of the 564 respective consortia. For study-specific acknowledgments, see Supplementary Materials. GWAS summary statistics 565 used in the paper are available either directly from, or via application submitted in, the web addresses listed below. 566 Data on coronary artery disease has been contributed by CARDIoGRAMplusC4D investigators and have been 567 downloaded from www.CARDIOGRAMPLUSC4D.ORG. matSpD is available at 568 neurogenetics.qimrberghofer.edu.au/matSpD/. This research has been conducted using the UK Biobank Resource 569 (application #18597). 570 Acknowledgments The authors wish to acknowledge Rosy Hoskins, Jaana Wessman and Joanna Martin for their 563 cromulent comments on the manuscript, Matthew Whittall for inspiration, and the patients and participants of the 564 respective consortia. References: 394 For study-specific acknowledgments, see Supplementary Materials. GWAS summary statistics 565 used in the paper are available either directly from, or via application submitted in, the web addresses listed below 566 Data on coronary artery disease has been contributed by CARDIoGRAMplusC4D investigators and have been 567 downloaded from www.CARDIOGRAMPLUSC4D.ORG. matSpD is available at 568 neurogenetics.qimrberghofer.edu.au/matSpD/. This research has been conducted using the UK Biobank Resource 569 (application #18597). 570 571 572 Data sources 573 Disorder or phenotype – Consortium or dataset identifier – web address: 574 Psychiatric disorders 575 ADHD – PGC-ADD2 - http://www.med.unc.edu/pgc/results-and-downloads 576 Anorexia nervosa(63) – PGC-ED - http://www.med.unc.edu/pgc/results-and-downloads 577 Anxiety disorder(64) – ANGST - http://www.med.unc.edu/pgc/results-and-downloads 578 Autism spectrum disorders(65) – PGC-AUT - http://www.med.unc.edu/pgc/results-and-downloads 579 Bipolar disorder – PGC-BIP2 - http://www.med.unc.edu/pgc/results-and-downloads (soon) 580 Major depressive disorder – PGC-MDD2 - http://www.med.unc.edu/pgc/results-and-downloads (soon) 581 OCD – PGC-OCDTS - http://www.med.unc.edu/pgc/results-and-downloads 582 PTSD – PGC-PTSD - http://www.med.unc.edu/pgc/results-and-downloads 583 Schizophrenia(22) – PGC-SCZ2 – http://www.med.unc.edu/pgc/results-and-downloads 584 Tourette Syndrome – TSAIGC – http://www.med.unc.edu/pgc/results-and-downloads 585 586 Neurological disorders 587 Alzheimer's disease(18) – IGAP - http://www.pasteur-lille.fr/en/recherche/u744/igap 588 Epilepsy and subtypes, focal and generalized(66) – ILAE – http://www.epigad.org/page/show/gwas_index 589 Intracerebral hemorrhage(67) – ISGC - http://www.strokegenetics.com/ 590 Ischemic stroke and subtypes (cardioembolic, early-onset, small-vessel and large-vessel)(68) – METASTROKE 591 dataset of the ISGC – http://www.strokegenetics.com/ 592 Migraine and subtypes, migraine with and without aura – IHGC – www.headachegenetics.org 593 Multiple sclerosis(69) – IMSGC - http://eaglep.case.edu/imsgc_web 594 Parkinson's disease(21) – IPDGC – www.pdgene.org 595 596 15 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint Figure 1. Genetic correlation matrix across psychiatric phenotypes. 610 611 Color of each box indicates the magnitude of the correlation, while size of the boxes indicates its significance, with 612 significant correlations filling each box completely. Asterisks indicate genetic correlations which are significant 613 after Bonferroni correction. References: 394 ADHD – attention deficit hyperactivity disorder; ASD – autism spectrum disorder; 614 MDD – major depressive disorder; OCD – obsessive-compulsive disorder; PTSD – post-traumatic stress disorder. 615 Color of each box indicates the magnitude of the correlation, while size of the boxes indicates its significance, with 612 significant correlations filling each box completely. Asterisks indicate genetic correlations which are significant 613 after Bonferroni correction. ADHD – attention deficit hyperactivity disorder; ASD – autism spectrum disorder; 614 MDD – major depressive disorder; OCD – obsessive-compulsive disorder; PTSD – post-traumatic stress disorder. 615 16 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint Figure 2. Genetic correlation matrix across neurological phenotypes. 616 617 Color of each box indicates the magnitude of the correlation, while size of the boxes indicates its significance, with 618 significant correlations filling each box completely. Asterisks indicate genetic correlations which are significant 619 after Bonferroni correction. Some phenotypes have substantial overlaps (see Table 1), e.g. all cases of generalized 620 epilepsy are also cases of epilepsy. Asterisks indicate significant genetic correlation after multiple testing 621 correction. ICH – intracerebral hemorrhage. 622 623 Color of each box indicates the magnitude of the correlation, while size of the boxes indicates its significance, with 618 significant correlations filling each box completely. Asterisks indicate genetic correlations which are significant 619 after Bonferroni correction. Some phenotypes have substantial overlaps (see Table 1), e.g. all cases of generalized 620 epilepsy are also cases of epilepsy. Asterisks indicate significant genetic correlation after multiple testing 621 correction. ICH – intracerebral hemorrhage. 622 623 17 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . References: 394 CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint 624 Figure 3. Genetic correlation matrix across neurological and psychiatric phenotypes. Figure 3. Genetic correlation matrix across neurological and psychiatric phenotypes. 625 Color of each box indicates the magnitude of the correlation, while size of the boxes indicates its significance, with 626 significant correlations filling each box completely. Asterisks indicate genetic correlations which are significant 627 after Bonferroni correction. ADHD – attention deficit hyperactivity disorder; ASD – autism spectrum disorder; ICH 628 – intracerebral hemorrhage; MDD – major depressive disorder; OCD – obsessive-compulsive disorder; PTSD – 629 post-traumatic stress disorder. 630 Color of each box indicates the magnitude of the correlation, while size of the boxes indicates its significance, with 626 significant correlations filling each box completely. Asterisks indicate genetic correlations which are significant 627 after Bonferroni correction. ADHD – attention deficit hyperactivity disorder; ASD – autism spectrum disorder; ICH 628 – intracerebral hemorrhage; MDD – major depressive disorder; OCD – obsessive-compulsive disorder; PTSD – 629 post-traumatic stress disorder. 630 18 18 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint Figure 4. Genetic correlation matrix across brain disorders and behavioral-cognitive phenotype 631 632 Color of each box indicates the magnitude of the correlation, while size of the boxes indicates its significance, with 633 significant correlations filling each box completely. Asterisks indicate genetic correlations which are significant 634 after Bonferroni correction. ADHD – attention deficit hyperactivity disorder; ASD – autism spectrum disorder; ICH 635 – intracerebral hemorrhage; MDD – major depressive disorder; OCD – obsessive-compulsive disorder; PTSD – 636 post-traumatic stress disorder; BMI –body-mass index. References: 394 637 Color of each box indicates the magnitude of the correlation, while size of the boxes indicates its significance, with 633 significant correlations filling each box completely. Asterisks indicate genetic correlations which are significant 634 after Bonferroni correction. ADHD – attention deficit hyperactivity disorder; ASD – autism spectrum disorder; ICH 635 – intracerebral hemorrhage; MDD – major depressive disorder; OCD – obsessive-compulsive disorder; PTSD – 636 post-traumatic stress disorder; BMI –body-mass index. 637 19 19 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint Table 1. Brain disorder phenotypes used in the Brainstorm project. Indented phenotypes are part of a larger whole, 638 e.g. the epilepsy study consists of the joint analysis of focal epilepsy and generalized epilepsy. Numbers in gray 639 denote a sample set which is non-unique, e.g. cardioembolic stroke samples are a subset of ischemic stroke samples. 640 ADHD – attention deficit hyperactivity disorder; OCD – obsessive-compulsive disorder. ‘Anxiety disorders’ refers 641 to a meta-analysis of five subtypes (generalized anxiety disorder, panic disorder, social phobia, agoraphobia and 642 specific phobias). Source details are listed under Data Sources and the references in Table S1. References: 394 643 644 645 646 Psychiatric disorders Neurological disorders Disorder Source Cases Controls Disorder Source Cases Controls ADHD PGC-ADD2 12,645 84,435 Alzheimer's disease IGAP 17,008 37,154 Anorexia nervosa PGC-ED 3,495 11,105 Epilepsy ILAE 7,779 20,439 Anxiety disorders ANGST 5,761 11,765 Focal epilepsy " 4,601 17,985 Autism spectrum disorder PGC-AUT 6,197 7,377 Generalized epilepsy " 2,525 16,244 Bipolar disorder PGC-BIP2 20,352 31,358 Intracerebral hemorrhage ISGC 1,545 1,481 Major depressive disorder PGC-MDD2 16,823 25,632 Ischemic stroke METASTROKE 10,307 19,326 OCD PGC-OCDTS 2,936 7,279 Cardioembolic stroke " 1,859 17,708 PTSD PGC-PTSD 2,424 7,113 Early-onset stroke " 3,274 11,012 Schizophrenia PGC-SCZ2 33,640 43,456 Large-vessel disease " 1,817 17,708 Tourette Syndrome PGC-OCDTS 4,220 8,994 Small-vessel disease " 1,349 17,708 Migraine IHGC 59,673 316,078 Migraine with aura " 6,332 142,817 Migraine without aura " 8,348 136,758 Multiple sclerosis IMSGC 5,545 12,153 Parkinson's disease IPDGC 5,333 12,019 Total psychiatric 108,493 238,514 Total neurologic 107,190 418,650 Table 1. Brain disorder phenotypes used in the Brainstorm project. Indented phenotypes are part of a larger whole, 638 e.g. the epilepsy study consists of the joint analysis of focal epilepsy and generalized epilepsy. Numbers in gray 639 denote a sample set which is non-unique, e.g. cardioembolic stroke samples are a subset of ischemic stroke samples. 640 ADHD – attention deficit hyperactivity disorder; OCD – obsessive-compulsive disorder. ‘Anxiety disorders’ refers 641 to a meta-analysis of five subtypes (generalized anxiety disorder, panic disorder, social phobia, agoraphobia and 642 specific phobias). Source details are listed under Data Sources and the references in Table S1. 643 Neurological disorders Total psychiatric 646 20 20 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint Table 2. Behavioral-cognitive and additional phenotypes used in the study. Numbers in gray denote overlapping 647 study sets, e.g. samples in the college attainment analysis are a subset of those in the analysis for years of education. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint References: 394 648 (d) – dichotomous phenotype, (q) – quantitative phenotype. BMI – body-mass index. Source details are listed under 649 Data Sources, while references are listed in Table S2. 650 Table 2. Behavioral-cognitive and additional phenotypes used in the study. Numbers in gray denote overlapping 647 study sets, e.g. samples in the college attainment analysis are a subset of those in the analysis for years of education. 648 (d) – dichotomous phenotype, (q) – quantitative phenotype. BMI – body-mass index. Source details are listed under 649 Data Sources, while references are listed in Table S2. 650 651 Phenotype Source Samples Behavioral-cognitive phenotypes Cognitive Years of education (q) SSGAC 293,723 College attainment (d) " 120,917 Cognitive performance (q) " 17,989 Intelligence (d) CTG 78,308 Personality measures Subjective well-being SSGAC 298,420 Depressive symptoms " 161,460 Neuroticism (q) " 170,911 Extraversion (q) GPC 63,030 Agreeableness (q) " 17,375 Conscientiousness (q) " 17,375 Openness (q) " 17,375 Smoking-related Never/ever smoked (d) TAG 74,035 Cigarettes per day (q) TAG 38,617 Additional phenotypes BMI (q) GIANT 339,224 Height (q) " 253,288 Coronary artery disease (d) Cardiogram 86,995 Crohn's disease (d) IIBDGC 20,883 Total 1,124,048 Source Samples 21 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted September 6, 2017. ; https://doi.org/10.1101/048991 doi: bioRxiv preprint Supplementary Materials 653 Materials and methods 654 Supplementary Text 655 Comparison with previous heritability estimates 656 Effect of phenotypic misclassification 657 Study-specific acknowledgements 658 Consortium memberships 659 Figures S1-10 660 Tables S1-10 661 22 22
https://openalex.org/W1741358326
http://personaybioetica.unisabana.edu.co/index.php/personaybioetica/article/download/3986/pdf
Spanish; Castilian
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EMPATHY IN THE PHYSICIAN-PATIENT RELATIONSHIP AS A DEMONSTRATION OF RESPECT FOR THE DIGNITY OF THE INDIVIDUAL.: A CONTRIBUTION BY EDITH STEIN
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ARTÍCULOS ARTÍCULOS Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? PREPROJECT OF SPANISH CONSTITUTIONAL LAW ON ABORTION: IMPROVEMENT OR RETROGRESSION? ANTEPROJETO DE LEI ORGÂNICA ESPANHOLA SOBRE O ABORTO: PROGRESSO OU RETROCESSO? Roberto Germán-Zurriaráin1 1 Universidad de La Rioja, España. roberto.german@unirioja.es Resumen El trabajo aborda el Anteproyecto español de Ley del aborto que aprobó el gobierno del Partido Popular a finales de diciembre de 2013. El Anteproyecto aprobado ha abierto un debate en la sociedad española que, según algunos, estaba cerrado. En efecto, para algunos este Anteproyecto de Ley Orgánica no es necesario, porque se acepta sin discusión la ley de plazos y, además, suprime el derecho de la mujer a decidir sobre su embarazo; para otros, en cambio, sí que es necesario, ya que el ser humano no nacido no queda protegido jurídicamente en la ley vigente. Sin embargo, en mi opinión, este Anteproyecto tampoco protege suficientemente al ser humano discapacitado. p Palabras clave: aborto, ley orgánica, anteproyecto de ley, mujeres embarazadas. (Fuente DeCS, Bireme DOI: 10.5294/pebi.2014.18.2.3 Para citar este artículo / To reference this article / Para citar este artigo Germán-Zurriaráin R. Anteproyecto de ley orgánica español sobre el aborto: ¿mejora o retroceso? pers.bioét. 2014; 18(2). 119-137. DOI: 10.5294/ pebi.2014.18.2.3 Fecha de Recepción: 2014-02-25 Fecha de envío a pares: 2014-02-27 Fecha de aprobación por pares: 2014-04-12 Fecha de aceptación: 2014-05-01 1 Universidad de La Rioja, España. roberto.german@unirioja.es I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 119 PERSONA Y BIOÉTICA • JULIO - DICIEMBRE 2014 Abstract The paper addresses the Preliminary draft of the Spanish Constitutional Law on Abortion passed by the PP Government by the end of December 2013. The approved preliminary draft has opened a debate within the Spanish society which, according to some, was closed. Indeed, for some, this draft of the Constitutional Law is not necessary because the deadline law had been accepted and not disputed, and, in addition, it suppresses the right women have to decide on their pregnancy; for others, however, it is indeed necessary, as the unborn human being is not legally protected in the current law. Nevertheless, it is my opinion that this Preliminary draft does not sufficiently protect the disabled human being. Keywords: Abortion, constitutional law, preliminary draft of law, pregnant women. (Source: DeCS, Bireme). I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 Resumo Este trabalho aborda o Anteprojeto espanhol de Lei do aborto que o governo do Partido Popular aprovou no final de dezembro de 2013. O Anteprojeto aprovado abriu um debate na sociedade espanhola que, segundo alguns, estava encerrado. De fato, para alguns, esse Anteprojeto de Lei Orgânica não é necessário, porque se aceita sem discussão a lei de prazos e, além disso, suprime o direito da mulher de decidir sobre sua gravidez. Para outros, em compensação, é necessário, já que o ser humano não nascido não está protegido juridicamente na lei vigente. Contudo, em minha opinião, esse Anteprojeto também não protege suficientemente o ser humano deficiente. i Palavras-chave: aborto, lei orgânica, anteprojeto de lei, mulheres grávidas. (Fonte: DeCS, Bireme). 120 Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto G Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin El tema del aborto estaba en el debate social desde que el Gobierno español aprobó el Anteproyecto de Ley Orgánica sobre Protección de la vida del concebido y de derechos de la embarazada que reformaba la Ley vigente en España 2/2010 sobre el aborto3. Decían algunos que este Anteproyecto era un paso atrás, pues con él desa- parecían muchos derechos conquistados por la mujer, y otros decían, en cambio, que recogía el espíritu de la ley del año 1985, aprobada por primera vez en España, la cual era una ley de supuestos o indicaciones. Con todo, el aborto es una realidad. Un ejemplo: en el 2012 en España, el 90,23 % de las mujeres que abortaron lo hicieron antes de la semana 12 de gestación; un 1,75 % más que en 2009. Entonces, ¿que habrá que hacer? Por tanto, la vida o la muerte de los inocentes concebidos no depende del juego político de mayorías y minorías democráticas, de ideologías de izquierdas o derechas. Es necesario afrontar la cuestión de fondo: si el aborto es la muerte de un ser humano inocente provocada por la libertad de quienes tienen la responsabilidad de protegerlo —el padre, la madre y los médicos—, avalada por las leyes de un “Estado de derecho” o no. 3 El Gobierno retiró, el 23 de septiembre de 2014, el Antepro- yecto de Ley. Resumo El aborto es un asunto que marca profundamente la vida de quien está implicado en él (los padres, la familia, los amigos…), abarca aspectos que interesan a diferentes áreas (política, religión, ciencia, etc.), por lo que es una cuestión tanto individual como social al mismo tiempo. Por consiguiente, este no es el momento del debate de pareceres o consensos extraños, sino de reconocer la vida humana y protegerla desde el inicio. Por eso, más allá de las soluciones pseudo-políticas, de cuestiones o intereses políticos y estrategias de partidos, en este artículo se reivindican los derechos de una conciencia capaz de conocer la verdad y obedecerla. Así también indican que las diferencias entre la Ley 2/2010 y el Anteproyecto aprobado eran irreconciliables. Pero, ¿eran opuestas? Es verdad que el aborto es malo para todos y todas; a nadie le gusta. El aborto es una tragedia y un drama para la madre, el padre y la sociedad, pero ¿por qué es un drama?, ¿solamente por la técnica que se va a utilizar? Lo calificamos como drama porque lo que está en juego no es un “grano”, sino la vida o muer- te de un ser humano inocente e indefenso. Hablamos de drama para la mujer embarazada porque el aborto conlleva mucho sufrimiento para la madre, el padre y los entornos de familiares y amigos. El tema del aborto tampoco es una cuestión de retroceso o de progreso. Hablamos de progreso siempre y cuando algo vaya en mejora de la persona o de la sociedad. Ade- más, el progreso no siempre se identifica con lo mejor, porque lo posterior, por el simple hecho de serlo, no siempre es mejor que lo anterior. Por otra parte, en este año 2014, por ejemplo, las cifras alcanzarán los dos millones, de abortos registrados en España desde 1985, y no podemos ni debemos permitir tantas muertes de seres humanos débiles e indefensos. La vida es un tema que nos afecta a todos sin distinción. Todos los hombres y mujeres de buena voluntad son muy sensibles a esta cuestión tan molesta y tan importante. Vamos a repasar las distintas leyes sobre el aborto en España, para terminar con una reflexión acerca del Anteproyecto presentado y retirado. 121 I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 En España, el aborto se despenalizó en 1985. En concreto, la Ley Orgánica 9/1985, de 5 de julio , que reformaba el artículo 417 bis del Código Penal, introdujo por vez primera en nuestro ordenamiento la despenalización del aborto realizado en determinadas y concretas situaciones. En España, el aborto se despenalizó en 1985. En concreto, la Ley Orgánica 9/1985, de 5 de julio4, que reformaba el artículo 417 bis del Código Penal, introdujo por vez primera en nuestro ordenamiento la despenalización del aborto realizado en determinadas y concretas situaciones5. En materia de aborto, desde 1985, hasta la entrada en vigor de la Ley Orgánica 2/2010, el sistema normativo en España seguía un modelo de indicaciones o supuestos: 1) tera- péutico, en caso de que haya un “grave peligro para la vida o la salud física o psíquica de la embarazada”, en cualquier momento de la gestación; 2) ético, en caso de violación, en las 12 primeras semanas, y 3) eugenésico, “que se presuma que el feto habrá de nacer con graves taras físicas o psíquicas”, en las primeras 22 semanas, respectivamente. El modelo de indicaciones reflejaba un conflicto real de intereses que el legislador resolvía ponderando, por un lado, la vida, la salud y la propia dignidad de la mu- jer embarazada y, por otro, la vida del embrión o feto. Efectivamente, mediante el modelo de supuestos lo que acaecía era que el legislador despenalizaba el aborto cuando confluían ciertas situaciones conflictivas objetivas, discrepancia de intereses o de valores entre la vida del embrión o feto, y la vida, la salud y la propia dignidad de la mujer embarazada. De forma sumaria, se puede afirmar que la solución de las indicaciones operaba de acuerdo con el principio: regla general-excepción. Para el modelo de indicaciones, el aborto consentido era delito (regla general) salvo que concurriese alguno de los supuestos excepcionales enumerados por la ley (excepción). , j , , p 5 “No será punible el aborto practicado por un médico, o bajo su dirección, en centro o establecimiento sanitario, público o privado, acreditado y con consentimiento expreso de la mu- jer embarazada, cuando concurra alguna de las circunstancias siguientes: 1º. Que sea necesario para evitar un grave peligro para la vida o la salud física o psíquica de la embarazada y así conste en un dictamen emitido con anterioridad a la in- tervención por un médico de la especialidad correspondiente, distinto de aquel por quien o bajo cuya dirección se practique el aborto. En caso de urgencia o riesgo vital para la gestante, podrá prescindirse del dictamen y del consentimiento expre- so. 2º. Resumo 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 PERSONA Y BIOÉTICA • JULIO - DICIEMBRE 2014 4 BOE, del 12 julio 1985, núm. 166, p. 22041. I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 8 Es necesario destacar que a los comités clínicos, a los que solo compete, según la Ley española, la confirmación del diagnósti- co, se les traslada la tarea de una evaluación de cada caso con- creto, con la responsabilidad “de decidir sobre la interrupción de la gestación”. De tal forma, que traslada la responsabilidad del médico, en el diagnóstico del feto y la rigurosa y respe- tuosa información a los padres, a un comité de expertos. Los aspectos relativos a la cooperación de los miembros del comité clínico están parcialmente regulados en el artículo 16 de la Ley 2/2010 y desarrollados en Real Decreto 825/2010, de 25 de junio, que desarrolla los artículos 16 y 17 de la Ley 2/2010. g 7 La Organización Mundial de la Salud establece la semana 22 de gestación como el plazo a partir del cual el feto es viable con independencia de la madre. Se entiende, entonces, que desde la viabilidad fetal no cabe el aborto, haya o no anomalía. No obstante, adviértase que hace cuarenta años un feto era viable a las 30 semanas. Hoy puede serlo a las 20 semanas, y sobran indicios para pensar que, en breve, lo pueda ser a las 12 o 15 semanas. 6 Ley Orgánica, de 3 de marzo; BOE, de 4 de marco de 2010, núm. 55: 21001-14. Para la aprobación de esta ley fue nece- saria la modificación de la Ley 10/1995, de 23 de noviembre, sobre el Código Penal. En España, el aborto se despenalizó en 1985. En concreto, la Ley Orgánica 9/1985, de 5 de julio , que reformaba el artículo 417 bis del Código Penal, introdujo por vez primera en nuestro ordenamiento la despenalización del aborto realizado en determinadas y concretas situaciones. Que el embarazo sea consecuencia de un hecho cons- titutivo de delito de violación del artículo 429, siempre que el aborto se practique dentro de las doce primeras semanas de gestación y que el mencionado hecho hubiese sido denuncia- do. 3º. Que se presuma que el feto habrá de nacer con graves taras físicas o psíquicas, siempre que el aborto se practique dentro de las 22 primeras semanas de gestación y que el dicta- men, expresado con anterioridad a la práctica del aborto, sea emitido por dos especialistas del centro o establecimiento sa- nitario, público o privado, acreditado al efecto, y distintos de aquel por quien o bajo cuya dirección se practique el aborto”. No obstante, la solución de la indicación terapéutica o de “grave peligro para la vida o la salud física o psíquica de la embarazada” cubría, en la práctica, cualquier su- puesto. Esta afirmación venía avalada por los datos del Ministerio de Sanidad español: en el 2009, último año de la ley de supuestos o indicaciones, de los 111.482 abortos practicados, el 96,74 % se realizaron bajo el 122 Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto G supuesto de “salud física o psíquica de la embarazada”, el 2,98 % de los abortos por riesgo fetal, el 0,02 % por violación y 0,27 % por varios motivos. embarazo solo en dos supuestos: si estuviera en riesgo la vida o la salud de la embarazada o si hubiera graves anomalías en el feto (art. 15). Por último, en el ámbito penal, la Ley 9/1985 penalizaba con tres años de cárcel a las mujeres que interrumpían su embarazo fuera de estos supuestos. En el primer supuesto debe presentar un dictamen emi- tido con anterioridad a la intervención por un médico o médica especialista distinto del que la practique o dirija. En caso de urgencia, por riesgo vital para la ges- tante, podrá prescindirse del dictamen. En el segundo supuesto, es decir, que exista riesgo de graves anomalías en el feto, se debe presentar un dictamen emitido con anterioridad a la intervención por dos médicos espe- cialistas distintos del que la practique o dirija. En España, el aborto se despenalizó en 1985. En concreto, la Ley Orgánica 9/1985, de 5 de julio , que reformaba el artículo 417 bis del Código Penal, introdujo por vez primera en nuestro ordenamiento la despenalización del aborto realizado en determinadas y concretas situaciones. Luego el segundo supuesto obliga a dos médicos distintos a los que fueran a practicar la intervención a evidenciar una “enfermedad extremadamente grave o incurable” del feto o “anomalías incompatibles con la vida”, para la que no se establecía ningún límite de tiempo. I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 Ley Orgánica 2/2010 La Ley Orgánica 2/2010, de Salud Sexual y Reproductiva y de Interrupción Voluntaria del Embarazo6, introduce en España el modelo o sistema de plazo, en virtud del cual se aprueba la práctica del aborto a petición de la mujer embarazada en las primeras 14 semanas de gestación sin necesidad de alegar causa alguna (art. 14) o situación característica de conflicto objetiva que permita justificar, al menos formalmente, la muerte de la vida humana del que va a nacer, es decir, el aborto es permitido por la voluntad de la madre, “a petición de la mujer”, cuando concurran un requisito temporal (que tenga lugar dentro de las primeras catorce semanas de gestación) y otro formal (que conste la existencia de un consentimiento informado de la madre y en el transcurso de tres días). De manera excepcional, hasta la semana 22 (5 meses y medio)7, la mujer puede interrumpir el A partir de esta fecha, solo un comité clínico (art. 16)8 puede autorizar un aborto en el que se detecten anoma- lías fetales incompatibles con la vida o una enfermedad extremadamente grave o incurable. Para ser más exactos, la Ley 2/2010 del aborto en España es un sistema de plazos que traza la frontera entre el 123 I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 PERSONA Y BIOÉTICA • JULIO - DICIEMBRE 2014 y con los derechos sexuales y reproductivos de las mu- jeres y el significado que el embarazo tiene para ellas. Por eso, la modificación fundamental que introduce la Ley Orgánica 2/2010 radica en que si durante las ca- torce primeras semanas se puede abortar sin ninguna responsabilidad social es porque se defiende que este es un derecho de la autonomía de la mujer (art. 3). 9 El Consejo Fiscal informó negativamente el Anteproyecto ar- gumentando, entre otras, la siguiente razón: 4) “La despena- lización no puede convertirse en un derecho […] No puede hablarse de un derecho al aborto, pues ello supondría el reco- nocimiento del derecho a eliminar a un ser humano distinto de la madre y titular del derecho a la vida humana”. Ley Orgánica 2/2010 En efecto, la norma legal tiene como objetivo que el aborto o “interrupción voluntaria del embarazo”, o utilizando la terminología americana “derecho a la libre elección”, deja de ser un delito en ciertos casos y es reconocido como un derecho personal e íntimo de toda mujer con independencia de su edad, o, lo que es lo mismo, que el aborto provocado se considera un derecho, protegi- do por el Estado, que forma parte de la salud sexual y reproductiva de la mujer9. aborto permitido y el aborto prohibido atendiendo al momento cronológico de las catorce primeras semanas. Transcurrido ese plazo, solamente se puede practicar el aborto si se dan ciertas circunstancias, esto es, la Ley española de 2010 es una ley de plazos hasta las prime- ras 14 semanas de gestación y una ley de supuestos a partir de ellas. Superadas esas 14 semanas, la Ley de 2010 solo permite el aborto en dos supuestos: si estu- viera en riesgo la vida o la salud de la embarazada, o si hubiera graves anomalías en el feto (art. 15), y a cada uno le da un tiempo, convirtiéndose entonces en una ley de supuestos. Fuera de esos motivos y esos límites temporales, el aborto es un delito. Esto quiere decir que a través del sistema de plazos se posibilitan los abortos que tengan lugar antes de un de- terminado periodo de tiempo fijado legalmente, es decir, las 14 primeras semanas, sin que sea necesario para ello que concurra ninguna causa o situación característica de conflicto objetiva, esto es, el aborto se convierte en una prestación a la que tiene derecho la mujer bajo ciertas condiciones. Por otra parte, otra de las novedades de la Ley 2/2010 reside en que la regulación del aborto se sustrae del contexto de Código Penal (1). F. J. Higuera (2), en cambio, estima inapropiado presentar el problema del aborto asociado al de igualdad y al margen de un contexto penal. La solución del plazo, dice el autor citado, no se pueden relacionar con la idea de igualdad. Los valedores del sistema de indicaciones opinan que es necesario un marco penal en lo relativo a la regulación del aborto, lo que no significa que se abogue por el encarcelamiento de las mujeres que abortan, pues no existen, realmente, mujeres internas en los centros penitenciarios españoles cumpliendo penas por delito de aborto. S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 10 La jurisprudencia constitucional posterior se remite a la Sen- tencia del Tribunal Constitucional 53/1985, como básica en la materia. Así, son especialmente significativas tanto la STC 212/1996, que resuelve el recurso de inconstitucionalidad con- tra la Ley 42/1988, de 28 diciembre 1988, de donación y utili- zación de embriones y fetos humanos, como la STC 116/1999, de 17 junio de 1999, por la que se resuelve el recurso de in- constitucionalidad contra la Ley de Reproducción Humana Asistida, en las que se afirma el valor de la doctrina de la Sen- tencia del Tribunal Constitucional 53/1985. En este sentido, el Tribunal Europeo de Justicia de Luxem- burgo ha dictado sentencia en favor de la dignidad del em- brión humano desde la concepción Cfr. Judgment de The Court (Gry Chamber), European Court de Justice, case Brüst- le v. Greenpeace, 18 october 2011. Ley Orgánica 2/2010 El objetivo de la sustitución del sistema de indicaciones por un sistema de plazos, sostienen los valedores de este sistema, es garantizar la autonomía de las mujeres para decidir sobre su embarazo durante la fase inicial de la gestación. Sostienen que el modelo de plazo garantiza a las mujeres la posibilidad de tomar una decisión libre e informada sobre la finalización del embarazo, de forma gratuita en la sanidad pública. Así es, los defensores de este modelo consideran que el sistema de indicaciones deja al margen el ámbito de autonomía de la mujer embarazada. Efectivamente, la Ley 2/2010 vincula el aborto con las estrategias públicas sobre salud sexual y reproductiva, I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 124 Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto G Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin Además, la primera ley del aborto en España no preveía nada en la formación de los futuros profesionales de la salud en la práctica de abortos. En cambio, la Ley 2/2010 prevé la regulación de la objeción de conciencia al aborto de los profesionales sanitarios, que entre 1985 y 2010 se vino ejerciendo pero sin que se regulara su ejercicio. Es verdad que la objeción de conciencia está reconocida por la Constitución española (art. 16) y por la Declaración Universal de Derechos Humanos (art. 18), pero también es cierto que lo importante es que dicho derecho se ejerza correctamente, es decir, que no se obre por razones de conveniencia ni vaya acompañado de la estigmatización o el rechazo del objetor. Entonces, la Ley 2/2010 permite el aborto de un feto que nacerá vivo, pero con una enfermedad muy grave e incurable. Sin embargo, ¿por qué la vida humana que va a vivir y sobrevivir tras el nacimiento, aun cuando padezca una enfermedad muy grave, no merece ser protegida? I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 Ley Orgánica 2/2010 ¿Este artículo es compatible con la doctrina constante y reiterada del Tribunal Constitucional sobre la aplicación del artículo 15 de la Constitución española a la vida humana incipiente, recogida en la Sentencia 53/1985 en la que la vida del que va a nacer es un bien jurídicamente protegido?10. En este mismo sentido, el Consejo Genético Prenatal debe facilitar la decisión libre de la madre o de la pareja. Supuestamente este consejo tiene como objetivo que la mujer tenga una información completa y asequible que le permita decidir entre llevar adelante la gestación o acabar con ella. La Ley del 2010 también permite el aborto llamado eugenésico en las primeras 22 semanas de gestación, aunque se admite un caso en que se puede practicar el aborto sin límite de tiempo (si el feto sufre anomalías graves incompatibles con la vida o una enfermedad extremadamente grave e incurable, arts. 15b y 15c respectivamente). Desgraciadamente, el diagnóstico prenatal es concebi- do, hoy día, con una doble intención y aunque también tenga una utilidad diagnóstica o terapéutica, esta queda vinculada a la eugenésica. Es más, la única intervención Ahora bien, el artículo mencionado de la Ley 2/2010 no está redactado en los mismos términos que el tercer supuesto de la Ley de 1985. En este último texto legal: 1) solo cabía practicar abortos ante el pronóstico de la existencia de graves anomalías, pero no en momentos avanzados del embarazo en los que ya se hubieran detectado tales anomalías o una enfermedad extrema- damente grave e incurable; 2) no cabía despenalización en ningún caso si se habían superado las 22 semanas de gestación; 3) siempre se requería el dictamen de al menos dos especialistas. 125 I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 PERSONA Y BIOÉTICA • JULIO - DICIEMBRE 2014 legítima en el feto es aquella encaminada a causarle un beneficio (3). 12 “…según lo establecido en el Consejo de Europa (Comité de Ministros N/90 del 13 al 21/6/1990) Comité de Bioética de la Unesco (Informe 29/08/1994) […] el diagnóstico prenatal solo puede tener finalidades terapéuticas y nunca eugenésicas. Se- ría en consecuencia ilógico que un diagnóstico prenatal que no puede lícitamente conducir a una IVE ajena a la salud de la madre fuera la base legitimadora de una IVE eugenésica, aunque el anteproyecto, por razones comprensibles expuestas en el expediente, no prefiera denominarla así” (6). p pi 13 La Convención de los Derechos de Personas con Discapaci- dad fue adoptada el 13 diciembre de 2006 en las Naciones Unidas en Nueva York, y fue abierta para firma el 30 marzo de 2007. Fue firmada por España el mismo 30 de marzo y ratifi- cada el 3 de diciembre de 2007. Entró en vigor el 3 de mayo de 2008 y, por consiguiente, es de aplicación interna según los artículos 10.2 y 96 de la Constitución española (BOE, de 21 de abril de 2008, núm. 96: 20648-59). La Convención, al formar parte del ordenamiento interno de España, hace ne- cesaria la adaptación y modificación de diversas normas para asegurar el pleno ejercicio de todos los derechos humanos y las libertades fundamentales de las personas con discapacidad sin discriminación alguna por estos motivos. En este sentido, el objetivo del Real Decreto 1276/2011, de 16 de septiembre, de Adaptación normativa a la Convención Internacional sobre los Derechos de las Personas con Discapacidad, es adecuar la regulación reglamentaria vigente en materia de discapacidad a las directrices de la Convención, en la línea marcada por Ley Orgánica 2/2010 Sin embargo, esta utilidad eugenésica del diagnóstico no obedece a causas médicas sino a otro tipo de utilidades (sociales, económicas, familiares, de política sanitaria…), es decir, que la eliminación del paciente fetal no es en realidad una terapia que redunde en su beneficio11. condiciona el marco biosanitario al promover el aborto de un ser humano con discapacidad. También es fun- damental que este diagnóstico se encuadre dentro de las clasificaciones de enfermedades psiquiátricas de la mujer, aceptadas por las academias de psiquiatría. Con todo, en la Ley 2/2010 el concepto de diagnóstico prenatal queda enmascarado en el artículo 15 b) y c), como con certera precisión advirtió el Consejo de Estado español12. Además, el aborto eugenésico del artículo 15 de la Ley 2/2010, en mi opinión, es incompatible con los artículos 14 y 15 de la Constitución española a la luz de la Convención sobre los Derechos de Personas con Discapacidad13. De estos artículos se desprende que Luego, un buen uso del diagnóstico prenatal se ve secuestrado por un uso con deriva eugenésica. La eva- luación ética negativa del diagnóstico sería aquella que conllevase una finalidad eugenésica y estableciese una conexión entre diagnóstico prenatal y aborto eugenésico en caso de un resultado positivo. Este último reforzaría la imagen de la persona con discapacidad como un indi- viduo que ha sido excluido por la sociedad (5). Es necesario, por tanto, un profundo debate ético con respecto al diagnóstico prenatal. Sin embargo, el marco sociopolítico en el que nos desenvolvemos en España, precisamente debido a la Ley 2/2010, hace inseparable el uso terapéutico de su uso eugenésico, lo que a su vez p pi 13 La Convención de los Derechos de Personas con Discapaci- dad fue adoptada el 13 diciembre de 2006 en las Naciones Unidas en Nueva York, y fue abierta para firma el 30 marzo de 2007. Fue firmada por España el mismo 30 de marzo y ratifi- cada el 3 de diciembre de 2007. Entró en vigor el 3 de mayo de 2008 y, por consiguiente, es de aplicación interna según los artículos 10.2 y 96 de la Constitución española (BOE, de 21 de abril de 2008, núm. 96: 20648-59). 11 La Organización Médica Española Colegial comparte esta mentalidad al suprimir el artículo 24.1 de su anterior Código Deontológico que establecía que “al ser humano embriofe- tal enfermo se le debe tratar de acuerdo con las mismas di- rectrices éticas, incluido el consentimiento informado de los progenitores, que se aplican a los demás pacientes”, e intro- duciendo uno nuevo que equipara el acto médico a lo legal, considerando el aborto un acto médico, redefiniendo así lo que es este acto, en contra de su propia doctrina sin ningún tipo de justificación. “Se entiende por acto médico toda actividad lícita, desarrollada por un profesional médico, legítimamente capacitado, sea en su aspecto asistencial, docente, investigador, pericial u otros, orientado a la curación de una enfermedad, al alivio de un padecimiento o a la promoción integral de la salud. Se incluyen actos diagnósticos, terapéuticos o de alivio del su- frimiento” (art. 7.1) (4). I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 Ley Orgánica 2/2010 La Convención, al formar parte del ordenamiento interno de España, hace ne- cesaria la adaptación y modificación de diversas normas para asegurar el pleno ejercicio de todos los derechos humanos y las libertades fundamentales de las personas con discapacidad sin discriminación alguna por estos motivos. En este sentido, el objetivo del Real Decreto 1276/2011, de 16 de septiembre, de Adaptación normativa a la Convención Internacional sobre los Derechos de las Personas con Discapacidad, es adecuar la regulación reglamentaria vigente en materia de discapacidad a las directrices de la Convención, en la línea marcada por I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 126 Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto G Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin sobre derechos humanos ratificados por España que, conforme al artículo 10.2 de la Constitución, deben ser utilizados para interpretar los derechos y las libertades fundamentales establecidos en la misma, en concreto, la Carta Fundamental Derechos de la Unión Europea (art. 21.1) de diciembre de 2000, el Convenio Interna- cional sobre Derechos Civiles y Políticos (arts 2.1 y 26) del 23 de mazo de 1976, y el Protocolo num. 12 de la Convención sobre la Protección de los Derechos Huma- nos y Libertades Fundamentales (art. 14)14. Por tanto, la Ley 2/2010 implica una diversidad de trato que debe ser considerada arbitraria, injustificada y no razonable. los nascituri con discapacidad deben tener también esa protección constitucional. Entender otra cosa supone una violación de la dignidad colectiva de las personas con discapacidad o capacitados diferentes, por considerar que dicha discapacidad puede ser causa de diferenciación en el equilibrio entre valores constitucionalmente protegidos, en contra de la dimensión institucional de la igualdad del artículo 14 de la Constitución y de la interpretación dada por el propio Tribunal Constitucional español. En este sentido, en la Sentencia 53/1985 el Tribunal Constitucional español se plantea en el fundamento jurídico 11, apartado c), la compatibilidad del supuesto del aborto eugenésico con la Constitución española. 14 Protocolo 12 de la Convención sobre la Protección de Dere- chos Humanos y Libertades Fundamentales (ETS núm. 177 del Consejo de Europa), Roma, 4 de noviembre de 2000. la Ley 26/2011, de 1 de agosto, de adaptación normativa a la Convención sobre los Derechos de las Personas con Discapa- cidad: 98872-79. I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 Ley Orgánica 2/2010 La sentencia, principalmente, habla de insuficiencia. Superar esta y eliminar la inseguridad de los padres acerca de la suerte del afectado por la grave tara en el caso de que les sobreviva es, en sí misma, responsabilidad del Estado que no cumple su obligación de eliminar las barreras sociales, económicas, políticas y jurídicas para que las personas con discapacidad o capacitados diferentes pue- dan ejercer sus derechos en condiciones de igualdad en el seno de una sociedad integradora e inclusiva, donde se respete y valore la diversidad humana. Por ello, esa situación no puede ser justificación para discriminar a los fetos con diversidad funcional, sino para exigir al Estado que cumpla las obligaciones que el respeto por la dignidad de todos los seres humanos le exige conforme al artículo 10 de la Constitución (7). Recapitulando estos textos legislativos, podemos afirmar que con el reconocimiento del aborto eugenésico de la Ley 2/2010, que establece que, en caso de riesgo de diversidad funcional severo puede rebajarse el nivel de protección de la vida del nasciturus, se está legalizando una desigualdad de trato según la diversidad funcional, discapacidad o capacitado diferente, violando de forma manifiesta el principio de no discriminación. Por eso, el colectivo de personas con diversidad funcional, tanto a nivel internacional como nacional, ha expresado su rechazo al aborto eugenésico por considerarlo una discriminación y una minusvaloración de la dignidad de la persona con diversidad funcional. En España, este colectivo sostiene que la Ley 2/2010, a través de la per- misión de un tipo de aborto fundado en la característica singular de discapacidad del sujeto no nacido, defiende que determinadas personas (personas con discapacidad o capacitados diferentes) no son iguales a las demás, pues Los artículos de la Ley 2/2010 también ponen de relieve la contradicción de esta con otros Tratados Internacionales 127 I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 PERSONA Y BIOÉTICA • JULIO - DICIEMBRE 2014 protección constitucional en el marco del resto de los derechos y las libertades fundamentales establecidos en la Constitución Española, aunque no sea titular de los mismos. Ley Orgánica 2/2010 Más aún, si consideramos que la dignidad es el primero de los valores constitucionales establecidos en el artículo 10 que sirve de pórtico al Título Primero de la Constitución relativo a los derechos y las libertades fundamentales. Ello explica, por ejemplo, la consideración del nasciturus como titular de derechos sucesorios. Es en este sentido que, aunque solo la persona sea titular del derecho a la no discriminación previsto en el artículo 14 de la Constitución espa- ñola, y aunque no sea titular del derecho fundamental a la igualdad y no discriminación, la dignidad inherente debe ser reconocida al nasciturus 0 transmite el saje: que las discapacidad o al nasciturus con discapacidad se le protege en menor grado su expectativa a nacer. En efecto, la redacción actual del artículo 15 b) y c) configura, según este colectivo, una situación discriminatoria de determinados seres humanos por razón de sus circunstancias personales, en concreto, por la posibilidad o realidad de adolecer de graves enfermedades o anomalías (8). De este modo, para la Ley 2/2010 las anomalías funcio- nales físicas, psíquicas, mentales o sensoriales pueden ser causa específica de aborto (por ejemplo, determi- nadas cegueras o sorderas de nacimiento, la falta de miembros superiores o inferiores) entre las semanas 14 y 22, y más allá de la semana 22 si esa diversidad funcional puede ser calificada de “enfermedad extremadamente grave e incurable en el momento del diagnóstico”, por ejemplo, en el caso de detectarse en el feto un síndrome de Down. La Ley 2/20 siguiente m personas con capacitados d iguales a la nasciturus di protege en me S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 15 “La condición constitucional del nasciturus, según se decla- ró en la STC 53/1985 (fundamento jurídico 7), nos recuer- da el citado fundamento jurídico 3 de la STC 212/1996, cuya protección implica, con carácter general, para el Estado el cumplimiento de una doble obligación: la de abstenerse de interrumpir o de obstaculizar el proceso natural de gestación, y la de establecer un sistema legal de defensa de la vida que suponga una protección efectiva de la misma y que, dado el ca- rácter fundamental de la vida, incluya también, como garantía última, las normas penales”. Así se recoge en la Exposición de Motivos del Anteproyecto de la Ley Orgánica sobre el aborto aprobado en diciembre de 2013. La Ley 2/2010 transmite el siguiente mensaje: que las personas con discapacidad o capacitados diferentes no son iguales a las demás, pues al nasciturus discapacitado se le protege en menor grado que a los demás. La Ley 2/2010 transmite el siguiente mensaje: que las personas con discapacidad o capacitados diferentes no son iguales a las demás, pues al nasciturus discapacitado se le protege en menor grado que a los demás. Por tanto, en mi opinión, la diferenciación realizada en el artículo 15 de la Ley Orgánica 2/2010 entre fetos en los “que exista riesgo de graves anoma- lías” o en los que se detecte “una enfermedad extremada- mente grave e incurable en el momento del diagnóstico” no tiene una justificación objetiva y razonable por lo que la utilización de estos elementos diferenciadores debe ser considerada arbi- traria, carente de fundamento aceptable constitucional- mente, y supone una violación manifiesta del principio constitucional de no discriminación, de la desigualdad de trato basada en la diversidad funcional. demás, pues al apacitado se le or grado que a emás. En definitiva, la cuestión que suscitan estos artículos de la Ley 2/2010 es su compatibilidad con el derecho a la no discriminación, el derecho a la vida y las obligaciones positivas exigibles constitucionalmen- te al Estado español en la defensa del colectivo de las personas con diversidad funcional. los De todas formas, al igual que el nasciturus está protegido como bien jurídico por el artículo 15 de la Constitución sin que sea titular del derecho fundamental a la vida, puede afirmarse que, por la dignidad inherente que le debe ser reconocida, debe reconocérsele igualmente la La Ley 2/2010 transmite el siguiente mensaje: que las personas con discapacidad o capacitados diferentes no I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 128 Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto G son iguales a las demás, pues al nasciturus discapacitado se le protege en menor grado que a los demás. Discri- mina a un feto de 14 o incluso más de 22 semanas, en razón de su diversidad funcional. 16 Consejo de Estado, “Dictamen del Consejo de Estado ante la ley de salud sexual reproductiva y de IVE”, 1384/2009, 17 de septiembre de 2009, basándose en la Sentencia del Tribunal Europeo de Derechos Humanos, de 13 de febrero de 2003 (TEDH/2003/ caso Odièvre contra Francia, par. 45). Por su parte, el Consejo Fiscal señalaba a este respecto, en su Informe sobre el Anteproyecto de esta Ley Orgánica: 1) “El “derecho a decidir”, como tal, en materia de terminación vo- luntaria del embarazo no está expresamente reconocido en los tratados internacionales invocados en la Exposición de Moti- vos. Por el contrario, debe ponerse de relieve la claridad de los I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 La Ley 2/2010 transmite el siguiente mensaje: que las personas con discapacidad o capacitados diferentes no son iguales a las demás, pues al nasciturus discapacitado se le protege en menor grado que a los demás. Ciertamente, evitar el nacimiento de un bebé con anomalías es claramente discriminatorio, pues otorga menor valor a la vida de una persona con discapacidad que a la de otra sin ella. vida humana del que va a nacer no es compatible con la doctrina constante y reiterada del Tribunal Constitucional sobre la aplicación del artículo 15 CE a la vida humana incipiente, en concreto, con la interpretación realizada por el Tribunal Constitucional en su Sentencia de 1985, pues, el modelo del plazo se dirige unilateralmente a favorecer a la mujer y no reconoce ningún bien jurídico digno de protección, es decir, no reconoce al embrión o feto como bien jurídico por proteger. Por tanto, si la doctrina del Tribunal Constitucional protege la vida como bien jurídico, parece una contradicción no protegerla en aquella etapa de su proceso que no solo es condición para la vida independiente del seno materno, sino que es también un momento del desarrollo de la misma. Dicha tutela jurídica debería contemplarse en nuestro ordenamiento tal y como se recoge también en lo dispuesto por el Tribunal Constitucional en la Sentencia 116/1999, Fundamento Jurídico 515. Este es el marco constitucional desde el que se debería articular una protección efectiva de la vida del embrión y de los fetos humanos con discapacidad o enfermedad. En mi opinión, en la Ley 2/2010, frente a la vida huma- na del feto, se da siempre prioridad a la voluntad de la mujer, como si de una parte de ella se tratara, de forma que no se requiere que esta acredite ninguna causa o circunstancia que justifique su decisión de acabar con la vida del feto. En esta misma línea, el Informe emitido por el Consejo de Estado, de 17 de septiembre de 2009, subrayaba que el aborto no es un derecho, ni siquiera en el caso de que el Estado renuncie en determinados supuestos a su punición o a su tipificación penal. No existe el derecho a causar un mal objetivo: la destrucción de la vida del aún no nacido. Un “derecho” al aborto desconocido en los ordenamientos de nuestro entorno susceptibles de ser tomados como modelos16. 20 Así también, el Tribunal Europeo de Derechos Humanos ha reconocido expresamente en Sentencia de 13 de febrero del 2003, que la vida del aún no nacido es un bien dotado de sus- tantividad propia, de relieve vital y, en consecuencia, de interés objetivo y general (TEDH/2003/ caso Odièvre contra Fran- cia, par. 45). La Ley 2/2010 transmite el siguiente mensaje: que las personas con discapacidad o capacitados diferentes no son iguales a las demás, pues al nasciturus discapacitado se le protege en menor grado que a los demás. Por eso, los autores a favor de un sistema de indicaciones manifiestan que la consideración del aborto como un asunto exclusivo del derecho de la autodeterminación de la mujer, de la que se hace depender la continuidad de la 129 I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 PERSONA Y BIOÉTICA • JULIO - DICIEMBRE 2014 Luego la Ley 2/2010 no articula un sistema de garantías que evite la desprotección absoluta de la vida del em- brión, ni las condiciones para que las mujeres puedan tomar una decisión libre e informada, pues esta está supeditada a los requisitos de entrega de información y periodo de reflexión, y no protegen de forma efectiva al embrión o feto. El modelo de plazo no sería acorde con la jurisprudencia constitucional pues, a tenor de las sentencias del Tribunal Constitucional 53/198517, 212/199618 y 116/199919, la Ley 2/2010 no protege de forma efectiva al nasciturus como exige, recurrentemente, la Jurisprudencia del Tribunal Constitucional. Se puede sostener que en el aborto la vida humana del nasciturus entra en conflicto con derechos relativos a valores constitucionales como la vida y la dignidad de la mujer. Ninguno de estos dos bienes puede afirmarse con carácter absoluto, por lo que se impone la ponderación (9). Por tanto, para la sentencia, la desprotección absoluta del nasciturus es incompatible con el derecho a la vida recogido por la Constitución20. Con otras palabras, el derecho de la mujer no puede tener primacía absoluta sobre la vida del nasciturus, dado que dicha prevalencia supone la desaparición, en todo caso, de un bien no solo constitucionalmente protegido, sino que encarna un valor central del ordenamiento constitucional21. Por último, la Ley 2/2010 permite que las menores de edad, pero mayores de 16 años, puedan abortar sin el conocimiento de sus padres en determinadas circunstan- cias. En la ley de 1985 solo podían abortar las mayores de edad o las menores con el consentimiento de sus padres. I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 p 21 Como afirma J. F. Higuera: “Como la solución del plazo pue- de abarcar supuestos en que exista realmente un elemento o motivo justificante y también supuestos, en hipótesis, de mera utilidad o conveniencia, entonces cuando estemos ante estos últimos supuestos se produce, a mi juicio, una grave incon- gruencia y contradicción, ya que primero se autorizan o justi- fican las intervenciones sobre el embrión o el feto vivos en el útero cuando tengan un propósito diagnóstico o terapéutico en su propio interés, concediéndole protección y después se podrá abortar sin que exista un elemento o motivo justificante, sin otorgar protección” (2). La Ley 2/2010 transmite el siguiente mensaje: que las personas con discapacidad o capacitados diferentes no son iguales a las demás, pues al nasciturus discapacitado se le protege en menor grado que a los demás. En conclusión, el modelo de plazo de la Ley 2/2010 instaura el aborto libre en la práctica desde el momen- principios del derecho internacional en materia de derechos humanos que protegen la vida del no nacido; 2) La tendencia legislativa mayoritaria en los Estados de la Unión Europea no admite el sistema de plazos en el aborto […] Entre los Estados que admiten un sistema de plazos se encuentran […] varios Estados recientemente incorporados a la Unión Europea en las dos últimas ampliaciones (Estonia, Letonia, Lituania, Bulgaria, Eslovenia) con legislaciones de aborto legatarias de regímenes no democráticos, claramente proabortistas con base en razones ideológicas y demográficas, incompatibles con los derechos fundamentales y donde existen unos porcentajes de abortos tan elevados que reflejan la utilización del aborto como un mé- todo anticonceptivo más, práctica absolutamente rechazable desde cualquier punto de vista; 3) El Anteproyecto no cumple con el deber de establecer un sistema legal para la defensa de la vida del nasciturus que pueda considerarse como una auténtica protección efectiva de la misma. La interrupción del embarazo a petición de la mujer […] durante las 14 primeras semanas de gestación […] sin necesidad del apoyo en otro tipo de causas, supondrá nada menos que el sacrificio del nascitu- rus. Nasciturus que posee vida humana distinta de la madre”. 7 STC de 11 de abril; BOE núm 119 de 18 de mayo de 1985: 21 Como afirma J. F. Higuera: “Como la solución del plazo pue- de abarcar supuestos en que exista realmente un elemento o motivo justificante y también supuestos, en hipótesis, de mera utilidad o conveniencia, entonces cuando estemos ante estos últimos supuestos se produce, a mi juicio, una grave incon- gruencia y contradicción, ya que primero se autorizan o justi- fican las intervenciones sobre el embrión o el feto vivos en el útero cuando tengan un propósito diagnóstico o terapéutico en su propio interés, concediéndole protección y después se podrá abortar sin que exista un elemento o motivo justificante, sin otorgar protección” (2). q p 17 STC, de 11 de abril; BOE, núm. 119, de 18 de mayo de 1985: 10211-34. q p 17 STC, de 11 de abril; BOE, núm. 119, de 18 de mayo de 1985: 10211-34. 18 STC, de 19 de diciembre; BOE, núm. 19, de 22 de enero de 1997: 32-36. 18 STC, de 19 de diciembre; BOE, núm. La nueva reforma Para aprobar el Anteproyecto de Ley Orgánica sobre Protección de la vida del concebido y de derechos de la embarazada22 también ha fue necesaria la reforma del Código Penal español, que despenalizaba el aborto en determinadas y concretas situaciones23. 22 Ministerio de Justicia, 20 de diciembre de 2013. El Gobierno lo retiró el 23 de septiembre de 2014. 23 “Dos. Se modifica el artículo 145, que queda redactado de la siguiente manera: 1) El que produzca el aborto de una mujer, con su consentimiento, fuera de los casos previstos en el artí- culo 145 bis, será castigado con la pena de prisión de uno a tres años e inhabilitación especial para ejercer cualquier profesión sanitaria, o para prestar servicios de toda índole en clínicas, establecimientos o consultorios ginecológicos, públicos o pri- vados, por tiempo de uno a seis años. El juez impondrá la pena en su mitad superior cuando los actos descritos en este aparta- do se realicen fuera de un centro o establecimiento público o privado acreditado. 2) El que indujere a una mujer a producirse su aborto o a consentir que otra persona se lo cause, fuera de los casos permitidos por la Ley, será castigado con la pena de prisión de uno a tres años e inhabilitación especial para ejercer cual- quier profesión sanitaria, o para prestar servicios de toda índole en clínicas, establecimientos o consultorios ginecológicos, pú- blicos o privados, por tiempo de uno a seis años. 3) En ningún caso será punible la conducta de la mujer embarazada.i En el caso de que el grave peligro para la salud psíquica de la mujer tenga su origen en la existencia en el feto de alguna anomalía incompatible con la vida, el informe exigido en el párrafo anterior será emitido por un solo médico, debiendo acreditarse, además, tal anomalía mediante otro informe moti- vado y emitido con anterioridad por un médico especialista en la materia, en quien concurran los mismos requisitos. A estos efectos, se entenderá por anomalía fetal incompatible con la vida aquella que previsible y habitualmente, en el momento del diagnóstico, se asocie con la muerte del feto o del recién nacido durante el periodo neonatal, aunque en condiciones excepcionales la supervivencia pueda ser mayor. La Ley 2/2010 transmite el siguiente mensaje: que las personas con discapacidad o capacitados diferentes no son iguales a las demás, pues al nasciturus discapacitado se le protege en menor grado que a los demás. narse el conflicto, desde el ámbito médico, de ninguna otra forma, para evitar un grave peligro para la vida o la salud física o psíquica de la embarazada, siempre que se practique dentro de las 22 primeras semanas de gestación. A estos efectos, se entenderá que existe grave peligro para la vida o la salud de la mujer cuando el embarazo produzca un menoscabo importan- te a su salud, con permanencia o duración en el tiempo, según los conocimientos de la ciencia médica en ese momento, y así se constate en un informe motivado y emitido con anteriori- dad por dos médicos de la especialidad correspondiente a la patología que genera el grave peligro para la mujer, distintos de aquel que practique el aborto o bajo cuya dirección este tenga lugar y que no desarrollen su actividad profesional en el centro o establecimiento en el que se lleve a cabo. I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 22 Ministerio de Justicia, 20 de diciembre de 2013. El Gobierno lo retiró el 23 de septiembre de 2014. La Ley 2/2010 transmite el siguiente mensaje: que las personas con discapacidad o capacitados diferentes no son iguales a las demás, pues al nasciturus discapacitado se le protege en menor grado que a los demás. 19, de 22 de enero de 1997: 32-36. 19 STC, de 17 de junio; BOE, núm. 162, de 8 de julio de 1999: 67-80. Remite a los fundamentos jurídicos 5 y 7 de la STC 53/1985 y de la STC 212/1996. 130 I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto G El Anteproyecto seguía un modelo de indicaciones como la ley de 1985, pero solo aceptaba dos de ellas, terapéutico y ética, y modificaba fundamentalmente la Ley de 2010 sobre el aborto, vigente en España, que es una ley de plazos. to en que queda protegido por una despenalización casi absoluta. Con la Ley 2/2010, el aborto, de delito despenalizado, se torna en un derecho de la mujer sin limitaciones hasta la semana catorce, quedando la vida del nasciturus desprotegida. Lejos queda lo dictado por la STC 53/1985 en su Fundamento Jurídico 9. narse el conflicto, desde el ámbito médico, de ninguna otra forma, para evitar un grave peligro para la vida o la salud física o psíquica de la embarazada, siempre que se practique dentro de las 22 primeras semanas de gestación. A estos efectos, se entenderá que existe grave peligro para la vida o la salud de la mujer cuando el embarazo produzca un menoscabo importan- te a su salud, con permanencia o duración en el tiempo, según los conocimientos de la ciencia médica en ese momento, y así se constate en un informe motivado y emitido con anteriori- dad por dos médicos de la especialidad correspondiente a la patología que genera el grave peligro para la mujer, distintos de aquel que practique el aborto o bajo cuya dirección este tenga lugar y que no desarrollen su actividad profesional en el centro o establecimiento en el que se lleve a cabo. p 23 “Dos. Se modifica el artículo 145, que queda redactado de la siguiente manera: 1) El que produzca el aborto de una mujer, con su consentimiento, fuera de los casos previstos en el artí- culo 145 bis, será castigado con la pena de prisión de uno a tres años e inhabilitación especial para ejercer cualquier profesión sanitaria, o para prestar servicios de toda índole en clínicas, establecimientos o consultorios ginecológicos, públicos o pri- vados, por tiempo de uno a seis años. El juez impondrá la pena en su mitad superior cuando los actos descritos en este aparta- do se realicen fuera de un centro o establecimiento público o privado acreditado. 2) El que indujere a una mujer a producirse su aborto o a consentir que otra persona se lo cause, fuera de los casos permitidos por la Ley, será castigado con la pena de prisión de uno a tres años e inhabilitación especial para ejercer cual- quier profesión sanitaria, o para prestar servicios de toda índole en clínicas, establecimientos o consultorios ginecológicos, pú- blicos o privados, por tiempo de uno a seis años. 3) En ningún caso será punible la conducta de la mujer embarazada. 22 Ministerio de Justicia, 20 de diciembre de 2013. El Gobierno lo retiró el 23 de septiembre de 2014. 23 “Dos. Se modifica el artículo 145, que queda redactado de la siguiente manera: 1) El que produzca el aborto de una mujer, con su consentimiento, fuera de los casos previstos en el artí- culo 145 bis, será castigado con la pena de prisión de uno a tres años e inhabilitación especial para ejercer cualquier profesión sanitaria, o para prestar servicios de toda índole en clínicas, establecimientos o consultorios ginecológicos, públicos o pri- vados, por tiempo de uno a seis años. El juez impondrá la pena en su mitad superior cuando los actos descritos en este aparta- do se realicen fuera de un centro o establecimiento público o privado acreditado. 2) El que indujere a una mujer a producirse su aborto o a consentir que otra persona se lo cause, fuera de los casos permitidos por la Ley, será castigado con la pena de prisión de uno a tres años e inhabilitación especial para ejercer cual- quier profesión sanitaria, o para prestar servicios de toda índole en clínicas, establecimientos o consultorios ginecológicos, pú- blicos o privados, por tiempo de uno a seis años. 3) En ningún caso será punible la conducta de la mujer embarazada. Tres. Se modifica el artículo 145 bis, que pasa a tener la si- guiente redacción: 1) No constituirá delito el aborto prac- ticado por un médico o bajo su dirección, en centro o esta- blecimiento sanitario, público o privado, acreditado y con el consentimiento expreso de la mujer embarazada, previamente informada y asesorada, cuando concurra alguna de las circuns- tancias siguientes: a) Que sea necesario, por no poder solucio- El Anteproyecto contemplaba la legalidad del aborto eugenésico en los casos de enfermedades del concebido “incompatibles con la vida”. Por tanto, también permitía el aborto eugenésico porque si el feto sufría anomalías graves incompatibles con la vida o una enfermedad extremadamente grave e incurable, se podía abortar. madre— hacen del aborto en el caso de la malformación del feto una práctica libre en ese periodo. Así, el Anteproyecto de Ley Orgánica despenalizaba el aborto en dos supuestos o indicaciones: si el embarazo era producto de una violación y el aborto se practicaba en las 12 primeras semanas; o si suponía un grave peli- gro para la vida o la salud psíquica o física de la mujer y el aborto se practicaba en las 22 primeras semanas. Y eso incluía, aunque superasen las 22 semanas primeras de gestación, las anomalías fetales incompatibles con la vida, siempre que dañasen psicológicamente a la gestante y siempre que no se hubiesen detectado o podido detectar anteriormente, con un diagnóstico certero. Me explico: que el discapacitado no nacido o capacitado diferente sea considerado un ser humano, o, dicho en términos jurídicos, que sea un bien jurídico digno de protección, según la Sentencia de 1985, está muy bien, pero hacerlo depender de la salud de la madre es afirmar que el aborto es un derecho absoluto de la mujer, aun- que el Anteproyecto no lo dijese o rechazase. Además, el Anteproyecto contemplaba la legalidad del aborto eugenésico en los casos de enfermedades del concebido “incompatibles con la vida”. Por tanto, también permitía el aborto eugenésico porque si el feto sufría anomalías graves incompatibles con la vida o una enfermedad extremadamente grave e incurable, se podía abortar. Se quitaba un tercer supuesto que aparecía en la ley de 1985 y que permitía abortar en el supuesto de mal- formación del feto. Se admitía en este Anteproyecto, escuchando así a muchas asociaciones de discapacitados, que estos tienen derecho a nacer. Así las cosas, ¿dónde siguen quedando los derechos de los nacidos con malformación? Lamentablemente, según la ley del 85, la indicación terapéutica o de “grave peligro para la salud psíquica de la embarazada” cubría, en la práctica, cualquier supuesto. Por eso es verdad que el Anteproyecto español recono- cía que el discapacitado era un ser humano y como tal necesitaba de protección jurídica, pero, en la práctica, dicho reconocimiento y protección eran baldíos, porque la madre tenía por el “mango” la posibilidad de abortar, esto es, la malformación del feto podía provocar en la madre una situación de riesgo para su salud psíquica y, en consecuencia, el aborto se convertía en un derecho de esta. La nueva reforma p p p y No será punible el aborto, aunque se superen las 22 semanas de gestación, siempre que no se hubiese detectado o podido detectar anteriormente, con un diagnóstico certero, la anoma- lía incompatible con la vida del feto y así conste en el informe emitido con anterioridad, conforme a lo exigido en este aparta- do, o cuando exista riesgo vital para la mujer que no sea posible evitar, dentro de lo clínicamente exigible, mediante la protec- ción de la vida del concebido a través de la inducción del parto. b) Que el embarazo sea consecuencia de un hecho constitutivo de delito contra la libertad o indemnidad sexual, siempre que el aborto se practique dentro de las doce primeras semanas de gestación y el mencionado hecho hubiese sido denunciado con anterioridad”. p j Tres. Se modifica el artículo 145 bis, que pasa a tener la si- guiente redacción: 1) No constituirá delito el aborto prac- ticado por un médico o bajo su dirección, en centro o esta- blecimiento sanitario, público o privado, acreditado y con el consentimiento expreso de la mujer embarazada, previamente informada y asesorada, cuando concurra alguna de las circuns- tancias siguientes: a) Que sea necesario, por no poder solucio- 131 PERSONA Y BIOÉTICA • JULIO - DICIEMBRE 2014 S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 El Anteproyecto contemplaba la legalidad del aborto eugenésico en los casos de enfermedades del concebido “incompatibles con la vida”. Por tanto, también permitía el aborto eugenésico porque si el feto sufría anomalías graves incompatibles con la vida o una enfermedad extremadamente grave e incurable, se podía abortar. Con todo, hasta la semana 14 ambas leyes —una por la vía de plazos y otra por el riesgo psíquico de la Con la ley de plazos pasa, en la práctica, lo mismo. Los datos del 2012 sobre los abortos practicados, esto es, 112.390, son los siguientes: “a petición de la mujer”, 91,26 %; “grave riesgo para la vida o la salud de la embarazada”, 5,67 %; “riesgos de graves anomalías en el feto”, 2,78 %; “ano- I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 132 Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto G malías fetales incompatibles con la vida o enfermedad extremadamente grave e incurable”, 0,27 %, y por varios motivos, 0,01 % (10). No es de extrañar que se diga que “a petición de la mujer” sea considerado el “coladero” de cualquier ley. los servicios sociales sobre las ayudas a la maternidad, cursos de formación y centros de atención a gestantes. Para llevar a cabo esta protección de la maternidad es imprescindible, por tanto, una ley con medidas de apoyo social y económico a las mujeres embarazadas, redes de apoyo y políticas públicas dirigidas a reducir el número de abortos, sobre todo, políticas de protec- ción que faciliten la conciliación entre vida profesional y vida familiar, especialmente a las que se encuentran en situaciones de dificultad. Para ello, se proponía que el Anteproyecto se acompañase de un Plan Integral de Apoyo a la Maternidad dotado con ficha financiera en los Presupuestos Generales del Estado. Por consiguiente, la Ley Orgánica 2/2010 permite el aborto por la voluntad de la madre —“a petición de la mujer”— las 14 primeras semanas de gestación. En cambio, el Anteproyecto autorizaba el aborto en las 22 primeras semanas de gestación. 24 Audiencia Provincial de Barcelona, Sección Sexta, Sentencia 30 de enero de 2013. I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 El Anteproyecto contemplaba la legalidad del aborto eugenésico en los casos de enfermedades del concebido “incompatibles con la vida”. Por tanto, también permitía el aborto eugenésico porque si el feto sufría anomalías graves incompatibles con la vida o una enfermedad extremadamente grave e incurable, se podía abortar. Además, para este era necesario un informe motivado y emitido con anterioridad por dos médicos de la especialidad, distintos de aquel que practique el aborto, o bajo cuya dirección este tenga lugar, y que no desarrollasen su actividad profesional en el centro o establecimiento en el que se lleve a cabo. Es verdad que el Anteproyecto exigía dos informes médicos. Pero hoy día, aun sin conseguir el informe médico, esto no supone mayor dificultad24, pues es bastante para aceptar el riesgo psíquico de la mujer el propio estrés que provoca el embarazo y, en consecuen- cia, no es necesaria la visita personal del facultativo al paciente para emitir el informe y ello solo constituye una falta administrativa que no tiene trascendencia penal puesto que no hay tercero perjudicado, es decir, al nasciturus no se le considera perjudicado aunque se la haya privado de la vida. No obstante, era suficiente que el informe sea realizado por un solo médico si el peligro para la salud psíquica de la mujer tiene su origen en la concurrencia en el embrión de una anomalía fetal incompatible con la vida, es decir, aquella que previsible y habitualmente aparezca asociada con la muerte del feto o del recién nacido durante el periodo neonatal, debiendo acredi- tarse la misma con otro informe emitido por un médico especialista en la materia. Efectivamente, toda mujer necesitaba dictámenes de dos especialistas. El proceso era el siguiente: la mujer que se acogía al supuesto de “grave peligro para su vida o su salud psíquica”, debía reunir dos informes que acrediten el daño que supondría seguir con la gestación; posteriormente, recibía una información verbal sobre las consecuencias médicas de la intervención. El médico tenía que certificar con su firma que ha proporcionado estos datos. A continuación, para que no hubiese pre- siones y se brindase apoyo, la mujer era informada por No olvidemos, por tanto, que el 97,8 % de los abortos practicados se han realizado bajo el supuesto de riesgo para la salud de la madre y un 2 % se han realizado por malformación del feto. Si sumamos, por un lado, el riesgo para la salud de la madre, y, por otro, la malformación del feto que puede convertirse en un riesgo para la salud 133 I S S N 0 1 2 3 - 3 1 2 2 • p e r s . La Ley 2/2010 permite que las menores de edad, mayores de 16 años, puedan abortar sin el conocimiento de sus padres. En cambio, en el Anteproyecto de Ley Orgánica, para la embarazada menor de 16 años o mayor de edad sujeta a tutela, además de su manifestación de voluntad, se precisaba el consentimiento expreso de sus padres. reconoce expresamente el aborto para el supuesto de riesgo para la salud psíquica de la madre, pareciendo que lo limita solo al “grave riesgo para la vida o la salud de la embarazada” (art. 15), mientras que el Anteproyecto de Ley incluía expresamente el riesgo de la salud psíquica de la madre hasta la semana 22 (art. 1, apartado 3), lo que daría a entender que el Anteproyecto español era más permisivo que la ley de plazos. La segunda diferen- cia es que la Ley 2/2010 incluye la malformación como supuesto; en cambio, el Anteproyecto no, pero, como ya hemos indicado, al mantener esta última el supuesto del riesgo psíquico de la madre en el caso de malformación —esto es, la madre puede abortar—, ambas leyes en la realidad eran equiparables. psíquica de la madre, hacen un total de 99,8 %, reser- vándose el 0,2 % para el supuesto de la incompatibilidad de la vida del feto. psíquica de la madre, hacen un total de 99,8 %, reser- vándose el 0,2 % para el supuesto de la incompatibilidad de la vida del feto. En el ámbito penal, la Ley 9/1985 penalizaba con tres años de cárcel a las mujeres que interrumpían su em- barazo fuera de los supuestos. La Ley Orgánica 2/2010 contempla la pena de multa; en cambio, el Anteproyecto de Ley no establecía ninguna sanción. En este sentido, según el Anteproyecto, la responsabilidad del aborto no recaía sobre las mujeres sino en los profesionales sanitarios. Volviendo al tema que tratábamos, de manera excep- cional, en el caso de que el grave peligro para la salud psíquica de la mujer tenga su origen en la existencia en el feto de alguna anomalía incompatible con la vida, aunque este supere la semana 22, y siempre que no se hubiese detectado o podido detectar anteriormente, solo se exigirá que el informe sea emitido por un solo médico, debiendo acreditarse, además, tal anomalía mediante otro informe motivado y emitido con anterioridad por un médico especialista en la materia, o cuando exista riesgo vital para la mujer que no sea posible evitar, dentro de lo clínicamente exigible, mediante la protección de la vida del concebido a través de la inducción del parto. Por otro lado, la Ley 2/2010 permite que las menores de edad, mayores de 16 años, puedan abortar sin el conoci- miento de sus padres. El Anteproyecto contemplaba la legalidad del aborto eugenésico en los casos de enfermedades del concebido “incompatibles con la vida”. Por tanto, también permitía el aborto eugenésico porque si el feto sufría anomalías graves incompatibles con la vida o una enfermedad extremadamente grave e incurable, se podía abortar. b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 PERSONA Y BIOÉTICA • JULIO - DICIEMBRE 2014 I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 25 Se añade el artículo 4 bis. 26 Modificación de la Ley Orgánica 10/1995, de 23 de noviem- bre, del Código Penal, Capítulo III bis, artículo 768 bis. 27 Artículo sexto. Modificación de la Ley 44/2003, de 21 de no- viembre, de ordenación de las profesiones sanitarias. La Ley 2/2010 permite que las menores de edad, mayores de 16 años, puedan abortar sin el conocimiento de sus padres. En cambio, en el Anteproyecto de Ley Orgánica, para la embarazada menor de 16 años o mayor de edad sujeta a tutela, además de su manifestación de voluntad, se precisaba el consentimiento expreso de sus padres. En cambio, en el Anteproyecto de Ley Orgánica, para la embarazada menor de 16 años o mayor de edad sujeta a tutela, además de su manifesta- ción de voluntad, se precisaba el consentimiento expreso de sus padres. Pero, cuando concurrían serios motivos que impidiesen o desaconsejasen que se consultara a los representantes legales o tutores de la mujer, o cuando interpelados negasen su consentimiento o asentimien- to, según procediera, o expresasen opiniones distintas a ella, el juez resolvería sobre la suficiencia y validez Luego, parece que la Ley 2/2010 y el Anteproyecto es- pañol de Ley del 2013 eran similares. En cambio había, al menos, dos diferencias: una, que la Ley 2/2010 no I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 134 Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto G del consentimiento prestado por la mujer conforme al procedimiento legalmente establecido. indicación y condiciones en las que se ha prestado la información clínica a la embarazada. Luego, en relación con la información previa al con- sentimiento del aborto, debía existir un asesoramiento obligatorio y reglado por parte de una entidad indepen- diente, que informase a la embarazada sobre ayudas a problemas que empujen al aborto, apoyos a la familia u otras opciones, así como sobre posibles riesgos y secuelas del aborto. Si, tras recibir el asesoramiento, la mujer mantenía la decisión de abortar, debería presentar las certificaciones emitidas en el centro o establecimiento. Por consiguiente, en caso de urgencia por peligro vital para la gestante, podía prescindirse del informe, ase- soramiento asistencial, información clínica personal y consentimiento, pudiendo el médico consultar, cuando las circunstancias lo permitiesen, a sus familiares o a las personas vinculadas de hecho a ella. I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 La Ley 2/2010 permite que las menores de edad, mayores de 16 años, puedan abortar sin el conocimiento de sus padres. En cambio, en el Anteproyecto de Ley Orgánica, para la embarazada menor de 16 años o mayor de edad sujeta a tutela, además de su manifestación de voluntad, se precisaba el consentimiento expreso de sus padres. 1 1 9 - 1 3 7 • 2 0 1 4 PERSONA Y BIOÉTICA • JULIO - DICIEMBRE 2014 llas leyes, regulaciones, costumbres y prácticas que constituyen discriminación y de la no contribución a la vulneración de la dignidad y el valor inherentes y los derechos iguales e inalienables de todos los miembros de la familia humana con independencia de sus diferencias. Esto exige que las autoridades públicas brinden todos los apoyos necesarios a los seres humanos con diversidad funcional para afrontar su situación de discapacidad o enfermedad. siguiente a comenzar la prestación de su servicio en un centro o establecimiento, público o privado, acreditado para la práctica, quedando esa decisión incorporada, con carácter reservado, a su expediente personal. Dicha infor- mación constituía un dato personal que, en ningún caso, podría ser objeto de tratamiento, registro o publicación, y estaría protegida con las garantías previstas en la Ley Orgánica 15/1999, de 13 de diciembre, de Protección de Datos de Carácter Personal. 2. El Anteproyecto español reconocía que el discapa- citado no nacido o capacitado diferente era un ser humano o, dicho en términos jurídicos, que era un bien jurídico digno de protección pero, en la prácti- ca, tales reconocimiento y protección eran baldíos, porque la madre tenía por el “mango” la posibilidad de abortar, esto es, la malformación del feto podía provocar en la madre una situación de riesgo para su salud psíquica, y, en consecuencia, el aborto se convertía en un derecho absoluto de la mujer, aunque el Anteproyecto no lo dijese o rechazase. No obstante, podría modificar su decisión en cualquier momento, poniéndolo en conocimiento del director del centro de forma inmediata o, en todo caso, antes de ini- ciarse la prestación. Además, los profesionales sanitarios que ejerzan ese derecho podían dispensar tratamiento y atención médica adecuados a las mujeres que lo preci- saran antes y después de haberse sometido a un aborto. La Ley 2/2010 permite que las menores de edad, mayores de 16 años, puedan abortar sin el conocimiento de sus padres. En cambio, en el Anteproyecto de Ley Orgánica, para la embarazada menor de 16 años o mayor de edad sujeta a tutela, además de su manifestación de voluntad, se precisaba el consentimiento expreso de sus padres. El Anteproyecto de Ley decía que la reflexión se podía realizar con el progenitor siempre que la embarazada lo admitiese y que la información fuese verbal, mientras que la Ley 2/2010 dice que se entregue físicamente y se firme su recepción, lo que parece da mayor credibilidad que el Anteproyecto. Con estas finalidades, se introducía en el Anteproyecto de Ley una modificación relevante en la Ley 41/2002, de 14 de noviembre, Básica Reguladora de la Autonomía del Paciente y de Derechos y Obligaciones en materia de Información y Documentación Clínica25. Este asesoramiento perseguía, según el Anteproyec- to español, por un lado, la protección de la vida del nasciturus y la oferta de las alternativas existentes al aborto para resolver los conflictos originados por el embarazo de la mujer, y, por otro lado, la transmisión de información clínica personal, dicha verbalmente o en la forma que fuese accesible atendiendo a la edad de la mujer, su madurez y circunstancias, sobre los posibles riesgos y secuelas de la intervención, debiendo mediar un plazo de reflexión de, al menos, siete días entre el asesoramiento asistencial, la información clínica y la prestación del consentimiento expreso de la mujer o ma- nifestación de su voluntad al aborto, o los asentimientos o consentimientos de sus progenitores, tutor o curador, cuando fueran necesarios. Este asesoramiento tendría que realizarse por médicos, profesionales sanitarios y de servicios sociales, distintos e independientes de aquel que realice el aborto o que lo dirija. El médico debía expedir una certificación por escrito sobre la fecha, También, si la embarazada fuera menor de edad o con la capacidad judicialmente complementada, se podía prescindir del consentimiento expreso o asentimiento de aquellos que tengan que prestarlo, si no pudieran efectuarlo26. Por último, el Anteproyecto de Ley Orgánica preveía el derecho a la objeción de conciencia de los profesionales sanitarios del aborto27, para inhibirse de cualquier parti- cipación o colaboración en los supuestos despenalizados en el Código Penal, el cual debía manifestarse anticipa- damente y por escrito al director, dentro de la semana 135 I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . Conclusiones 1. Era muy positiva, por tanto, una nueva legislación en la que los seres humanos con discapacidad funcional o capacitados diferentes tuviesen o no la conside- ración de persona en el ordenamiento jurídico, no recibiesen un estándar de protección inferior que los seres humanos de igual naturaleza que no tuviesen esa discapacidad funcional. La alternativa a cualquier aborto y, en especial, al aborto del discapacitado funcional o, en el caso de violación, es intensificar el acompañamiento a la mujer y facilitarle la entrega del hijo en adopción. Para ello, es necesario incluir una revisión del procedimiento de adopciones, con el fin de agilizarlo. 1. Era muy positiva, por tanto, una nueva legislación en la que los seres humanos con discapacidad funcional o capacitados diferentes tuviesen o no la conside- ración de persona en el ordenamiento jurídico, no recibiesen un estándar de protección inferior que los seres humanos de igual naturaleza que no tuviesen esa discapacidad funcional. La alternativa a cualquier aborto y, en especial, al aborto del discapacitado funcional o, en el caso de violación, es intensificar el acompañamiento a la mujer y facilitarle la entrega del hijo en adopción. Para ello, es necesario incluir una revisión del procedimiento de adopciones, con el fin de agilizarlo. 3. Estas preguntas siguen en pie: ¿qué pasa con aquellos seres humanos que tienen anomalías incompatibles con la vida, es decir, que morirán inexorablemente nada más nacer?, ¿podemos hacer que una madre geste a ese ser humano que va a morir?, ¿cualquier otro, aunque sea la madre, tiene la capacidad para decidir si estamos ante una vida humana? Una ma- dre, ¿puede decidir la muerte de otro, aunque este presente graves anomalías incompatibles con la vida? Las respuestas a estas preguntas constituyen el núcleo de la cuestión, pero estas preguntas ¿son de índole jurídica? No estamos hablando de una madre y su feto que sufren una enfermedad extre- madamente grave o incurable y que la madre morirá Por tanto, la derogación de la Ley 2/2010 se debe afirmar desde el objetivo de la eliminación de aque- I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 3. AEBI. Conclusiones del VII Congreso Nacional de Bioética. Bioética y con-ciencia. Cuadernos de Bioética. 2009;70:503-6. I S S N 0 1 2 3 - 3 1 2 2 • p e r s . b i o é t . • V o l . 1 8 • N ú m e r o 2 • P á g s . 1 1 9 - 1 3 7 • 2 0 1 4 Conclusiones 1 1 9 - 1 3 7 • 2 0 1 4 136 Anteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin nteproyecto de Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Ger Ley Orgánica español sobre el aborto: ¿mejora o retroceso? l Roberto Germán-Zurriaráin 4. La Organización Médica Española Colegial Código Deontoló- gico. Guía de Ética Médica. España; 2011. irremediablemente si sigue con el embarazo, sino de aquellos fetos que poseen anomalías incompatibles con la vida, es decir, que morirán inexorablemente nada más nacer. En mi opinión, mientras exista el supuesto de grave riesgo para la salud psíquica de la madre habrá algún aborto con malformación del feto o sin ella. ¿Por qué en esos casos esas vidas no merecen ser protegidas? ¿Puede sostenerse, con el artículo 15 de la Constitución Española en la mano, que esas vidas no deben ser amparadas por el Estado? 5. Gónzalez-Melado FJ, Di Pietro ML. Diagnóstico prenatal ge- nético no invasivo: reflexión bioética sobre la utilización del diagnóstico prenatal no invasivo a partir del análisis de ácidos nucleicos presentes en sangre periférica materna. Cuadernos de Bioética. 2011;74:50. 6. Consejo de Estado español. Dictamen del Consejo de Es- tado ante la ley de salud sexual reproductiva y de IVE. Ma- drid; 2009. 7. Sanjosé Gil A, Cardona Llorens L. Foro de Vida Indepen- diente. Informe sobre la inconstitucionalidad del supuesto de “aborto eugenésico” previsto en la Ley orgánica de salud sexual y reproductiva y de la interrupción voluntaria del em- barazo. Madrid; 2010. R e f e r e n c i a s R e f e r e n c i a s 1. Laurenzo Copello P. Aborto y derecho a la sexualidad: un nue- vo marco para un viejo debate. El Cronista del Estado Social y Democrático de Derecho. 2009,7:27. 8. CERMI. Derechos humanos y discapacidad. Informe España 2008. Madrid: Ediciones Cinca; 2009. 2. Higuera Guimerá JF. Las propuestas de introducción de “la solución del plazo” con indicaciones en el delito de aborto: sus problemas constitucionales. Revista General de Derecho Penal. 2009;11:28 9. Cuerda Riezu A. La colisión de deberes en Derecho Penal. Madrid: Tecnos; 1984. 10. Ministerio de Sanidad, Servicios Sociales e Igualdad. Datos sobre la Interrupción voluntaria del embarazo.Madrid; 2013. 3. AEBI. Conclusiones del VII Congreso Nacional de Bioética. Bioética y con-ciencia. Cuadernos de Bioética. 2009;70:503-6. 137
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Observed positive vegetation-rainfall feedbacks in the Sahel dominated by a moisture recycling mechanism
Nature communications
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1 Nelson Institute Center for Climatic Research, University of Wisconsin-Madison, Madison, WI 53706, USA. 2 Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 91109, USA. 3 Environmental Sciences Division and Climate Change Science Institute, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. Correspondence and requests for materials should be addressed to Y.Y. (email: yuyan06@gmail.com) NATURE COMMUNICATIONS| 8: 1873 | DOI: 10.1038/s41467-017-02021-1| www.nature.com/naturecommunications ARTICLE ARTICLE ARTICLE Third, regional climate is affected by variability in slowly-evolving for- cings from both the oceans and vegetation, making it difficult to distinctly separate their individual contributions through tradi- tional regression-based analyses. T T he Sahel is characterized by substantial interannual to decadal variability in rainfall and high socio-economic vulnerability to hydrologic extremes1. Attribution of this pronounced rainfall variability to either oceanic3–5 or terrestrial6– 13 drivers has proven to be an elusive challenge. The Sahel experienced one of the most extreme and prolonged droughts in the global observational record during the 1970s-1990s2, 14, 15, which was believed to be mainly driven by external forcing from sea-surface temperature (SST) anomalies and amplified by local feedbacks associated with land degradation and desertification according to past modeling studies1, 5, 10, 12, 16–18. Despite vege- tation’s apparent significant role in amplifying and extending this drought episode10, 12, the current understanding of vegetation’s impacts on climate across the Sahel, through proposed albedo, moisture, and momentum feedbacks, originates from biased and highly parameterized climate models, with land-atmosphere interactions that remain insufficiently tested against observa- tions. Charney6 first hypothesized a positive vegetation-rainfall feedback regarding Sahel desertification, in which reduced greenness leads to increased surface albedo, resulting in low-level cooling, increased atmospheric stability, low-level subsidence, and drying. Other modeling studies have also simulated this proposed positive feedback but disagreed in terms of the relative dominance of the albedo and moisture mechanisms10, 12, 13, likely due to the exaggerated albedo forcing applied in the earlier modeling study6. Moreover, none of the previous observational studies have quantified the influence of observational uncertainty on the identified vegetation-rainfall feedbacks. Therefore, both the observed positive vegetation-rainfall feedback and the underlying mechanisms need more sophisticated and comprehensive investigations. g The apparent recovery from the multi-decadal drought during the early 21st century and the underlying recovery mechanism remain highly debated, partly due to increased interannual variability in Sahel rainfall27. In particular, the recent interannual variability in Sahel rainfall was not successfully predicted by the SST forcings that explained the late 20th century decadal drought in most state-of-the-art climate models, implying either recent changes in oceanic drivers of the interannual Sahel rainfall variability or elevated importance of other regulators, including land surface feedbacks28. Despite this model-based hypothesis regarding the change in oceanic regulations, there has never been rigorous observational quantification of the relative contribution from oceanic versus terrestrial drivers of the recent interannual variability in Sahel rainfall. ARTICLE Although the multiple regression- based study attempted to quantify the relative contribution of variability in Atlantic SST and Sahel Normalized Difference Vegetation Index (NDVI) to Sahel rainfall variability25, the results are uncertain due to the limited reliability of multiple regression in separating impacts from individual forcings and are not directly comparable with previous modeling studies due to the absence of remote oceanic impacts assessed in their study. Most studies on biophysical vegetation feedbacks have been restricted to running and analyzing coupled vegetation-climate model simulations, which have several key limitations19. Simu- lated feedbacks are model dependent, given that climate models vary in terms of dynamical core, numerical schemes, para- meterizations, and spatial resolution20. Many modeling studies have applied extreme sensitivity experiments, such as complete, instantaneous regional deforestation, which have limited real- world relevance, as observed vegetation changes are typically heterogeneous in space and transient in time. Owing to these modeling study limitations, observational studies of vegetation feedbacks are critically needed for testing the model-based hypotheses21 but would have to address several key challenges. First, observational records are generally short in duration and marred by uncertainty from measurement errors. Second, it is challenging to extract the observed signal of vegetation forcing on the atmosphere from the large atmospheric internal noise, espe- cially given that the atmospheric forcing on vegetation clearly outweighs vegetation’s feedback to the atmosphere20, 22. Third, regional climate is affected by variability in slowly-evolving for- cings from both the oceans and vegetation, making it difficult to distinctly separate their individual contributions through tradi- tional regression-based analyses. p y A multivariate, lagged covariance statistical method, the Gen- eralized Equilibrium Feedback Assessment (GEFA) (Methods), was developed to address the aforementioned challenges in the observational analysis of the oceanic and terrestrial forcings on the atmosphere19, 29, 30. GEFA’s reliability at extracting key oceanic and terrestrial drivers on North African climate, even with short data records, was successfully demonstrated through dynamical experiments with modified regional SST or leaf area index (LAI) using the National Center for Atmospheric Research (NCAR) Community Earth System Model (CESM)31, 32. Fur- thermore, it was shown that GEFA, when applied to assess land surface feedbacks, captures the combined effects of coupled vegetation and soil moisture anomalies32. ARTICLE In order to quantify uncertainty in estimated observed terrestrial feedbacks, GEFA is applied here to a spectrum of gridded observations, remote sen- sing products, and reanalyses, while weighting different datasets according to their estimated regional reliability across the Sahel in order to minimize the impacts of measurement errors (Methods). This multi-data set approach is particularly valuable over the data-sparse Sahel. On the basis of GEFA as applied to multiple observational data sets, we identify positive vegetation-rainfall feedbacks across the Sahel during the late to post-monsoon periods associated with a moisture recycling mechanism. The classic albedo-based mechanism is not supported by the observational data. We fur- ther reveal that diminished vegetation growth and accompanying dry soils lead to enhanced dust emissions across the Sahel, which potentially contributes to the positive vegetation-rainfall feedback. Previous observational studies on vegetation feedbacks across the Sahel are largely based on multiple linear regression and apply a single observational or reanalysis product, leading to limited credibility in their conclusions and no quantification of uncer- tainty. Evidence of positive vegetation-rainfall feedbacks has been found from previous observational studies using a statistical vegetation index simulation23 and Granger causality analysis24– 26. However, these observational investigations, based on multiple linear regression, do not fully tease out potential oceanic impacts on the variability in both the vegetation and precipitation, either due to the absence of oceanic predictors in their analysis or limitations of the applied methodology itself, namely that mul- tiple linear regression-based methods are unable to address highly correlated predictors, thereby biasing the assessed terrestrial impacts. Furthermore, their analyses are generally based on a single observational or reanalysis product for each variable, e.g., precipitation from the Climatic Research Unit (CRU), which has limited gauge coverage and thus limited reliability in the Sahel. ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 T he Sahel is characterized by substantial interannual to decadal variability in rainfall and high socio-economic vulnerability to hydrologic extremes1. Attribution of this pronounced rainfall variability to either oceanic3–5 or terrestrial6– 13 drivers has proven to be an elusive challenge. The Sahel experienced one of the most extreme and prolonged droughts in the global observational record during the 1970s-1990s2, 14, 15, which was believed to be mainly driven by external forcing from sea-surface temperature (SST) anomalies and amplified by local feedbacks associated with land degradation and desertification according to past modeling studies1, 5, 10, 12, 16–18. Despite vege- tation’s apparent significant role in amplifying and extending this drought episode10, 12, the current understanding of vegetation’s impacts on climate across the Sahel, through proposed albedo, moisture, and momentum feedbacks, originates from biased and highly parameterized climate models, with land-atmosphere interactions that remain insufficiently tested against observa- tions. Charney6 first hypothesized a positive vegetation-rainfall feedback regarding Sahel desertification, in which reduced greenness leads to increased surface albedo, resulting in low-level cooling, increased atmospheric stability, low-level subsidence, and drying. Other modeling studies have also simulated this proposed positive feedback but disagreed in terms of the relative dominance of the albedo and moisture mechanisms10, 12, 13, likely due to the exaggerated albedo forcing applied in the earlier modeling study6. Most studies on biophysical vegetation feedbacks have been restricted to running and analyzing coupled vegetation-climate model simulations, which have several key limitations19. Simu- lated feedbacks are model dependent, given that climate models vary in terms of dynamical core, numerical schemes, para- meterizations, and spatial resolution20. Many modeling studies have applied extreme sensitivity experiments, such as complete, instantaneous regional deforestation, which have limited real- world relevance, as observed vegetation changes are typically heterogeneous in space and transient in time. Owing to these modeling study limitations, observational studies of vegetation feedbacks are critically needed for testing the model-based hypotheses21 but would have to address several key challenges. First, observational records are generally short in duration and marred by uncertainty from measurement errors. Second, it is challenging to extract the observed signal of vegetation forcing on the atmosphere from the large atmospheric internal noise, espe- cially given that the atmospheric forcing on vegetation clearly outweighs vegetation’s feedback to the atmosphere20, 22. NATURE COMMUNICATIONS| 8: 1873 | DOI: 10.1038/s41467-017-02021-1| www.nature.com/naturecommunications Observed positive vegetation-rainfall feedbacks in the Sahel dominated by a moisture recycling mechanism Yan Yu 1,2, Michael Notaro1, Fuyao Wang1, Jiafu Mao 3, Xiaoying Shi3 & Yaxing Wei3 Classic, model-based theory of land-atmosphere interactions across the Sahel promote positive vegetation-rainfall feedbacks dominated by surface albedo mechanism. However, neither the proposed positive vegetation-rainfall feedback nor its underlying albedo mechanism has been convincingly demonstrated using observational data. Here, we present observational evidence for the region’s proposed positive vegetation-rainfall feedback on the seasonal to interannual time scale, and find that it is associated with a moisture recycling mechanism, rather than the classic albedo-based mechanism. Positive anomalies of remotely sensed vegetation greenness across the Sahel during the late and post-monsoon periods favor enhanced evapotranspiration, precipitable water, convective activity and rainfall, indi- cative of amplified moisture recycling. The identified modest low-level cooling and anomalous atmospheric subsidence in response to positive vegetation greenness anomalies are counter to the responses expected through the classic vegetation-albedo feedback mechanism. The observational analysis further reveals enhanced dust emissions in response to diminished Sahel vegetation growth, potentially contributing to the positive vegetation-rainfall feedback. 1 Nelson Institute Center for Climatic Research, University of Wisconsin-Madison, Madison, WI 53706, USA. 2 Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 91109, USA. 3 Environmental Sciences Division and Climate Change Science Institute, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. Correspondence and requests for materials should be addressed to Y.Y. (email: yuyan06@gmail.com) 1 NATURE COMMUNICATIONS| 8: 1873 | DOI: 10.1038/s41467-017-02021-1| www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 Indeed, Sahel rainfall variability during SON of 1982–2010 is largely successfully reconstructed by the GEFA-based prediction model containing only terrestrial NDVI forcings, with a temporal cor- relation of 0.79 between the multi-data set average predicted and observed time series, compared with that of 0.81 using both SST and NDVI forcings, and 0.56 using only SST forcings (Supple- mentary Fig. 1b). In contrast to the late-to-post monsoon season and consistent with previous model-based findings10, 12, the monsoon rainfall over the Sahel during June–August is mostly regulated by oceanic drivers with a temporal correlation between the predicted and observed rainfall of 0.84 using only oceanic forcings, compare with 0.88 using both oceanic and terrestrial forcings (Supplementary Fig. 1a). was one of the wettest years during the study period, an anomalously wet Sahel monsoon was mainly supported by La Niña conditions, and extended into the post-monsoon season, when oceanic impacts largely diminished and terrestrial forcings were allowed to dominate. The relatively enhanced contribution from land-atmosphere interactions in autumn is likely attributed to two factors. One likely explanation is the reduced amplitude and broad-scale atmospheric circulation impacts of key ocean-atmosphere tele- connection patterns33, including El Niño-Southern Oscillation (ENSO)34–36 and Atlantic Niño mode37 (Supplementary Fig. 3). Another contributing factor involves seasonally wet soils38 and consequential vegetation growth in response to the antecedent monsoon that support significant evapotranspiration (ET) fluxes (Fig. 2a). Indeed, the explained variance in Sahel ET associated with terrestrial drivers peaks during SON at 51%, according to the multi-data set average. Note that the apparent greater contribu- tion from oceanic forcings is not an artifact of the imbalanced number of oceanic versus terrestrial forcings, as confirmed by additional sensitivity tests that modulate the number of either type of forcing. Similar to the prior observational study based on a statistical vegetation index23, the combined oceanic and terrestrial drivers considered here through GEFA explain only 20–52% and 35–75%, by month, of the total variance in observed Sahel precipitation and air temperature, respectively. The residual portion of the total variance in precipitation and air temperature is comprised of the atmospheric internal variability (equation 1 in Methods section), non-linear impacts of oceanic and terrestrial forcings which are not detected by the linear GEFA statistical method, and impacts from other oceanic (e.g., higher order SST empirical orthogonal functions (EOFs) or SST EOFs from other basins) or terrestrial (e.g., NDVI from other ecoregions) forcings absent from the forcing matrix. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 a b Ocean 70 60 50 Explained variance (%) Explained variance (%) 40 0 10N 20N 30N 40N 30 E 30 20 10 0 70 60 50 40 30 20 10 0 FMA JFM MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Land Precipitation Air temperature Season FMA JFM MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Season Fig. 1 Total percent variance in observed atmospheric conditions across the Sahel explained by oceanic versus terrestrial forcings in each season during 1982–2011. a precipitation and b 2-m air temperature. The lines (red: terrestrial forcings, blue: oceanic forcings) represent the multi-data set average, and the shading represents the 10th and 90th percentiles of the multi-data set uncertainty range. Labels on the x axis stand for 3-month seasons, e.g., JFM for January, February, and March. The inserted map in a shows the Sahel region (12˚ N–17˚ N, 20˚ W–40˚ E). The smaller multi-data set uncertainty range in precipitation is apparently due to a greater number of datasets included in the analysis, compared to the temperature analysis. By randomly selecting two out of the four precipitation datasets, the typical multi-data set uncertainty largely increases and is comparable with that of air temperature a Ocean 70 60 50 Explained variance (%) 40 0 10N 20N 30N 40N 30 E 30 20 10 0 FMA JFM MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Land Precipitation Season b Explained variance (%) 70 60 50 40 30 20 10 0 Air temperature FMA JFM MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Season a b Season Season Fig. 1 Total percent variance in observed atmospheric conditions across the Sahel explained by oceanic versus terrestrial forcings in each season during 1982–2011. a precipitation and b 2-m air temperature. The lines (red: terrestrial forcings, blue: oceanic forcings) represent the multi-data set average, and the shading represents the 10th and 90th percentiles of the multi-data set uncertainty range. Labels on the x axis stand for 3-month seasons, e.g., JFM for January, February, and March. The inserted map in a shows the Sahel region (12˚ N–17˚ N, 20˚ W–40˚ E). The smaller multi-data set uncertainty range in precipitation is apparently due to a greater number of datasets included in the analysis, compared to the temperature analysis. Results Terrestrial versus oceanic contributions to Sahel climate. According to GEFA, oceanic forcings largely overwhelm terres- trial forcings on the observed variability in the Sahel’s regional precipitation and air temperature, with the exception of the post- monsoon season (SON), when vegetation feedbacks on local precipitation appear to dominate (Fig. 1). The multi-data set, average percent variance of Sahel precipitation explained by NATURE COMMUNICATIONS| 8: 1873 | DOI: 10.1038/s41467-017-02021-1| www.nature.com/naturecommunications 2 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 By randomly selecting two out of the four precipitation datasets, the typical multi-data set uncertainty largely increases and is comparable with that of air temperature oceanic forcings is 22% on the annual mean (mainly from tropical Pacific and tropical Atlantic SSTs), ranging from 4% in September–November (SON) to 44% in July–September (JAS). Oceanic forcings impose a greater regulation on Sahel air tem- perature, explaining 36% of the total variance on the annual mean and ranging from 26% in SON to 53% in JAS. The explained variance in precipitation by terrestrial forcings is 8% on the annual mean and ranges from 0% in April-June (AMJ) to 18% in August-October (ASO) and SON, which is comparable in mag- nitude with their regulation of air temperature. SON is the only season in which the land surface forcings dominate over oceanic forcings, with land surface variability explaining 17.5–18.2% among datasets of the total variance in precipitation. Indeed, Sahel rainfall variability during SON of 1982–2010 is largely successfully reconstructed by the GEFA-based prediction model containing only terrestrial NDVI forcings, with a temporal cor- relation of 0.79 between the multi-data set average predicted and observed time series, compared with that of 0.81 using both SST and NDVI forcings, and 0.56 using only SST forcings (Supple- mentary Fig. 1b). In contrast to the late-to-post monsoon season and consistent with previous model-based findings10, 12, the monsoon rainfall over the Sahel during June–August is mostly regulated by oceanic drivers with a temporal correlation between the predicted and observed rainfall of 0.84 using only oceanic forcings, compare with 0.88 using both oceanic and terrestrial forcings (Supplementary Fig. 1a). oceanic forcings is 22% on the annual mean (mainly from tropical Pacific and tropical Atlantic SSTs), ranging from 4% in September–November (SON) to 44% in July–September (JAS). Oceanic forcings impose a greater regulation on Sahel air tem- perature, explaining 36% of the total variance on the annual mean and ranging from 26% in SON to 53% in JAS. The explained variance in precipitation by terrestrial forcings is 8% on the annual mean and ranges from 0% in April-June (AMJ) to 18% in August-October (ASO) and SON, which is comparable in mag- nitude with their regulation of air temperature. SON is the only season in which the land surface forcings dominate over oceanic forcings, with land surface variability explaining 17.5–18.2% among datasets of the total variance in precipitation. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 Climatology (cm per month) Climatology (g kg–1) Climatology (kg m–2) Response (cm per month σ–1 NDVI) Response (cm per month σ–1 NDVI) Response (g kg–1 σ–1 NDVI) a b e Climatology (W m–2) Response (W m–2 σ–1 NDVI) Response (fraction σ–1 NDVI) Climatology (fraction) d f c ET 8.0 0.8 18 2.0 1.0 0.0 –1.0 –2.0 Response (kg m–2 σ–1 NDVI) 2.0 50 16 12 0.8 0.30 0.012 0.008 0.004 0.000 –0.004 0.20 0.10 0.00 –0.10 0.4 0.0 –0.4 –0.8 8 4 0 –4 30 20 10 –10 –20 –30 0 300 280 260 240 220 200 40 30 20 10 0 1.0 0.0 –1.0 –2.0 15 12 9 6 3 0 0.4 0.0 –0.4 –0.8 Season 6.0 4.0 2.0 0.0 JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF 2-m specific humidity Precipitable water OLR Precipitation Wet day frequency Climatology (cm per month) g. 2 Multi-data set observational moisture responses to positive local NDVI anomalies across the Sahel during 1982–2011. a evapotranspiration 2-m specific humidity, c precipitable water, d outgoing longwave radiation (OLR), e precipitation, and f frequency of wet days. In a–e, dots ind tistically significant (p < 0.1) multi-data set average responses, referring to the right y-axis; open circles represent the 10th and 90th percentiles ulti-data set responses, regardless of their statistical significance. In f, dots and open circles indicate significant and insignificant responses, respec ng daily station precipitation data. Dashed lines indicate a response of zero. Bars indicate the multi-data set mean climatology of the response va erring to the left y-axis. σNDVI in the units of the response variables refers to one standard deviation in the Sahel NDVI anomaly. Labels on the xis stand for 3-month seasons, e.g., JFM for January, February, and March. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 The statistical significance is determined based on Monte Carlo boo ting RTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-0 Climatology (cm per month) Response (cm per month σ–1 NDVI) a ET 8.0 0.8 0.4 0.0 –0.4 –0.8 Season 6.0 4.0 2.0 0.0 JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Climatology (g kg–1) Response (g kg–1 σ–1 NDVI) b 18 2.0 1.0 0.0 –1.0 –2.0 15 12 9 6 3 0 Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF 2-m specific humidity b Climatology (kg m–2) c Response (kg m–2 σ–1 NDVI) 2.0 50 40 30 20 10 0 1.0 0.0 –1.0 –2.0 Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Precipitable water Climatology (W m–2) Response (W m–2 σ–1 NDVI) d 30 20 10 –10 –20 –30 0 300 280 260 240 220 200 Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF OLR d d c Climatology (W m–2) Season Season Response (cm per month σ–1 NDVI) e Response (fraction σ–1 NDVI) Climatology (fraction) f 16 12 0.8 0.30 0.012 0.008 0.004 0.000 –0.004 0.20 0.10 0.00 –0.10 0.4 0.0 –0.4 –0.8 8 4 0 –4 Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Precipitation Wet day frequency Climatology (cm per month) Response (fraction σ–1 NDVI) Climatology (fraction) f 0.30 0.012 0.008 0.004 0.000 –0.004 0.20 0.10 0.00 –0.10 Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Wet day frequency Response (cm per month σ–1 NDVI) e 16 12 0.8 0.4 0.0 –0.4 –0.8 8 4 0 –4 Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Precipitation Climatology (cm per month) f e Climatology (cm per month) Response (cm per month σ–1 NDVI) Response (fraction σ–1 NDVI) Climatology (fraction) Climatology (frac Season Season Season Fig. 2 Multi-data set observational moisture responses to positive local NDVI anomalies across the Sahel during 1982–2011. a evapotranspiration (ET), b 2-m specific humidity, c precipitable water, d outgoing longwave radiation (OLR), e precipitation, and f frequency of wet days. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 Measurement errors, which have been partly accounted for in the analysis, potentially contribute to this residual explained variance as well. In contrast to the g y g The contribution from each oceanic and terrestrial forcing on individual drought and pluvial cases can be decomposed by GEFA. For example, during 1984 (Supplementary Fig. 2a, b), which was the driest year in the Sahel during 1982–2011, an anomalously dry monsoon was favored by the Atlantic and Indian SST anomalies, as well as anomalously sparse vegetation cover in the Sahel, West African monsoon region, and Horn of Africa. This drought continued during the post-monsoon season, mainly driven by the anomalies in Sahel vegetation cover. As another example, during 1999 (Supplementary Fig. 2c, d), which NATURE COMMUNICATIONS| 8: 1873 | DOI: 10.1038/s41467-017-02021-1| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 This proposed link between an anomalously wet and extended monsoon season is supported by a temporal correlation of 0.58 between the observed detrended JJA rainfall and September rainfall anomalies over the Sahel during 1901–2014. previous observational study based on Granger causality analysis25, the current GEFA-based analysis includes both nearby and remote oceanic forcings and considers multiple observational datasets, thereby providing a more convincing quantification of oceanic versus terrestrial contributions to Sahel rainfall varia- bility. Furthermore, by considering both nearby and remote oceanic impacts, the GEFA-based assessment of oceanic versus terrestrial contributions is directly comparable with previous modeling studies that modify global SSTs through dynamical experiments10, 12. Vegetation-climate feedbacks in the Sahel. The observational analysis verifies the proposed positive vegetation-rainfall feedback across the Sahel from prior modeling studies6, 10, 12, 13 and regression-based observational studies23–26. However, the feed- back is seemingly confined to the post-monsoon autumn season and largely due to a moisture recycling mechanism (Fig. 2). Positive NDVI anomalies favor enhanced ET, precipitable water, convective activity [reduced outgoing longwave radiation (OLR)], and total precipitation amount, indicative of amplified moisture recycling. The positive vegetation-rainfall feedback in the Sahel is confirmed with station rainfall observations (Supplementary Fig. 4a) and regional downscaling of global reanalysis (Supple- mentary Note 1, Supplementary Fig. 5a). The ET and precipita- tion responses are of comparable magnitude in SON, namely +0.43 (0.40–0.44 among data sets) cm month−1 per standard deviation of NDVI anomalies (σ−1NDVI) and +0.49 (0.48–0.50 among data sets) cm month−1 σ−1NDVI, respectively, implying the dominance of the moisture recycling mechanism underlying the positive vegetation-rainfall feedback in the post-monsoon season across the Sahel (Fig. 2a, e). Vegetation imposes a greater influ- ence on the frequency, rather than intensity, of convective activity across the Sahel, consistent with previous observational findings regarding enhanced probability of afternoon precipitation in eastern United States and Mexico by high evaporation39. Speci- fically, the increase in precipitation amount of +7.7 (7.5–7.9 The observational analysis here, focused on the seasonal to interannual time scale, challenges the proposed classic albedo- based mechanism6 for Sahel vegetation feedbacks. Low-level cooling (due to increased ET and latent heat flux and thus decreased Bowen ratio), weakened lapse rate (Fig. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 In a–e, dots indicate statistically significant (p < 0.1) multi-data set average responses, referring to the right y-axis; open circles represent the 10th and 90th percentiles of the multi-data set responses, regardless of their statistical significance. In f, dots and open circles indicate significant and insignificant responses, respectively, using daily station precipitation data. Dashed lines indicate a response of zero. Bars indicate the multi-data set mean climatology of the response variable, referring to the left y-axis. σNDVI in the units of the response variables refers to one standard deviation in the Sahel NDVI anomaly. Labels on the x-axis stand for 3-month seasons, e.g., JFM for January, February, and March. The statistical significance is determined based on Monte Carlo bootstrap testing NATURE COMMUNICATIONS| 8: 1873 | DOI: 10.1038/s41467-017-02021-1| www.nature.com/naturecommunications 4 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 ARTICLE among data sets) % σ−1NDVI during SON is largely due to a +7.8% σ−1NDVI increase in precipitation frequency, with minimal response in precipitation event intensity to NDVI anomalies (Fig. 2e, f). A likely explanation for the differential response in precipitation frequency versus intensity is that the surface tur- bulent flux partitioning, associated with the vegetation and soil moisture anomalies, shifts the local atmosphere from a non- convective to convective state, while other broad-scale controls, such as free tropospheric moisture content or large-scale moisture convergence, largely determine the rainfall intensity40. Further- more, positive NDVI anomalies favor a higher frequency of moderately low OLR days in the Sahel (not shown), implying that anomalously enhanced vegetation growth supports an increased chance of moderate-intensity convective events. Although the GEFA-based analysis agrees with previous regression-based analyses on the existence of a positive vegetation-rainfall feed- back in the Sahel, the statistical vegetation index analysis con- cluded that the vegetation feedback peaks during the monsoon season23, rather than the post-monsoon season as identified by GEFA. A potential reason for this inconsistent conclusion is that the regression-based analysis failed to account for the oceanic impacts, which peak during the monsoon season (Fig. 1) and likely bias the estimated vegetation impacts. The identified posi- tive vegetation-rainfall feedback in the Sahel during the late-to- post monsoon season suggests that an anomalous wet monsoon tends to persist longer, since enhanced vegetation growth and wet soil associated with anomalously abundant rainfall likely cause an extended monsoon season over the Sahel. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 3a), higher Climatology (°C) Response (°C σ–1 NDVI) Regression (fraction σ– NDVI) a b Evergreen broadleaf Shrublands Grasslands Savannas Woody Savannas Air temperature 33.0 0.40 0.040 0.020 0.000 –0.020 –0.040 0.20 0.00 –0.20 –0.40 30.0 27.0 24.0 21.0 Surface albedo Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Fig. 3 Observed local energy responses to Sahel NDVI anomalies during 1982–2011. a surface air temperature and b temporal regression coefficient of monthly surface albedo upon standardized NDVI anomalies, averaged by biome type. In a, dots indicate statistically significant (p < 0.1, based on Monte Carlo bootstrap test) multi-data set average responses, referring to the right y axis; open circles represent the 10th and 90th percentiles of the multi-data set responses, regardless of their statistical significance; the dashed line indicates a response of zero; bars indicate the multi-data set mean climatology of the response variable, referring to the left y axis. In b, filled dots indicate statistically significant (p < 0.1) correlations, according to the Student’s t-test; the size of the circles and thickness of lines denote the annual abundance of the corresponding biome across the Sahel, which is based on the remotely sensed land cover type from the International Satellite Land Surface Climatology Project (ISLSCP) initiative II International Geosphere-Biosphere Project (IGBP) DISCover and Simple Biosphere (SiB) Land Cover data set. The surface albedo is from the Global Land Surface Satellites data set66. In both a and b, labels on the x axis stand for 3-month seasons, e.g., JFM for January, February, and March Climatology (°C) Response (°C σ–1 NDVI) a Air temperature 33.0 0.40 0.20 0.00 –0.20 –0.40 30.0 27.0 24.0 21.0 Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Regression (fraction σ– NDVI) b Evergreen broadleaf Shrublands Grasslands Savannas Woody Savannas 0.040 0.020 0.000 –0.020 –0.040 Surface albedo Season JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF b a Regression (fraction σ– NDVI) Season Season Season Season Fig. 3 Observed local energy responses to Sahel NDVI anomalies during 1982–2011. a surface air temperature and b temporal regression coefficient of monthly surface albedo upon standardized NDVI anomalies, averaged by biome type. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 In a, dots indicate statistically significant (p < 0.1, based on Monte Carlo bootstrap test) multi-data set average responses, referring to the right y axis; open circles represent the 10th and 90th percentiles of the multi-data set responses, regardless of their statistical significance; the dashed line indicates a response of zero; bars indicate the multi-data set mean climatology of the response variable, referring to the left y axis. In b, filled dots indicate statistically significant (p < 0.1) correlations, according to the Student’s t-test; the size of the circles and thickness of lines denote the annual abundance of the corresponding biome across the Sahel, which is based on the remotely sensed land cover type from the International Satellite Land Surface Climatology Project (ISLSCP) initiative II International Geosphere-Biosphere Project (IGBP) DISCover and Simple Biosphere (SiB) Land Cover data set. The surface albedo is from the Global Land Surface Satellites data set66. In both a and b, labels on the x axis stand for 3-month seasons, e.g., JFM for January, February, and March Fig. 3 Observed local energy responses to Sahel NDVI anomalies during 1982–2011. a surface air temperature and b temporal regression coefficient of monthly surface albedo upon standardized NDVI anomalies, averaged by biome type. In a, dots indicate statistically significant (p < 0.1, based on Monte Carlo bootstrap test) multi-data set average responses, referring to the right y axis; open circles represent the 10th and 90th percentiles of the multi-data set responses, regardless of their statistical significance; the dashed line indicates a response of zero; bars indicate the multi-data set mean climatology of the response variable, referring to the left y axis. In b, filled dots indicate statistically significant (p < 0.1) correlations, according to the Student’s t-test; the size of the circles and thickness of lines denote the annual abundance of the corresponding biome across the Sahel, which is based on the remotely sensed land cover type from the International Satellite Land Surface Climatology Project (ISLSCP) initiative II International Geosphere-Biosphere Project (IGBP) DISCover and Simple Biosphere (SiB) Land Cover data set. The surface albedo is from the Global Land Surface Satellites data set66. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 In both a and b, labels on the x axis stand for 3-month seasons, e.g., JFM for January, February, and March 5 NATURE COMMUNICATIONS| 8: 1873 | DOI: 10.1038/s41467-017-02021-1| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 Climatology (kg m–2 per month) Response (kg m–2 per month σ–1 NDVI) a –60 –36 –12 12 36 60 Dust concentration Dust emission (% mean σ–1 NDVI) >0 –0.12 –0.04 0.04 0.12 b Bodélé Station dust frequency (fraction σ–1 NDVI) Season 8.0 0.60 0.40 30 N 1.5 Dust transport (kg m m–2 σ–1 NDVI) 20 N 10 N 0 10 S 30 W 0 30 E 0.20 0.00 –0.20 –0.40 6.0 4.0 2.0 0.0 JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF Fig. 4 Observed dust responses to negative Sahel NDVI anomalies and corresponding dry soil anomalies during 1982–2011. a total dust emission (bars: climatology, referring to the left y axis; dots and open circles: significant (p < 0.1 based on Monte Carlo bootstrap test) and insignificant responses, respectively, referring to the right y axis). In a, labels on the x axis stand for 3-month seasons, e.g., JFM for January, February, and March. b spatial pattern of GEFA responses in dust emission (% climatology σNDVI−1, green-brown color), column dust concentration (kg m−2 σNDVI−1, stitching and hatching), column dust transport (kg m−1 per month σNDVI−1, vector), and regional dust frequency (defined in Methods section, fraction σNDVI−1, circles in blue-red color) in September-November. Only statistical significant (p < 0.1) responses are shown in b, according to the Monte Carlo bootstrap test. Open circles in b represent stations with insignificant responses in dust frequency Climatology (kg m–2 per month) 2 1 a Season 8.0 0.60 0.40 0.20 0.00 –0.20 –0.40 6.0 4.0 2.0 0.0 JFM FMA MAM AMJ MJJ JJA JAS ASO SON OND NDJ DJF –60 –36 –12 12 36 60 Dust concentration Dust emission (% mean σ–1 NDVI) >0 –0.12 –0.04 0.04 0.12 b Bodélé Station dust frequency (fraction σ–1 NDVI) 30 N 1.5 Dust transport (kg m m–2 σ–1 NDVI) 20 N 10 N 0 10 S 30 W 0 30 E b a Season Fig. 4 Observed dust responses to negative Sahel NDVI anomalies and corresponding dry soil anomalies during 1982–2011. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 6c), however, suggests that both the albedo and roughness mechanisms are swamped by the dominant moisture recycling feedback mechanism in the Sahel on seasonal to interannual time scales. However, at long time scales associated with land use change, the albedo impact might be significantly more important if pronounced grass–desert conversion or soil degradation occurs. The surface cooling and weakened surface wind speed associated with positive NDVI anomalies are confirmed with station observations (Supplementary Fig. 4b, c) and regional downscaling of global reanalysis (Supplementary Note 1, Supplementary Fig. 5b–d) in terms of both sign and magnitude. surface pressure, and anomalous lower-tropospheric to mid- tropospheric subsidence (Supplementary Fig. 6) in response to positive NDVI anomalies support an active stability mechanism, consistent with findings in the regional modeling study regarding the impacts of vegetation cover on heat and moisture fluxes over the Sahel and West African monsoon region41. The increased atmospheric stability in response to positive NDVI anomalies, although swamped by the moisture recycling mechanism, is counter to the dynamic responses expected through the classic vegetation-albedo feedback mechanism. During autumn, positive NDVI anomalies trigger modest declines in surface albedo, confined to the savanna, woody savanna, and broadleaf evergreen forest portions of the Sahel (Fig. 3b), consistent with previous model-based findings42. However, the magnitudes of the albedo anomalies, on the order of 0.01–0.02 σ−1NDVI, are too small, compared with the imposed change in surface albedo of 0.21 applied in the pioneering experiments of Charney6, to trigger the instability responses necessary for the albedo-based positive vegetation-rainfall feedback. The albedo responses are trivial over the more widespread grasslands or shrublands of the Sahel, probably because the seasonal to interannual time scale is too short for the grass-desert or shrub-desert conversions proposed by Charney6. There is observed evidence of a weak vegetation roughness mechanism43, with slightly diminished 10-m wind speed on the order of −0.05 (−0.04 to −0.08 among data sets) m s −1 σ−1NDVI during July–September, in response to positive NDVI anomalies (Supplementary Fig. 6a). The lack of anomalous ascending motion in response to enhanced vegetation abundance (Supplementary Fig. 6c), however, suggests that both the albedo and roughness mechanisms are swamped by the dominant moisture recycling feedback mechanism in the Sahel on seasonal to interannual time scales. However, at long time scales associated with land use change, the albedo impact might be significantly more important if pronounced grass–desert conversion or soil degradation occurs. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 a total dust emission (bars: climatology, referring to the left y axis; dots and open circles: significant (p < 0.1 based on Monte Carlo bootstrap test) and insignificant responses, respectively, referring to the right y axis). In a, labels on the x axis stand for 3-month seasons, e.g., JFM for January, February, and March. b spatial pattern of GEFA responses in dust emission (% climatology σNDVI−1, green-brown color), column dust concentration (kg m−2 σNDVI−1, stitching and hatching), column dust transport (kg m−1 per month σNDVI−1, vector), and regional dust frequency (defined in Methods section, fraction σNDVI−1, circles in blue-red color) in September-November. Only statistical significant (p < 0.1) responses are shown in b, according to the Monte Carlo bootstrap test. Open circles in b represent stations with insignificant responses in dust frequency surface pressure, and anomalous lower-tropospheric to mid- tropospheric subsidence (Supplementary Fig. 6) in response to positive NDVI anomalies support an active stability mechanism, consistent with findings in the regional modeling study regarding the impacts of vegetation cover on heat and moisture fluxes over the Sahel and West African monsoon region41. The increased atmospheric stability in response to positive NDVI anomalies, although swamped by the moisture recycling mechanism, is counter to the dynamic responses expected through the classic vegetation-albedo feedback mechanism. During autumn, positive NDVI anomalies trigger modest declines in surface albedo, confined to the savanna, woody savanna, and broadleaf evergreen forest portions of the Sahel (Fig. 3b), consistent with previous model-based findings42. However, the magnitudes of the albedo anomalies, on the order of 0.01–0.02 σ−1NDVI, are too small, compared with the imposed change in surface albedo of 0.21 applied in the pioneering experiments of Charney6, to trigger the instability responses necessary for the albedo-based positive vegetation-rainfall feedback. The albedo responses are trivial over the more widespread grasslands or shrublands of the Sahel, probably because the seasonal to interannual time scale is too short for the grass-desert or shrub-desert conversions proposed by Charney6. There is observed evidence of a weak vegetation roughness mechanism43, with slightly diminished 10-m wind speed on the order of −0.05 (−0.04 to −0.08 among data sets) m s −1 σ−1NDVI during July–September, in response to positive NDVI anomalies (Supplementary Fig. 6a). The lack of anomalous ascending motion in response to enhanced vegetation abundance (Supplementary Fig. NATURE COMMUNICATIONS| 8: 1873 | DOI: 10.1038/s41467-017-02021-1| www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 The surface cooling and weakened surface wind speed associated with positive NDVI anomalies are confirmed with station observations (Supplementary Fig. 4b, c) and regional downscaling of global reanalysis (Supplementary Note 1, Supplementary Fig. 5b–d) in terms of both sign and magnitude The observational GEFA analysis further verifies that dimin- ished vegetation growth and accompanying dry soils across the Sahel lead to enhanced dust emissions and dust storm activity during the mid- to post-monsoon season (Fig. 4a), as suggested by previous correlation-based observational studies44. In addition, the current observational analysis reveals the remote impacts of Sahel vegetation and soil moisture on dust concentration over the tropical Atlantic Ocean. The enhancement in dust emissions is most pronounced across the southern boundary of North Africa’s major dust source regions within the Sahel, including the Bodélé Depression45, where dust emissions increase by more than 60% during SON in response to a negative NDVI anomaly on the order of one standard deviation (Fig. 4b). These enhanced emissions across the Sahel support increased southwestward dust transport and thus elevated surface and column dust concentra- tions, as well as greater frequency of dust days according to station observations (defined in Methods section), across tropical and subtropical North Africa and the eastern tropical Atlantic Ocean (Fig. 4b). This observed vegetation-dust feedback acts as a potential secondary mechanism for the positive vegetation- rainfall feedback in the Sahel, as proposed by previous modeling studies46, 47, given the direct effects of dust aerosols that cause low-level cooling and atmospheric stabilization, and the indirect radiative effects of dust aerosols that increase the number of cloud condensation nuclei and inhibit precipitation efficiency. The current study presents the first convincing observational evidence for the model-hypothesized positive vegetation-rainfall feedbacks in the Sahel, by successfully isolating terrestrial feedbacks from oceanic drivers and systematically examining multiple observational datasets in order to quantify observational uncertainty in feedback response estimates. The identification of key oceanic and terrestrial drivers will aid in successful seasonal predictions of regional climate in Sahel, a region that is highly vulnerable to hydrological extremes. Future projections of Sahel rainfall, in response to the anthropogenically enhanced greenhouse effect, remain highly uncertain in terms of both sign and magnitude within phases three and five of the Coupled Model Intercomparison Project (CMIP3 and CMIP5)1, 14, 15, 48, 49. Indeed, the Sahel is one of the most uncertain regions for rainfall projections worldwide. Methods GEFA d In this way, the number of forcings to be assessed by GEFA decreases, thereby allowing more reliable estimates of the feedbacks associated with the remaining, significant forcings. The length of data needed to obtain reliable seasonal estimates of the feedback matrix (B), such that GEFA can capture the seasonal cycle of area-average responses for most variables, including ET, surface air temperature, planetary boundary layer height, surface specific humidity, surface wind, and rainfall32, with a temporal correlation of at least 0.8 (N = 12 months) with the dynamical experiments, is reduced to about 30 years by applying this stepwise selection procedure32. B ¼ CAOðτÞ  C1 OOðτÞ: ð4Þ ð4Þ The estimated feedback matrix represents the instantaneous influence of slowly- evolving oceanic and terrestrial variables on an atmospheric variable. In theory, the estimate of B does not depend on τ, but due to insufficient L, the sampling error always increases with greater τ, resulting in unrealistic estimates of the magnitude of the feedback response at large τ. On the basis of the GEFA evaluation work within CESM31, 32, larger τ leads to deteriorating magnitude estimates of the oceanic and terrestrial feedbacks compared to the dynamical experiments, especially with shorter data records that are comparable in length with most observational data sets. Therefore, τ is assigned to be one month in the current observational analysis. g g g g g contribution to the variability of the atmospheric variable and can subsequently remain excluded from the forcing matrix. In this way, the number of forcings to be assessed by GEFA decreases, thereby allowing more reliable estimates of the feedbacks associated with the remaining, significant forcings. The length of data needed to obtain reliable seasonal estimates of the feedback matrix (B), such that GEFA can capture the seasonal cycle of area-average responses for most variables, including ET, surface air temperature, planetary boundary layer height, surface specific humidity, surface wind, and rainfall32, with a temporal correlation of at least 0.8 (N = 12 months) with the dynamical experiments, is reduced to about 30 years by applying this stepwise selection procedure32. y In the current study, the GEFA forcing matrix is comprised of the leading two SST EOF modes from eight non-overlapping basins, area-average Mediterranean SSTs (Supplementary Fig. Methods GEFA d GEFA and stepwise selection. In the current study, a multivariate statistical method, GEFA, is used to study the oceanic and terrestrial regulators of the Sahel’s climate. The statistical GEFA approach extracts the forcing of a slowly-evolving environmental variable, such as SST or NDVI, on the rapidly-evolving atmosphere, either in climate model output or observational data. The GEFA methodology29, based on stochastic climate theory50, addresses the local and non-local feedbacks simultaneously, which is critical given that vegetation and SST anomalies can affect atmospheric conditions both locally and remotely19, 50, 51. GEFA can largely separate the individual impacts of different ocean basins and vegetated regions on climate in select regions. For example, GEFA’s capability of extracting the oceanic and terrestrial impacts on North American regional climate was previously demonstrated using the NCAR Community Climate System Model Version 3.519, 30. In particular, GEFA’s reliability in isolating the oceanic and land surface feedbacks on the North African climate was successfully demonstrated by com- paring the statistical assessments with dynamical experiments in CESM31, 32. In the GEFA validation regarding the assessed terrestrial impacts on North African cli- mate, the statistically assessed atmospheric responses were evaluated against those assessed from two ensembles of dynamical experiments developed for the Sahel or West African Monsoon (WAM) region, i.e., EXPLAI, in which regional leaf area index (LAI) was modified, and EXPSOIL, in which regional LAI and soil moisture were modified together during winter-spring, motivated by the strong soil moisture-LAI coupling across the Sahel and the WAM region in CESM. g g g p g determined through rotated EOF analysis of detrended monthly remotely sensed NDVI anomalies. Regional average responses are obtained by applying GEFA to the atmospheric fields averaged across the Sahel (12˚ N–17˚ N, 20˚ W–40˚ E). The analysis is performed for 1982–2011. Before applying GEFA, the seasonal cycle and linear trend are removed from all forcing and response fields. The statistical significance of GEFA feedback matrices is assessed using the Monte Carlo bootstrap method with 1000 random iterations in which the time series of the response variable is scrambled30. In order to achieve sufficient length of data and obtain reliable estimates of the feedback matrices (explained later), seasonal feedbacks are estimated by aggregating data from the consecutive three months so that the effective sample size is three times the number of years, or 90 months. Methods GEFA d p y The short duration of the remotely sensed NDVI record, covering about three decades, remains a challenge to the application of GEFA to understand land- atmosphere feedbacks, especially when simultaneously considering numerous potential forcings. In order to obtain reliable seasonal estimates of the feedback matrix (B) in terms of about 20 forcings (17 SST fields and 3 NDVI fields) for the Sahel, at least 100 years of data is needed for most response variables so that GEFA can accurately capture the seasonal cycle of area-average seasonal responses with a temporal correlation of 0.8 or greater (N = 12 months) with the dynamical experiments, which are regarded as the truth and provide a benchmark for GEFA evaluation32. In order to minimize the sampling error associated with relatively short datasets, it is necessary to reduce the number of forcings under consideration before estimating the feedback matrix. Here negligible forcings for the Sahel are eliminated using a backward-selection stepwise method58, which compares the relative contribution from each forcing to the atmospheric variability and retains the truly important ones as predictors through an automated procedure. Stepwise selection has been widely applied to predictor selection in developing linear prediction models for the climate or ecosystems59, 60. Akaike information criterion (AIC)61, which measures the relative quality of a statistical model by estimating the goodness of fit and penalizing the complexity of the model (number of predictors), is used as the criterion in the stepwise selection. 30. In particular, GEFA’s reliability in isolating the oceanic and land surface feedbacks on the North African climate was successfully demonstrated by com- paring the statistical assessments with dynamical experiments in CESM31, 32. In the GEFA validation regarding the assessed terrestrial impacts on North African cli- mate, the statistically assessed atmospheric responses were evaluated against those assessed from two ensembles of dynamical experiments developed for the Sahel or West African Monsoon (WAM) region, i.e., EXPLAI, in which regional leaf area index (LAI) was modified, and EXPSOIL, in which regional LAI and soil moisture were modified together during winter-spring, motivated by the strong soil moisture-LAI coupling across the Sahel and the WAM region in CESM. p g g At time scales longer than the atmospheric memory (about 1 week), the atmospheric variable (e.g. precipitation) at time t, A(t), can be expressed as the sum of feedback responses to an array of slowly-evolving variables (e.g. Methods GEFA d SST, NDVI), O(t), and the atmospheric internal noise, N(t)29: A tð Þ ¼ B  O tð Þ þ NðtÞ; ð1Þ ð1Þ where B is the feedback matrix. Right multiplying OT(t-τ) on both sides of equation (1) and applying the covariance yield: CAO τð Þ ¼ B  COO τð Þ þ CNOðτÞ; ð2Þ ð2Þ AIC ¼ 2 ´ Nf  2 ´ ln ^L   ; ð5Þ ð5Þ AIC ¼ 2 ´ Nf  2 ´ ln ^L   ; where τ is the time scale, exceeding the atmospheric adjustment time, and C(τ) represents a covariance matrix at lag τ. Given the time series’ length L of the atmospheric and oceanic variables, the lagged covariance matrices are estimated as: ^L ¼  L 2 ln X L t¼1 ^A tð Þ  A tð Þ  2=L ! þ C1; ð6Þ ð6Þ CAO τð Þ ¼ 1 L A tð ÞOT t  τ ð Þ; COO τð Þ ¼ 1 L O tð ÞOT t  τ ð Þ; CNO τð Þ ¼ 1 L N tð ÞOT t  τ ð Þ: ð3Þ ð3Þ ^AðtÞ ¼ B  OðtÞ: ð7Þ ^AðtÞ ¼ B  OðtÞ: ð7Þ The superscript T indicates a transposed matrix. Since oceanic or terrestrial variability cannot be forced by an atmospheric internal noise at a later time, and the atmospheric internal noise is not affected by oceanic or terrestrial variability by definition in equation (1), CNO(τ) = 0, which results in an estimate of the feedback matrix as: In equation (5), Nf represents the number of forcings in the forcing matrix, and ^L stands for the maximized likelihood function of the statistical model in (1), which represents the goodness of fit of the statistical model in (1) with the B matrix obtained from equation (4) and is estimated by equations (6) and (7), based on the linear theory62. In equation (6), L is the length of the data record, and C1 is a constant independent of the statistical model. In equation (7), ^AðtÞ stands for the predicted atmospheric condition at time t, based on equation (1). If AIC does not increase after removing a select forcing, then this forcing has negligible contribution to the variability of the atmospheric variable and can subsequently remain excluded from the forcing matrix. ARTICLE ARTICLE forcing matrix. The purpose of performing GEFA in truncated SST EOF space is to reduce the sampling error from highly correlated forcing fields51, 52. Past studies have suggested the potential impacts of SST variability across a vast multitude of ocean basins, including the tropical Pacific (TP)3, 53, North Pacific (NP), tropical Atlantic (TA)5, 53–55, tropical Indian (TI)53, 56, North Atlantic (NA)1, 54, South Pacific (SP)3, South Indian (SI)3, South Atlantic (SA)3, and Mediterranean Sea57, on Sahel rainfall. Moreover, for most basins, the leading two EOF modes have clear physical meanings, such as ENSO, Indian Ocean Basin Mode, and Atlantic Niño mode. In terms of terrestrial forcings, the Sahel, WAM region, and HOA represent unique North African landscapes, i.e. mainly savanna and grasslands across the Sahel, savanna and woody savanna across the WAM region, and shrubs and bare ground across the HOA. In order to obtain reliable estimates of vegetation feedbacks in the Sahel, land forcings that are moderately-to-highly correlated with NDVI in the Sahel, such as NDVI in the WAM and HOA, also need to be included in the forcing matrix32. The geographic extent of the three ecoregions is determined through rotated EOF analysis of detrended monthly remotely sensed NDVI anomalies. Regional average responses are obtained by applying GEFA to the atmospheric fields averaged across the Sahel (12˚ N–17˚ N, 20˚ W–40˚ E). The analysis is performed for 1982–2011. Before applying GEFA, the seasonal cycle and linear trend are removed from all forcing and response fields. The statistical significance of GEFA feedback matrices is assessed using the Monte Carlo bootstrap method with 1000 random iterations in which the time series of the response variable is scrambled30. In order to achieve sufficient length of data and obtain reliable estimates of the feedback matrices (explained later), seasonal feedbacks are estimated by aggregating data from the consecutive three months so that the effective sample size is three times the number of years, or 90 months. further studies, the GEFA-based assessment of the key observed oceanic and terrestrial drivers of North African regional climate will serve as an observational benchmark for evaluating the representation of ocean-land-atmosphere interactions within state-of-the-art climate models as applied by the Intergovern- mental Panel on Climate Change. This innovative approach will foster model evaluation and development, along with the formulation of process-based model performance metrics for weighting future climate projections for the Sahel and reducing associated uncertainty. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 In NATURE COMMUNICATIONS| 8: 1873 | DOI: 10.1038/s41467-017-02021-1| www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 NATURE COMMUNICATIONS| 8: 1873 | DOI: 10.1038/s41467-017-02021-1| www.nature.com/naturecommunications calculated by The GEFA-based response is first obtained from each data set, and then a probability distribution function (PDF) of the weighted-average response of all datasets is generated by the Monte Carlo bootstrap approach with 1000 random iterations. With each iteration, weights are randomly generated from a uniform distribution and assigned to the datasets in the order of their regional reliability, with the highest weight assigned to the most reliable data set. On the basis of the multi-data set PDF of the 1000 weighted averages, the multi-data set average and uncertainty range of the responses to oceanic and terrestrial forcings are obtained. The regional reliability of each observational data set across the Sahel is evaluated based on the criteria outlined in Supplementary Tables 2–6, leading to a practical ranking of all data sets, specifically for the Sahel, to be applied in the Monte Carlo bootstrap approach7. 11. Clark, D. B., Xue, Y. K., Harding, R. J. & Valdes, P. J. Modeling the impact of land surface degradation on the climate of tropical North Africa. J. Clim. 14, 1809–1822 (2001). 12. Wang, G., Eltahir, E. A. B., Foley, J. A., Pollard, D. & Levis, S. Decadal variability of rainfall in the Sahel: results from the coupled GENESIS-IBIS atmosphere-biosphere model. Clim. Dyn. 22, 625–637 (2004). 13. Foley, J. A., Coe, M. T., Scheffer, M. & Wang, G. Regime shifts in the Sahara and Sahel: Interactions between ecological and climatic systems in Northern Africa. Ecosystems 6, 524–532 (2003). 14. Giannini, A., Biasutti, M. & Verstraete, M. M. A climate model-based review of drought in the Sahel: Desertification, the re-greening, and climate change. Glob. Planet Change. 64, 119–128 (2008). g 15. Giannini, A., Biasutti, M., Held, I. M. & Sobel, A. H. A global perspective on African climate. Clim. Change 90, 359–383 (2008). g 16. Held, I. M., Delworth., T. L., Lu, J., Findell, K. & Knutson, T. R. Simulation of Sahel drought in the 20th and 21st centuries. Proc. Natl Acad. Sci. USA 102, 17891–17896 (2005). 17. Scaife, A. A. et al. The CLIVAR C20C project: selected twentieth century climate events. Clim. Dyn. 33, 603–614 (2009). Station dust observations and MERRA-2 dust reanalysis. Dust observations are retrieved from the National Climatic Data Center (NCDC) hourly global and U.S. Integrated Surface hourly data set for 1982–2011 at 502 stations across North Africa. calculated by 3. Folland, C. K., Palmer, T. N. & Parker, D. E. Sahel rainfall and worldwide sea temperatures, 1901-85. Nature 320, 602–607 (1986). temperatures, 1901-85. Nature 320, 602–607 (1986). VO ¼ CAOA=VA; ð8Þ 4. Rowell, D. P., Folland, C. K., Maskell, K. & Ward, M. N. Variability of summer rainfall over tropical North Africa (1906-92): observations and modeling. Quart. J. R. Meteorol. Soc. 121, 669–704 (1995). where CAOA is the covariance between the observed atmospheric time series (A) and predicted atmospheric time series by oceanic forcings (AO), and VA is the variance of the atmospheric time series. The predicted atmospheric time series is reconstructed by 5. Giannini, A., Saravanan, R. & Change, P. Oceanic forcing of Sahel rainfall on interannual to decadal time scales. Science 302, 1027–1030 (2003). 6. Charney, J. G. Dynamics of desert and drought in the Sahel. Quart. J. R. Meteorol. Soc. 101, 193–202 (1975). ð9Þ AO ¼ BO  O; AO ¼ BO  O; ð9Þ 7. Charney, J. G., Quirk, W. J., Chow, S. H. & Kornfield, J. A comparative study of the effects of albedo change on drought in semi-arid regions. J. Atmos. Sci. 34, 1366–1385 (1977). where BO is the GEFA feedback matrix when only oceanic forcings are included in the forcing matrix, and O is the forcing matrix containing the oceanic forcings. The percentage of explained variance by terrestrial forcings is calculated similarly. 8. Shukla, J. & Mintz, Y. Influence of land-surface evapotranspiration on Earth’s climate. Science 215, 1498–1501 (1982). 9. Xue, Y.-K. Biosphere feedback on regional climate in tropical North Africa. Quart. J. R. Meteorol. Soc. 123, 1483–1515 (1997). 10. Zeng, N., Neelin, J. D., Lau, K.-M. & Tucker, C. J. Enhancement of interdecadal climate variability in the Sahel by vegetation interaction. Science 286, 1537–1540 (1999). Multi-data set bootstrapping method. Multiple observational, remote sensing, and reanalysis datasets (Supplementary Table 1) are analyzed for the Sahel in the GEFA framework. By applying the Monte Carlo bootstrapping approach63, the potential impacts of observational measurement errors across data-limited North Africa on estimated GEFA response fields are reduced. Furthermore, this approach facilitates a reliable estimation of the multi-data set mean and quantification of observational uncertainty in the GEFA-based atmospheric responses to oceanic and terrestrial forcings. calculated by The MERRA-2 reanalysis of aerosols includes assimilation of bias corrected Aerosol Optical Depth (AOD) from Advanced Very High Resolution Radiometer (AVHRR) over ocean, Moderate Resolution Imaging 23. Los, L. O. et al. An observation-based estimate of the strength of rainfall- vegetation interactions in the Sahel. Geophys. Res. Lett. 33, L–16402 (2006). 24. Lee, E., He, Y., Zhou, M. & Liang, J. Potential feedback of recent vegetation changes on summer rainfall in the Sahel. Phys. Geography 36, 449–470 (2015). Spectroradiometer (MODIS) sensors on both Terra and Aqua satellites, Multi- angle Imaging SpectroRadiometer (MISR) over bright surfaces and Aerosol Robotic Network (AERONET) data. The vertical structure of MERRA-2 aerosol reanalysis has been successfully validated using Cloud-Aerosol Lidar with Orthogonal Polarization (CALIOP) data over a number of regions, including North Africa during 200865. Furthermore, the MERRA-2 surface dust concentration reanalysis exhibits similar seasonal cycle and interannual variability with the station dust frequency across the Sahel (not shown). 25. He, Y. & Lee, E. Empirical relationships of sea surface temperature and vegetation activity with summer rainfall variability over the Sahel. Earth Inter. 20, 6–23 (2016). 26. Green, J. K. et al. Regionally strong feedbacks between the atmosphere and terrestrial biosphere. Nat. Geosci. 10, 1038 (2017). 27. Nicholson, S. E. The West African Sahel: A review of recent studies on the rainfall regime and its interannual variability. ISRN Meteorol. 2013, 453521 (2013). Data availability. The data that support the findings of this study are available from the corresponding authors upon request. 28. Biasutti, M., Held, I. M., Sobel, A. H. & Giannini, A. SST forcings and Sahel rainfall variability in simulations of the twentieth and twenty-first centuries. J. Clim. 21, 3471–3486 (2008). Received: 14 April 2017 Accepted: 1 November 2017 Received: 14 April 2017 Accepted: 1 November 2017 29. Liu, Z., Wen, N. & Liu, Y. On the assessment of nonlocal climate feedback. Part I: The Generalized Equilibrium Feedback Assessment. J. Clim. 21, 134–148 (2008). 30. Wang, F., Liu, Z. & Notaro, M. Extracting the dominant SST modes impacting North America’s observed climate. J. Clim. 26, 5424–5452 (2013). 31. Wang, F. et al. Advancing a model-validated statistical method for decomposing the key oceanic drivers of regional climate: Focus on North African climate variability in CESM, J. Climate. https://doi.org/10.1175/JCLI-D- 17-0219.1 (2017). 2. Nicholson, S. E. Revised rainfall series for the West African subtropics. Mon. Weather Rev. 107, 620–623 (1979). 1. Rodriguez-Fonseca, B. et al. Variability and predictability of West African droughts: A review on the role of sea surface temperature anomalies. J. Clim. 28, 4034–4080 (2015). calculated by At each station, a dust day is defined as a day in which either dust/sand storm or severe dust/sand storm is reported at least once, or dust suspension is reported for at least a quarter of the total number of observations during the daytime60, 64. Therefore, the dust day metric is a combined measure of the fre- quency and intensity of dust activity. 18. Kucharski, F., Zeng, N. & Kalnay, E. A further assessment of vegetation feedback on decadal Sahel rainfall variability. Clim. Dyn. 40, 1453 (2013). 19. Wang, F., Notaro, M., Liu, Z. & Chen, G. Observed local and remote influences of vegetation on the atmosphere across North America using a model-validated statistical technique that first excludes oceanic forcings. J. Clim. 27, 362–382 (2014). 20. Liu, Z., Notaro, M., Kutzbach, J. & Liu, N. Assessing global vegetation-climate feedbacks from observations. J. Clim. 19, 787–814 (2006). Station observations are first gridded to a spatial resolution of 0.25˚ × 0.25˚. In each grid cell, a regional dust day is defined if at least one station within that grid cell indicates a dust day. Monthly dust frequency in each grid cell is calculated when dust observations are available on more than half of the days during that month, or otherwise left as a missing value. 21. O’Brien, K. L. Tropical deforestation and climate change. Prog. Phys. Geogr. 20, 311–335 (1996). 22. Notaro, M., Liu, Z. & Williams, J. W. Observed vegetation-climate feedbacks in the United States. J. Clim. 19, 763–786 (2006). g In addition to station dust observations, dust aerosol reanalysis from MERRA-2 is also analyzed. The MERRA-2 reanalysis of aerosols includes assimilation of bias corrected Aerosol Optical Depth (AOD) from Advanced Very High Resolution Radiometer (AVHRR) over ocean, Moderate Resolution Imaging Spectroradiometer (MODIS) sensors on both Terra and Aqua satellites, Multi- angle Imaging SpectroRadiometer (MISR) over bright surfaces and Aerosol Robotic Network (AERONET) data. The vertical structure of MERRA-2 aerosol reanalysis has been successfully validated using Cloud-Aerosol Lidar with Orthogonal Polarization (CALIOP) data over a number of regions, including North Africa during 200865. Furthermore, the MERRA-2 surface dust concentration reanalysis exhibits similar seasonal cycle and interannual variability with the station dust frequency across the Sahel (not shown). g In addition to station dust observations, dust aerosol reanalysis from MERRA-2 is also analyzed. Methods GEFA d 7), and time series of area-average NDVI across the Sahel (12˚ N–17˚ N, 20˚ W–40˚ E), WAM region (5˚ N–12˚ N, 20˚ W–30˚ E), and Horn of Africa (HOA) (5˚ S–10˚ N, 30˚ E–52˚ E) (Supplementary Fig. 8). Higher order SST EOF modes are less important, as evidenced by the trivial increase in total explained variance by adding the third to fifth EOF modes of basin SSTs to the According to GEFA, the percent variance in a select response variable, as explained by either an oceanic or terrestrial forcing, is calculated similarly to the analysis of variance (ANOVA) approach in multiple linear regression62. For example, the percentage of explained variance by oceanic forcings, VO, is 7 7 NATURE COMMUNICATIONS| 8: 1873 | DOI: 10.1038/s41467-017-02021-1| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 calculated by ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02021-1 58. Hocking, R. R. 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Clim. 24, 3878 (2011). p y 61. Akaike, H. A new look at the statistical model identification. IEEE Trans. Autom. Control 19, 716–723 (1974). cycle of the West African Monsoon. J. Clim. 24, 3878 (2011). 37. Latif, M. & Grotzner, A. The equatorial Atlantic oscillation and its response to ENSO. Clim. Dyn. 16, 213–218 (2000). 62. Wonnacott, T. H. & Wonnacott, R. J. Introductory Statistics, Vol. 19690 (Wiley, New York, 1972). y 38. Liu, Y. et al. Trend-preserving blending of passive and active microwave soil moisture retrieval. Remote Sens. Environ. 123, 280–297 (2013). 63. Efron, B. The Jackknife, the Bootstrap and Other Resampling Plans. (Society for Industrial and Applied Mathematics, 1982). 39. Findell, K. L., Gentine, P., Lintner, B. R., & Kerr, C. Probability of afternoon precipitation in eastern United States and Mexico enhanced by high evaporation. Nat. Geosci. 4, 434-439 (2011). 64. Yu, Y. et al. Assessing temporal and spatial variations in atmospheric dust over Saudi Arabia through satellite, radiometric, and station data. J. Geophys. Res. Atmos. 118, 13253 (2013). 40. Romps, D. M. & Kuang, Z. Nature versus Nurture in shallow convection. J. Atmos. Sci. 67, 1655–1666 (2010). 65. Buchard, V. & Da Silva, A. Ensemble-based assimilation of aerosol observations in GEOS-5. Available at: https://ntrs.nasa.gov/search.jsp?R=20160005006 (2016). 41. Klein, C. et al. Feedback of observed internnual vegetation change: a regional climate model analysis for the West African monsoon. Clim. Dyn. Author contributions 46. Rosenfeld, D., Rudich, Y. & Lahav, R. Desert dust suppressing precipitation: A possible desertification feedback loop. Proc. Natl Acad. Sci. USA 98, 5975–5980 (2001). Y.Y. led the data collection, data analysis, and writing, with suggestions and comments from M.N. F.W., J.M., X.S., and Y.W. contributed comments and edits. M.N. conceived the original ideas for the work and directed the overall project. Y.Y. led the data collection, data analysis, and writing, with suggestions and comments from M.N. F.W., J.M., X.S., and Y.W. contributed comments and edits. M.N. conceived the original ideas for the work and directed the overall project. 47. Gu, Y., Liou, K. N., Jiang, J. H., Su, H. & Liu, X. Dust aerosol impact on North African climate: a GCM investigation of aerosol-cloud-radiation interactions using A-Train satellite data. Atmos. Chem. Phys. 12, 1667–1679 (2015). Acknowledgements 43. Sud, Y. C., Shukla, J. & Mintz, Y. Influence of land-surface roughness on atmospheric circulation and precipitation: A sensitivity study with a general circulation model. J. Appl. Meteorol. 27, 1036–1054 (1988). This work is funded by Department of Energy (DOE) Regional and Global Climate Modeling (RGCM) program and National Science Foundation (NSF) Climate and Large- Scale Dynamics (CLD) program. Computer resources are provided by DOE National Energy Research Scientific Computing Center (NERSC). The authors are thankful for helpful discussions with Drs. Zhengyu Liu, Yongkang Xue, Ankur Desai, Daniel Vimont, and Tristan L’Ecuyer. This work is funded by Department of Energy (DOE) Regional and Global Climate Modeling (RGCM) program and National Science Foundation (NSF) Climate and Large- Scale Dynamics (CLD) program. Computer resources are provided by DOE National Energy Research Scientific Computing Center (NERSC). The authors are thankful for helpful discussions with Drs. Zhengyu Liu, Yongkang Xue, Ankur Desai, Daniel Vimont, and Tristan L’Ecuyer. pp 44. Kim, D. et al. Role of surface wind and vegetation cover in multi-decadal variations of dust emission in the Sahara and Sahel. Atmos. Environ. 148, 282–296 (2017). 45. Evan, A. T. et al. Derivation of an observation-based map of North African dust emission. Aeolian Res. 16, 153–162 (2015). ARTICLE 48, 2837–2858 (2017). 66. Liu, Q. et al. Preliminary evaluation of the long-term GLASS albedo product. Int. J. Dig. Earth 6, 69–95 (2013). 42. Notaro, M. & Liu, Z. Joint statistical and dynamical assessment of simulated vegetation feedbacks on climate over the boreal forests. Clim. Dyn. 31, 691–712 (2008). Additional information 48. Roehrig, R., Bounioi, D., Guichard, F., Hourdin, F. & Redelsperger, J.-L. The present and future of the West African monsoon: A process-orientated assessment of CMIP5 simulations along the AMMS transect. J. Clim. 26, 6471–6504 (2013). Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 017-02021-1. Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 017-02021-1. Competing interests: The authors declare no competing financial interests. Competing interests: The authors declare no competing financial interests. References 32. Yu, Y. et al. Validation of a statistical methodology for extracting vegetation feedbacks: focus on North African ecosystems in the Community Earth System Model, J. Climate. https://doi.org/10.1175/JCLI-D-17-0220.1 (2017). NATURE COMMUNICATIONS| 8: 1873 | DOI: 10.1038/s41467-017-02021-1| www.nature.com/naturecommunications 8 8 Competing interests: The authors declare no competing financial interests. 49. Schewe, J. & Levermann, A. Non-linear intensification of Sahel rainfall as a possible dynamic response to future warming. Earth Syst. Dyn. 8, 495–505 (2017). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ 50. Frankignoul, C. & Hasselmann, K. Stochastic climate models, Part II: Application to sea-surface temperature anomalies and thermocline variability. Tellus 29, 289–305 (1977). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 51. Wen, N., Liu, Z. & Liu, Q. Observational assessment of nonlocal heat flux feedback in the North Atlantic by GEFA. J. Appl. Meteorol. Climatol. 52, 645–653 (2013). 52. Sun, S. & Wang, G. The complexity of using a feedback parameter to quantify the soil moisture-precipitation relationship. J. Geophys. Res. 117, D11113 (2012). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 53. Sheen, K. L. et al. Skillful prediction of Sahel summer rainfall on inter-annual and multi-year timescales Nat. Commun., 8, 14966 (2017). 54. Giannini, A., Saravanan, R. & Change, P. Dynamics of the boreal summer African monsoon in the NSIPPI atmospheric model. Clim. Dyn. 25, 517–535 (2005). 55. Hoerling, M. P., Hurrell, J. W., Eischeid, J. & Philips, A. Detection and attribution of twentieth-century northern and southern African rainfall change. J. Clim. 19, 3989-4008. h 56. Lu, J. & Delworth, T. L. Oceanic forcing of the late 20th century Sahel drought. Geophys. Res. Lett. 32, L 22706 (2005). 57. Rowell, D. P. The impact of Mediterranean SSTs on the Sahelian rainfall season. J. Clim. 16, 849–862 (2003). © The Author(s) 2017 NATURE COMMUNICATIONS| 8: 1873 | DOI: 10.1038/s41467-017-02021-1| www.nature.com/naturecommunications 9 9
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Dual Targeting of Hypoxia and Homologous Recombination Repair Dysfunction in Triple-Negative Breast Cancer
Molecular cancer therapeutics
2,014
cc-by
4,549
Supplementary figures for Hunter et al., “Dual targeting of hypoxia and homologous recombination repair dysfunction in triple-negative breast cancer”. Figure S1. Uncropped replicate RAD51 immunoblots of TNBC cell lines annotated with molecular size markers. Figure S1. Uncropped replicate RAD51 immunoblots of TNBC cell lines annotated with Figure S1. Uncropped replicate RAD51 immunoblots of TNBC cell lines annotated with molecular size markers. Figure S1. Uncropped replicate RAD51 immunoblots of TNBC cell lines annotated with molecular size markers. 1 Figure S2. Absolute hypoxic cytotoxicity ratios (HCR) of HAP in TNBC cell lines. HCR was defined as the intra-experiment quotient (IC50Aerobic/IC50Hypoxic) and the mean + SEM of 3-6 independent determinations is plotted. Blue bars represent BRCA1 wild type cell lines whereas red bars indicate lines carrying BRCA1 mutations. TPZ SN30000 PR-104A TH-302 nitroCBI Hypoxic cytotoxicity ratio 100 101 102 103 104 BT549 D3H2LN MDA-MB-468 SUM159PT HCC1937 MDA-MB-436 SUM1315MO2 SUM149PT TPZ SN30000 PR-104A TH-302 nitroCBI Hypoxic cytotoxicity ratio 100 101 102 103 104 BT549 D3H2LN MDA-MB-468 SUM159PT HCC1937 MDA-MB-436 SUM1315MO2 SUM149PT Figure S2. Absolute hypoxic cytotoxicity ratios (HCR) of HAP in TNBC cell lines. HCR was defined as the intra-experiment quotient (IC50Aerobic/IC50Hypoxic) and the mean + SEM of 3-6 independent determinations is plotted. Blue bars represent BRCA1 wild type cell lines whereas red bars indicate lines carrying BRCA1 mutations. 2 Figure S3. Quantitation of POR band intensity on Western blots of TNBC cell lines. BRCA1 mutant cell lines are colored in red, BRCA1 wild type lines are in blue. MDAMB436 MDAMB468 HCC1937 D3H2LN BT549 SUM159PT SUM1315MO2 SUM149PT POR:ACTIN ratio 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Figure S3. Quantitation of POR band intensity on Western blots of TNBC cell lines. BRCA1 mutant cell lines are colored in red, BRCA1 wild type lines are in blue. MDAMB436 MDAMB468 HCC1937 D3H2LN BT549 SUM159PT SUM1315MO2 SUM149PT POR:ACTIN ratio 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Figure S3. Quantitation of POR band intensity on Western blots of TNBC cell lines. BRCA1 mutant cell lines are colored in red, BRCA1 wild type lines are in blue. 3 3 Figure S4. Uncropped replicate POR immunoblots of TNBC cell lines annotated with molecular size markers. Figure S4. Uncropped replicate POR immunoblots of TNBC cell lines annotated with molecular size markers. Figure S4. Uncropped replicate POR immunoblots of TNBC cell lines annotated with molecular size markers. Figure S4. Uncropped replicate POR immunoblots of TNBC cell lines annotated with molecular size markers. Supplementary figures for Hunter et al., “Dual targeting of hypoxia and homologous recombination repair dysfunction in triple-negative breast cancer”. 4 Cisplatin 0 2 4 6 8 10 PR-104H anoxic 0 2 4 6 8 10 12 14 16 18 20 Cisplatin 0 2 4 6 8 10 HN2 anoxic 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Olaparib 0 20 40 60 80 100120140160180 TH-302 aerobic 20 40 60 80 100 Olaparib 0 20 40 60 80 100120140160180 TH-302 anoxic 0.00 0.05 0.10 0.15 0.20 0.25 Cisplatin 0 2 4 6 8 10 SN30000 aerobic 0 200 400 600 800 1000 1200 1400 Cisplatin 0 2 4 6 8 10 SN30000 anoxic 0 2 4 6 8 10 Cisplatin 0 2 4 6 8 10 TPZ aerobic 0 100 200 300 400 500 600 700 Cisplatin 0 2 4 6 8 10 TPZ anoxic 0 1 2 3 4 5 6 Cyt. c 0 2 4 6 8 10 12 14 16 18 SN30000 anoxic 0 2 4 6 8 10 POR:ACTIN 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 SN30000 anoxic 0 2 4 6 8 10 Cisplatin 0 2 4 6 8 10 HN2 anoxic 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Cisplatin 0 2 4 6 8 10 PR-104H anoxic 0 2 4 6 8 10 12 14 16 18 20 Cisplatin Olaparib 0 20 40 60 80 100120140160180 TH-302 aerobic 20 40 60 80 100 0 20 40 60 80 100120140160180 TH-302 anoxic 0.00 0.05 0.10 0.15 0.20 0.25 0 20 40 60 80 100120140160180 Olaparib Cisplatin 0 2 4 6 8 10 SN30000 anoxic 0 2 4 6 8 10 Olaparib Olaparib Olaparib Cisplatin 0 2 4 6 8 10 SN30000 aerobic 0 200 400 600 800 1000 1200 1400 Cisplatin Cisplatin 0 2 4 6 8 10 TPZ anoxic 0 1 2 3 4 5 6 Cisplatin 0 2 4 6 8 10 TPZ aerobic 0 100 200 300 400 500 600 700 Cisplatin Cyt. c 0 2 4 6 8 10 12 14 16 18 SN30000 anoxic 0 2 4 6 8 10 POR:ACTIN 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 SN30000 anoxic 0 2 4 6 8 10 Cyt. c 5 5 Figure S5 (previous page). Spearman correlations of phenotypic measures of one-electron reductase activity or HR repair activity with sensitivity of TNBC cell lines to HAP and their cytotoxic metabolites. Supplementary figures for Hunter et al., “Dual targeting of hypoxia and homologous recombination repair dysfunction in triple-negative breast cancer”. IC50 values of cisplatin and olaparib under normoxic conditions, both in µM, cyanide-resistant and NADPH dependent reduction of cytochrome c by POR (cyt c, nmol.min-1.mg-1) and POR:ACTIN ratios are plotted on the abscissae. IC50 values of HAP or cytotoxic effectors, under the indicated gas condition, are plotted in µM on the ordinates. Spearman correlation coefficients and P values are indicated. The sub-plots within the correlations of olaparib and TH-302 sensitivity show the correlation recomputed after excluding the HCC1937 cells, which are highly resistant to both these agents. A simple decay function is fitted to model the association between SN30000 sensitivity under anoxia and POR enzymatic activity or expression. 6 6 SUM149PT HCC1937 MDA-436 BT549 D3H2LN SUM1315MO2 SUM159PT MDA-468 RAD51:ACTIN ratio 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Figure S6. Quantitation of RAD51 band intensity on Western blots of TNBC cell lines. BRCA1 mutant cell lines are colored in red, BRCA1 wild type lines are in blue. SUM149PT HCC1937 MDA-436 BT549 D3H2LN SUM1315MO2 SUM159PT MDA-468 RAD51:ACTIN ratio 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Figure S6. Quantitation of RAD51 band intensity on Western blots of TNBC cell lines. Figure S6. Quantitation of RAD51 band intensity on Western blots of TNBC cell lines Figure S6. Quantitation of RAD51 band intensity on Western blots of TNBC cell lines. BRCA1 mutant cell lines are colored in red, BRCA1 wild type lines are in blue. 7 + 0.5 g/mL doxycycline TRIPZ control V2THS_254648 V2THS_254609 V2THS_90880 V2THS_238864 V2THS_198913 V2THS_238842 V2THS_980987 V3THS_305107 V3THS_369349 V3THS_369350 V2THS_157188 Fluorescence intensity 0 10 20 30 40 50 No doxycycline TRIPZ control V2THS_254648 V2THS_254609 V2THS_90880 V2THS_238864 V2THS_198913 V2THS_238842 V2THS_980987 V3THS_305107 V3THS_369349 V3THS_369350 V2THS_157188 Fluorescence intensity 0 10 20 30 40 50 BRCA1 PALB2   Figure S7. Induction of RFP expression by doxycycline in HEK293 cells transiently transfected with TRIPZ lentiviral plasmids. HEK293 cells were seeded in flat-bottomed, transparent 96-well microplates (Nunc) and transfected with lentiviral plasmids by lipid- based transfection. The cells were induced with 0.5 µg/mL doxycycline (A) or blank medium (B) for 48 h, beginning 24 h after transfection. To minimize variation in transfection efficiency, 4 independent transfections were performed for each condition and the mean plus SEM of these determinations is plotted. Fluorescence was measured with a Spectra Max M2 fluorescent plate reader (Molecular Devices) using an excitation wavelength of 544 nm and detection wavelength of 590 nm. Fluorescence is calibrated in arbitrary units. The BRCA1 + 0.5 g/mL doxycycline TRIPZ control V2THS_254648 V2THS_254609 V2THS_90880 V2THS_238864 V2THS_198913 V2THS_238842 V2THS_980987 V3THS_305107 V3THS_369349 V3THS_369350 V2THS_157188 Fluorescence intensity 0 10 20 30 40 50 No doxycycline TRIPZ control V2THS_254648 V2THS_254609 V2THS_90880 V2THS_238864 V2THS_198913 V2THS_238842 V2THS_980987 V3THS_305107 V3THS_369349 V3THS_369350 V2THS_157188 Fluorescence intensity 0 10 20 30 40 50 BRCA1 PALB2   Figure S7. Induction of RFP expression by doxycycline in HEK293 cells transiently transfected with TRIPZ lentiviral plasmids. HEK293 cells were seeded in flat-bottomed, + 0.5 g/mL doxycycline TRIPZ control V2THS_254648 V2THS_254609 V2THS_90880 V2THS_238864 V2THS_198913 V2THS_238842 V2THS_980987 V3THS_305107 V3THS_369349 V3THS_369350 V2THS_157188 Fluorescence intensity 0 10 20 30 40 50 No doxycycline TRIPZ control V2THS_254648 V2THS_254609 V2THS_90880 V2THS_238864 V2THS_198913 V2THS_238842 V2THS_980987 V3THS_305107 V3THS_369349 V3THS_369350 V2THS_157188 Fluorescence intensity 0 10 20 30 40 50 BRCA1 PALB2   Figure S7 Ind ction of RFP e pression b do c cline in HEK293 cells transientl + 0.5 g/mL doxycycline TRIPZ control V2THS_254648 V2THS_254609 V2THS_90880 V2THS_238864 V2THS_198913 V2THS_238842 V2THS_980987 V3THS_305107 V3THS_369349 V3THS_369350 V2THS_157188 Fluorescence intensity 0 10 20 30 40 50 BRCA1 PALB2 Figure S7. Induction of RFP expression by doxycycline in HEK293 cells transiently transfected with TRIPZ lentiviral plasmids HEK293 cells were seeded in flat bottomed Figure S7. Induction of RFP expression by doxycycline in HEK293 cells transiently Figure S7. Induction of RFP expression by doxycycline in HEK293 cells transiently transfected with TRIPZ lentiviral plasmids. 7 Effects of 72 h doxycycline exposure on RFP expression (A) and cell growth (B) were assayed using a fluorometric plate reader and SRB staining, respectively. Values plotted are mean + SEM of 8 replicate wells Fluorescence was measured with a Spectra Max M2 fluorescent plate reader (Molecular Devices) using an excitation wavelength of 544 nm and detection wavelength of 590 nm. Fluorescence is calibrated in arbitrary units. The doxycycline induction concentration that was chosen for induction of cells in experiments, 2 µg/mL, is indicated by blue dashed lines. Doxycycline (g/mL) 0 2 4 6 8 10 12 14 16 RFP fluorescence intensity Doxycycline (g/mL) 0.1 1 10 Optical density (490 - 450 nm) 0 20 40 60 80 100 120 A B Doxycycline (g/mL) 0 2 4 6 8 10 12 14 16 RFP fluorescence intensity Doxycycline (g/mL) 0.1 1 10 Optical density (490 - 450 nm) 0 20 40 60 80 100 120 A B Doxycycline (g/mL) 0 2 4 6 8 10 12 14 16 RFP fluorescence intensity A A Doxycycline (g/mL) Doxycycline (g/mL) 0.1 1 10 Optical density (490 - 450 nm) 0 20 40 60 80 100 120 B B B Figure S10. Titration of doxycycline concentration for induction of sorted, stably transduced D3H2LN-TRIPZ control cells. Effects of 72 h doxycycline exposure on RFP expression (A) and cell growth (B) were assayed using a fluorometric plate reader and SRB staining, respectively. Values plotted are mean + SEM of 8 replicate wells Fluorescence was measured with a Spectra Max M2 fluorescent plate reader (Molecular Devices) using an excitation wavelength of 544 nm and detection wavelength of 590 nm. Fluorescence is calibrated in arbitrary units. The doxycycline induction concentration that was chosen for induction of cells in experiments, 2 µg/mL, is indicated by blue dashed lines. Figure S10. Titration of doxycycline concentration for induction of sorted, stably transduced D3H2LN-TRIPZ control cells. Effects of 72 h doxycycline exposure on RFP expression (A) and cell growth (B) were assayed using a fluorometric plate reader and SRB staining, respectively. Values plotted are mean + SEM of 8 replicate wells Fluorescence was measured with a Spectra Max M2 fluorescent plate reader (Molecular Devices) using an excitation wavelength of 544 nm and detection wavelength of 590 nm. Fluorescence is calibrated in arbitrary units. 7 HEK293 cells were seeded in flat-bottomed, transparent 96-well microplates (Nunc) and transfected with lentiviral plasmids by lipid- based transfection. The cells were induced with 0.5 µg/mL doxycycline (A) or blank medium (B) for 48 h, beginning 24 h after transfection. To minimize variation in transfection Figure S7. Induction of RFP expression by doxycycline in HEK293 cells transiently transfected with TRIPZ lentiviral plasmids. HEK293 cells were seeded in flat-bottomed, transparent 96-well microplates (Nunc) and transfected with lentiviral plasmids by lipid- based transfection. The cells were induced with 0.5 µg/mL doxycycline (A) or blank medium (B) for 48 h, beginning 24 h after transfection. To minimize variation in transfection efficiency, 4 independent transfections were performed for each condition and the mean plus SEM of these determinations is plotted. Fluorescence was measured with a Spectra Max M2 fluorescent plate reader (Molecular Devices) using an excitation wavelength of 544 nm and detection wavelength of 590 nm. Fluorescence is calibrated in arbitrary units. The BRCA1 and PALB2 shRNA plasmids that were ultimately chosen for experimental use are colored in red. 8 8 TRIPZ control V2THS_254648 V2THS_254609 V2THS_90880 V2THS_238864 V2THS_198913 V2THS_238842 V2THS_980987 V3THS_305107 Relative BRCA1 mRNA 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Non-induced + doxycycline TRIPZ control V3THS_369349 V3THS_369350 V2THS_157188 Relative PALB2 mRNA 0.0 0.2 0.4 0.6 0.8 1.0 1.2 A B TRIPZ control V2THS_254648 V2THS_254609 V2THS_90880 V2THS_238864 V2THS_198913 V2THS_238842 V2THS_980987 V3THS_305107 Relative BRCA1 mRNA 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Non-induced + doxycycline A A A TRIPZ control V3THS_369349 V3THS_369350 V2THS_157188 Relative PALB2 mRNA 0.0 0.2 0.4 0.6 0.8 1.0 1.2 B B Figure S8. Suppression of target mRNA in HEK293 cells transiently transfected with TRIPZ shRNA plasmids. HEK293 cells were transfected and induced with doxycycline as in Fig. S1. RNA was prepared using Trizol-chlorophorm extraction. Complementary DNA was synthesized using a SuperScript III First-Strand Synthesis kit and amplified using SYBR green (both from Invitrogen). Abundance of BRCA1 (A) and PALB2 mRNA (B) is shown relative to non-induced cells transfected with TRIPZ control plasmid. 9 10 10 Figure S9 (previous page). Fluorescence-activated sorting of stably transduced D3H2LN cells. FACS Diva graphical outputs of flow cytometry gates for aseptic sorting of high expressing D3H2LN-TRIPZ control (A), shBRCA1 (B) and shPALB2 (C) cells based on doxycycline-induced RFP signal. 11 11 Figure S10. Titration of doxycycline concentration for induction of sorted, stably transduced D3H2LN-TRIPZ control cells. 7 The doxycycline induction concentration that was chosen for induction of cells in experiments, 2 µg/mL, is indicated by blue dashed lines. 12 12 D3H2LN SUM149PT HCT116 SUM159PT HCC1937 MDA-MB-468 MDA-MB-436 DLD-1 SUM1315MO2 DLD-1 BRCA2-/- Geometric mean of FSL-61 fluorescence area (relative to HCT116) 0.0 0.5 1.0 1.5 2.0 2.5 3.0     Figure S11. Reductive activation of the fluorogenic probe FSL-61 by DLD-1 and DLD-1 BRCA2-/- cells under hypoxia, and comparison to TNBC cell lines. Values are mean + SEM of geometric mean of fluorescence area from 3 independent determinations TNBC cell lines D3H2LN SUM149PT HCT116 SUM159PT HCC1937 MDA-MB-468 MDA-MB-436 DLD-1 SUM1315MO2 DLD-1 BRCA2-/- Geometric mean of FSL-61 fluorescence area (relative to HCT116) 0.0 0.5 1.0 1.5 2.0 2.5 3.0     Figure S11. Reductive activation of the fluorogenic probe FSL-61 by DLD-1 and DLD-1 BRCA2-/- cells under hypoxia, and comparison to TNBC cell lines. Values are mean + SEM of geometric mean of fluorescence area from 3 independent determinations. TNBC cell lines are colored in blue or red, depending on BRCA1 status, DLD-1 and DLD-1 BRCA2-/- cells in black HCT116 was used an internal control for comparability across experiments and is D3H2LN SUM149PT HCT116 SUM159PT HCC1937 MDA-MB-468 MDA-MB-436 DLD-1 SUM1315MO2 DLD-1 BRCA2-/- Geometric mean of FSL-61 fluorescence area (relative to HCT116) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Figure S11. Reductive activation of the fluorogenic probe FSL-61 by DLD-1 and DLD-1 igure S11. Reductive activation of the fluorogenic probe FSL-61 by DLD-1 and DLD-1 Figure S11. Reductive activation of the fluorogenic probe FSL-61 by DLD-1 and DLD-1 BRCA2-/- cells under hypoxia and comparison to TNBC cell lines Values are mean + SEM Figure S11. Reductive activation of the fluorogenic probe FSL-61 by DLD-1 and DLD-1 BRCA2-/- cells under hypoxia, and comparison to TNBC cell lines. Values are mean + SEM of geometric mean of fluorescence area from 3 independent determinations. TNBC cell lines are colored in blue or red, depending on BRCA1 status, DLD-1 and DLD-1 BRCA2-/- cells in black. HCT116 was used an internal control for comparability across experiments, and is shown in the white crosshatched bar. 13 Figure S12. Evaluation of AKR1C3 expression in DLD-1 and DLD-1 BRCA2-/- cell lines by Western blot. H460 and LAPC4 cells, which have been characterized for AKR1C3 expression (Yin Y., Fu M.B., Brooke D.G., Heinrich D.M., Denny W.A., Jamieson S.M.F. 7 Small molecule inhibition of AKR1C3 by SN33638 prevents 11β-PGF2α formation, but has little activity on steroid hormone production or function in castration-resistant prostate cancer and ER-positive breast cancer cell lines; in preparation), were used as positive and negative controls, respectively. 14 Figure S13. Uncropped replicate AKR1C3 immunoblots of in DLD-1 and DLD-1 BRCA2-/- cell lines annotated with molecular size markers. H460 and LAPC4 cells, in which we have thoroughly characterized AKR1C3 expression (Yin Y., Fu M.B., Brooke D.G., Heinrich D.M., Denny W.A., Jamieson S.M.F. Small molecule inhibition of AKR1C3 by SN33638 prevents 11β-PGF2α formation, but has little activity on steroid hormone production or function in castration-resistant prostate cancer and ER-positive breast cancer cell lines; in preparation), were used as positive and negative controls, respectively. 7 15 TH-302 HCC1937 DLD-1 D3H2LN SUM1315MO2 BT549 MDA-MB-468 SUM159PT MDA-MB-436 DLD-1 BRCA2-/- SUM149PT Hypoxic IC50 (M) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 PR-104A DLD-1 PC3 D3H2LN MDA-MB-231 C-33A HCC1937 A375 OVCAR-3 SUM159PT HT29 BT549 SUM149PT A2780 A549 SKOV-3 SiHa MIA PaCa2 PANC1 SUM1315MO2 H1299 MDA-MB-468 HCT 116 22Rv1 MDA-MB-436 DLD-1 BRCA2-/- H460 Hypoxic IC50 (M) 0 5 10 15 20 25 SN30000 SUM1315MO2 SUM159PT PC3 DLD-1 HCC1937 SUM149PT MDA-MB-231 HT29 A549 MIA PaCa2 BT549 MDA-MB-436 MDA-MB-468 22Rv 1 SiHa DLD-1 BRCA2-/- A2780 H460 C-33A HCT 116 H1299 Hypoxic IC50 (M) 0 2 4 6 8 10 12 Cisplatin DLD-1 SUM1315MO2 D3H2LN BT549 MDA-MB-436 SUM159PT C-33A DLD-1 BRCA2-/- MDA-MB-468 SUM149PT Hypoxic IC50 (M) 0 2 4 6 8 10 12 14 16 18 TH-302 HCC1937 DLD-1 D3H2LN SUM1315MO2 BT549 MDA-MB-468 SUM159PT MDA-MB-436 DLD-1 BRCA2-/- SUM149PT Hypoxic IC50 (M) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 PR-104A DLD-1 PC3 D3H2LN MDA-MB-231 C-33A HCC1937 A375 OVCAR-3 SUM159PT HT29 BT549 SUM149PT A2780 A549 SKOV-3 SiHa MIA PaCa2 PANC1 SUM1315MO2 H1299 MDA-MB-468 HCT 116 22Rv1 MDA-MB-436 DLD-1 BRCA2-/- H460 Hypoxic IC50 (M) 0 5 10 15 20 25 SN30000 SUM1315MO2 SUM159PT PC3 DLD-1 HCC1937 SUM149PT MDA-MB-231 HT29 A549 MIA PaCa2 BT549 MDA-MB-436 MDA-MB-468 22Rv 1 SiHa DLD-1 BRCA2-/- A2780 H460 C-33A HCT 116 H1299 Hypoxic IC50 (M) 0 2 4 6 8 10 12 Cisplatin DLD-1 SUM1315MO2 D3H2LN BT549 MDA-MB-436 SUM159PT C-33A DLD-1 BRCA2-/- MDA-MB-468 SUM149PT Hypoxic IC50 (M) 0 2 4 6 8 10 12 14 16 18 PR-104A DLD-1 PC3 D3H2LN MDA-MB-231 C-33A HCC1937 A375 OVCAR-3 SUM159PT HT29 BT549 SUM149PT A2780 A549 SKOV-3 SiHa MIA PaCa2 PANC1 SUM1315MO2 H1299 MDA-MB-468 HCT 116 22Rv1 MDA-MB-436 DLD-1 BRCA2-/- H460 Hypoxic IC50 (M) 0 5 10 15 20 25 TH-302 HCC1937 DLD-1 D3H2LN SUM1315MO2 BT549 MDA-MB-468 SUM159PT MDA-MB-436 DLD-1 BRCA2-/- SUM149PT Hypoxic IC50 (M) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 PR-104A DLD-1 PC3 D3H2LN MDA-MB-231 C-33A HCC1937 A375 OVCAR-3 SUM159PT HT29 BT549 SUM149PT A2780 A549 SKOV-3 SiHa MIA PaCa2 PANC1 SUM1315MO2 H1299 MDA-MB-468 HCT 116 22Rv1 MDA-MB-436 DLD-1 BRCA2-/- H460 Hypoxic IC50 (M) 0 5 10 15 20 25 SN30000 SUM1315MO2 SUM159PT PC3 DLD-1 HCC1937 SUM149PT MDA-MB-231 HT29 A549 MIA PaCa2 BT549 MDA-MB-436 MDA-MB-468 22Rv 1 SiHa DLD-1 BRCA2-/- A2780 H460 C-33A HCT 116 H1299 Hypoxic IC50 (M) 0 2 4 6 8 10 12 Cisplatin DLD-1 SUM1315MO2 D3H2LN BT549 MDA-MB-436 SUM159PT C-33A DLD-1 BRCA2-/- MDA-MB-468 SUM149PT Hypoxic IC50 (M) 0 2 4 6 8 10 12 14 16 18   Figure S14. 7 Comparison of antiproliferative potency of TH-302, PR-104A, SN30000 and cisplatin in DLD-1 and DLD-1 BRCA2-/- under hypoxia with TNBC cells and other cancer cell lines. IC50 values for DLD-1 and DLD-1 BRCA2-/- cell lines, which relate to Fig. 5C of this manuscript, are in black bars. IC50 values for TNBC cell lines, which relate to Fig. 1B of this manuscript, are colored purple. Other miscellaneous human cancer cell lines for which we have historical IC50 data generated using identical methodology in our laboratory are shown in grey. Values are mean + SEM of ≥3 independent experiments. HCC193 DLD D3H2L SUM1315MO BT54 MDA-MB-46 SUM159 MDA-MB-43 DLD-1 BRCA2 SUM149 DL P D3H2 MDA-MB- C-3 HCC1 A OVCA SUM159 HT BT SUM149 A2 A SKO S MIA PaC PAN SUM1315M H1 MDA-MB- HCT 22 MDA-MB- DLD-1 BRCA H SN30000 SUM1315MO2 SUM159PT PC3 DLD-1 HCC1937 SUM149PT MDA-MB-231 HT29 A549 MIA PaCa2 BT549 MDA-MB-436 MDA-MB-468 22Rv 1 SiHa DLD-1 BRCA2-/- A2780 H460 C-33A HCT 116 H1299 Hypoxic IC50 (M) 0 2 4 6 8 10 12 Cisplatin DLD-1 SUM1315MO2 D3H2LN BT549 MDA-MB-436 SUM159PT C-33A DLD-1 BRCA2-/- MDA-MB-468 SUM149PT Hypoxic IC50 (M) 0 2 4 6 8 10 12 14 16 18 Figure S14. Comparison of antiproliferative potency of TH-302, PR-104A, SN30000 and cisplatin in DLD-1 and DLD-1 BRCA2-/- under hypoxia with TNBC cells and other cancer cell lines. IC50 values for DLD-1 and DLD-1 BRCA2-/- cell lines, which relate to Fig. 5C of this manuscript, are in black bars. IC50 values for TNBC cell lines, which relate to Fig. 1B of this manuscript, are colored purple. Other miscellaneous human cancer cell lines for which we have historical IC50 data generated using identical methodology in our laboratory are shown in grey. Values are mean + SEM of ≥3 independent experiments. 7 16 16 Mechlorethamine HCC1937 BT549 D3H2LN SUM1315MO2 SUM159PT DLD-1 SUM149PT MDA-MB-468 MDA-MB-436 DLD-1 BRCA2-/- Aerobic IC50 (M) 0 2 4 6 8 PR-104H D3H2LN MDA-MB-468 A549 PC3 MDA-MB-231 MIA PaCa2 H1299 SiHa HT29 HCC1937 PANC1 BT549 SUM1315MO2 HEP G2 DLD-1 Hep 3B SUM159PT 22Rv1 HCT 116 OVCAR-3 H460 MDA-MB-436 C-33A SUM149PT A2780 DLD-1 BRCA2-/- Aerobic IC50 (M) 0 10 20 30 40 Chlorambucil SiHa PC3 HT29 SKOV-3 MDA-MB-231 DLD-1 A549 H1299 HCT 116 MIA PaCa2 A375 22Rv1 H460 A2780 C-33A DLD-1 BRCA2-/- Aerobic IC50 (M) 0 5 10 15 20 25 Cisplatin DLD-1 BT549 Hep 3B SUM159PT D3H2LN SUM1315MO2 HCC1937 HCT 116 SUM149PT MDA-MB-468 HEP G2 DLD-1 BRCA2-/- A2780 MDA-MB-436 SiHa Aerobic IC50 (M) 0 5 10 15 20 25 Figure S15. Comparison of antiproliferative potency of mechlorethamine, PR-104H, chlorambucil and cisplatin in DLD-1 and DLD-1 BRCA2-/- under normoxia with TNBC cell and other cancer cell lines. IC50 values for DLD-1 and DLD-1 BRCA2-/- cell lines, which PR-104H D3H2LN MDA-MB-468 A549 PC3 MDA-MB-231 MIA PaCa2 H1299 SiHa HT29 HCC1937 PANC1 BT549 SUM1315MO2 HEP G2 DLD-1 Hep 3B SUM159PT 22Rv1 HCT 116 OVCAR-3 H460 MDA-MB-436 C-33A SUM149PT A2780 DLD-1 BRCA2-/- Aerobic IC50 (M) 0 10 20 30 40 Mechlorethamine HCC1937 BT549 D3H2LN SUM1315MO2 SUM159PT DLD-1 SUM149PT MDA-MB-468 MDA-MB-436 DLD-1 BRCA2-/- Aerobic IC50 (M) 0 2 4 6 8 Mechlorethamine Aerobic IC50 (M) D Chlorambucil SiHa PC3 HT29 SKOV-3 MDA-MB-231 DLD-1 A549 H1299 HCT 116 MIA PaCa2 A375 22Rv1 H460 A2780 C-33A DLD-1 BRCA2-/- Aerobic IC50 (M) 0 5 10 15 20 25 Cisplatin DLD-1 BT549 Hep 3B SUM159PT D3H2LN SUM1315MO2 HCC1937 HCT 116 SUM149PT MDA-MB-468 HEP G2 DLD-1 BRCA2-/- A2780 MDA-MB-436 SiHa Aerobic IC50 (M) 0 5 10 15 20 25 Cisplatin DLD-1 BT549 Hep 3B SUM159PT D3H2LN SUM1315MO2 HCC1937 HCT 116 SUM149PT MDA-MB-468 HEP G2 DLD-1 BRCA2-/- A2780 MDA-MB-436 SiHa Aerobic IC50 (M) 0 5 10 15 20 25 Chlorambucil Cisplatin Chlorambucil SiHa PC3 HT29 SKOV-3 MDA-MB-231 DLD-1 A549 H1299 HCT 116 MIA PaCa2 A375 22Rv1 H460 A2780 C-33A DLD-1 BRCA2-/- Aerobic IC50 (M) 0 5 10 15 20 25 Figure S15. Comparison of antiproliferative potency of mechlorethamine, PR-104H, chlorambucil and cisplatin in DLD-1 and DLD-1 BRCA2-/- under normoxia with TNBC cells and other cancer cell lines. IC50 values for DLD-1 and DLD-1 BRCA2-/- cell lines, which relate to Fig. 5D of this manuscript, are in black bars. IC50 values for TNBC cell lines, which relate to Fig. 7 1A of this manuscript, are colored purple. Other miscellaneous human cancer cell lines for which we have historical IC50 data generated using identical methodology in our laboratory are shown in grey. Values are mean + SEM of ≥3 independent experiments. 17 17 Figure S16. Inhibition of growth of in DLD-1 and DLD-1 BRCA2-/- tumors by radiotherapy. A, B, growth kinetics of individual DLD-1 and DLD-1 BRCA2-/- tumors, respectively, treated with sham radiation or 10 Gy radiotherapy. C, D, Kaplan-Meier survival curves of the same cohorts. Survival was defined as tumor volume less than three times volume on the day DLD-1 wild type Days post treatment -10 0 10 20 30 40 50 Tumour volume (mm3) 0 500 1000 1500 2000 2500 3000 3500 Sham 10 Gy DLD-1 BRCA2-/- Days post treatment -20 -10 0 10 20 30 40 50 Tumour volume (mm3) 0 500 1000 1500 2000 2500 3000 Sham 10 Gy DLD-1 wild type Days post treatment 0 5 10 15 20 25 30 Survival (%) 0 20 40 60 80 100 Sham 10 Gy DLD-1 BRCA2-/- Days post treatment 0 10 20 30 40 Survival (%) 0 20 40 60 80 100 Sham 10 Gy DLD-1 wild type Days post treatment -10 0 10 20 30 40 50 Tumour volume (mm3) 0 500 1000 1500 2000 2500 3000 3500 Sham 10 Gy DLD-1 BRCA2-/- Days post treatment -20 -10 0 10 20 30 40 50 Tumour volume (mm3) 0 500 1000 1500 2000 2500 3000 Sham 10 Gy DLD-1 wild type Days post treatment -10 0 10 20 30 40 50 Tumour volume (mm3) 0 500 1000 1500 2000 2500 3000 3500 Sham 10 Gy 3 DLD-1 BRCA2-/- Days post treatment -20 -10 0 10 20 30 40 50 Tumour volume (mm3) 0 500 1000 1500 2000 2500 3000 Sham 10 Gy Tumour volume (mm3) Tumour volume (mm3) DLD-1 wild type Days post treatment 0 5 10 15 20 25 30 Survival (%) 0 20 40 60 80 100 Sham 10 Gy DLD-1 BRCA2-/- Days post treatment 0 10 20 30 40 Survival (%) 0 20 40 60 80 100 Sham 10 Gy Figure S16. Inhibition of growth of in DLD-1 and DLD-1 BRCA2-/- tumors by radiotherapy. A, B, growth kinetics of individual DLD-1 and DLD-1 BRCA2-/- tumors, respectively, treated with sham radiation or 10 Gy radiotherapy. C, D, Kaplan-Meier survival curves of the same cohorts. 7 Survival was defined as tumor volume less than three times volume on the day of treatment. Statistical significance of effects of radiotherapy on survival was evaluated using log-rank tests. Radiosensitivity of DLD-1 and DLD-1 BRCA2-/- tumors was compared using median endpoint time ratios (10 Gy/sham) for each model. N = 4 for all cohorts with the exception of the DLD-1 BRCA2-/- radiotherapy cohort, for which N = 5. Figure S16. Inhibition of growth of in DLD-1 and DLD-1 BRCA2-/- tumors by radiotherapy. A, B, growth kinetics of individual DLD-1 and DLD-1 BRCA2-/- tumors, respectively, treated with sham radiation or 10 Gy radiotherapy. C, D, Kaplan-Meier survival curves of the same cohorts. Survival was defined as tumor volume less than three times volume on the day of treatment. Statistical significance of effects of radiotherapy on survival was evaluated using log-rank tests. Radiosensitivity of DLD-1 and DLD-1 BRCA2-/- tumors was compared using median endpoint time ratios (10 Gy/sham) for each model. N = 4 for all cohorts with the exception of the DLD-1 BRCA2-/- radiotherapy cohort, for which N = 5. Figure S16. Inhibition of growth of in DLD-1 and DLD-1 BRCA2-/- tumors by radiotherapy. A, B, growth kinetics of individual DLD-1 and DLD-1 BRCA2-/- tumors, respectively, Figure S16. Inhibition of growth of in DLD-1 and DLD-1 BRCA2-/- tumors by radiotherapy. A, B, growth kinetics of individual DLD-1 and DLD-1 BRCA2-/- tumors, respectively, treated with sham radiation or 10 Gy radiotherapy. C, D, Kaplan-Meier survival curves of the same cohorts. Survival was defined as tumor volume less than three times volume on the day of treatment. Statistical significance of effects of radiotherapy on survival was evaluated using log-rank tests. Radiosensitivity of DLD-1 and DLD-1 BRCA2-/- tumors was compared using median endpoint time ratios (10 Gy/sham) for each model. N = 4 for all cohorts with the exception of the DLD-1 BRCA2-/- radiotherapy cohort, for which N = 5. 18 18
https://openalex.org/W2590954794
https://discovery.ucl.ac.uk/id/eprint/1542970/1/jech-2016-208037.full.pdf
English
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Health and social exclusion in older age: evidence from Understanding Society, the UK household longitudinal study
Journal of epidemiology and community health
2,017
cc-by
9,355
ABSTRACT k domain, where poor health is often considered a predictor or risk factor,5–7 an indicator8–10 or an outcome of exclusion.6 11–13 The variety of ways that health is used in social exclusion research, and the many pathways through which social exclusion and health interact,14 constrain our understanding of the process and consequently possible solutions to resultant health inequality. domain, where poor health is often considered a predictor or risk factor,5–7 an indicator8–10 or an outcome of exclusion.6 11–13 The variety of ways that health is used in social exclusion research, and the many pathways through which social exclusion and health interact,14 constrain our understanding of the process and consequently possible solutions to resultant health inequality. Background Social exclusion of the elderly is a key policy focus but evidence on the processes linking health and social exclusion is hampered by the variety of ways that health is used in social exclusion research. We investigated longitudinal associations between health and social exclusion using an analytical framework that did not conflate them. 1Department of Epidemiology and Public Health, University College London, London, UK 2Dementia Services Development Centre Wales, Bangor University, Bangor, UK 3Institute for Health and Human Development, University of East London, London, UK As people grow older, the chance that they will become socially excluded is greater than the chance that they will move out of or become less excluded.7 This highlights the severity and continu- ity of social exclusion for older adults. Current healthy ageing strategies in Europe are designed to try to address issues including social exclusion,15 by providing an environment in which people can engage in a process of ‘active ageing’, allowing them to “realize their potential for physical, social and mental well-being throughout the life course and to participate in society according to their needs, desires and capacities, while providing them with adequate protection, security and care when they require assistance”.(ref. 16 p. 12) While such policies are clearly designed to reduce the chances of people becoming socially excluded, the current lack of understanding about the pathways and mechanisms through which social exclusion exists is likely to inhibit their overall success. Methods Data employed in this study came from 4 waves of Understanding Society, the UK Household Longitudinal Study 2009–2013. ABSTRACT k The sample comprised all adults who took part in all 4 waves, were 65 years or more in Wave 3, and had complete data on our variables of interest for each analysis. We used linear regression to model the relationship between Wave 2/3 social exclusion and Wave1–2 health transitions (N=4312) and logistic regression to model the relationship between Wave2/3 social exclusion and Wave 4 health states, conditional on Wave 3 health (N=4244). Results There was a dose–response relationship between poor health in Waves 1 and 2 and later social exclusion. Use of a car, mobile phone and the internet moderated the association between poor health and social exclusion. Given the health status in Wave 3, those who were more socially excluded had poorer outcomes on each of the three domains of health in Wave 4. Correspondence to Professor Amanda Sacker, ESRC International Centre for Lifecourse Studies in Society and Health, Research Department of Epidemiology and Public Health, University College London, 1-19 Torrington Pace, London, WC1E 6BT, UK; a.sacker@ucl.ac.uk Received 5 July 2016 Revised 5 December 2016 Accepted 29 January 2017 Conclusions Use of the internet and technology protected older adults in poor health from social exclusion. Age-friendly hardware and software design might have public health benefits. Using 4 waves of data from a large UK house- hold panel survey,17 we explore the process of social exclusion in later life. With health being a particularly important correlate of social exclusion for older adults, this paper focuses on the associ- ation between health and social exclusion, examin- ing (1) whether poor health is a predictor of social exclusion in people aged 65 years and over; (2) whether health is an outcome of social exclusion; and (3) factors that might modify these relationships. INTRODUCTION l l Social exclusion is a multidimensional process through which individuals become disengaged from mainstream society, depriving people of the rights, resources and services available to the majority.1 A key priority for policymakers in Europe,2 social exclusion manifests through a number of inter- linked and mutually reinforcing problems that deny people the opportunities available to most in society. There are a number of drivers of social exclusion including poverty, lower levels of educa- tional attainment, unemployment, ill health, poor housing or homelessness, poor transport access, increased levels of crime and limited social support, all of which can have long-lasting effects.3 The interlinked nature of social exclusion makes it diffi- cult to understand the relationships between differ- ing domains, and to tease apart those that are direct risk factors, mediating or moderating factors, indicators or outcomes of exclusion.4 This complex relationship can clearly be seen with the health Amanda Sacker,1 Andy Ross,1 Catherine A MacLeod,2 Gopal Netuveli,3 Gill Windle2 Amanda Sacker,1 Andy Ross,1 Catherine A MacLeod,2 Gopal Netuveli,3 Gill Wind ▸Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/jech- 2016-208037). Research JECH Online First, published on February 22, 2017 as 10.1136/jech-2016-208037 Rese JECH Online First, published on February 22, 2017 as 10.1136/jech-2016-2080 Research report 6-208037 To cite: Sacker A, Ross A, MacLeod CA, et al. J Epidemiol Community Health Published Online First: [please include Day Month Year] doi:10.1136/jech- 2016-208037 Received 5 July 2016 Revised 5 December 2016 Accepted 29 January 2017 Correspondence to Professor Amanda Sacker, ESRC International Centre for Lifecourse Studies in Society and Health, Research Department of Epidemiology and Public Health, University College London, 1-19 Torrington Pace, London, WC1E 6BT, UK; a.sacker@ucl.ac.uk Copyright Article author (or their employer) 2017. Produced by BMJ Publishing Group Ltd under licence. Covariates Covariates were split into two groups: (1) confounders and (2) mediators and/or modifiers. To overcome the problem that not all indicators of social exclusion were available in the same wave, social exclusion was measured using data from two consecutive waves of Understanding Society (Wave 2 (2010/2011) and Wave 3 (2011/ 2012)). A summary of the indicators and methods used for con- structing social exclusion is given below. For a more in-depth overview, see MacLeod et al.1 Confounders: gender;25 age; age-squared;26 ethnicity (White or non-White);27 28 place of birth (born in the UK or else- where);27 28 marital status (married/in civil partnership, living as a couple, single never married/in civil partnership, separated or divorced, or widowed);25 job status (whether the respondent was in work or not);29 highest qualification (degree, other higher, A level or equivalent, GCSE or equivalent, other, or no qualifications);28 30 social class (NS-SEC managerial and profes- sional, intermediate, small employer and own account, lower supervisor and technical, semiroutine and routine occupations, or whether the respondent never had a job);26 30 region (whether the respondent lived in one of nine Government Office Regions of England, or in Scotland, Wales or Northern Ireland).31 Service provision and access: respondents were allocated a point for each of the following: reporting that they were not able to access all services such as healthcare, food shops or learning facilities when they needed to; rating the quality of local medical facilities as ‘fair’ or ‘poor’; rating local shopping facilities as ‘fair’ or ‘poor’; rating local leisure facilities as ‘poor’; and/or reporting that they found it ‘difficult’ or ‘very dif- ficult’ to get to a sports or leisure facility if they wanted to, including a leisure centre, recreation ground or park. Scores were summed to give an overall scale from 0 to 5 with high scores indicating poorer service provision and access. Covariates Mediators/moderators: Potential modification of the health and social exclusion association was assessed in relation to: area type (rural/urban);14 32 33 car access (whether the respondent lived in a household that owns or has continuous use of a car or not);34 35 mobile phone ownership, and internet use (whether the respondent used the internet often (daily or several times a week), sometimes (several times a month or less), or never (never used it or no access at home, work or elsewhere).36 Civic participation: Respondents identified activities they had participated in during the past 12 months from predefined lists of cultural, sport and leisure activities, and reported the fre- quency with which they participated in each set of activities. Two items were derived to give the breadth (number of acti- vities) and frequency of participation. Respondents scored a point for each indicator where they were in the bottom quartile. Respondents were also allocated 1 point for not regularly par- ticipating in the work of an organisation or group (from 16 listed organisations), and 1 point if they did not volunteer. Scores on the 4 items were summed and recalibrated to give an overall scale from 0 to 5 with high scores indicating poorer civic participation. Social exclusion Following Walsh et al,19 a social exclusion index was con- structed with three underlying domains: (1) Service provision and access; (2) Civic participation; and (3) Social relations and resources. Each subdomain comprised 4–5 indicators capturing relevant aspects of social exclusion pertaining to that domain. The guiding principle for the selection and construction of these indicators was that each should identify the most excluded quartile of individuals. METHODS Participants p Data come from the first four waves of Understanding Society, the UK Household Longitudinal Study (UKHLS).17 The UKHLS is a nationally representative study, which began in 2009 with an aim of recruiting over 100 000 indi- viduals in 40 000 households. The data collection period takes 2 years to complete one wave of the study. All persons in the household aged 10 years and older are eligible to be surveyed annually. Adults, 16 and older, are offered a combination of computer-assisted personal interview and self- completion questionnaire. After July 2012, To cite: Sacker A, Ross A, MacLeod CA, et al. J Epidemiol Community Health Published Online First: [please include Day Month Year] doi:10.1136/jech- 2016-208037 Sacker A, et al. J Epidemiol Community Health 2017;0:1–10. doi:10.1136/jech-2016-208037 Sacker A, et al. J Epidemiol Community Health 2017;0:1–10. doi:10.1136/jech-2016-208037 Sacker A, et al. J Epidemiol Community Health 2017;0:1–10. doi:10.1136/jech-2016-208037 or (or their employer) 2017. Produced by BMJ Publishing Group Ltd under licence. Research report out socially or visiting friends when they felt like it. Scores were summed to give an overall scale from 0-5 with high scores indi- cating poorer social relations and resources. computer-assisted telephone interviews were offered to non- responders. The topics covered include subjective well-being, employment, health and various other economic and social topics. More detailed information on the sampling frame and data collection procedures are available.18 The social exclusion index was derived by summing scores for the three subdomains, measured on a scale of 0–15 with higher scores indicating greater social exclusion. The sample for our study includes members of the general population sample of Understanding Society who took part in Waves 1–4 and was aged 65 years or more in Wave 3. Of the 6473 aged 65+ in Wave 3, 5475 were interviewed in each of the first four waves. Item non-response reduced the sample to 4312 and 4244 for research questions 1 and 2, respectively. Online supplementary tables A1 and A2 in the online appendix show that the analysis samples were more advantaged and in better health than the samples of excluded respondents. Prior to construction of the social exclusion subdomains, imputation using chained equations (ICE) was employed to impute missing values if respondents were missing a single item within a subdomain.20–22 The table in MacLeod et al1 shows the prevalence for indicators preimputation and postimputation. Health Health measures include poor self-rated health (SRH: excellent, very good, good vs fair or poor); limiting long-term illness or disability (LLTI: no vs yes); and psychological distress, measured using General Health Questionnaire (GHQ) with the bimodal scoring method and a cut-off of 3 or more signifying distress (no vs yes).23 24 Derived health transition variables (Wave 1 to Wave 2) took values 0 stable good; 1 declining; 2 improving; and 3 stable poor health. Sacker A, et al. J Epidemiol Community Health 2017;0:1–10. doi:10.1136/jech-2016-208037 Data analysis Th ff f doi:10.1136/jech-2016-208037 2 Research report Table 1 Mean SEI and 95% CIs at Wave 2/3 by explanatory factors (N=4312) SEI Explanatory factor Mean 95% CI Wave 1/2 SRH Stable good 3.80 3.69 to 3.91 Good→poor 4.99*** 4.68 to 5.30 Poor→good 4.93*** 4.61 to 5.25 Stable poor 5.75*** 5.54 to 6.96 LLTI Stable no LLTI 3.83 3.72 to 3.95 LLTI onset 4.50*** 4.19 to 4.82 LLTI recovery 4.47*** 4.20 to 4.73 Stable LLTI 5.34*** 5.16 to 5.51 GHQ Stable low 4.13 4.02 to 4.24 Low→high 4.99*** 4.68 to 5.30 High→low 4.86*** 4.54 to 5.17 Stable high 5.87*** 5.54 to 6.19 Wave 1 Gender Female 4.60 4.47 to 4.72 Male 4.20*** 4.08 to 4.32 Age (years) <75 4.04 3.94 to 4.14 ≥75 5.19*** 4.99 to 5.39 Ethnicity White 4.40 4.30 to 4.49 Non-white 5.26** 4.71 to 5.81 Country of birth UK 4.39 4.29 to 4.49 Elsewhere 4.80* 4.43 to 5.18 Marital status Married 3.68 3.56 to 3.79 Living as a couple 3.89 3.38 to 4.41 Single never married 6.36*** 5.95 to 6.77 Separated or divorced 5.78*** 5.54 to 6.03 Widowed 5.75*** 5.54 to 5.95 Job status In work 3.77 3.58 to 3.96 Not in work 4.51*** 4.40 to 4.62 Education Degree 3.07 2.87 to 3.27 Other higher 3.60** 3.36 to 3.84 A level 3.93*** 3.70 to 4.16 GCSE 3.68*** 3.46 to 3.90 Other 4.71*** 4.50 to 4.92 None 5.41*** 5.23 to 5.59 Social class Man and Prof 3.61 3.46 to 3.77 Intermediate 4.22*** 4.00 to 4.44 Small emp. and own acc. 4.48*** 4.16 to 4.80 Lower supervisory and tech. 4.80*** 4.51 to 5.10 Semiroutine and routine 5.09*** 4.93 to 5.26 Never had a job 4.65*** 4.25 to 5.05 Government office region South East 3.93 3.64 to 4.22 North East 4.66** 4.30 to 5.02 gender, results are not stratified. Modification of the relation by area type, car access, mobile phone ownership and internet use was assessed by adding interaction terms to model 4. Second, the effect of social exclusion on subsequent health was assessed using logistic regression. Three models were esti- mated for each of the three health measures examined: (1) Base model adjusting for gender, age and age-squared and all three health measures; (2) further adjustment for ethnicity, country of birth, marital status, job status, highest qualification, social class and region; (3) further adjustment for area type, car access, mobile phone ownership and internet use. Data analysis Th ff f The effect of health transitions on social exclusion was assessed using linear regression. Four models were estimated for each of the three health transition measures examined (SRH, LLTI, GHQ): (1) Base model adjusting for gender, age and age-squared; (2) further adjustment for ethnicity, country of birth, marital status, job status, highest qualification, social class and region; (3) further adjustment for area type, car access, mobile phone ownership and internet use; and (4) further adjustment for the remaining two health measures. Social exclu- sion was measured in Waves 2 and 3 of Understanding Society, health was measured over Waves 1 to 2 and all other measures were from Wave 1. The gender invariance of the association between health transitions and social exclusion was assessed using an interaction term in model 1. Since we found no evi- dence of an interaction between the health transitions and Social relations and resources: Respondents who lived alone were allocated 2 points, and respondents living with a spouse or partner were allocated 1 point if they scored within the bottom quartile of a relationship closeness scale. Respondents were allo- cated 1 point if they did not have a child living outside of the home or their level of contact with that child was especially low. One point was allocated if the respondent reported having one or no close friendships, and 1 point if they reported not going Sacker A, et al. J Epidemiol Community Health 2017;0:1–10. Data analysis Th ff f The health outcomes were measured in Wave 4 and social exclusion in Waves 2 and 3. Time-invariant measures (ethnicity, country of birth, highest qualification) were measured in Wave 1 and time-varying mea- sures (health, marital status, job status, social class and region in Wave 3. The gender invariance of the association between social exclusion and health was assessed using an interaction term in model 1: no evidence of interactions between social exclusion and gender were found. Modification of the relation by area type, car access, mobile phone ownership and internet use was assessed by adding interaction terms to model 3. All analyses used survey methods in Stata V14.1(Stata Statistical Software: Release 14.1 [program]. College Station, Texas: StataCorp LP., 2015) to provide cluster-robust SEs and Wave 4 longitudinal weights applied to take account of unequal selection probabilities, attrition and the non-response of eligible participants. Further models assessed whether the association between health transitions and social exclusion, and social exclusion and subsequent health, varied across the three subdomains of social exclusion (service provision and access, civic participation and social relations and resources). Social exclusion and prior health Table 1 shows mean differences in the social exclusion index (SEI) by the health and covariate measures. Mean SEI was of a similar magnitude irrespective of the health measure, ranging from around 4 for those in stable good health to almost 6 for those in stable poor health. SEI means also differed across values of the covariates in expected directions with the excep- tion of area type; there were no urban/rural differences in SEI. The results for the linear regression models predicting SEI by the three health transition measures are shown in table 2. The association between SRH and SEI after adjustment for age and gender could still be seen (model 0) and suggests a dose– response relationship with similar increases in SEI (≈1 point) for transitions from good to poor and poor to good SRH and a larger increase (≈2) for those with poor SRH at both time points. There was some attenuation in the relationships after controlling for the hypothesised confounders (model 1) and more limited attenuation after including the set of potential mediators (area type, car access, mobile phone ownership and internet use). Model 3 confirmed an independent relationship between SRH and the SEI after accounting for the other health transition measures (LLTI and GHQ). The regression coefficients for LLTI transitions in model 0 showed similar trends in the LLTI to SEI relationship, although the magnitude of the differences in SEI was smaller than that seen for SRH. Again, there was some attenuation after account- ing for the confounders but little further change after adjusting for the mediators. In model 3, the coefficients were reduced further and only stable LLTI was associated with an increase in SEI (b=0.22). Sacker A, et al. J Epidemiol Community Health 2017;0:1–10. Social exclusion and subsequent health Table 3 shows the relationship of the covariates and the SEI with poor health at Wave 4. First, there was a clear gradient in poor health by the SEI for all three health measures. Second, there was confirmation of continuity in poor health in Wave 3 to poor health 1-year later, especially for poor self-rated health and limit- ing long-term illness. Third, differences in rates of poor health across categories of all the covariates were found for at least one of the three health outcomes with the exception of country of birth. For consistency across health measures, we include the same set of covariates in the modelling of the relationship between social exclusion and subsequent health (table 4). The baseline model 0 adjusted for health in Wave 3, age and gender. There was a 16% increase in the odds of poor SRH for each unit increase in the SEI conditional on health at Wave 3. After adjustment for the control variables in model 1, the odds were essentially unchanged. The potential mediators did not attenuate this association between social exclusion and subse- quent health. GHQ transitions were also related to social exclusion: SEI scores were raised for those with high GHQ at one or 2 time points. Independent of the confounders, mediators and other health measures, only stable high GHQ and a transition from low to high GHQ remained predictive of higher SEI scores. A similar set of results can be seen for limiting long-term illness (table 4, second row). The odds of LLTI in Wave 4 were increased by 8% for each unit increase in the SEI. This remained unchanged with the addition of the control variables and the hypothesised mediators. For the GHQ baseline model, the odds were 1.04 per unit increase in the SEI. However, these odds were revealed to be slightly higher and statistically significant after the adjustments in models 1 and 2. Social exclusion subdomains and prior health Results using the social exclusion subdomains (see online supplementary appendix table A3) indicated a much stronger relationship between SRH and civic participation than between SRH and service provision/access or social relations and resources. For LLTI, associations were strongest for civic partici- pation, then service provision/access and weakest for social rela- tions and resources. Like SRH, GHQ was more strongly associated with civic participation than with the service provi- sion/access and social relations and resources subdomains, although the magnitude of the differences was not so great. Modification of social exclusion and prior health relationship We investigated whether there was any moderation of the associ- ation between health transitions and SEI by car access, mobile phone ownership and internet use. There was a significant inter- action between car access and SRH transitions (p=0.007) and between car access and LLTI transitions (p=0.03): Car access made no difference to SEI scores for those in stable good health but respondents who transitioned to poor health (SRH only) or had stable poor health had lower scores on the SEI when they had access to a car than when not (see figure 1). There was no evidence of any moderation of the positive association between poor health and subsequent SEI by area type, car access, mobile phone ownership or internet use. The subdomain analyses (see online supplementary appendix table A4) indicated that conditional on health in Wave 3, social exclusion in the domains of civic participation and social rela- tions and resources increased the probability of poor SRH. The domains of civic participation and service provision and access, but not social relations and resources, were related to subse- quent LLTI. GHQ was associated with prior civic participation only. Modification of the SRH and SEI relationship by internet use (p=0.003) and of the LLTI and SEI relationship by mobile phone ownership (p=0.0001) was also observed. Again, no dif- ference in SEI scores was observed for those in stable good health. However, respondents with stable poor SRH had higher scores on the SEI if they never used the internet than if they did (figure 2A) and SEI scores were higher for respondents with stable LLTI without a phone than with (figure 2B). Furthermore, occasional internet use was associated with lower social exclusion scores than regular use for those whose SRH improved and having a mobile phone was associated with lower social exclusion scores for those no longer reporting LLTI. Social exclusion and prior health doi:10.1136/jech-2016-208037 3 Research report Table 1 Continued SEI Explanatory factor Mean 95% CI Yorkshire and the Humber 4.41* 4.13 to 4.69 East Midlands 4.80*** 4.45 to 5.15 West Midlands 4.57** 4.22 to 4.91 East of England 4.16 3.96 to 4.36 London 4.40* 4.09 to 4.71 South West 4.22 3.90 to 4.54 Scotland 4.59* 4.17 to 5.01 Wales 4.72** 4.37 to 5.07 Northern Ireland 4.76** 4.22 to 5.29 Area type Urban 4.41 4.29 to 4.53 Rural 4.43 4.24 to 4.61 Car access Yes 3.92 3.83 to 4.02 No 6.30*** 6.07 to 6.53 Mobile phone Yes 4.09 3.99 to 4.19 No 5.50*** 5.27 to 5.73 Internet use Often 3.41 3.29 to to 3.54 Sometimes 3.77** 3.55 to 3.99 Never 5.14*** 5.01 to 5.27 unweighted N; weighted means. Linear regression models test for significance of mean differences: * p<0.05, ** p<0.01, *** p<0.001. GHQ, 12 item General Health Questionnaire; LLTI, limiting long-term illness/disability; SEI, Social Exclusion Index; SRH, self-rated health. Unlike the other two health transition measures, there was no evidence of moderation of the GHQ to SEI relationship, although those with no access to a car and stable high GHQ had higher SEI scores than those with access to a car and stable low GHQ (p=0.02). DISCUSSION h d d This study adopted a new analytical framework for understand- ing health and social exclusion in older age by explicitly separat- ing measures of health from those of social exclusion. Previous work has confounded the two concepts making it difficult to understand the dynamics between health and social exclusion. Using this approach, we have both confirmed and extended Sacker A, et al. J Epidemiol Community Health 2017;0:1–10. doi:10.1136/jech-2016-208037 4 Research report Table 2 Linear regression estimates and 95% CIs for the Social Exclusion Index (Wave 2/3) regressed on health transitions (Wave 1-2) Model 0 Model 1 Model 2 Model 3 Table 2 Linear regression estimates and 95% CIs for the Social Exclusion Index (Wave 2/3) regressed on health transitions (Wave 1-2) Model 0 Model 1 Model 2 Model 3 SRH Stable good 0.00 Reference 0.00 Reference 0.00 Reference 0.00 Reference Good→poor 1.06*** (0.77 to 1.34) 0.83*** (0.56 to 1.10) 0.76*** (0.49 to 1.02) 0.58*** (0.30 to 0.86) Poor→good 1.10*** (0.79 to 1.41) 0.81*** (0.52 to 1.10) 0.76*** (0.47 to 1.04) 0.61*** (0.32 to 0.90) Stable poor 1.81*** (1.59 to 2.03) 1.39*** (1.19 to 1.60) 1.31*** (1.11 to 1.51) 0.95*** (0.72 to 1.18) LLTI Stable no LLTI 0.00 Reference 0.00 Reference 0.00 Reference 0.00 Reference LLTO onset 0.45** (0.17 to 0.74) 0.31* (0.07 to 0.56) 0.30* (0.05 to 0.54) 0.00 (−0.25 to 0.24) LLTI recovery 0.51*** (0.25 to 0.78) 0.37** (0.13 to 0.62) 0.33** (0.10 to 0.56) 0.05 (−0.17 to 0.28) Stable LLTI 1.30*** (1.10 to 1.49) 0.96*** (0.78 to 1.14) 0.91*** (0.74 to 1.09) 0.22* (0.02 to 0.42) GHQ Stable low 0.00 Reference 0.00 Reference 0.00 Reference 0.00 Reference Low→high 0.73*** (0.40 to 1.06) 0.58*** (0.30 to 0.86) 0.60*** (0.32 to 0.88) 0.28* (0.01 to 0.54) High→low 0.67*** (0.38 to 0.97) 0.54*** (0.27 to 0.82) 0.52*** (0.25 to 0.78) 0.27 (0.00 to 0.54) Stable high 1.59*** (1.27 to 1.91) 1.41*** (1.13 to 1.70) 1.41*** (1.15 to 1.68) 0.91*** (0.64 to 1.18) Model 0: Baseline model adjusted for Wave 1 gender, age and age2. Model 1: M0+Wave 1 controls (ethnicity, UK born, marital status, job status, education, social class, region). Model 2: M1+Wave 1 mediators (urban/rural, car access, mobile phone ownership, internet use). Model 3: M2+other Wave 1-2 health transition measures. *p<0.05, **p<0.01,***p<0.001. GHQ, 12 item General Health Questionnaire; LLTI, limiting long-term illness/disability; SEI, Social Exclusion Index; SRH, self-rated health. Model 3: M2+other Wave 1-2 health transition measures. *p<0.05, **p<0.01,***p<0.001. DISCUSSION h d d Once data are avail- able, it should be possible to elucidate the longer term dynamics hinted at by the observed differences in SEI scores across 1-year health transition categories. Somewhat surprisingly, there was no evidence that urban/rural location, car access, mobile phone ownership or internet use explained the prior health with later SEI relationship, even though both health and SEI were related to each of these potential mediators. There are a number of other mechanisms that might explain the relationship between health and social exclusion that could also be amenable to policy intervention. Examples include the material and financial consequences of poor health,5 28 37 discrimination38 and envir- onmental factors beyond those considered here.5 28 38 Our study has some distinct strengths: we used data from a large contemporary panel study, which meant that we were able to take advantage of the longitudinal design to investigate the relationship between health and social exclusion unfolding over time; we tested a new analytical framework for our analysis; and we considered multiple domains of health and social exclu- sion. On the other hand, some limitations must be acknowl- edged. First, as in all longitudinal studies, there were missing data which may have affected our results. The vast majority of missing data was for the GHQ-12 scores as these were com- pleted mainly as part of a self-completion module. We repeated the analyses for those with complete data over Waves 1–4 (not shown); the substantive findings were unchanged but lacked pre- cision due to the smaller sample size, so we present results using complete cases for each research question. We used longitudinal weights to account for dropout. Any bias introduced by non- response is likely to have underestimated effects. Second, the SEI index is specific to Understanding Society and of necessity its construction was limited by the data available, although many government departments and third sector agencies rely on these data for evidence-based policy development. Nevertheless, the SEI may not have fully captured all dimensions of the sub- domains. Finally, Walsh et als’ framework that guided our ana- lysis was conceptualised for a specific rural context and may not generalise to the UK population. The evidence for effect modification also suggests points for intervention. The role of car access suggests that alternatives to the car, such as improved public transport and taxi schemes for the elderly, might also be able to prevent social exclusion. DISCUSSION h d d GHQ, 12 item General Health Questionnaire; LLTI, limiting long-term illness/disability; SEI, Social Exclusion Index; SRH, self-rated health. *p<0.05, **p<0.01,***p<0.001. GHQ, 12 item General Health Questionnaire; LLTI, limiting long-term illness/disability; SEI, Social Exclusion Index; SRH, self-rated health. p<0.05, p<0.01, p<0.001. GHQ, 12 item General Health Questionnaire; LLTI, limiting long-term illness/disability; SEI, Social Exclusion Index; SRH, self-rated health. Figure 1 Modification of relationship between health transitions Wave 1-2 and the Social Exclusion Index in Wave 2/3 by access to car(s) in Wave 1. LLTI, limited long-term illness/disability. Figure 1 Modification of relationship between health transitions Wave 1-2 and the Social Exclusion Index in Wave 2/3 by access to car(s) in Wave 1. LLTI, limited long-term illness/disability. Sacker A, et al. J Epidemiol Community Health 2017;0:1–10. doi:10.1136/jech-2016-208037 5 Research report Research report Figure 2 Modification of relationship between health transitions Wave 1-2 and the Social Exclusion Index in Wave 2/3 by technology use in Wave 1. LLTI, limited long-term illness/disability. Research report Figure 2 Modification of relationship between health transitions Wave 1-2 and the Social Exclusion Index in Wave 2/3 by technology use in Wave 1. LLTI, limited long-term illness/disability. whose SRH improved might also suggest that those in poor health were seeking information online. existing knowledge on social exclusion in older age. Consistent with expectations, we found that poor health predicted social exclusion 1–2 years later. Given the health status at baseline, we also found that social exclusion predicted later declines in health. Finally, we identified use of a car, mobile phone and the internet as factors that might support older adults in poor health and help break the downward spiral in well-being. Civic participation was the subdomain most strongly and con- sistently associated with health, both as an outcome of health transitions and as a predictor of subsequent health change. That there is a bidirectional association is consistent with findings from Europe,13 but our findings go further in showing the dom- inance of civic participation over service provision and access and social relations and resources. Another study found that over 20% of Canadian seniors wanted to be more involved in social activities,40 highlighting the extent of need in this area. We failed to find much evidence that improvements in health reduced social exclusion in the short term. DISCUSSION h d d doi:10.1136/jech-2016-208037 6 Research report Table 3 Weighted per cent (95% CI) in poor health at Wave 4 by explanatory factors (N=4244) Poor SRH LLTI High GHQ Wave 2/3 SEI 0—5 23.77 (21.99 to 25.66) 34.03 (31.98 to 6—10 47.64*** (44.31 to 50.98) 51.51*** (48.33 to 11—15 72.22*** (44.31 to 50.98) 78.67*** (63.76 to Wave 1 Gender Male 31.58 (29.24 to 34.01) 37.72 (35.20 to Female 32.04 (29.76 to 34.41) 42.05* (39.70 to Age (years) <75 25.56 (23.85 to 27.34) 35.38 (33.51 to ≥75 30.30*** (27.09 to 33.72) 50.37*** (46.74 to Ethnicity White 31.60** (29.89 to 33.37) 40.14 (38.33 to Non-white 41.29 (34.72 to 48.19) 36.82 (29.65 to Country of birth UK 31.69 (29.94 to 33.50) 40.29 (38.43 to Elsewhere 34.10 (28.75 to 39.89) 36.21 (30.71 to Education Degree 20.15 (16.79 to 23.99) 33.40 (29.13 to Other higher 22.36 (18.11 to 27.27) 34.47 (29.61 to A level 31.89*** (27.55 to 36.58) 35.40 (30.89 to GCSE 27.11* (22.94 to 31.73) 32.69 (28.57 to Other 32.74*** (29.08 to 36.63) 42.68** (38.50 to None 41.05*** (37.75 to 44.43) 47.77*** (44.44 to Wave 3 Poor SRH No 13.01 (11.62 to 14.54) 26.01 (24.07 to Yes 75.89*** (73.10 to 78.48) 72.98*** (69.92 to LLTI No 12.02 (10.31 to 13.97) 13.92 (12.14 to Yes 47.68*** (45.22 to 50.15) 60.98*** (58.67 to High GHQ No 27.99 (26.25 to 29.79) 36.33 (34.47 to Yes 60.16*** (55.13 to 64.98) 67.59*** (62.86 to Marital status Married 28.18 (26.12 to 30.33) 35.90 (33.64 to Living as a couple 22.53 (14.31 to 33.64) 41.70 (34.37 to 23.77 (21.99 to 25.66) 34.03 (31.98 to 36.14) 13.53 (12.03 to 15.17) 47.64*** (44.31 to 50.98) 51.51*** (48.33 to 54.67) 21.92*** (19.27 to 24.83) 72.22*** (44.31 to 50.98) 78.67*** (63.76 to 88.54) 42.68*** (27.24 to 59.69) 31.58 (29.24 to 34.01) 37.72 (35.20 to 40.30) 12.45 (10.89 to 14.20) 32.04 (29.76 to 34.41) 42.05* (39.70 to 44.43) 19.99*** (18.04 to 22.09) 25.56 (23.85 to 27.34) 35.38 (33.51 to 37.31) 14.85 (13.44 to 16.37) 30.30*** (27.09 to 33.72) 50.37*** (46.74 to 54.00) 20.26** (17.41 to 23.44) 31.60** (29.89 to 33.37) 40.14 (38.33 to 41.98) 16.50 (15.12 to 17.98) 41.29 (34.72 to 48.19) 36.82 (29.65 to 44.63) 18.01 (13.08 to 24.28) 31.69 (29.94 to 33.50) 40.29 (38.43 to 42.18) 16.49 (15.07 to 18.00) 34.10 (28.75 to 39.89) 36.21 (30.71 to 42.10) 17.42 (13.97 to 21.50) 20.15 (16.79 to 23.99) 33.40 (29.13 to 37.96) 13.57 (10.83 to 16.88) 22.36 (18.11 to 27.27) 34.47 (29.61 to 39.67) 13.87 (10.65 to 17.87) 31.89*** (27.55 to 36.58) 35.40 (30.89 to 40.19) 14.57 (11.54 to 18.23) 27.11* (22.94 to 31.73) 32.69 (28.57 to 37.10) 16.48 (13.19 to 20.41) 32.74*** (29.08 to 36.63) 42.68** (38.50 to 46.97) 17.28 (14.54 to 20.42) 41.05*** (37.75 to 44.43) 47.77*** (44.44 to 51.11) 18.96* (16.51 to 21.68) 13.01 (11.62 to 14.54) 26.01 (24.07 to 28.05) 10.70 (9.51 to 12.03) 75.89*** (73.10 to 78.48) 72.98*** (69.92 to 75.83) 30.21*** (27.15 to 33.45) 12.02 (10.31 to 13.97) 13.92 (12.14 to 15.91) 10.64 (9.19 to 12.29) 47.68*** (45.22 to 50.15) 60.98*** (58.67 to 63.25) 21.26*** (19.32 to 23.33) 27.99 (26.25 to 29.79) 36.33 (34.47 to 38.24) 10.88 (9.75 to 12.13) 60.16*** (55.13 to 64.98) 67.59*** (62.86 to 71.98) 58.29*** (53.02 to 63.38) 28.18 (26.12 to 30.33) 35.90 (33.64 to 38.23) 15.27 (13.56 to 17.15) 22.53 (14.31 to 33.64) 41.70 (34.37 to 49.42) 11.15 (5.74 to 20.55) Continued 23.77 (21.99 to 25.66) 34.03 (31.98 to 36.14) 47.64*** (44.31 to 50.98) 51.51*** (48.33 to 54.67) 72.22*** (44.31 to 50.98) 78.67*** (63.76 to 88.54) 31.58 (29.24 to 34.01) 37.72 (35.20 to 40.30) 32.04 (29.76 to 34.41) 42.05* (39.70 to 44.43) 25.56 (23.85 to 27.34) 35.38 (33.51 to 37.31) 30.30*** (27.09 to 33.72) 50.37*** (46.74 to 54.00) 31.60** (29.89 to 33.37) 40.14 (38.33 to 41.98) 41.29 (34.72 to 48.19) 36.82 (29.65 to 44.63) 31.69 (29.94 to 33.50) 40.29 (38.43 to 42.18) 34.10 (28.75 to 39.89) 36.21 (30.71 to 42.10) 20.15 (16.79 to 23.99) 33.40 (29.13 to 37.96) 22.36 (18.11 to 27.27) 34.47 (29.61 to 39.67) 31.89*** (27.55 to 36.58) 35.40 (30.89 to 40.19) 27.11* (22.94 to 31.73) 32.69 (28.57 to 37.10) 32.74*** (29.08 to 36.63) 42.68** (38.50 to 46.97) 41.05*** (37.75 to 44.43) 47.77*** (44.44 to 51.11) 13.01 (11.62 to 14.54) 26.01 (24.07 to 28.05) 75.89*** (73.10 to 78.48) 72.98*** (69.92 to 75.83) 12.02 (10.31 to 13.97) 13.92 (12.14 to 15.91) 47.68*** (45.22 to 50.15) 60.98*** (58.67 to 63.25) 27.99 (26.25 to 29.79) 36.33 (34.47 to 38.24) 60.16*** (55.13 to 64.98) 67.59*** (62.86 to 71.98) 28.18 (26.12 to 30.33) 35.90 (33.64 to 38.23) 22.53 (14.31 to 33.64) 41.70 (34.37 to 49.42) 33.40 (29.13 to 37.96) Sacker A, et al. DISCUSSION h d d The import- ance of internet use and technology highlights the need for further research to understand which capabilities constrain older adults’ use of these forms of communication. There was no modification of the relationship between GHQ and social exclusion. On the one hand, this is unsurprising since symptoms of depression and anxiety include diminished interest and loss of pleasure in social activities.39 On the other, this distinction between physical and psychological health may provide a clue that it is physical and cog- nitive capabilities, rather than psychosocial capabilities, driving use of the internet and technology. The finding that occasional inter- net use was more beneficial than regular use for respondents Sacker A, et al. J Epidemiol Community Health 2017;0:1–10. DISCUSSION h d d J Epidemiol Community Health 2017;0:1–10. doi:10.1136/jech-2016-208037 Research report Table 3 Continued Poor SRH LLTI High GHQ Single never married 37.58* (30.21 to 45.57) 48.60 (43.32 to 53.92) 17.67 (13.42 to 22.90) Separated or divorced 37.42** (32.49 to 42.63) 47.79*** (43.93 to 51.68) 18.68 (14.88 to 23.19) Widowed 38.74*** (35.11 to 42.51) 33.08*** (23.75 to 43.96) 19.28* (16.28 to 22.67) Job status In work 14.27 (10.77 to 18.66) 20.16 (15.92 to 25.18) 7.83 (5.49 to 11.05) Not in work 33.51*** (31.72 to 35.36) 41.97*** (40.07 to 43.90) 17.37*** (15.93 to 18.92) Social class Man and Prof 24.81 (22.14 to 27.69) 35.46 (32.39 to 38.64) 14.18 (12.16 to 16.48) Intermediate 29.39 (25.32 to 33.82) 39.42 (34.98 to 44.04) 19.64* (16.13 to 23.70) Small emp. and own acc. 27.84*** (22.38 to 34.06) 37.76 (32.10 to 43.77) 18.68 (14.33 to 23.99) Lower supervisory and tech. DISCUSSION h d d 40.50*** (34.17 to 47.16) 50.10*** (43.66 to 56.54) 16.01 (11.70 to 21.53) Semiroutine and routine 38.45 (35.51 to 41.48) 43.34** (40.28 to 46.44) 17.35* (15.17 to 19.76) Never had a job 36.19** (28.57 to 44.58) 39.97 (32.02 to 48.48) 14.58 (10.01 to 20.74) Region South East 26.19 (21.88 to 31.01) 37.72 (32.99 to 42.69) 15.98 (12.25 to 20.57) North East 35.58* (27.83 to 44.17) 42.04 (32.74 to 51.94) 17.93 (11.45 to 26.95) North West 32.78 (27.43 to 38.62) 40.99 (35.74 to 46.45) 14.75 (11.30 to 19.04) Yorkshire and the Humber 34.17* (28.38 to 40.47) 39.79 (33.81 to 46.10) 15.93 (11.83 to 21.13) East Midlands 33.77 (27.71 to 40.42) 41.50 (35.01 to 48.30) 17.60 (13.50 to 22.63) West Midlands 31.28 (26.80 to 36.14) 36.87 (31.51 to 42.57) 21.96 (17.13 to 27.69) East of England 31.53 (26.78 to 36.70) 42.43 (37.36 to 47.67) 15.19 (11.85 to 19.28) London 29.80 (24.32 to 35.93) 33.31 (28.16 to 38.89) 21.50 (17.30 to 26.38) South West 28.12 (23.24 to 33.57) 40.39 (34.54 to 46.52) 12.37 (9.28 to 16.32) Wales 42.15*** (36.00 to 48.56) 47.43* (39.52 to 55.46) 17.30 (11.50 to 25.18) Scotland 35.40* (29.58 to 41.68) 40.87 (35.73 to 46.22) 17.51 (13.20 to 22.84) Northern Ireland 33.43 (23.87 to 44.59) 41.40 (30.48 to 53.23) 12.85 (7.13 to 22.05) Area type Urban 33.86 (31.86 to 35.92) 42.00 (39.91 to 44.13) 17.98 (16.32 to 19.77) Rural 27.19 (24.15 to 30.46) 35.66** (32.43 to 39.02) 13.26** (11.15 to 15.71) Car access Research report LLTI 48.60 (43.32 to 53.92) 17.67 (13.42 to 22.90) 47.79*** (43.93 to 51.68) 18.68 (14.88 to 23.19) 33.08*** (23.75 to 43.96) 19.28* (16.28 to 22.67) 20.16 (15.92 to 25.18) 7.83 (5.49 to 11.05) 41.97*** (40.07 to 43.90) 17.37*** (15.93 to 18.92) 35.46 (32.39 to 38.64) 14.18 (12.16 to 16.48) 39.42 (34.98 to 44.04) 19.64* (16.13 to 23.70) 37.76 (32.10 to 43.77) 18.68 (14.33 to 23.99) 50.10*** (43.66 to 56.54) 16.01 (11.70 to 21.53) 43.34** (40.28 to 46.44) 17.35* (15.17 to 19.76) 39.97 (32.02 to 48.48) 14.58 (10.01 to 20.74) 37.72 (32.99 to 42.69) 15.98 (12.25 to 20.57) 42.04 (32.74 to 51.94) 17.93 (11.45 to 26.95) 40.99 (35.74 to 46.45) 14.75 (11.30 to 19.04) 39.79 (33.81 to 46.10) 15.93 (11.83 to 21.13) 41.50 (35.01 to 48.30) 17.60 (13.50 to 22.63) 36.87 (31.51 to 42.57) 21.96 (17.13 to 27.69) 42.43 (37.36 to 47.67) 15.19 (11.85 to 19.28) 33.31 (28.16 to 38.89) 21.50 (17.30 to 26.38) 40.39 (34.54 to 46.52) 12.37 (9.28 to 16.32) 47.43* (39.52 to 55.46) 17.30 (11.50 to 25.18) 40.87 (35.73 to 46.22) 17.51 (13.20 to 22.84) 41.40 (30.48 to 53.23) 12.85 (7.13 to 22.05) 42.00 (39.91 to 44.13) 17.98 (16.32 to 19.77) 35.66** (32.43 to 39.02) 13.26** (11.15 to 15.71) 36.67 (34.73 to 38.64) 15.10 (13.66 to 16.67) 52.52*** (48.55 to 56.46) 21.80*** (18.81 to 25.13) 37.72 (35.85 to 39.62) 15.88 (14.46 to 17.42) 50.45*** (45.95 to 54.94) 19.43* (16.17 to 23.17) Continued 37.58* (30.21 to 45.57) 48.60 (43.32 to 53.92) 17.67 (13.42 to 22.90) 37.42** (32.49 to 42.63) 47.79*** (43.93 to 51.68) 18.68 (14.88 to 23.19) 38.74*** (35.11 to 42.51) 33.08*** (23.75 to 43.96) 19.28* (16.28 to 22.67) 14.27 (10.77 to 18.66) 20.16 (15.92 to 25.18) 7.83 (5.49 to 11.05) 33.51*** (31.72 to 35.36) 41.97*** (40.07 to 43.90) 17.37*** (15.93 to 18.92) 24.81 (22.14 to 27.69) 35.46 (32.39 to 38.64) 14.18 (12.16 to 16.48) 29.39 (25.32 to 33.82) 39.42 (34.98 to 44.04) 19.64* (16.13 to 23.70) 27.84*** (22.38 to 34.06) 37.76 (32.10 to 43.77) 18.68 (14.33 to 23.99) 40.50*** (34.17 to 47.16) 50.10*** (43.66 to 56.54) 16.01 (11.70 to 21.53) 38.45 (35.51 to 41.48) 43.34** (40.28 to 46.44) 17.35* (15.17 to 19.76) 36.19** (28.57 to 44.58) 39.97 (32.02 to 48.48) 14.58 (10.01 to 20.74) 26.19 (21.88 to 31.01) 37.72 (32.99 to 42.69) 15.98 (12.25 to 20.57) 35.58* (27.83 to 44.17) 42.04 (32.74 to 51.94) 17.93 (11.45 to 26.95) 32.78 (27.43 to 38.62) 40.99 (35.74 to 46.45) 14.75 (11.30 to 19.04) 34.17* (28.38 to 40.47) 39.79 (33.81 to 46.10) 15.93 (11.83 to 21.13) 33.77 (27.71 to 40.42) 41.50 (35.01 to 48.30) 17.60 (13.50 to 22.63) 31.28 (26.80 to 36.14) 36.87 (31.51 to 42.57) 21.96 (17.13 to 27.69) 31.53 (26.78 to 36.70) 42.43 (37.36 to 47.67) 15.19 (11.85 to 19.28) 29.80 (24.32 to 35.93) 33.31 (28.16 to 38.89) 21.50 (17.30 to 26.38) 28.12 (23.24 to 33.57) 40.39 (34.54 to 46.52) 12.37 (9.28 to 16.32) 42.15*** (36.00 to 48.56) 47.43* (39.52 to 55.46) 17.30 (11.50 to 25.18) 35.40* (29.58 to 41.68) 40.87 (35.73 to 46.22) 17.51 (13.20 to 22.84) 33.43 (23.87 to 44.59) 41.40 (30.48 to 53.23) 12.85 (7.13 to 22.05) 33.86 (31.86 to 35.92) 42.00 (39.91 to 44.13) 17.98 (16.32 to 19.77) 27.19 (24.15 to 30.46) 35.66** (32.43 to 39.02) 13.26** (11.15 to 15.71) 28.78 (26.93 to 30.70) 36.67 (34.73 to 38.64) 15.10 (13.66 to 16.67) 43.00 (39.14 to 46.94) 52.52*** (48.55 to 56.46) 21.80*** (18.81 to 25.13) 29.79 (28.03 to 31.61) 37.72 (35.85 to 39.62) 15.88 (14.46 to 17.42) 40.83 (36.50 to 45.30) 50.45*** (45.95 to 54.94) 19.43* (16.17 to 23.17) Continued 37.58* (30.21 to 45.57) 37.42** (32.49 to 42.63) 38.74*** (35.11 to 42.51) 14.27 (10.77 to 18.66) 33.51*** (31.72 to 35.36) 24.81 (22.14 to 27.69) 29.39 (25.32 to 33.82) 27.84*** (22.38 to 34.06) 40.50*** (34.17 to 47.16) 38.45 (35.51 to 41.48) 36.19** (28.57 to 44.58) 26.19 (21.88 to 31.01) 35.58* (27.83 to 44.17) 32.78 (27.43 to 38.62) 34.17* (28.38 to 40.47) 33.77 (27.71 to 40.42) 31.28 (26.80 to 36.14) 31.53 (26.78 to 36.70) 29.80 (24.32 to 35.93) 28.12 (23.24 to 33.57) 42.15*** (36.00 to 48.56) 35.40* (29.58 to 41.68) 33.43 (23.87 to 44.59) 33.86 (31.86 to 35.92) 27.19 (24.15 to 30.46) 28.78 (26.93 to 30.70) 43.00 (39.14 to 46.94) 29.79 (28.03 to 31.61) 40.83 (36.50 to 45.30) 37.58* (30.21 to 45.57) 48.60 (43.32 to 53.92) 37.42** (32.49 to 42.63) 47.79*** (43.93 to 51.68) 38.74*** (35.11 to 42.51) 33.08*** (23.75 to 43.96) 14.27 (10.77 to 18.66) 20.16 (15.92 to 25.18) 33.51*** (31.72 to 35.36) 41.97*** (40.07 to 43.90) 24.81 (22.14 to 27.69) 35.46 (32.39 to 38.64) 29.39 (25.32 to 33.82) 39.42 (34.98 to 44.04) 27.84*** (22.38 to 34.06) 37.76 (32.10 to 43.77) 40.50*** (34.17 to 47.16) 50.10*** (43.66 to 56.54) 38.45 (35.51 to 41.48) 43.34** (40.28 to 46.44) 36.19** (28.57 to 44.58) 39.97 (32.02 to 48.48) 26.19 (21.88 to 31.01) 37.72 (32.99 to 42.69) 35.58* (27.83 to 44.17) 42.04 (32.74 to 51.94) 32.78 (27.43 to 38.62) 40.99 (35.74 to 46.45) 34.17* (28.38 to 40.47) 39.79 (33.81 to 46.10) 33.77 (27.71 to 40.42) 41.50 (35.01 to 48.30) 31.28 (26.80 to 36.14) 36.87 (31.51 to 42.57) 31.53 (26.78 to 36.70) 42.43 (37.36 to 47.67) 29.80 (24.32 to 35.93) 33.31 (28.16 to 38.89) 28.12 (23.24 to 33.57) 40.39 (34.54 to 46.52) 42.15*** (36.00 to 48.56) 47.43* (39.52 to 55.46) 35.40* (29.58 to 41.68) 40.87 (35.73 to 46.22) 33.43 (23.87 to 44.59) 41.40 (30.48 to 53.23) 33.86 (31.86 to 35.92) 42.00 (39.91 to 44.13) 27.19 (24.15 to 30.46) 35.66** (32.43 to 39.02) 28.78 (26.93 to 30.70) 36.67 (34.73 to 38.64) 43.00 (39.14 to 46.94) 52.52*** (48.55 to 56.46) 29.79 (28.03 to 31.61) 37.72 (35.85 to 39.62) 40.83 (36.50 to 45.30) 50.45*** (45.95 to 54.94) 39.79 (33.81 to 46.10) 41.50 (35.01 to 48.30) Sacker A, et al. Sacker A, et al. J Epidemiol Community Health 2017;0:1–10. doi:10.1136/jech-2016-208037 REFERENCES 1 MacLeod C, Ross A, Windle G, et al. Measuring Later Life Social Exclusion in Understanding Society. 2016, ISBN 978-0-9527377-7-3. https:// www.ucl.ac.uk/icls/ publications/working-papers (accessed 5 Jul 2016). 1 MacLeod C, Ross A, Windle G, et al. Measuring Later Life Social Exclusion in Understanding Society. 2016, ISBN 978-0-9527377-7-3. https:// www.ucl.ac.uk/icls/ publications/working-papers (accessed 5 Jul 2016). Among non-community living older people in the UK, poor health is associated with greater social exclusion and, in turn, social exclusion is linked to health decline. Use of a car, mobile phone and the internet are factors that protected older adults in poor health from social exclusion. Designing age-friendly hardware and software might have public health benefits. 2 European Commission. The European Platform Against Poverty and Social Exclusion: a European Framework for Social and Territorial Cohesion. 2011 12 August 2011. http://ec.europa.eu/social/main.jsp?catId=738&langId=en&pubId=6028&type= 2&furtherPubs=yes (accessed 5 Jul 2016). 3 Bradshaw J, Kemp P, Baldwin S, et al. The drivers of social exclusion: a review of the literature. London: Social Exclusion Unit, Office of the Deputy Prime Minister, 2004. 4 Levitas R, Pantazis C, Fahmy E, et al. The multi-dimensional analysis of social exclusion. Bristol: Department of Sociology and School for Social Policy Townsend Centre for the International Study of Poverty and Bristol Institute for Public Affairs, University of Bristol, Bristol, 2007. Acknowledgements Understanding Society is an initiative by the Economic and Social Research Council, with scientific leadership by the Institute for Social and Economic Research, University of Essex and survey delivery by the National Centre for Social Research and TNS BRMB, and survey management by the UK Data Archive. y 5 Barnes M, Blom A, Cox K, et al. The social exclusion of older people. Evidence from the first wave of the English Longitudinal Study of Ageing (ELSA): Final report: London, 2006. 6 Burchardt T. Being and becoming: social exclusion and the onset of disability. CASE reports: centre for analysis of social exclusion, LSE, 2003. Contributors AS, AR, GW, CAM and GN developed the idea and contributed to the study design. AR and AS carried out the analysis and AS wrote the manuscript. AR, GW, CAM and GN commented on the paper and have seen and accepted the final version. AS takes responsibility for the overall content of the paper. y 7 Kneale D. Is social exclusion still important for older people? London. 20 8 Aldridge H, Parekh A, MacInnes T, et al. What is already known on this subject Open Access This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/ by/4.0/ Poor health and social exclusion cluster in older adults but the causal mechanism is less clear. Poor health and social exclusion cluster in older adults but the causal mechanism is less clear. What this study adds DISCUSSION h d d J Epidemiol Community Health 2017;0:1–10. doi:10.1136/jech-2016-208037 Sacker A, et al. J Epidemiol Community Health 2017;0:1–10. doi:10.1136/jech-2016-208037 8 Table 3 Continued Poor SRH LLTI High GHQ Internet use Often 22.78 (20.70 to 25.00) 32.16 (29.86 to 34.55) 13.73 (12.07 to 15.57) Sometimes 29.52* (24.88 to 34.63) 37.93* (32.74 to 43.41) 13.65 (10.39 to 17.72) Never 40.20*** (37.53 to 42.94) 47.41*** (44.64 to 50.19) 19.60*** (17.51 to 21.87) Logistic regression models test for difference in proportions: * p<0.05, ** p<0.01,*** p<0.001. GHQ, 12 item General Health Questionnaire; LLTI, limited long-term illness/disability; poor SRH, fair/poor self-rated health; SEI, Social Exclusion Index. Research report Research report GHQ, 12 item General Health Questionnaire; LLTI, limited long-term illness/disability; poor SRH, fair/poor self-rated health; SEI, Social Exclusion Index. Table 4 Logistic regression estimates (ORs and 95% CIs) for poor health outcomes in Wave 4 regressed on the Social Exclusion Index at Waves 2/3 Model 0 Model 1 Model 2 Poor SRH 1.16*** (1.11 to 1.21) 1.14*** (1.09 to 1.20) 1.15*** (1.09 to 1.21) LLTI 1.08*** (1.04 to 1.12) 1.08** (1.03 to 1.13) 1.07** (1.02 to 1.12) High GHQ 1.04 (0.99 to 1.09) 1.07** (1.02 to 1.13) 1.07* (1.02 to 1.13) Model 0: Baseline model adjusted for Wave 3 SRH, LLTI, GHQ, gender, age and age2. Model 1: M0+Wave 1 controls (ethnicity, UK born, education)+Wave 3 controls (marital status, job status, social class, region). Model 2: M1+Wave 3 mediators (urban/rural, car access, mobile phone ownership, internet use). * p<0.05, ** p<0.01, *** p<0.001. GHQ, 12 item General Health Questionnaire; LLTI, limited long-term illness/disability; SRH, self-rated health. imates (ORs and 95% CIs) for poor health outcomes in Wave 4 regressed on the Social Exclusion Index at Waves Funding This research was supported by Centre grants from the Economic and Social Research Council (AS, AR and GN: grant number ES/J019119/1; GW and CAM: grant number RES-060-25-0060). Competing interests None declared. Ethics approval The Understanding Society study was approved by the University of Essex Ethics Committee and the National Research Ethics Service. No additional ethical approval was necessary for this secondary data analysis. CONCLUSION There is synergy between health and social exclusion among older people living independently in the UK. Our findings suggest that it might be more effective to target the prior health to exclusion relationship than the exclusion to later health rela- tionship. Designing age-friendly hardware and software might support social inclusion in later life. There is synergy between health and social exclusion among older people living independently in the UK. Our findings suggest that it might be more effective to target the prior health to exclusion relationship than the exclusion to later health rela- tionship. Designing age-friendly hardware and software might support social inclusion in later life. Competing interests None declared. Provenance and peer review Not commissioned; externally peer reviewed. Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement A working paper is referenced in the paper that gives detailed instructions on how we constructed our social exclusion indices from the Understanding Society data that are publicly available from the UK Data Service. Research report Research report 25 Arber S. Gender, marital status, and ageing: linking material, health, and social resources. J Ageing Stud 2004;18:91–108. 9 Hrast MF, Hlebec V, Kavcic M. The social exclusion of the elderly: a mixed-methods study in Slovenia. Sociologicky Casopis-Czech Sociological Review 2012;48:1051–74. 9 Hrast MF, Hlebec V, Kavcic M. The social exclusion of the elderly: a mixed methods study in Slovenia. Sociologicky Casopis-Czech Sociological Review 2012;48:1051–74. g g 26 McNiece R, Majeed A. Socioeconomic differences in general practice consultation rates in patients aged 65 and over: prospective cohort study. BMJ 1999; 319:26–8. y g y g 10 Ogg J. Social exclusion and insecurity among older Europeans: the influence of welfare regimes. Ageing Soc 2005;25:69–90. g g g 11 Grundy E, Sloggett A. Health inequalities in the older population: the role of personal capital, social resources and socio-economic circumstances. Soc Sci Med 2003;56:935–47. 27 Ingleby D. Ethnicity, migration and the ‘Social Determinants of Health’ agenda. Psychosoc Interv 2012;21:331–41. 12 Hawton A, Green C, Dickens AP, et al. The impact of social isolation on the health status and health-related quality of life of older people. Qual Life Res 2011;20:57–67. y 28 Scharf T, Phillipson C, Smith A. Social exclusion of older people in deprived urban communities of England. European J Ageing 2005;2:76–87. 29 Levitas R. The concept of social exclusion and the new Durkheimian hegemony. Crit Soc Policy 1996;16:5–20. 13 Leone T, Hessel P. The effect of social participation on the subjective and objective health status of the over-fifties: evidence from SHARE. Ageing oc 2016;36:968–87. 30 Grundy E, Holt G. The socioeconomic status of older adults: how should we measure it in studies of health inequalities?. J Epidemiol Community Health 2001;55:895–904. g g 14 Burholt V, Scharf T. Poor health and loneliness in later life: the role of depressive symptoms, social resources, and rural environments. J Gerontol B Psychol Sci Soc Sci 2014;69:311–24. 31 Scharf T, Phillipson C, Kingston P, et al. Social exclusion and older people: exploring the connections. Educ Ageing 2001;16:303–20. 15 World Health Organisation. Strategy and action plan for healthy ageing in Europe, 2012–2020. (Report No. EUR/RC62/10 Rev.1). Copenhagen, Denmark: World Health Organisation Regional Office for Europe, 2012. 32 Shucksmith M, Chapman P. Rural development and social exclusion. Sociologia Ruralis 1998;38:225–42. g g 16 World Health Organisation. Active ageing: A policy framework. (Report No. WHO/ NMH/NPH02.8). Geneva, Switzerland: World Health Organisation, 2002. REFERENCES Monitoring poverty and social exclusion 2011. York, UK: The Joseph Rowntree Foundation, 2011. Sacker A, et al. J Epidemiol Community Health 2017;0:1–10. doi:10.1136/jech-2016-208037 Sacker A, et al. J Epidemiol Community Health 2017;0:1–10. doi:10.1136/jech-2016-208037 9 Research report 33 Myck M, Najsztub M, Oczkowska M. Measuring social deprivation and social exclusion. In: Börsch-Supan A, Kneip T, Litwin H, et al., eds. Ageing in Europe— supporting policies for an inclusive society. Berlin: De Gruyter, 2015: pp 67–78. 17 University of Essex. Institute for Social and Economic Research and NatCen Social Research, Understanding Society: Waves 1-4 2009-2013, [computer file]. 4th edn. Colchester, Essex: UK Data Archive [distributor], 2014. , SN: 6676, . 34 Lucas K. Transport and social exclusion: where are we now? Transport Policy 2012;20:105–13. Colchester, Essex: UK Data Archive [distributor], 2014. , SN: 6676 18 Buck N, McFall S. Understanding Society: design overview. Longitudinal and Life Course Stud 2012;3:5–17. 35 Shergold I, Parkhurst G. Transport-related social exclusion among older people in rural Southwest England and Wales. J Rural Stud 2012;28:412–21. 19 Walsh K, O’Shea E, Scharf T. Social exclusion and ageing in diverse rural communities: findings from a cross-border study in Ireland and Northern Ireland. February 2012 ed. Galway, Ireland: Irish Centre for Social Gerontology, 2012. 36 Haddon L. Social exclusion and information and communication technologies: lessons from studies of single parents and the young elderly. New Media Society 2000;2:387–406. 20 Royston P. Multiple imputation of missing values. Stata J 2004;4:227–41. 37 Najsztub M, Bonfatti A, Duda D. Material and social deprivation in the macroeconomic context. In: Börsch-Supan A, Kneip T, Litwin H, et al. eds. Ageing in Europe—supporting policies for an inclusive society. Walter de Gruyter, 2015: 79–89. 21 Royston P. Multiple imputation of missing values: update of ice. Stata J 2005;5:527–36. 22 Royston P. Multiple imputation of missing values: further update of ice, with an emphasis on categorical variables. Stata J 2009;9:466–77. 38 Guberman N, Lavoie JP. Equipe Vies: Framework on Social Exclusion. Montréal, QC: Montréal, QC., Centre de recherche et d’expertise de gérontologie sociale – CAU/CSSS Cavendish, 2004. p g 23 Goldberg D, Williams P. A user’s guide to the General Health Questionnaire. Windsor, UK: NFER-Nelson, 1988. 39 Alexopoulos GS. Depression in the elderly. Lancet 2005;365:1961–70. 39 Alexopoulos GS. Depression in the elderly. Lancet 2005;365:1961–70. 24 Goldberg DP, Gater R, Sartorius N, et al. The validity of two versions of the GHQ in the WHO study of mental illness in general healthcare. Psychol Med 1997;27:191–7. 1997/01/01. 40 Gilmour H. Social participation and the health and well-being of Canadian seniors. Health Rep 2012;23:23–32. 2013/01/30. 40 Gilmour H. Research report Social participation and the health and well-being of Canadian seniors. Health Rep 2012;23:23–32. 2013/01/30. 40 Gilmour H. Social participation and the hea Health Rep 2012;23:23–32. 2013/01/30. Sacker A, et al. J Epidemiol Community Health 2017;0:1–10. doi:10.1136/jech-2016-208037 10
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Detection of blaTEM Gene of Klebsiella pneumoniae Isolated from Swab of Food-Producing Animals in East Java
Tropical Animal Science Journal
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EFFENDI ET AL. / Trop p-ISSN 2615-787X e-ISSN 2615-790X Accredited by Directorate General of Research and Development Strengthening No: 36b/E/KPT/2016 EFFENDI ET AL. / Trop p-ISSN 2615-787X e-ISSN 2615-790X Accredited by Directorate General of Research and Development Strengthening No: 36b/E/KPT/2016 p-ISSN 2615-787X e-ISSN 2615-790X al Science Journal 41(3):174-178 Tropical Animal Science Journal, December 2018, 41(3):174-178 DOI: https://doi.org/10.5398/tasj.2018.41.3.174 Available online at http://journal.ipb.ac.id/index.php/tasj al Science Journal 41(3):174-178 Tropical Animal Science Journal, December 2018, 41(3):174-178 DOI: https://doi.org/10.5398/tasj.2018.41.3.174 Available online at http://journal.ipb.ac.id/index.php/tasj Accredited by Directorate General of Research and Development Strengthening No: 36b/E/KPT/2016 INTRODUCTION lactamase. The beta-lactamase enzyme is firstly identified in Escherichia coli, which is encoded by the blaTEM gene. In addi­ tion, blaTEM gene was also currently found in K. pneumoniae. (Lalzampuia et al., 2014). Treatments of bacterial infections of beta-lactamase producers have so far involved the use of cephalosporins and aztreonam which also belong to the group of beta-lactam antibiotics. In fact, this drug can not kill lactamase producing bacteria as a results of it spread of its resistance spectrum, to penicillin, cephalosporins, and aztreonam so that it called as the Extended Spectrum Beta-Lactamase (ESBL) bacteria. The ability of ESBL strains to hydrolyze betalactam antibiotics is generally due to a number of mutations in the gene, and one of which is bla­ TEM gene. These mutations are generally found to be at the active site of the enzyme that leads to a higher enzymatic activity (Yuwono, 2011). The existance of ESBL strain in food-producing animals is reported by Overdevest et al. (2011). Klebsiella pneumoniae is a bacterium belonging to the genus of Klebsiella a member of the family of Enterobacteriaceae. The bacterium is a normal organ­ ism living in traktus digestivus so that it can be iso­ lated from animal or human feces (Susilo et al., 2004). K. pneumoniae is a Gram-negative, basil, nonmotile, and one of the important pathogenic bacteria. This species is the agents of various diseases, such as pneumonia, urinary tract infection, bakteremia, infection in wounds, and abscesses of the liver (Rahamathulla et al., 2016). K. pneumoniae pose a great impact on the health sector. In a report on global surveillance on antimicrobial resistance carried out by the World Health Organization, K. pneu­ moniae is one of nine bacteria concerned in resistance to antibiotics (WHO, 2014). K. pneumoniae was found to be capable of being resistant towards many of the third-generation of cephalosporin antibiotics especially cefotaxime, ceftazidine, and ceftriaxone (Yeh et al., 2007).f Animals can carry harmful bacteria in their intes­ tines. When antibiotics are given to animals, antibiotics kill most of the bacteria. However, resistant bacteria are survive and multiply. Food-producing animals have been known as reservoirs for ESBL-producing bacteria. Food-producing animals are capable of spread­ ing bacteria that are resistant to antibiotics through feces. Through feces, resistant bacteria contained in Each bacteria employs different mechanisms in causing resistance to antibiotics. Detection of blaTEM Gene of Klebsiella pneumoniae Isolated from Swab of Food- Producing Animals in East Java M. H. Effendia,*, I. G. Bintarib, E. B. Aksonoc, & I. P. Hermawanb aDepartment of Veterinary Public Health, Faculty of Veterinary Medicine, Airlangga University bStudent, Faculty of Veterinary Medicine, Airlangga University cDepartment of Basic Veterinary Medicine, Faculty of Veterinary Medicine, Airlangga University Jalan Mulyorejo, Kampus C Mulyorejo Surabaya 60115, Indonesia *Corresponding author: mheffendi@yahoo.com (Received 22-01-2018; Reviewed 06-04-2018; Accepted 03-08-2018) p gf y (Received 22-01-2018; Reviewed 06-04-2018; Accepted 03-08-2018) ABSTRACT Klebsiella pneumoniae is one of 9 bacteria resistance to antibiotics in concern. This research aimed to detect any gene of blaTEM in bacteria of the K. pneumoniae isolated from swab of food-producing ani­ mals. In this study, 195 swab samples were taken from 17 sampling locations. Samples obtained were cultivated on selective medium and had several tests including identification, antibiotic sensitivity test using Kirby-Bauer method against antibiotics of ampicillin, cefotaxime, amoxicillin, meropenem, and trimetrophrim-sulfamethoxazole, and followed by PCR test for detecting the gene that was re­ sponsible for the antibiotic resistances. The results showed that 10 out of 195 samples were found to be K. pneumonia, those were 4 samples originated from dairy cows (SP-S1, SP-S3, SP-B2, SP-G4), 2 samples originated from beef (SPT-K1, SPT-K2), 1 sample originated from chickens (A-W5), and 3 samples originated from fish (IN-P2, IN-P3, IN-S3). Most of isolates (9/10) were found to be resistant toward amoxicillin. These isolates were SP-S3, SP-B2, SP-G4, SPT-KI, SPT-K2, A-W5, IN-P2, IN-P3, and the IN-S3 and all of them also showed to be positive of blaTEM gene. It could be concluded that most of K. pneumoniae isolates from food animals harbour had Extended Spectrum Beta-Lactamase (ESBL) encoding gene. Keywords: antibiotic resistance, ESBL, food-producing animals, Klebsiella pneumonie, amoxicillin 174 December 2018 Sample Collection and Preparation DNA extraction by adding K. pneumoniae bacte­ ria was performed using QIAamp DNA mini kit 50 (Qiagen, USA) according to manufacturer protocol. Briefly, samples were added with 5 μL lyzozyme (5 mg/mL) enzyme and incubated for 30 min at 56ºC. Furthermore, the extracted DNA was diluted to 100 μL with a buffer kit. DNA solution used for PCR amplifica­ tion was as much as 1 μL. In this study 195 swab samples were obtained from 17 locations in East Java. Sampling was done by using swab aseptically from dairy cows, beef cattle, broiler, and tilapia in 17 sampling locations. The samples were directly analysed within 30-40 min or then tranfered to the lab for further analysis in a cool box.t As many as 195 samples of dairy cows, beef cattle, broilers, and tilapia were cultivated by taking 1 ose and then scrawled by streak plate technique method on se­ lective media of Mac Conkey and EMBA then incubated at 37˚C for 24 h. In Mac Conkey media, K. pneumoniae looks pink with colonies culture looks very mucoid and in EMBA media K. pneumoniae looks red to brick (Masruroh et al., 2016). Characterization of Isolates Pure bacterial isolates were identified based on morphological characters that included colony mor­ phology, cell morphology, and gram staining tests. Observation of colony morphology was based on the shape, color, and the edge of bacterial colonies. The morphological observations of bacterial cells include the shape and structure of bacterial cells. Furthermore, each isolate was biochemically characterized including carbohydrate fermentation test (glucose, lactose, man­ nitol, maltose, and sucrose), indole, motility, and citrate (Lestari et al., 2016). Amplification product was then separated on 2% gel agarose, stained with gel-red, and visualized using UV light. blaTEM gene detection was considered to be positive when bands at 445 bp of apparent size was observed in the gel. INTRODUCTION The transfer of antibiotic- resistant genes, especially through plasmids is considered to be one of the important mechanisms in the spreading of antibiotic resistance in bacteria (Apriliani & Pinatih, 2017). This event is known to be mediated by an enzyme of beta- 174 December 2018 EFFENDI ET AL. / Tropical Animal Science Journal 41(3):174-178 et al., 2016). Selection of antibiotic discs (disks) used previous research and based on some journals for reference. The types of antibiotics used were am­ picillin, amoxicillin, cefotaxim, meropenem, and sulfamethonazole-trimetrophrim. animal waste and they can migrate around the farms, slaughterhouses or poultry slaughterhouses, and dur­ ing meat processing. The surroundings of farms and slaughterhouses or chicken slaughterhouses will also be contaminated even though it is far from the source of contamination (Doosti et al., 2014).t Pure cultures were prepared in suspensions with an equivalent of 0.5 McFarland (1-2 x 108 CFU/mL) tur­ bidity. The cultures were taken using sterile swab cotton and distributed by means of a diole on the surface of the Mueller Hinton agar (MHA), and allowed to stand for ± 5 min. The antibiotic-containing discs were placed on the top of the MHA, which had been dispersed with pure cultures, at a distance of 25-30 mm. Furthermore, the culture was incubated at 35ºC for 24 h (Masruroh et al., 2016). Attempts to detect the existence of resistant genes in bacteria from food-producing animals in Indonesia, to our knowledge, remains limited. In fact, the gene detection should allows us to understand the pattern of genes spreading and possibility of the bacteria to gain further antibiotic resistance from certain groups. Accordingly, detection of the blaTEM gene group in K. pneumonia strain is important to do. Amplification of blaTEM gene: Polymerase Chain Reaction For the amplification was perfomed using Qiagen HotStarTag Master Mix (Qiagen, USA) according to manufacturer protocol with pure genomic DNA of K. pneumonia was used as a template. The primers used in this study were shown in Table 1. The amplification steps involved a denaturation process at 95°C for 15 min, 30 cycles denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extention at 72°C for 2 min followed by the final extension on temperature of 72°C for 10 min (Moenstein et al., 2007).i Antibiotic Test The result showed that 10 out of 195 samples isolated from swab dairy cows, beef cattle, broiler chick­ ens, and fish tilapia were positive K. pneumoniae. The presence of these bacteria on swab samples consisted of dairy cows by 4.8% (4/83), beef cattle by 40% (2/5), chicken broiler by 10% (1/10), and fish by 3% (3/97). All isolates also showed 90% resistance to the amoxicillin The antibiotic sensitivity test was performed using Kirby-Bauer agar diffusion method (Ningrum et al., 2016). The resulting clear zone was then grouped into sensitive groups (S), intermediates (I) or resistant (R) (Sagita et al., 2015; Kusumaningrum December 2018 175 Table 1. The primers used in this study Gene target Primary sequences Amplicon (bp) Reference blaTEM F- 5'-TCGCCGCATACACTATTCTCAGAATGA-3' 445 Monstein et al., 2007 R-5'-ACGCTCACCGGCTCCAGATTTAT-3' December 2018 175 EFFENDI ET AL. / Tropical Animal Science Journal 41(3):174-178 (9/10) and sensitive to other types of antibiotics (Table 2, Figure 1). (9/10) and sensitive to other types of antibiotics (Table 2, Figure 1). Based on the morphology of colonies grown on Mac Conkey agar and biochemical test, K. pneumoniae isolates were found from a dairy cow feces, chicken broiler, and tilapia fish as much as 10 out of 45 stool samples. The presence of bacteria of the K. pneumoniae on swab samples were 4.8% (4/83) in dairy cows, 40% (2/5) in beef cattle, 10% (1/10) in the broiler chicken, and 3% (3/97) in the tilapia fish. Examination of DNA from samples using agarose electrophoresis gel (Figure 2) showed that the blaTEM gene was successfully amplified with blaTEM-F and blaTEM- R primaries. The 10 samples tested by PCR in the study showed a positive result of 9 samples (90%) of the blaTEM gene (Figure 2 and Figure 3). K. pneumoniae bacteria resistance test against antibiotics indicated as much as 90% (9/10) resistant to amoxicillin. The results are similar to the results found by Sagita et al. (2015) that the bacteria K. pneumoniae was resistant to the antibiotic amoxicillin. Resistance occurs due to the ability of the bacteria to produce penisilinase enzymes that are capable of breaking down the beta lactam ring. With this effect, penicillin is converted into penicilloid acid that is not so active. Resistance is pro­ duced by taking action against degraded penicillin by beta-lactamase. Beta lactamase enzymes protect Gram- positive and Gram-negative bacteria. Antibiotic Test In a Gram-positive bacteria, the enzyme is liberated in the medium and de­ stroys antibiotics before it reached the cell and in gram negative it is located on the route where antibiotics must DISCUSSION The results of biochemical identification showed that K. pneumoniae bacteria did not contain indole (-) and Methyl Red (-), contained urea (+), Simmon’s Citrate (+), Voges Proskauer (+), and positive fermentation test of carbohydrate (+). The isolation and identification results were confirmed by K. pneumoniae character according to Holt et al. (2000). K. pneumoniae bacteria is a bacteria with a size of 2.0-3.0 x 0.6 μm and this bacterium is a normal flora in the intestinal and respiratory tracts. K. pneumoniae has a large capsule so that in its colonies culture looks very mucoid (Brooks et al., 2005). Table 2. Antibiotic inhibition zone interpretation Isolate code Antibiotic discs SAM AML SXT MEM CTX D (mm) R,I,S D (mm) R,I,S D (mm) R,I,S D (mm) R,I,S D (mm) R,I,S SP-S1 25 S 17 S 28 S 29 S 34 S SP-S3 24 S 11 R 25 S 30 S 36 S SP-B2 24 S 13 R 29 S 30 S 35 S SP-G4 22 S 13 R 23 S 32 S 34 S SPT-K1 25 S 13.7 R 19 S 34.5 S 40 S SPT-K2 24.5 S 10 R 20.1 S 30.2 S 30 S AW-5 22 S 11.2 R 29.7 S 27.7 S 33 S IN-P2 23 S 12 R 19.9 S 29 S 28 S IN-P3 21.1 S 10.5 R 22.9 S 27.8 S 29.4 S IN-S3 24 S 11 R 18 S 34 S 37 S Note: R (resistant), I (intermediates), S (sensitive), SP-S (dairy cow in Senduro), SP-B (dairy cow in Batu), SP-G (dairy cow in Grati), SPT-K (beef cattle), AW (broiler in Wonokromo), IN-P (customs tilapia), IN-S (tilapia in Sedila), SAM (ampicillin 10µg), SXT (sulfamethonazole-trimetrophrim 27,75 µg), CTX (cefotaxime 30 µg), MEM (meropenem 10 µg), AML (amoxycilin 15 µg). MP-1809_English check MP-1809_English 319 Table 2. Antibiotic inhibition zone interpretation Note: R (resistant), I (intermediates), S (sensitive), SP-S (dairy cow in Senduro), SP-B (dairy cow in Batu), SP-G (dairy cow in Grati), SPT-K (beef cattle), AW (broiler in Wonokromo), IN-P (customs tilapia), IN-S (tilapia in Sedila), SAM (ampicillin 10µg), SXT (sulfamethonazole-trimetrophrim 27,75 µg), CTX (cefotaxime 30 µg), MEM (meropenem 10 µg), AML (amoxycilin 15 µg). MP-1809_English check 319 Figure 2. Agarose gel electrophoresis 2% product used primer for blaTEM gene detection in Klebsiella pneumoniae bacte­ ria. Lane M: marker; lane 1-6 sample; lane K (-): nega­ tive control. REFERENCES Ahmed, A.M. & T. Shimammoto. 2011. Molecular char­ acterization of antimicrobial resistance in gram- negative bacteria isolated from bovine mastitis in Egypt. Microbiol. Immunol. 55: 318-327. https://doi. org/10.1111/j.1348-0421.2011.00323.x Figure 3. Agarose gel electrophoresis 2% product used primer for blaTEM gene detection in Klebsiella pneumoniae bacte­ ria. Lane M: marker; lane 7-10 sample; lane K (-): nega­ tive control. Note: lane 7= SPT-K2; lane 8= IN-P3; lane 9=IN-S3; lane 10=AW-5. gure 3. Agarose gel electrophoresis 2% product used primer for blaTEM ge on in Klebsiella pneumoniae bacteria. Lane M: marker; lane 7-10 sample (-): negative control. Aljanaby, A.A.J & A.H.A. Alhasani. 2016. Virulence factors and antibiotic susceptibility patterns of multidrug resistance Klebsiella pneumoniae isolated from different clinical infec­ tions. African Journal of Microbiology Research 10: 829- 843. https://doi.org/10.5897/AJMR2016.8051 ne K proceed to reach the target (Sagita et al., 2015). The bac­ terial isolate that were resistance in this study only oc­ curred against amoxicillin and showed sensitive against ampicillin sulbactam, cefotaxim, meropenem, and sulfa­ methonazole trimethoprim. These results are contrary to the study conducted by Sagita et al. (2015) and Ghasemi et al. (2013) stating that the bacteria K. pneumoniae are resistant to cefotaxime antibiotics. Ghasemi et al. (2013) also states that the bacterium K. pneumoniae are 100% resistant to ampicillin antibiotics. Note: lane 7= SPT-K2; lane 8= IN-P3; lane 9=IN-S3; lane 10=AW-5. Apriliani, N. P. E. U. & K. J. P. Pinatih. 2017. Prevalensi kelom­ pok gen blactx-m-1 pada Klebsiella pneumoniae di Rumah Sakit Umum Pusat Sanglah Denpasar. E-Jurnal Medika 6: 1-7. Brooks, G. F., J.S. Butel, & S.A. Morse. 2005. Medical Microbiology. Edisi I. Alih Bahasa: Bagian Mikrobiologi, FKU Unair. Salemba Medika. Jakarta. Doosti, A., M. Pourabbas, A. Arshi, M. Chehelgerdi, & H. Kabiri. 2014. TEM and SHV genes in Klebsiella pneumoniae isolated from cockroaches and their antimicrobial resistance pattern. Osong Public Health and Research Perspectives 6: 3-8. https://doi.org/10.1016/j.phrp.2014.10.011 Research conducted by Ahmed & Shimamoto (2011) shows that blaTEM genes as antimicrobial re­ sistance found as many as 23 isolates (67. 6%) of 34 isolates Gram-negative in case of mastitis in Egypt, and research conducted by Aljanaby & Alhasani (2016) also showed 30 isolates (93.75%) found blaTEM genes from 32 bacterial isolates K. pneumoniae isolated from patients with different clinias infections in Iraq. The majority of ESBL enzymes derived from the TEM type decoded by gene blaTEM. REFERENCES blaTEM gene is a gene causes antibiotic resis­ tance in the plasmids, and it is most often detected in clinical populations of Gram-negative microorganisms (Wilopo et al., 2015). Ghasemi, Y., T.Archin, M.Kargar, & M.Mohkam. 2013. A Simple multiplex PCR for assecing prevalence of extended spectrum beta lactamases producing Klebsiella pneumoniae in Intensive Care Units of a referral hospital in Shiraz Iran. Asian Pasific Journal of Tropical Medicine. 703-708. l h Holt, S.R., E.G.D. Murray, & R.N. Smith. 2000. Bergey’s Manual Determinative of Bacteriology. 9th ed. Waverly Press, Baltimore. Kusumaningrum, H. D., L. Handayani, & R. Novrianti. 2016. Partial sequencing of 16S rRNA gene of selected Staphylococcus aureus isolates and its antibiotic resis­ tance. Med. Pet. 39: 67-74. http://dx.doi.org/10.5398/ medpet.2016.39.2.67t 17 Lalzampuia, H., T. K. Dutta, I. Warjri & R. Chandra. 2014. Detection of extended-spectrum β-lactamases (blaCTX-M-1 and blaTEM) in Escherichia coli, Salmonella spp., and Klebsiella pneumoniae isolated from poultry in North Eastern India. Veterinary World 7: 1026-1031. https://doi.org/10.14202/ vetworld.2014.1026-1031 CONCLUSION K. pneumoniae can be isolated from swab samples of food-producing animals that is equal to 5.12% (10/195). All of the isolates showed a tendency to be resistant to amoxicillin 90% (9/10). Their resistances also be con­ firmed by detecting the ESBL-encoding gene ie blaTEM genes. Further research needs to be conducted to study that these bacteria have other ESBL-encoding genes such as CTX and SHV genes. In addition, it is necessary to detect the ESBL-producing K. pneumoniae bacteria from the meat of food-producing animals, farm waste, and slaughterhouses, as well as human feces. Lestari, N. W., A. Budiharjo, & A. Pangastuti. 2016. Bakteri Heterotrof aerobic asal saluran pencernaan ikan sidat (Anguilla bicolor bicolor) dan potensinya sebagai probiotik. Bioteknologi 13: 9-17. Masruroh, C. A., M. B. Sudarwanto, & H. Latif. 2016. The Occurence of extended spectrum B-Lactamase-producing Escherichia coli from broiler feces in Bogor. JSV 34: 42-49 Monstein, H.J., A. Ostholm-Bulkhed, M.V. Nilsson, M. Dombusch, & L.E. Nilsson. 2007. Multiplex PCR amplifi­ cation assay for the detection of blaSHV, blaTEM, and blaC­ TX-M genes in Enterobacteriaceae. APMIS. 115:1400-1408. https://doi.org/10.1111/j.1600-0463.2007.00722.xi ACKNOWLEDGEMENT Thanks to the Integrated Applied Research Flagship Universities of the Ministry of Research, Technology and Higher Education of the Republic of Indonesia the 2017 Budget that supported this research. DISCUSSION / Tropical Animal Science Journal 41(3):174-178 500 bp 400 bp 300 bp 200 bp 100 bp Figure 3. Agarose gel electrophoresis 2% product used primer for blaTEM gene detection in Klebsiella pneumoniae bacte­ ria. Lane M: marker; lane 7-10 sample; lane K (-): nega­ tive control. Note: lane 7= SPT-K2; lane 8= IN-P3; lane 9=IN-S3; lane 10=AW-5. Figure 3. Agarose gel electrophoresis 2% product used primer for blaTEM ge tion in Klebsiella pneumoniae bacteria. Lane M: marker; lane 7-10 sample; (-): negative control. 445 bp with other people or organization related to the material discussed in the manuscript. DISCUSSION Note: lane 1= SP-S1; lane 2= SP-S3; lane 3= SP-B2; lane 4= SP-G4; lane 5= IN-P2; lane 6= SPT-K1. , 4) Figure 2. Agarose gel electrophoresis 2% product used primer for blaTEM gen ection in Klebsiella pneumoniae bacteria. Lane M: marker; lane 1-6 sample; la → → → → → → 500 bp 400 bp 300 bp 200 bp 100 bp 445 bp Figure 1. Inhibition zone diameter interpretation. (1) SAM (ampicillin 10µg), (2) SXT (sulfamethonazole-trime­ trophrim 27,75 µg), (3) CTX (cefotaxime 30 µg), (4) MEM (meropenem 10 µg), (5) AML (amoxycilin 15 µg). Figure 2. Agarose gel electrophoresis 2% product used primer for blaTEM gene detection in Klebsiella pneumoniae bacte­ ria. Lane M: marker; lane 1-6 sample; lane K (-): nega­ tive control. Note: lane 1= SP-S1; lane 2= SP-S3; lane 3= SP-B2; lane 4= SP-G4; lane 5= IN-P2; lane 6= SPT-K1. gure 1. Inhibition Zone Diameter Interpretation. (1) SAM (ampicillin10µg), sulfamethonazole-trimetrophrim 27,75 µg), (3) CTX (cefotaxime 30 µg), (4) 320 321 Figure 2. Agarose gel electrophoresis 2% product used primer for blaTEM gene 322 detection in Klebsiella pneumoniae bacteria. Lane M: marker; lane 1-6 sample; lane 323 → → → → → → 500 bp 400 bp 300 bp 200 bp 100 bp 445 bp Figure 1. Inhibition zone diameter interpretation. (1) SAM (ampicillin 10µg), (2) SXT (sulfamethonazole-trime­ trophrim 27,75 µg), (3) CTX (cefotaxime 30 µg), (4) MEM (meropenem 10 µg), (5) AML (amoxycilin 15 µg). gure 1. Inhibition Zone Diameter Interpretation. (1) SAM (ampicillin10µ ulfamethonazole-trimetrophrim 27,75 µg), (3) CTX (cefotaxime 30 µg), 320 321 322 d 323 Figure 1. Inhibition zone diameter interpretation. (1) SAM (ampicillin 10µg), (2) SXT (sulfamethonazole-trime­ trophrim 27,75 µg), (3) CTX (cefotaxime 30 µg), (4) MEM (meropenem 10 µg), (5) AML (amoxycilin 15 µg). ure 1. Inhibition Zone Diameter Interpretation. (1) SAM (ampicillin10µ ulfamethonazole-trimetrophrim 27,75 µg), (3) CTX (cefotaxime 30 µg) 320 321 322 d 323 Figure 2. Agarose gel electrophoresis 2% product used primer for blaTEM gene detection in Klebsiella pneumoniae bacte­ ria. Lane M: marker; lane 1-6 sample; lane K (-): nega­ tive control. Note: lane 1= SP-S1; lane 2= SP-S3; lane 3= SP-B2; lane 4= SP-G4; lane 5= IN-P2; lane 6= SPT-K1. ), 4) Figure 2. Agarose gel electrophoresis 2% product used primer for blaTEM gen ection in Klebsiella pneumoniae bacteria. Lane M: marker; lane 1-6 sample; l 176 December 2018 MEM (meropenem EFFENDI ET AL. CONFLICT OF INTEREST t p g j Ningrum, S.G., R. D. Soejoedono, H. Latif, W. Arnafia, & I. W. T. Wibawan. 2016. Prevalence and characterization of shiga toxin-producing Escherichia coli isolated from The Authors declare that there is no conflict of in­ terest with any financial, personal, or other relationships December 2018 177 December 2018 177 EFFENDI ET AL. / Tropical Animal Science Journal 41(3):174-178 slaughtered qurban animal in Jakarta Province. Med. Pet. 39: 90-94. http://dx.doi.org/10.5398/medpet.2016.39.2.90 slaughtered qurban animal in Jakarta Province. Med. Pet. 39: 90-94. http://dx.doi.org/10.5398/medpet.2016.39.2.90 berat molekul 40 kDA Klebsiella pneumoniae sebagai anti­ bodi. Jurnal Kedokteran Brawijaya 20: 12-18. https://doi. org/10.21776/ub.jkb.2004.020.01.3 Overdevest, I., I. Willemsen, M. Rijnsburger, A. Eustace, X. Li, P. Hawkey, M. Heck, P. Savelkoul, C. Vandenbroucke- Grauls, K. van der Zwaluw, X. Huijsdens, & J. Klutmans. 2011. Extended-Spectrum β-laktamase genes of Escherichia coli in chicken meat and humans, the Netherlands. Emerg. Infect. Dis.17: 1216-1222. https://doi.org/10.3201/ eid1707.110209 Wilopo, B.A.P., S. Sudigdoadi, E. Sahiratmadja, & I.M.W. Dewi. 2015. Loop-mediated isothermal amplification un­ tuk mendeteksi gen blaTEM sebagai penyandi extended- spectrum beta-lactamase pada isolat enterobacteriaceae. Majalah Kedokteran Bandung 47: 243-244. https://doi. org/10.15395/mkb.v47n4.618 g World Health Organization. 2014. Antimicrobial resistance: global report on surveillance. Rahamathulla, M.P., B.N. Harish, L. Mataseje, & M.R. Mulvey. 2016. Carbapenem resistance mechanisms among blood isolates of Klebsiella pneumoniae and Escherichia coli. Journal. Microbiol. 10:45-53.i Yeh, K, M., A. Kurup, L.K. Siu, Y.L.Koh, C.P. Fung, J.C. Lin, T.L. Chen, F.Y. Chang, & T.H. Koh. 2007. Capsular sero­ type K1 or K2, rather than magA and rmpA, is a major virulence determinant for Klebsiella pneumoniae liver ab­ scess in singapore and Taiwan J. Clin. Microbiol. 45:466- 471. https://doi.org/10.1128/JCM.01150-06 Sagita, D., L. Azizah, & Y. Sari. 2015. Identification of bacteria and antibiotic sensivity test of pus of post surgical wound infection in Jambi Public Hospital in the period August- October 2014. Sains Farmasi 1: 6-7. t Yuwono, J. 2011. Prevalensi gen TEM pada extended spec­ trum beta lactamase producing enterobactericeae. Jurnal Kedokteran dan Kesehatan 43: 3098-3102. Susilo, J., T. R. Sartono, & Sumarno. 2004. Deteksi bakteri Klebsiella pneumoniae pada sputum dengan metode imu­ nohistokimia menggunakan anti outer membrane protein 178 December 2018 178 December 2018
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Controls on the geotechnical response of sedimentary rocks to weathering
Earth surface processes and landforms
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de Vilder Saskia (Orcid ID: 0000-0002-4531-2070) de Vilder Saskia (Orcid ID: 0000-0002-4531-2070) de Vilder, S.J¹,²*., Brain, M.J¹., Rosser, N.J¹ de Vilder, S.J¹,²*., Brain, M.J¹., Rosser, N.J¹ Department of Geography, Durham University, Lower Mountjoy, South Road, Durham DH1 3LE UK Department of Geography, Durham University, Lower Mountjoy, South Road, Durham DH1 3LE UK ² Now at GNS Science, 1 Fairway Drive, Avalon 5010, NZ ² Now at GNS Science, 1 Fairway Drive, Avalon 5010, NZ *Corresponding author: s.devilder@gns.cri.nz *Corresponding author: s.devilder@gns.cri.nz *Corresponding author: s.devilder@gns.cri.nz Keywords: unconfined compression, rock strength, stress history, failure style, rockfall, rock bridges Keywords: unconfined compression, rock strength, stress history, failure style, rockfall, rock bridges This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/esp.4619 This article is protected by copyright. All rights reserved. Abstract Weathering reduces the strength of rocks and so is a key control on the stability of rock slopes. Recent research suggests that the geotechnical response of rocks to weathering varies with ambient stress conditions resulting from overburden loading and/or stress concentrations driven by near-surface topography. In addition, the stress history experienced by the rock can influence the degree to which current weathering processes cause rock breakdown. To address the combined effect of these potential controls, we conducted a set of weathering experiments on two sedimentary lithologies in laboratory and field conditions. We firstly defined the baseline geotechnical behaviour of each lithology, characterising surface hardness and stress-strain behaviour in unconfined compression. Weathering significantly reduced intact rock strength, but this was not evident in measurements of surface hardness. The ambient compressive stress applied to samples throughout the experiments did not cause any observable differences in the geotechnical behaviour of the samples. We created a stress history effect in sub-sets of samples by generating a population of microcracks that could be exploited by weathering processes. We also geometrically modified groups of samples to cause near-surface stress concentrations that may allow greater weathering efficacy. However, even these pronounced sample modifications resulted in insignificant changes in geotechnical behaviour when compared to unmodified samples. The observed reduction in rock strength changed the nature of failure of the samples, which developed post-peak strength and underwent multiple stages of brittle failure. Although weakened, these samples could sustain greater stress and strain following exceedance of peak strength. On this basis, the multi-stage failure style exhibited by Weathering reduces the strength of rocks and so is a key control on the stability of rock slopes. Recent research suggests that the geotechnical response of rocks to weathering varies with ambient stress conditions resulting from overburden loading and/or stress concentrations driven by near-surface topography. In addition, the stress history experienced by the rock can influence the degree to which current weathering processes cause rock breakdown. To address the combined effect of these potential controls, we conducted a set of weathering experiments on two sedimentary lithologies in laboratory and field conditions. We firstly defined the baseline geotechnical behaviour of each lithology, characterising surface hardness and stress-strain behaviour in unconfined compression. Weathering significantly reduced intact rock strength, but this was not evident in measurements of surface hardness. This article is protected by copyright. All rights reserved. Abstract The ambient compressive stress applied to samples throughout the experiments did not cause any observable differences in the geotechnical behaviour of the samples. We created a stress history effect in sub-sets of samples by generating a population of microcracks that could be exploited by weathering processes. We also geometrically modified groups of samples to cause near-surface stress concentrations that may allow greater weathering efficacy. However, even these pronounced sample modifications resulted in insignificant changes in geotechnical behaviour when compared to unmodified samples. The observed reduction in rock strength changed the nature of failure of the samples, which developed post-peak strength and underwent multiple stages of brittle failure. Although weakened, these samples could sustain greater stress and strain following exceedance of peak strength. On this basis, the multi-stage failure style exhibited by This article is protected by copyright. All rights reserved. weaker weathered rock may permit smaller-magnitude, higher-frequency events to trigger fracture through intact rock bridges as well as influencing the characteristics of pre-failure deformation. These findings are consistent with patterns of behaviour observed in field monitoring results. weaker weathered rock may permit smaller-magnitude, higher-frequency events to trigger fracture through intact rock bridges as well as influencing the characteristics of pre-failure deformation. These findings are consistent with patterns of behaviour observed in field monitoring results. This article is protected by copyright. All rights reserved. Introduction Rock slope failures are a significant hazard (Dussauge-Peisser et al., 2002; Fell et al., 2008; Guzzetti et al., 1999) and contribute to landscape evolution over a variety of timescales (Clarke and Burbank, 2010; Korup et al., 2007; Moore et al., 2009). The susceptibility of a rock slope to failure is controlled by its intrinsic properties, such as the strength of intact rock bridges (Jennings, 1970), the nature of joint sets (Einstein et al., 1983; Goodman and Shi, 1985), and its physical setting (slope angle, aspect and curvature) (e.g. Matsuoka and Sakai, 1999; Messenzehl et al., 2017; Sass, 2005). Slopes can be destabilised rapidly in response to sudden and short- lived changes in stress conditions that trigger failure, such as those resulting from strong earthquake ground shaking or heavy rainfall (Iverson, 2000; Keefer et al., 1987). Rock slope instability can also develop over longer (100 - 103 years) timescales in response to incremental and cumulative reductions in rock mass strength driven by micro-fracture development and/or weathering processes that reduce the cohesional strength of rocks (Collins and Stock, 2016; Eppes and Keanini, 2017; Gunzburger et al., 2005). The significance of weathering in modifying rock strength has been widely observed in a number of studies (e.g. Durgin, 1977; Fookes et al., 1988; Hencher and This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. McNicholl, 1995; Migon, 2010; Thomson et al., 2014; Yatsu, 1988) and its importance for rock slope stability has been demonstrated in numerical and analogue studies (e.g. Bachmann et al., 2004; Huisman et al., 2011). Engineering classifications provide descriptive insight into the nature of weathering along discontinuities (e.g. Selby, 1980; Hoek, 1983), but such schemes do not sufficiently consider weathering-induced strength degradation of intact rock bridges that critically influence shallow rock slope failures and rockfall activity, which are our focus here (de Vilder et al., 2017; Jennings, 1970; Kemeny, 2005). Weathering processes operate concurrently and/or interact with a range of other processes that prepare slopes for macro-scale fracture (Aldred et al., 2016; Atkinson, 1984; Collins and Stock, 2016; Eppes and Keanini, 2017b; Eppes et al., 2016; Gischig et al., 2011; Lamp et al., 2017; Rosser et al., 2013; Stock et al., 2012) but their combined effect on rock mass strength and failure style remains poorly constrained. This article is protected by copyright. All rights reserved. Introduction Laboratory experiments have improved our understanding of weathering processes and their influence on surficial changes to rocks (Goudie, 2016; e.g. Moses et al., 2014). However, these studies are not sufficient to fully constrain the influence of weathering on changing intact rock strength and rock slope stability for a variety of reasons. Firstly, few of these studies consider changes in rock strength at a scale relevant to rock slope failures, particularly for small and shallow rockfalls where stability is controlled by one critical rock bridge (de Vilder et al., 2017). Secondly, weathering can also cause changes to rock rheology (Fookes et al., 1988). In turn, this may result in a change in the nature and style of failure (Basu et al., 2009; Gupta and Seshagiri Rao, 2000; Viles, 2013). This aspect of rock response is rarely directly considered in weathering studies. Thirdly, conventional weathering studies This article is protected by copyright. All rights reserved. undertaken under laboratory conditions replicate environments where ambient compressive, shear or tensile stress conditions are considered negligible, such as desert surfaces or foreshore platforms (e.g. Coombes et al., 2013; Mottershead, 2013; Viles, 2005; Warke, 2007). However, stress concentrations resulting from temporal and spatial variations in topography, overburden load and macro- and local-scale slope geometry (Brain et al., 2014; Leith et al., 2014a, 2014b; Martel, 2006) can occur in rock slopes. In turn, these elevated stress conditions can cause, for example, an increased density of microcracks (Eberhardt et al., 1998) that can subsequently be exploited by weathering processes. Recent analogue experiments have suggested that the effects of weathering on rock mass strength may differ where ‘ambient’ stress concentrations exist (Bruthans et al., 2014; Rihosek et al., 2016; Zhang et al., 2015). Fourthly, the significance of stress history on weathering rates and effects has only recently been directly considered (e.g. Røyne et al., 2008; Viles et al., 2018; Warke, 2007). Viles et al. (2018), for example, demonstrated that rocks that have previously been exposed to physical and chemical weathering processes may be more susceptible to weathering than rocks with no previous stress history. However, this effect has not been considered in the context of potential rheological changes and stress concentrations noted above. To improve our understanding of the links between weathering and shallow rock slope failure, we undertook a suite of experiments that subjected cylindrical rock samples to weathering processes typically experienced by coastal rock cliffs. Sample lithology We used two lithologies in our study: Staithes Formation Siltstone (‘siltstone’) and Catcastle Buff Sandstone (‘sandstone’) (Figure 1). These rocks have different grain size characteristics, strength properties and, hence, associated differences in their potential susceptibility to weathering-driven weakening (cf. Eberhardt et al., 1999). The Siltstone forms part of the Lower Jurassic Staithes sandstone formation, which were deposited within the shallow seas of the Cleveland Basin (Rawson and Wright, 2000). It is light grey-blue, with 2 mm to 6 mm thick banding inclined at 6° to 15° (classification based on ISRM, 2015). The Catcastle Buff Sandstone forms part of the Carboniferous Millstone Grit Group, deposited via fluvial processes in the Central Pennine sub-basin (BGS, 2017). It is light grey-brown, massive and medium grained with minor (≤10%) coarse grains (ISRM, 2015). Introduction Our experimental design allowed us to determine the effects, if any, of ambient compressive stresses on the nature of weathering and its effect(s) on rock strength and failure style. Within our experimental program, we also assessed the influence of This article is protected by copyright. All rights reserved. stress history, via generating pre-existing microcrack populations, and stress concentrations, via modifying sample geometries, on the strength and deformation behaviour of rock. stress history, via generating pre-existing microcrack populations, and stress concentrations, via modifying sample geometries, on the strength and deformation behaviour of rock. This article is protected by copyright. All rights reserved. Overview and experimental design The first stage of our experimental program involved determining the baseline geotechnical characteristics of the siltstone and sandstone lithologies. We provide an overview to describe the context and rationale of our experimental design, and then provide details on the specific methods applied in the subsequent sections (these This article is protected by copyright. All rights reserved. include; unconfined compression strength testing, sample modification, surface hardness measurements and visual appearance, baseline characterisation, laboratory and field weathering experiments). To assess changes in intact rock strength, we determined the unconfined compressive strength (UCS) of samples. UCS is a widely-used measurement of strength in rock mechanics and slope engineering (e.g. Jaeger et al., 2007), and also is closely related to other key measurements of intact rock strength (Perras and Diederichs, 2014). UCS testing also allowed us to obtain a detailed understanding of stress-strain and, hence, fundamental rheological behaviour of the sample, including the nature of failure. For the latter, we considered the strain value coincident with peak strength (UCS); we term this ‘strain-at-failure’. In addition, we also noted the number and nature of failure ‘events' that occurred until near or total strength loss had occurred in each sample. These failure events were defined in stress-strain curves as substantial, near-instantaneous reductions in recorded compressive stress with no or limited strain accumulation evident For our baseline dataset, we used cylindrical samples that are typical of standard geotechnical testing procedures (ASTM, 2008). These standard, unmodified samples are henceforth referred to as U (unmodified) samples. We also measured the surface hardness of samples used for baseline characterisation, since this has been used as an indication of rock strength (Aoki and Matsukura, 2007). To consider the influence of stress history on susceptibility to weathering processes, rates and associated changes in behaviour (Røyne et al., 2008; Viles et al., 2018; Warke, 2007), we pre-loaded a separate set of samples to a predetermined value of This article is protected by copyright. All rights reserved. UCS (observed in baseline tests – see results) that was sufficient to cause micro- cracking, but insufficient to cause full failure. This created an elevated density of micro-cracks (and so the stress history, or damage condition). We refer to these ‘pre- damaged’ samples using the notation P. This article is protected by copyright. All rights reserved. Overview and experimental design To consider the effects that variations in cliff-face surface topography and resultant stress concentrations may have on the effectiveness of weathering processes, we cut vertical notches into cylindrical samples and characterised their baseline behaviour. This allowed us to assess if resultant stress concentrations in the areas surrounding these notches created any evidence that resultant enhanced micro- cracking can be subsequently exploited by weathering processes (Lajtai and Lajtai, 1974). In addition, the increase in surface area of the sample as a result of the notch may affect the nature, rate and effectiveness of weathering (Robinson et al., 1982). Samples with modified geometry are referred to using the notation G. We considered the combined effects of both modified geometry (notches) and stress history on susceptibility to weathering using a combination of the pre-treatment types outlined above; these are referred to as PG samples. The second stage of our testing program involved assessing the effects of weathering on the key geotechnical properties determined in our baseline characterisation stage, namely strength and rheological behaviour. There were two elements to our experiments. Firstly, we considered the effects of weathering in a controlled laboratory environment. These tests focussed on the effects of salt-water wetting and drying cycles on rock properties, typical of conditions experienced in coastal rock slopes (Mottershead, 2013). Secondly, since weathering processes do not operate in isolation (Viles, 2013), we also undertook a set of field-based weathering experiments where rock samples were exposed to weather conditions at a coastal cliff-top location in Staithes, North Yorkshire, UK. For both laboratory and field experiments, we considered the effects of weathering on U, G, P and PG samples. In addition, our experimental design allowed us to assess the effects of an elevated ambient compressive stress on weathering impacts on U, G, P and PG samples. To do so, we placed samples under a constant vertical compressive stress for the full duration of the experiments in both laboratory and field weathering experiments. The magnitude of compressive stress was selected to be representative of the stress conditions experienced at the base of the coastal cliffs of North Yorkshire. For every sample placed under stress, there was an equivalent control sample that was not subjected to vertical stresses but had been subjected to the same pre-test modifications. This article is protected by copyright. All rights reserved. Unconfined compression tests We determined the UCS of samples in broad accordance with ASTM D7012-14 (2014) using a compressive load frame manufactured by GDS Instruments Ltd. (Barla et al., 2010). Deviations from this standard reflect our experimental design, which involved modifications of sample geometry. For the banded siltstone formation, cores were drilled perpendicular to banding. Samples were loaded under compressive strain control at a rate of 0.1% min-1; this strain rate reflects the net strain recorded by the apparatus and is comprised of both deformation of the rock sample and the apparatus itself in response to load (‘net strain’). The magnitude of deformation of the apparatus is constant for a given applied stress. As such, we This article is protected by copyright. All rights reserved. could directly compare strain values between samples using net strain. This was an important consideration because use of direct, local measurements of rock sample deformation were not always possible following completion of weathering tests, where the fragile and highly-friable nature of the weathered core surface prevented appropriate attachment of ‘local’ displacement transducers (LVDTs). However, for all baseline samples and for suitable post-weathering samples, we directly monitored sample deformation using two vertically-mounted and diametrically-opposed LVDTs on the rock surface. Local strain measurements were used to calculate Young’s Modulus of Elasticity and characterise the local stress-strain behaviour of the rock (ASTM D7012-14, 2014). We normalised stress-stain curves relative to the mean value of UCS and net strain at failure of the baseline UCS tests, due to the inherent variability in UCS and strain behaviour in the baseline dataset, and the need to compare baseline tests with weathered samples. Normalised stress and strain values of 1 are equal to the mean values recorded in baseline tests. Normalised axial strain values above and below 1 indicate increases and decreases, respectively, in strain values at failure relative to those observed in baseline tests. This article is protected by copyright. All rights reserved. Surface hardness measurements and visual appearance We measured baseline surface hardness of samples using a standard (d-type) Equotip portable hardness testing device (Viles et al., 2011). We measured the surface hardness of the rock in Leeb numbers (L); a higher L value indicates a greater rock surface hardness. For each sample, we recorded the mean of ten measurements, obtained at random locations on the sample. At the end of each weathering experiment the samples were air dried and then weighed to determine if mass loss or mass gain had occurred. Additionally, we recorded qualitative descriptions and photographs of the condition of each sample, noting how the surface texture and colour changed through time This article is protected by copyright. All rights reserved. Sample modifications We created pre-existing damage within the samples by loading designated P samples in unconfined compression to 75% of the median UCS observed in standard baseline tests (see results). This magnitude of loading was chosen as it typically considered to exceed the crack initiation threshold, ci, and, hence generate a population of distributed micro-cracks, but without causing macro-scale fracture (Figure 2 a) (Eberhardt et al., 1998). For G-type samples, we cut three 5 mm wide by 5 mm deep vertical ‘notches’ spaced 50 mm apart at a 120° circumferential offset between notches (Figure 2 b). The reduction in cross-sectional area was accounted for in the calculation of compressive stress. PG-type samples were firstly modified in terms of geometry and then pre-damaged using the same procedures as above. Surface hardness measurements and visual appearance This article is protected by copyright. All rights reserved. Baseline characterisation We determined baseline UCS and stress-strain behaviour of standard (U) siltstone (n = 12) and sandstone (n = 11) samples. We also measured baseline UCS and stress- strain behaviour of modified geometry (G) samples for siltstone (n = 2) and sandstone (n = 3). All baseline samples were instrumented with two axial LVDTs to record the axial strain response of the samples. Laboratory weathering experiments Laboratory weathering experiments were undertaken in a climate-controlled laboratory (temperature: 20.9˚C ± 0.24 ˚C; relative humidity: 45% ± 5.3%), allowing us to isolate the effects of saltwater wetting and drying on the samples. We subjected 32 (16 sandstone and 16 siltstone) rock samples to laboratory-controlled weathering conditions for a total of 90 days. All samples experienced 360 wetting and drying cycles. A summary of sample types considered (U, P, G or PG) is provided in Table 1. For each type, vertical compressive stress was applied to two samples, and two samples acted as control (non-stressed) samples that experienced the same weathering cycles, allowing us to isolate the effects of ambient compressive stress on weathering. We used front-loading oedometers (Head and Epps, 2011, Figure 3) to place appropriate samples under a constant vertical compressive stress of 3.8 MPa, equivalent to approximately 150 m to 200 m of vertical overburden. For the siltstone, this compressive stress represented 11.1% of mean UCS, and for the sandstone 6.8% of mean UCS. Using the pump system detailed in Figure. 3, rock samples were subjected to six-hour wetting and drying cycles consisting of 30 minutes of submersion in sodium chloride solution (200 g/l), followed by drainage of the cell and subsequent exposure to air for 5.5 hours. These six-hour cycles mimic semi-diurnal tidal flooding conditions experienced at the coastal cliff toe at Boulby. We monitored the net vertical deformation of the four ‘stressed’ samples with LVDTs (Figure 3). Vertical displacement of each sample was recorded as the mean of measurements observed over a one-minute interval. We also monitored the surface appearance and texture of rock samples and measured surface hardness using the Equotip device on a weekly basis. Following completion of the weathering experiments, we measured the mass of the air-dried samples. We then determined the UCS and associated stress-strain behaviour of all 32 samples. Baseline characterisation This allowed us to quantify any resultant changes in strength and failure style in response to weathering and in terms of each sample type (U, P, G or PG). Half (n = 16) of these samples were instrumented with two axially-mounted LVTDs to characterise local strain; one specimen of each pre- treatment type was selected for LVDT instrumentation. For the remaining half (n = 16) only net strain values were obtained. This article is protected by copyright. All rights reserved. Field weathering experiments We undertook a year-long (19th August 2016 – 30th August 2017) field experiment in which we used a purpose-built loading frame at the cliff top at Boulby, North Yorkshire, UK (Figure 4) to subject 32 (16 sandstone and 16 siltstone) rock samples to cliff-top field conditions. The length of time for the field experiments was longer than that of the laboratory experiments, as the samples were subject to a variety of natural environmental cycles rather than the increased frequency of the saline brine wetting and drying compared to tidal cycles. Based on data collected 3 km to the north at Loftus (Meteorological Office weather station), our field site experiences mean annual precipitation of 467 mm; peak rainfall intensities reach 79.1 mm hr-1. In 2016, mean daily air temperature ranged from -1.99°C in January to 21.0°C in September. However, the potential for frost weathering was limited, with only one day recording a mean daily temperature below freezing. A summary of the type of samples tested (U, P, G or PG) is detailed in Table 1. For each type, a vertical compressive stress of 2 MPa, equivalent to approximately 80 m to 100 m of vertical overburden, was applied to two samples using the loading frame, and two samples acted as non-stressed control samples that experienced the same environmental conditions. The 2 MPa vertical compressive stress was equivalent to 5.9% of mean UCS for siltstone samples, and 3.6% of mean UCS for sandstone samples. During the field experiment, we qualitatively monitored and described the surface appearance and texture of rock samples and measured surface strength using the Equotip device monthly. Following completion of the field experiments, we measured the post-test mass and bulk density of the samples. We then determined the UCS and associated stress- strain behaviour of all 32 samples. Half (n = 16) of these samples were instrumented with two axially-mounted LVTDs to characterise local strain; one specimen of each pre-treatment type was selected for LVDT instrumentation. For the remaining half (n = 16) only net strain values were obtained. Analysis Methods To determine the effects of weathering on rock strength and failure style, we grouped and compared samples based on:  Lithology: siltstone or sandstone;  Experimental setting: laboratory or field;  Ambient compressive stress conditions: ‘control’ (non-stressed) or ‘stressed’ samples; and  Pre-treatment type: U, P, G or PG.  Pre-treatment type: U, P, G or PG. This article is protected by copyright. All rights reserved. Field weathering experiments This article is protected by copyright. All rights reserved. We tested for statistically-significant differences groups using the Wilcoxon Rank- Sum test for non-normally distributed datasets that displayed equal variance, as determined using Lilliefors tests and Levene’s tests respectively (Hollander et al., 2015). For normally distributed datasets with equal variance, as determined using Lilliefors tests and Bartlett’s tests respectively (Hollander et al., 2015), we used one- way Analysis of Variance (ANOVA) and Student’s t-tests to test for significant difference(s) in the mean values of key variables of weathered samples and their baseline counterparts. We tested for statistical significance between baseline groups and the broad groupings of lithology, experimental setting, stress conditions and pre- treatment type, rather than sub-sets of the groupings. Our statistical tests are supplemented by semi-quantitative, graphical displays of differences in sample strength relative to baseline conditions. Results Baseline siltstone characterisation Results Baseline siltstone characterisation Baseline siltstone characterisation We recorded a mean Equotip L-value of 397.5 ± 126.7. The mean UCS was 34.15 MPa ± 6.43 MPa (Table 2). Failure occurred at a mean net strain of 1.47% ± 0.07% and mean local strain of 0.46% ± 0.21% (Figure 5). We calculated a mean Young’s Modulus of 8.99 GPa ± 3.2 GPa. Most samples (n =10) displayed one or two stages of brittle failure before residual or zero strength was reached. For the modified geometry (G) samples, we observed mean UCS vales of 33.69 MPa ±1.57 MPa. We undertook linear regression analysis of UCS as a function of pre-test bulk density and found a reasonably-strong, statistically-significant relationship (r² = 0.61, p = 0.003; Figure 6). The regression model envelope (Figure 6) allowed us to determine the representative baseline mean from which to compare the effect of weathering in absolute and percentage terms. If the initial starting bulk density of the post- test/weathered siltstone samples was less than or greater than the range of bulk density values measured in the baseline tests, they were not used in subsequent comparisons to weathered samples. This permitted more direct comparison of the effects of weathering on UCS and ensured the rock samples had comparable physical and geotechnical properties at the start of the experiments. This article is protected by copyright. All rights reserved. Siltstone visual appearance and surface hardness The visual appearance of all samples changed during laboratory weathering experiments (Figure 7). We observed iron leaching, grain loss and slaking events Iron leaching was present for all stressed samples and in four control samples. Slaking (Figure 7 a, b) was characterised by loss of fragments of rock (typical long axis of 5 mm, typical short axis of 2 mm and 2 mm thick) (Figure 7 b). These fragments could be identified several weeks prior to detachment, characterised by sub-vertical cracks with a 1 to 2 mm aperture (Figure 7 b). We also observed tight (< 2 mm aperture), stepped, sub-horizontal cracks up to 50 mm long (Figure 7 c). All field samples displayed surface grain loss that resulted in a ‘powdery’ surface texture (Figure 7 d). Field samples also developed tight (< 2 mm aperture), stepped sub- horizontal cracks (2 to 10 mm in length) and tight, sub-vertical cracks (5 to 20 mm in length) (Figure 7 b, d). We did not observe any consistent relationships between This article is protected by copyright. All rights reserved. observed changes in the nature of weathering effects on surface texture and pre- treatment type (U, P, G or PG) and/or ambient compressive stress conditions (stressed vs. control) samples. All laboratory samples displayed a positive change in mass, with a mean of 5% ± 2.27%, while the field samples displayed a mean negative change in mass of -2.11% ± 3.08%. Of the field samples, we measured a positive change in mass for only three of the 16 samples. For the laboratory experiments we assessed the relationship between surface hardness and week number (and, hence, time) using Pearson correlation. We did not observe a strong or statistically-significant relationship between these variables for both control (r = -0.21, p = 0.08) and stressed (r = -0.18, p = 0.14) samples. However, for the field experiments we observed a statistically-significant increase in surface hardness in both control (r = 0.21, p = 0.04) and stressed (r = 0.22, p = 0.03) samples. General changes in siltstone compressive strength and strain values General changes in siltstone compressive strength and strain values This article is protected by copyright. All rights reserved. General changes in siltstone compressive strength and strain values Following completion of laboratory weathering experiments, control siltstone samples (all pre-treatment types) displayed a mean UCS of 16.72 MPa ± 1.64 MPa and failed at 1.15% ± 0.13% and 0.38% ±0.17% net and local strain, respectively (Table 3). We observed a mean Young’s Modulus of 3.41 GPa ± 1.73 GPa. Stressed siltstone samples (all pre-treatment types) displayed a mean UCS of 18.89 MPa ± 3.95 MPa, failing at 1.19% ± 0.16% and 0.27% ± 0.18% net and local strain respectively. We observed a mean Young’s Modulus of 1.69 GPa ± 0.03 GPa (Table 3). Normalised stress-strain curves for laboratory experiments relative to baseline tests are displayed in Figure 8 a. Stressed U-type samples displayed a single stage of brittle This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. failure, while all other samples displayed at least two stages of brittle failure. (Table 3). 3). For field weathering experiments, control siltstone samples (all pre-treatment types) displayed a mean UCS value of 36.71 MPa ± 14.93 MPa, failing at 1.4% ± 0.09% and 0.26% ± 0.17% net and local strain respectively. We observed a mean Young’s Modulus value of 5.0 GPa ± 1.52 GPa (Table 4). Stressed siltstone samples (all pre- treatment types) displayed a UCS value of 37.30 MPa ± 13.74 MPa, failing at 1.12% ± 0.1% and 0.29% ± 0.17% net and local strain respectively. We observed a mean Young’s Modulus value of 3.63 GPa ± 2.79 GPa, (Table 4). Normalised stress-strain curves relative to baseline tests are displayed in Figure 8 b. Stressed P-type samples displayed a single stage of brittle failure, while all other samples displayed at least two stages of brittle failure. (Table 4). This article is protected by copyright. All rights reserved. Siltstone: Experimental setting For siltstone samples weathered under laboratory conditions we observed a mean UCS of 17.81 MPa ± 3.10 MPa, equivalent to an absolute reduction in mean UCS of 12.70 MPa, or 41.36% (p < 0.001) (Table 5). We recorded a reduction in mean net strain at failure of 20.69% (p < 0.001), and mean reduction in Young’s Modulus of 69.7% (p = 0.001). These changes in mean conditions are also indicated in Figure 9 a & b, which demonstrates a shift in the kernel density estimates of normalised axial stress and strain to values lower than those observed in baseline tests. For siltstone samples weathered in field conditions, we observed a mean UCS of 37.10 MPa ± 13.57 MPa, which is not statistically-significantly different from baseline values (p = 0.583; Figure 9 b). Strain at failure values decreased (p > 0.001) relative to those This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. observed in baseline tests (12.55%), but overall variability increased (Figure 9 b). with Young’s Modulus values also decreasing by 54.54% (p =0.006). Siltstone: Effect of ambient compressive stress Siltstone control samples displayed a mean UCS of 25.81 MPa ± 14.13 MPa, equivalent to an absolute reduction in mean UCS of 6.34 MPa, or 22.26% (p = 0.043) (Table 5). We recorded a reduction in mean net strain at failure and Young’s Modulus of 14.31% (p > 0.001) and 54.96% (p = 0.0065), respectively. Figure 9 c & d demonstrates a shift in the kernel density estimates of normalised axial stress and strain to values lower than those observed in baseline tests, with overall variability in normalised axial stress (UCS) increased (Table 5). For stressed siltstone samples, we observed a mean UCS of 28.80 MPa ± 13.86 MPa, equivalent to a reduction of 4.3 MPa, or 14.52% (p = 0.043; Figure 9 c). Strain-at-failure values decreased (p = 0.002) relative to those observed in baseline tests (18.57 %) (Figure 9 d), as did Young’s Modulus (p = 0.001, 66.82%) (Table 5). No statistically-significant differences exist between the mean UCS, strain-at-failure, and Young’s Modulus values of stressed and control siltstone samples (p = 0.262, p = 0.044, and p = 0.300, respectively) (Figure 9 c). Vertical strain measurements recorded during the weathering experiments indicated that each of the 8 stressed samples in the laboratory experiments compressed over the duration of the laboratory weathering test. We observed small-scale expansion events (-0.01 % to -0.02 % strain) on time-scales greater than that of the wetting and drying cycles that lasted for week long periods. We also observed elastic rebound of the samples at the end of the tests when the load was removed. U and P samples This article is protected by copyright. All rights reserved. displayed no permanent strain over the course of the experiment, in contrast to the G and PG samples, which displayed permanent strain values of 0.01 % to 0.16 % at the end of the weathering experiment. Siltstone: Effect of pre-treatment type For U- and P--type samples, mean UCS values were statistically-significantly (p = 0.001 and p = 0.042, respectively) lower than those observed in baseline tests; we recorded reductions in mean UCS of 38.28% and 20.28% respectively (Table 5; Figure 9 e). G- and PG-type samples did not show statistically-significant changes (p = 0.159 and p = 0.116 respectively) in mean UCS values relative to those observed in baseline tests. Siltstone sample modifications displayed lower kernel density distributions for strain-at-failure values relative to baseline (p <0.022) (Figure 9 f). Young’s Modulus values were all lower than those observed in baseline tests, indicating a decrease in sample stiffness (p < 0.05) (Table 5). This article is protected by copyright. All rights reserved. Baseline sandstone characterisation We recorded a mean Equotip L- value of 564.87 ± 68.73. The mean UCS was 55.69 MPa ± 7.61 MPa (Table 2). Failure occurred at a mean net strain of 1.25% ± 0.07% and mean local strain of 0.24% ± 0.14%. We calculated a mean Young’s Modulus of 5.69 GPa ± 0.86 GPa. All baseline sandstone samples exhibited a single-stage brittle failure (Figure 5). For the modified geometry (G) samples, we observed mean UCS values of 48.75 MPa ± 3.2 MPa. We did not observe a strong or statistically- significant relationship between UCS and bulk density for the sandstone samples (r² = 0.1626, p = 0.2188). To consider the effects of weathering on the strength of sandstone, we therefore compared absolute and percent changes in UCS for weathered samples to the overall baseline mean value. Sandstone visual appearance and surface hardness The visual appearance of all samples changed during laboratory weathering experiments, principally by surficial grain loss, with occasional sub-horizontal cracking. Iron leaching was evident for all stressed laboratory samples (Figure 7 e). Surficial grain loss was observed in all field samples; this occurred in concentrated ‘pockets’ for three samples (Figure 7 f), though there was no relationship with pre- treatment type of ambient stress conditions. We did not observe surface cracking or slaking in control or stressed samples for both laboratory or field experiments. We measured a positive change in mass for all laboratory samples, with a mean of 1.72% ± 0.72%. We measured a positive change in mass for 10 field samples, with the remaining 6 samples displaying a negative change in mass. As such, field samples displayed a mean change in mass of -0.62% ± 3.32%. For laboratory samples we observed a statistically-significant net decrease in surface hardness through time for both control (r = -0.26, p = 0.023) and stressed (r = -0.36, p = 0.001) samples. For field samples we observed a statistically-significant increase in surface hardness in both control (r = 0.4, p < 0.001) and stressed (r = 0.44, p < 0.001) samples. General changes in Sandstone compressive strength and strain values General changes in Sandstone compressive strength and strain values Following completion of laboratory weathering experiments, control sandstone samples displayed a mean UCS of 35.76 MPa ± 7.5 MPa and failed 1.23% ± 0.09% This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. and 0.22% ± 0.2% net and local strain, respectively (Table 3). We observed a mean Young’s Modulus of 6.58 GPa ± 1.47 GPa (Table 3). Stressed sandstone samples (all pre-treatment types) displayed a mean UCS value of 38.73 MPa ± 7.32 MPa, failing at 1.25% ± 0.09% and 0.27% ± 0.16% net and local strain, respectively. Normalised stress-strain curves relative to baseline tests are displayed in Figure 8 c. We observed a mean Young’s Modulus value of 5.49 GPa ± 0.38 GPa (Table 3). All samples displayed at least two stages of brittle failure (Table 3). For field weathering experiments, control sandstone samples displayed a mean UCS value of 49.93 MPa ± 7.84 MPa and failed at 1.23% ± 0.19% and 0.22% ± 0.16% net and local strain, respectively (Table 4). Normalised stress-strain curves relative to baseline tests are displayed in Figure 8 d. We observed a mean Young’s Modulus value of 7.07 GPa ± 2.39 GPa (Table 4). All field control sandstone samples displayed two stages of brittle failure (Table 4). Field stressed sandstone samples displayed a mean UCS value of 44.53 MPa ± 14.16 MPa and failed at 1.23% ± 0.19% and 0.17% ± 0.14% net and local strain, respectively. We observed a mean Young’s Modulus value of 6.90 GPa ± 0.86 GPa (Table 4). Stressed PG-type samples displayed a single stage of brittle failure. U- and G-type samples displayed two stages of brittle failure. P-type samples displayed three stages of brittle failure (Table 4). Sandstone: Experimental setting For sandstone samples weathered under laboratory conditions, we observed a mean UCS of 37.24 MPa ± 7.32 MPa, equivalent to an absolute reduction in mean UCS of 18.45 MPa, or 33.12% (Table 5). This overall reduction in UCS (p < 0.001) relative to This article is protected by copyright. All rights reserved. those observed in baseline tests is statistically significant (Figure 10 a). We recorded a reduction in mean net strain at failure of 0.38% and an increase in mean Young’s Modulus of 6.06%, which were not statistically significantly (p = 0.884 and p = 0.460, respectively) different from baseline tests (Figure 10 b) (Table 5). For sandstone samples weathered in field conditions, we observed a mean UCS of 47.23 MPa ± 11.40 MPa, which is statistically-significantly lower (8.46 MPa, or 15.19%) than baseline values (p = 0.042) (Figure 10 a). We recorded a reduction in mean net strain at failure of 1.81%, which was not statistically significant (p = 0.632) from baseline tests (Figure 10 b), along with a statistically significant increase in mean Young’s Modulus of 22.8% (p = 0.04) (Table 5). This article is protected by copyright. All rights reserved. Sandstone: Effect of ambient compressive stress Sandstone control samples displayed a mean UCS of 42.85 MPa ± 10.41 MPa, equivalent to an absolute reduction in mean UCS of 12.84 MPa, or 23.06% (Table 5). This statistically-significant change in mean UCS (p = 0.043 and p = 0.001) is also evident in Figure 10 c. We recorded a reduction in mean net strain at failure of 1.53% and an increase in mean Young’s Modulus of 19.98%, which were not statistically-significantly different (p = 0.538 and p = 0.089, respectively) from baseline tests (Figure 10 d) (Table 5). For sandstone stressed samples we observed a mean UCS of 41.63 MPa ± 11.29 MPa, which is statistically-significantly lower (14.06 MPa, or 25.25%) than baseline values (p = 0.042) (Figure 10 c). We recorded a reduction in mean net strain at failure of 0.66 % and an increase in mean Young’s Modulus of 8.88%, which were not statistically-significantly different (p = 0.847 and p = 0.250, respectively) from baseline tests (Figure 10 d) (Table 5). No statistically- This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. significant differences exist between the mean UCS, strain-at-failure values, and Young’s Modulus of stressed and control sandstone samples (p = 0.006, p = 0.668, and p = 0.204, respectively) (Figure 10 c). The same behaviour as seen for the monitored strain measurements for stressed siltstone samples is observed for the stressed sandstone samples, with six samples compressing in the direction of load over test duration. We observed expansion events on the order of days to weeks, which exerted –0.025 % to -0.1 % strain (Figure 11). We recorded rebound of samples at the end of the tests when the load was removed, with U and P displaying no permanent strain while G and PG samples displayed permanent strain values of 0.01% to 0.05% strain. This article is protected by copyright. All rights reserved. Sandstone: Effect of pre-treatment type For U-, P- and G--type samples, mean UCS values were statistically-significantly (p < 0.001, p = 0.008 and p = 0.008, respectively) lower than those observed in baseline tests; we recorded mean reductions in UCS of 13.69%, 12.77% and 11.58%, respectively (Table 5; Figure 10 e). PG-type samples did not show statistically-significant changes (p = 0.089) in mean UCS values relative to those observed in baseline tests. Sandstone sample modifications displayed a similar distribution in strain-at-failure values to baseline (Figure 10 f), with no statistically- significant changes observed. Young’s Modulus values were all higher than those observed in baseline tests, indicating an increase in sample stiffness (Table 5). For U samples this increase was statistically significant (p = 0.023). Failure mode Failure mode This article is protected by copyright. All rights reserved. For both siltstone and sandstone samples tests, single-stage failures occurred in samples that displayed the highest UCS values. As UCS sample strength decreased, failure mode changed to include more brittle stages of failure or ‘stress- drops’ before residual strength was achieved. Both lithologies displayed this behaviour (Figure 12), with significant differences in strength for each failure style existing for sandstone (p < 0.010; Figure 12b). These multi-stage failures may often sustain stresses slightly lower (~1 MPa to 2 MPa) than peak strength of the sample until further or final failure occurs (Figure 12c). Along with changes in stress-strain behaviour, a greater number of cracks and associated complexity of failure morphology were observed within the samples with increasing number of brittle failure stages (Figure 12d). No correlations existed between failure mode and environmental setting, test conditions or sample pre-treatments. This article is protected by copyright. All rights reserved. Discussion Controls on visual appearance and surface hardness Our weathering experiments were designed to understand controls on weathering intensity, including the influence of ambient compressive stress, stress history and local stress concentrations. Our monitoring during the experiments revealed that changes in the visual appearance of samples and surface hardness showed little sensitivity to pre-treatment. We note surface textures and degradation processes that are common to both lithologies, notably surficial grain loss, iron leaching and surface cracking. However, we also observed consistent patterns in surface texture that highlight the importance of lithology and the mechanism of weathering as acting as a control on the type and nature of surface appearance and texture. For example, This article is protected by copyright. All rights reserved. siltstone samples experienced slaking events in response to laboratory saltwater submergence and exposure cycles, as is commonly observed in argillaceous lithologies (Qi et al., 2015). In contrast, slaking was not observed in the field tests on siltstone; in this weathering environment where full saltwater submergence did not occur and where wetting resulted from precipitation, samples weathered by surficial grain loss that resulted in a distinctive ‘powdery’ surface texture. Critically, the pre- treatment type and/or ambient compressive stress condition did not affect the surficial weathering processes operating in the siltstone samples. For the sandstone samples, there was no obvious difference in the mechanism of weathering and the resultant surface response between laboratory and field tests; grain loss and cracking dominated in both settings. However, the presence of an ambient compressive stress resulted in more pronounced iron leaching, possibly due to sample dilatancy and permeability in response to stress application (Mitchell and Faulkner, 2009; Nicholson, 2001; Oda et al., 2002; Zoback and Byerlee, 1975). In turn, this likely facilitated oxidation, solution and water-borne removal of iron present in the sandstone samples. The controls of lithology and mechanism of weathering were also manifest in the nature of changes in the surface hardness of samples throughout the weathering experiments. Siltstone samples weathered in the laboratory did not show any change in surface hardness. In contrast, siltstone samples weathered in field conditions showed an increase in surface hardness. Sandstone samples weathered in the laboratory displayed a reduction in surface hardness, possibly resulting from a more dispersed loss of grains and a loss of near-surface cement. Discussion In contrast, field samples displayed an increase in surface hardness, in part due to the less-widely distributed This article is protected by copyright. All rights reserved. grain loss in isolated ‘pockets’, permitting case hardening to develop in areas not experiencing concentrated grain loss (Mol and Viles, 2012; Viles et al., 2011). Biogenic case hardening may also explain the increase in surface hardness for field samples, while the saline brine of the laboratory experiments prevented the development of such a crust (Slavík et al., 2017). This article is protected by copyright. All rights reserved. Controls on weathering induced strength degradation Our geotechnical analysis indicates that pre-existing micro-crack damage, modified sample geometries and/or samples subjected to a constant compressive vertical stress do not result in enhanced strength degradation relative to samples that have undergone no modification. We note that not all reductions in mean values of UCS were statistically-significant in some pre-treatment groups, but we observed decreases in the modal value(s) of UCS occurring in all samples, indicative of a general trend (Figures 9 and 10). A greater density of initial micro-cracks, as present in P samples, does not necessarily result in a greater degree of strength reduction, resulting from exploitation of micro-crack populations by weathering processes. This is in contrast to other studies, where increased surface area as a result of micro-cracking, or pre- existing damage within a sample due other weathering processes have been observed to accelerate the rate of weathering (Røyne et al., 2008, Viles et al., 2018). The removal of the high compressive stresses used in the pre-damaging process prior to commencing the weathering experiments may have permitted any newly- created microcracks to contract. High, damaging stresses may need to be maintained during operation of weathering processes for intergranular flaws to be This article is protected by copyright. All rights reserved. exploited and a more obvious control on strength reduction to be observed. We suggest that the nature of stress history and if and how this stress is maintained may be important. The degree of geometric modification in P and PG sample may have been insufficient to create stress concentrations of sufficient magnitude to result in enhanced micro-cracking (Lajtai and Lajtai, 1974). We suggest that differing cliff-face surface geometries at a range of scales may significantly generate greater stress concentrations that can be preferentially weathered than created by our sample modifications (Martel, 2006; Brain et al., 2014). However, within this experimental set-up and over the time-scale of the experiments considered, a constant compressive stress has a negligible effect on strength degradation resulting from weathering processes, with no statistically significant differences between the UCS values of control and stressed samples for both lithologies. This indicates that the ambient stress environment does not affect the intensity of weathering and its effect on compressive strength within the ambient stress range and experimental set-up considered here. This article is protected by copyright. All rights reserved. Controls on weathering induced strength degradation This result is in contrast to experiments conducted using sediments with no cementation (Bruthans et al., 2014; Zhang et al., 2015) or weak cementation (Bruthans et al., 2016; Rihosek et al., 2016). These previously-published experiments showed a temporal component of stress influence on weakening, displaying faster erosion rates (Bruthans et al., 2014) and strength degradation (Zhang et al., 2015) until a ‘critical’ stress value was reached. The interlocking strength of the grains was great enough to slow or prevent further erosion and weathering from occurring. The frictional properties of these materials were the dominant components of strength, rather than cohesional properties of intact rock. Martin and Chandler (1994) proposed that the strength of intact rock is controlled primarily by cohesion up until 75% to 80% of UCS. Our findings suggest that gravitationally-induced compressive stress (here 2 MPa and 3.8 MPa) has a limited impact on the processes which result in cohesional strength reduction. This imposed topographic stress may also be of an insufficient magnitude when combined with stresses generated by weathering to cause any volumetric changes via contraction or dilation of cracks in the rock samples that could subsequently be exploited by weathering processes. This is despite the enhanced leaching observed, and greater permeability postulated, in stressed sandstone samples. Higher ambient stresses that cause crack initiation (σci) and/or propagation (σcd) may cause weathering and sub-critical crack growth processes to drive greater differences in compressive strength between control and stressed samples. The significance of the magnitude of the ambient stress environment has been observed in laboratory tests where ambient stress environments are tensile (Voigtländer et al., 2018). This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Effect of weathering on compressive rock strength Overall, we demonstrate that weathering results in a significant reduction of strength for all laboratory samples and even for rock that has been exposed to natural environmental conditions for a year, as demonstrated by the 15.19% loss in strength for sandstone samples placed at the cliff-top. The observed reductions in intact rock strength are likely to result from a combination of factors, in addition to those that are evident in the observed surficial changes in the rock, such as slaking, cracking and grain loss. For example, sub-critical crack growth throughout the rock samples may be a key driver, where micro-crack growth occurs at stresses lower than the crack initiation threshold within a rock mass (Atkinson, 1984). Small amplitude stress as a result of environmental processes such as insolation or wetting and drying can therefore drive micro-crack growth (Eppes and Keanini, 2017). This can occur via stress corrosion cracking where molecular bonds are strained and stretched at crack tips by a chemically active environmental agent, such as water (Atkinson, 1984; Eppes and Keanini, 2017; Voigtländer et al., 2018). This process is enhanced where chemical conditions are conducive to corrosion; changes in the geochemistry, as evidenced by leaching, may subsequently change sub-critical cracking characteristics, though little is known about the exact controls on this process (Atkinson, 1984; Dunning and Huf, 1983; Freiman, 1984). We noted a greater reduction in UCS in both lithologies for samples weathered under laboratory conditions, highlighting the importance of weathering process in driving degradation of rock strength. In particular, we note the relative efficacy of salt as a weathering agent and in driving strength loss of intact rocks (cf. Goudie et al., 1970). We observed significant periods of expansion within the laboratory strain data, indicating that such expansion was able to counteract the 3.8 MPa vertical stress acting on the sample. This potentially explains the limited influence of topographic stress on strength as weathering can generate stresses that counteract those generated by overburden loading. We observed a mismatch between the two measures of strength that we used. Notably, the reductions in UCS recorded following the completion of weathering experiments were not evident in any statistically-significant trends in surface hardness during the experiment in all samples. In field-weathering sandstone samples, surface hardness increased, despite a decrease in ultimate UCS. Effect of weathering on compressive rock strength This inconsistency indicates that our interpretation of rock strength from visual observations and surface hardness data may not capture the ‘internal’ weakening of intact rock. This internal weakening is of critical importance to shallow rock slope failures, where release of an incipient failure mass is contingent on brittle fracture through intact rock (Collins and Stock, 2016). Our results indicate that use of surface hardness measurements in constraining the influence of weathering on intact rock strength and, hence, shallow rock slope failure that show visible evidence of weathering may be limited and/or not appropriate in all situations (see Aoki and Matsukura, 2007; Coombes et al., 2013; Viles et al., 2011). This article is protected by copyright. All rights reserved. Effects of weathering on failure style Our results suggest that the two lithologies display differing responses to weathering in terms changes to strain-at-failure values. We observed no change in strain-at- failure values in weathered sandstone samples when compared to baseline tests. In contrast, siltstone samples displayed statistically-significant reductions in strain-at- failure values relative to baseline values. Weathered siltstone samples also displayed mean Young’s modulus values that are statistically-significantly lower than baseline values. An explanation of these differences in strain and elasticity between lithologies is enigmatic but may result from a greater degree of softening and ductility in the siltstone samples due to the presence of clay minerals (Fabre and Pellet, 2006). The siltstone may be more susceptible to granular rearrangement than the highly-cemented sandstone, which undergoes considerably less plastic deformation at values less than UCS. Our analysis indicates that a reduction in strength is linked to and manifest in a change in failure style. Weaker rocks display a more distributed multi-stage failure process reflected in their stress-strain behaviour and the resultant failure morphology (as observed also in studies by Basu et al., 2009; Gupta and Seshagiri Rao, 2000). Multi-stage failures involve several stages of macro-scale fracture and strength loss until residual or total loss of strength occurs. These types of failure can temporally sustain high stress levels even after a peak stress level has been reached. It is only after sufficient post-peak strain has accumulated within the sample that subsequent failure event(s) occur. Weakening of the rock sample by weathering may lead to more diffuse micro-cracking that eventually results in an increased number of macro- cracks, as seen in cyclic loading tests, which can be used as a proxy for environmental fluctuations and associated weathering processes (Cerfontaine and Collin, 2017). These distributed micro-cracks do not result in the same pattern of coalescence required for unstable ‘run-away’ macro-scale fracture, as normally predicted for a similar point on stress-strain curves (Eberhardt et al., 1998; Martin and Chandler, 1994). The failure events observed in the multi-failure stage failures instead may represent mini-coalescence events in weaker zones to form relatively smaller macro-scale fractures, which do not connect in the first instance. This article is protected by copyright. All rights reserved. Implications for shallow rock slope failures We suggest that a change in failure style and strength loss over time (Figure 13) will determine the nature of triggers required for failure to occur and will hence dictate This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. the timing of ultimate failure. Intact rock characterised by single-stage brittle fracture likely requires a higher magnitude loading event that will result in near-instantaneous failure, as indicated in Figure 13. Two-stage brittle failure likely still require a relatively higher magnitude loading event for an initial fracture event to occur and may require another similar magnitude loading event for final failure to occur (Figure 13). The timing between two such events may be potentially be prolonged, resulting in a quasi-stable state (Leroueil, 2001). As damage accumulates through time, the magnitude of environmental stresses required for fracture to occur decreases (Figure 13), but the frequency of such events will likely increase, resulting in a positive feedback and potential pattern of acceleration towards ultimate failure. Figure 14 illustrates this process, with each ‘new’ failure event weakening the rock, so that the next stage of brittle fracturing requires a lower magnitude of stress to act as a trigger for further failure. This multi-stage failure processes may be represented as ‘step- wise’ fracture through multiple rock bridges or partial fracturing through an individual rock bridge, where fracture represents an initial failure stage of the stress–strain graph (Brideau et al., 2009; Eberhardt et al., 2004b; de Vilder et al., 2017). For final failure to occur, only a low magnitude stress perturbation may be required (Figure 14) due to the critical concentration of micro-cracks and accumulated damage within the rock (Main, 2000). In the context of rock slope failure, this final stress perturbation may reflect stress-redistribution of the slope following progressive failure (e.g. Eberhardt et al., 2004a; Rosser et al., 2007; Stock et al., 2012), environmental stress fluctuations (Collins and Stock, 2016; Gischig et al., 2011; Gunzburger et al., 2005; Moore et al., 2011) or topographic stress concentrations within the slope (Brain et al., 2014). In such a scenario, even though topographic stress is not a This article is protected by copyright. All rights reserved. control on the rate of weakening, as we have determined from our experimental datasets, it may therefore control the location of rock failure. This article is protected by copyright. All rights reserved. Implications for shallow rock slope failures As final failure occurs a distinct period of time after the initial damaging loading event (such as an earthquake or storm), this may explain the low observed correlations between environmental variables and failure (Lim et al., 2010; Rosser et al., 2007; Stock et al., 2012), with a ‘lag’ between initial and final failure. Monitoring observations have revealed that external precipitation triggering events can initiate extended periods of increased and accelerated deformation indicating a critical damage threshold has been reached in the rock, with previous observations showing that in some settings that this can accelerate to final failure within a week (Carlà et al., 2018; Kromer et al., 2017a; Loew et al., 2016). Observations of pre-failure deformation that did not result in a hyperbolic acceleration towards failure may represent a failure sequence where a significant ‘lag’ effect exists between the initial and final failure events (Carlà et al., 2018). Such a ‘lag’ effect suggests that weathered rock slopes may adjust more slowly to changing environmental conditions. This multi-stage and prolonged failure sequence may also be reflected in the resulting failure scar surface, where greater roughness coupled with surficial weathering provide an indication of failure history. Weathered and broken rock bridges can represent an initial failure event, which has been followed by a sufficiently long period for substantial surficial weathering (i.e. damage accumulation in Figure 14) to occur before the final loss of strength and collapse (de Vilder et al., 2017). In addition, the greater strain that can be sustained in the weathered samples This article is protected by copyright. All rights reserved. increases, suggesting that weathered rock bridges can sustain greater levels of strain before final detachment occurs. This may have important implications for monitoring of pre-failure deformation; our results suggest that a universal value of ‘critical strain’ is unlikely to be valid for a given rock type and will depend on the degree of weathering experienced by any individual section of rockslope. Pre-failure deformation may also record several stages of macro-scale brittle failure related to rock bridge fracture (Figure 14)(Carlà et al., 2017; Kromer et al., 2015; Royan et al., 2015). Implications for shallow rock slope failures The change in failure style may dictate the degree of discernible pre-failure deformation, with multi-failure events potentially displaying higher degrees of pre- failure deformation than compared to single stage events as the period of time over which total loss of strength occurs is longer (Kromer et al., 2017b; Petley et al., 2005; Williams et al., 2018). The time-scales over which such deformation could be observed is also dependent on the exact rock bridge attributes within an incipient failure mass, with the amount of deformation increasing for larger rockfalls (Kromer et al., 2017b), reflecting the breakage of multiple rock bridges (de Vilder et al., 2017). We, therefore, hypothesise that as a rock slope weakens through time, the mechanics of rockfall detachment are likely to change. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Conclusion We conducted a series of experiments on coarse- and fine-grained sedimentary rocks under constant uniaxial compressive stress to constrain the relationship between exposure to various environmental conditions, compressive stress and ultimate failure behaviour. We modified samples to account for pre-existing micro- crack damage within the rock, as well as increased surface area and localised stress This article is protected by copyright. All rights reserved. concentrations created by slope geometry. Our experimental dataset reveals that weathering does result in a reduction in unconfined compressive strength, though the applied compressive stresses of 2 and 3.8 MPa neither enhance nor dampen the degree of weathering-induced strength loss. In isolation, this result suggests that sections of rock slope subjected to high stresses will not preferentially weaken in response to weathering relative to sections under no ambient stress. However, we suggest that our findings may be specific to the lithologies considered here, where cohesional strength dominates over friction. In addition, greater ambient stresses as a proportion of ultimate strength may be possible in certain topographic settings, influencing mechanisms of weathering. Alongside this, pre-existing damage and increased surface roughness also have no discernible influence on the magnitude of strength reduction resulting from weathering. The reduction in UCS strength is not consistently reflected in a reduction of surface hardness, placing limits on the interpretation of intact rock strength made solely based on surface hardness monitoring. In our experiments, weathering resulted in considerable changes in the style of failure, even over relatively short (three months to one year) timescales. Weathered weaker samples developed post-peak strength and so several stages of macro-scale fracture were required before a total loss of strength was observed. We suggest that these multiple fracture events may be representative of complete or partial rock bridge fracture. As post peak strength evolves, we theorise that the magnitude of triggering events that result in brittle facture, and ultimately rockfall release, decreases. This provides further explanation of rockfall activity that occurs in response low-magnitude environmental forcing that are conventionally considered to This article is protected by copyright. All rights reserved. be too low to cause shallow rock slope failure. The associated prolonged sequence of brittle fracture may be manifested in step-wise patterns of pre-failure deformation data. Weathering induced strength degradation, therefore, may alter the rock slope response to environmental loading events and the subsequent mechanical evolution of failure. be too low to cause shallow rock slope failure. This article is protected by copyright. All rights reserved. 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DOI: 10.1029/JB080i005p00752 Acknowledgments The authors gratefully acknowledge support for this research from ICL Fertilizers (UK) Ltd. We thank Neil Tunstall and Chris Longley for help with laboratory testing, Dave Hodgson for help with the installation of the laboratory and field experiments, Sam Waugh for help with field monitoring, Simon Varley and Richard Boothroyd for help with sample collection, and Zuzanna Swirad for help with laboratory monitoring. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Figure 1. a) Example siltstone sample core. b) Example sandstone sample core. Both cores are 96 mm high, and 48 mm in diameter. Figure 1. a) Example siltstone sample core. b) Example sandstone sample core. Both cores are 96 mm high, and 48 mm in diameter. Figure 1. a) Example siltstone sample core. b) Example sandstone sample core. Both cores are 96 mm high, and 48 mm in diameter. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Figure 2. Detail of sample modifications for a) preloaded (P) and b) geometrically- altered (G) samples. a) Stress-strain curve showing the different stages of micro- crack development under conditions of uniaxial compression (adapted from: Eberhardt et al., 1998). Samples were preloaded to 75% of peak strength in order to exceed the crack initiation threshold but not exceed that of the crack damage threshold. b) Geometry of notches cut into G samples, showing the plan view and an example notch within a sandstone core. Figure 2. Detail of sample modifications for a) preloaded (P) and b) geometrically- altered (G) samples. a) Stress-strain curve showing the different stages of micro- crack development under conditions of uniaxial compression (adapted from: Eberhardt et al., 1998). Samples were preloaded to 75% of peak strength in order to exceed the crack initiation threshold but not exceed that of the crack damage threshold. b) Geometry of notches cut into G samples, showing the plan view and an example notch within a sandstone core. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Figure 3. Detail of the laboratory weathering set-up, showing modified oedomete that subjected stressed samples to constant vertical compressive stress an corresponding control (non-stressed) samples (a and b). Vertical compressive stres is applied by fixed hanging weights and a lever system (a) The saltwater wetting an Figure 3. Detail of the laboratory weathering set-up, showing modified oedometers that subjected stressed samples to constant vertical compressive stress and corresponding control (non-stressed) samples (a and b). Vertical compressive stress is applied by fixed hanging weights and a lever system (a). The saltwater wetting and drying system is evident in a) and summarised conceptually in c). The system This article is protected by copyright. All rights reserved. operates by pumping water into the containers via a pipe (i) from the saltwater reservoir, and draining via a valve (ii) after 30 minutes of inundation. Over-topping of the containers was prevented through the use of an overflow pipe (iii). A small amount of standing water was present below the valve line within the container, and so samples were placed on a pedestal (iv) to ensure that they were located above the standing water to allow full drainage. operates by pumping water into the containers via a pipe (i) from the saltwater reservoir, and draining via a valve (ii) after 30 minutes of inundation. Over-topping of the containers was prevented through the use of an overflow pipe (iii). A small amount of standing water was present below the valve line within the container, and so samples were placed on a pedestal (iv) to ensure that they were located above the standing water to allow full drainage. This article is protected by copyright. All rights reserved. Figure 4. Photograph (a) and schematic summary (b) of the field compression apparatus used to place samples under a constant vertical compressive stress. Non- stressed samples were placed on top of the beam (c), while ‘stressed’ samples were placed under load using a simple lever system (c, d). The weights for each lever arm were contained within protective tubes to prevent the hanging weights from moving due to wind (a). Vertical compressive stress was applied to samples via level system (b). Samples were compressed against the levelling jack when weights were applied on the opposite lever arm. Figure 4. This article is protected by copyright. All rights reserved. Photograph (a) and schematic summary (b) of the field compression apparatus used to place samples under a constant vertical compressive stress. Non- stressed samples were placed on top of the beam (c), while ‘stressed’ samples were placed under load using a simple lever system (c, d). The weights for each lever arm were contained within protective tubes to prevent the hanging weights from moving due to wind (a). Vertical compressive stress was applied to samples via level system (b). Samples were compressed against the levelling jack when weights were applied on the opposite lever arm. Figure 4. Photograph (a) and schematic summary (b) of the field compression apparatus used to place samples under a constant vertical compressive stress. Non- stressed samples were placed on top of the beam (c), while ‘stressed’ samples were placed under load using a simple lever system (c, d). The weights for each lever arm were contained within protective tubes to prevent the hanging weights from moving due to wind (a). Vertical compressive stress was applied to samples via level system (b). Samples were compressed against the levelling jack when weights were applied on the opposite lever arm. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Figure 5. Stress-strain curves for unconfined compression tests on siltstone (a and b) and sandstone (c and d) samples for baseline property characterisation. a) and c) display net axial strain response; b) and d) display local (LVDT) strain response. Figure 5. Stress-strain curves for unconfined compression tests on siltstone (a and b) and sandstone (c and d) samples for baseline property characterisation. a) and c) display net axial strain response; b) and d) display local (LVDT) strain response. Figure 5. Stress-strain curves for unconfined compression tests on siltstone (a and b) and sandstone (c and d) samples for baseline property characterisation. a) and c) display net axial strain response; b) and d) display local (LVDT) strain response. Figure 6. Scatter plot of UCS values against initial bulk density values for siltstone samples. The modelled regression envelope (fit plus 95% confidence bounds) and statistical summary information represent samples used for baseline characterisation. Results for samples weathered during field and laboratory experiments which plot outside of these bounds are considered to significantly different to baseline UCS values. Figure 6. Scatter plot of UCS values against initial bulk density values for siltstone samples. The modelled regression envelope (fit plus 95% confidence bounds) and statistical summary information represent samples used for baseline characterisation. Results for samples weathered during field and laboratory experiments which plot outside of these bounds are considered to significantly different to baseline UCS values. Figure 6. Scatter plot of UCS values against initial bulk density values for siltstone samples. The modelled regression envelope (fit plus 95% confidence bounds) and statistical summary information represent samples used for baseline characterisation. Results for samples weathered during field and laboratory experiments which plot outside of these bounds are considered to significantly different to baseline UCS values. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Figure 7. Photographs of weathered samples taken during monitoring experiments. a) Siltstone sample displaying slaking with detached fragments evident around the Figure 7. Photographs of weathered samples taken during monitoring experiments. a) Siltstone sample displaying slaking, with detached fragments evident around the base of the container. b) Siltstone core displaying an incipient slaking event characterised by a vertical shallow crack with a narrow aperture. c) Siltstone sample with a ‘powdery’ surface to touch, with many individual grains at the base of the core. d) Siltstone sample with multiple sub-horizontal cracks at the top and mid-point of the core. e) A sandstone sample with modified geometry (G) displaying iron leaching. f) Sandstone sample displaying an area of concentrated grain loss. a), b), and e) were Figure 7. Photographs of weathered samples taken during monitoring experiments. a) Siltstone sample displaying slaking, with detached fragments evident around the base of the container. b) Siltstone core displaying an incipient slaking event characterised by a vertical shallow crack with a narrow aperture. c) Siltstone sample with a ‘powdery’ surface to touch, with many individual grains at the base of the core. d) Siltstone sample with multiple sub-horizontal cracks at the top and mid-point of the core. e) A sandstone sample with modified geometry (G) displaying iron leaching. f) Sandstone sample displaying an area of concentrated grain loss. a), b), and e) were Figure 7. Photographs of weathered samples taken during monitoring experiments. a) Siltstone sample displaying slaking, with detached fragments evident around the base of the container. b) Siltstone core displaying an incipient slaking event characterised by a vertical shallow crack with a narrow aperture. c) Siltstone sample with a ‘powdery’ surface to touch, with many individual grains at the base of the core. d) Siltstone sample with multiple sub-horizontal cracks at the top and mid-point of the core. e) A sandstone sample with modified geometry (G) displaying iron leaching. f) Sandstone sample displaying an area of concentrated grain loss. a), b), and e) were re 7. Photographs of weathered samples taken during monitoring experiments subjected to laboratory weathering conditions. c), d) and f) were subjected to field weathering conditions. Figure 8. Normalised stress-strain curves for UCS tests of siltstone (a and b, laboratory and field respectively) and sandstone (c and d, laboratory and field respectively) samples following the completion of weathering experiments. This article is protected by copyright. All rights reserved. Normalised values of 1 are equivalent to the mean UCS and strain-at-failure values observed in baseline tests. The shaded areas represent the envelope of the range of baseline stress values for a given strain value. Figure 8. Normalised stress-strain curves for UCS tests of siltstone (a and b, laboratory and field respectively) and sandstone (c and d, laboratory and field respectively) samples following the completion of weathering experiments. Normalised values of 1 are equivalent to the mean UCS and strain-at-failure values observed in baseline tests. The shaded areas represent the envelope of the range of baseline stress values for a given strain value. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Figure 9. Kernel density plots displaying the distributions of normalised UCS and normalised axial strain at failure for siltstone samples of different groupings compared to baseline tests. Normalised values of 1 are equivalent to the mean UCS and strain-at-failure values observed in baseline tests. a) & b) Siltstone samples weathered in laboratory and field conditions compared to baseline tests. c) & d) ‘Stressed’ and ‘control’ siltstone samples compared to baseline tests. e) & f) Each sample pre-treatment group of siltstone subjected to weathering compared to baseline test results. Figure 9. Kernel density plots displaying the distributions of normalised UCS and normalised axial strain at failure for siltstone samples of different groupings compared to baseline tests. Normalised values of 1 are equivalent to the mean UCS and strain-at-failure values observed in baseline tests. a) & b) Siltstone samples weathered in laboratory and field conditions compared to baseline tests. c) & d) ‘Stressed’ and ‘control’ siltstone samples compared to baseline tests. e) & f) Each sample pre-treatment group of siltstone subjected to weathering compared to baseline test results. Figure 9. Kernel density plots displaying the distributions of normalised UCS and normalised axial strain at failure for siltstone samples of different groupings compared to baseline tests. Normalised values of 1 are equivalent to the mean UCS and strain-at-failure values observed in baseline tests. a) & b) Siltstone samples weathered in laboratory and field conditions compared to baseline tests. c) & d) ‘Stressed’ and ‘control’ siltstone samples compared to baseline tests. e) & f) Each sample pre-treatment group of siltstone subjected to weathering compared to baseline test results. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Figure 10. Kernel density plots displaying the distributions of normalised UCS and normalised axial strain at failure for sandstone samples of different groupings compared to baseline tests. Normalised values of 1 are equivalent to the mean UCS and strain-at-failure values observed in baseline tests. a) & b) Sandstone samples weathered in laboratory and field conditions compared to baseline tests. c) & d) ‘Stressed’ and ‘control’ sandstone samples compared to baseline tests. e) & f) Each sample pre-treatment group of sandstone subjected to weathering compared to baseline test results. Figure 10. Kernel density plots displaying the distributions of normalised UCS and normalised axial strain at failure for sandstone samples of different groupings compared to baseline tests. Normalised values of 1 are equivalent to the mean UCS and strain-at-failure values observed in baseline tests. a) & b) Sandstone samples weathered in laboratory and field conditions compared to baseline tests. c) & d) ‘Stressed’ and ‘control’ sandstone samples compared to baseline tests. e) & f) Each sample pre-treatment group of sandstone subjected to weathering compared to baseline test results. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Figure 11. Strain response of four samples subjected to a constant uniaxial compressive load within the laboratory experiments. a) Daily averaged fluctuations in temperature and relative humidity within the climate-controlled laboratory over the duration of the experiment. b) Strain response for each of the four stressed siltstone samples within the laboratory experiments (Table 1). Stress 1 and 2 are PG samples, and Stress 3 and 4 are G samples. An increase in strain values reflects compression of the sample, while decreases in strain values reflect expansion of sample. Rebound occurred at the end of the experiment for all samples once the constant uniaxial compressive stress was removed. b) First 24 hours of experiment, displaying an initial compression for all samples, followed by either further compression as is the case for Stress 3, or expansion as seen for Stress 1, 2 and 4. Figure 11. Strain response of four samples subjected to a constant uniaxial compressive load within the laboratory experiments. a) Daily averaged fluctuations in temperature and relative humidity within the climate-controlled laboratory over the duration of the experiment. b) Strain response for each of the four stressed siltstone samples within the laboratory experiments (Table 1). Stress 1 and 2 are PG samples, and Stress 3 and 4 are G samples. An increase in strain values reflects compression of the sample, while decreases in strain values reflect expansion of sample. Rebound occurred at the end of the experiment for all samples once the constant uniaxial compressive stress was removed. b) First 24 hours of experiment, displaying an initial compression for all samples, followed by either further compression as is the case for Stress 3, or expansion as seen for Stress 1, 2 and 4. This article is protected by copyright. All rights reserved. This article is protected by copyright. A This article is protected by copyright. All rights rese This article is protected by copyright. All rights reserved. Figure 12. a) Boxplots displaying changes in UCS in relation to the number of stress- drop failure events observed for siltstone samples. b) Boxplots displaying changes in UCS in relation to the number of stress-drop failure events observed for sandstone samples. c) Conceptual diagrams of typical stress-strain plots associated with differences in the number of stress-drop failure events. d) Photographs of typical failure styles and the associated degree of fragmentation associated with differences in the number of stress-drop failure events. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. p This article is protected by copyright. All rights reserved. Figure 13. Conceptual diagram of the impact of incremental strength decrease over time in response to environmental conditions (modified from: Gunzburger et al., 2005). Over time, as rock strength decreases the failure style will transition from a purely brittle failure (a) to a multi-stage (c) and brittle-ductile failure (d). Each stress- strain curve represents the type of failure style expected given the strength of the rock, with the loading events, such as earthquakes and storms, representing the required stresses necessary for failure to occur. As weathering proceeds, the magnitude of the event required to cause failure decreases. Figure 13. Conceptual diagram of the impact of incremental strength decrease ove time in response to environmental conditions (modified from: Gunzburger et al Figure 13. Conceptual diagram of the impact of incremental strength decrease over Figure 13. Conceptual diagram of the impact of incremental strength decrease over time in response to environmental conditions (modified from: Gunzburger et al., 2005). Over time, as rock strength decreases the failure style will transition from a purely brittle failure (a) to a multi-stage (c) and brittle-ductile failure (d). Each stress- strain curve represents the type of failure style expected given the strength of the rock, with the loading events, such as earthquakes and storms, representing the required stresses necessary for failure to occur. As weathering proceeds, the magnitude of the event required to cause failure decreases. Figure 13. Conceptual diagram of the impact of incremental strength decrease over time in response to environmental conditions (modified from: Gunzburger et al., 2005). Over time, as rock strength decreases the failure style will transition from a purely brittle failure (a) to a multi-stage (c) and brittle-ductile failure (d). Each stress- strain curve represents the type of failure style expected given the strength of the rock, with the loading events, such as earthquakes and storms, representing the required stresses necessary for failure to occur. As weathering proceeds, the magnitude of the event required to cause failure decreases. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Figure 14. Conceptual stress-strain diagram of the stages and drivers of weathered brittle rock failure, displaying initial micro-crack initiation and propagation thresholds for intact rock (adapted from Eberhardt et al., 1998). For macro-scale fracture resulting in eventual final failure to occur, weathered rock bridges must experience sustained stress and strain, resulting in failure events. Figure 14. Conceptual stress-strain diagram of the stages and drivers of weathered brittle rock failure, displaying initial micro-crack initiation and propagation thresholds for intact rock (adapted from Eberhardt et al., 1998). For macro-scale fracture resulting in eventual final failure to occur, weathered rock bridges must experience sustained stress and strain, resulting in failure events. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Tables Table 1: Sample types tested for each weathering experiment. For each of the sample types (e.g. U, P, G, PG), two of the samples were placed under a constant vertical stress, while the other two samples were controls. Unmodified samples (U) Pre- damaged samples (P) Modified geometry samples (G) Pre-damaged and modified geometry samples (PG) Laboratory– Sandstone 4 4 4 4 Laboratory - Siltstone 4 4 4 4 Cliff – Sandstone 4 4 4 4 Cliff - Siltstone 4 4 4 4 Tables Tables Table 1: Sample types tested for each weathering experiment. For each of the sample types (e.g. U, P, G, PG), two of the samples were placed under a constant vertical stress, while the other two samples were controls. Unmodified samples (U) Pre- damaged samples (P) Modified geometry samples (G) Pre-damaged and modified geometry samples (PG) Laboratory– Sandstone 4 4 4 4 Laboratory - Siltstone 4 4 4 4 Cliff – Sandstone 4 4 4 4 Cliff - Siltstone 4 4 4 4 Unmodified samples (U) Pre- damaged samples (P) Modified geometry samples (G) Pre-damaged and modified geometry samples (PG) Laboratory– Sandstone 4 4 4 4 Laboratory - Siltstone 4 4 4 4 Cliff – Sandstone 4 4 4 4 Cliff - Siltstone 4 4 4 4 Table 2: Baseline geotechnical characteristics derived from UCS testing. Table 2: Baseline geotechnical characteristics derived from UCS testing. Table 2: Baseline geotechnical characteristics derived from UCS testing. UCS (MPa) Mean bulk density (g cmˉ³) Mean Young’s modulus (GPa) Mean axial strain at failure (%) Brittl e failu re eve nts Me an Stand ard deviat ion Me an Stand ard deviat ion Me an Stand ard deviat ion Machine strain Local strain Mea n Me an Stand ard deviat ion Me an Stand ard deviat ion Siltsto ne 34. 15 6.43 2.3 1 0.15 8.9 9 3.2 1.4 7 0.07 0.4 6 0.21 1.7 Sandst one 55. 69 7.61 2.4 0.04 5.6 9 0.86 1.2 5 0.07 0.2 4 0.14 1 This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Table 2: Baseline geotechnical characteristics derived from UCS testing. UCS (MPa) Mean bulk density (g cmˉ³) Mean Young’s modulus (GPa) Mean axial strain at failure (%) Brittl e failu re eve nts Me an Stand ard deviat ion Me an Stand ard deviat ion Me an Stand ard deviat ion Machine strain Local strain Mea n Me an Stand ard deviat ion Me an Stand ard deviat ion Siltsto ne 34. 15 6.43 2.3 1 0.15 8.9 9 3.2 1.4 7 0.07 0.4 6 0.21 1.7 Sandst one 55. 69 7.61 2.4 0.04 5.6 9 0.86 1.2 5 0.07 0.2 4 0.14 1 This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Table 3: Geotechnical characteristics obtained from UCS testing of both lithologies following laboratory weathering experiments. Table 3: Geotechnical characteristics obtained from UCS testing of both lithologies following laboratory weathering experiments. Litholo gy Ambie nt stress conditi ons Sam ple type No. of sampl es Mean values UCS (MPa) Net axial strain at failure (%) Local axial strain at failure (%) Young's Modulus (GPa) Brittl e failur e even ts Siltstone Control All 6 16.72 (±1.64) 1.15 (±0.13) 0.38 (±0.17) 3.41 (±1.73) 2 U 2 18.73 1.17 0.24 5.40 2 P 1 16.57 1.36 NA* NA* 2 G 1 15.55 1.14 0.27 2.18 3 PG 2 16.74 1.02 0.20 2.66 3 Stress ed All 6 18.89 (±3.95) 1.19 (±0.16) 0.27 (±0.18) 1.69 (±0.03) 2 U 1 18.23 1.39 NA* NA* 1 P 1 20.19 1.13 NA* NA* 2 G 2 22.00 1.18 0.15 1.70 3 PG 2 15.45 1.12 0.12 1.70 2 Sandstone Control All 8 35.76 (±7.5) 1.23 (±0.09) 0.22 (±0.2) 6.58 (±1.47) 2 U 2 27.73 1.18 0.13 8.60 2 P 2 31.11 1.20 0.22 6.67 3 G 2 42.00 1.34 0.15 5.85 2 PG 2 42.21 1.22 0.37 5.22 3 Stress ed All 8 38.73 (±7.32) 1.25 (±0.09) 0.27 (±0.16) 5.49 (±0.38) 2 U 2 42.73 1.33 0.28 5.78 2 P 2 39.39 1.31 0.28 5.47 2 G 2 36.01 1.14 0.33 5.74 2 PG 2 36.79 1.23 0.21 4.96 2 *No local axial data and associated Young’s modulus values were obtained. This article is protected by copyright. All rights reserved. Table 4: Geotechnical characteristics obtained from UCS testing of sandstone and siltstone samples following field weathering experiments. p g g p Litholo gy Ambie nt stress conditi ons Sam ple type No. This article is protected by copyright. All rights reserved. of sampl es Mean values UCS (MPa) Net axial strain at failure (%) Local axial strain at failure (%) Young's modulus (GPa) Brittl e failur e stag es Siltstone Control All 5 36.71 (±14.93) 1.40 (±0.09) 0.26 (±0.17) 5.00 (±1.52) 2 U 1 37.51 1.30 NA* NA 2 P 2 25.30 1.41 0.15 3.93 3 G 0 - - - - - PG 2 47.73 1.44 0.38 6.08 2 Stress ed All 7 37.30 (±13.74) 1.21 (±0.1) 0.29 (±0.17) 3.63 (±2.79) 2 U 1* 23.72 1.12 0.41 1.81 2 P 2 39.92 1.28 0.25 0.73 1 G 2 47.27 1.24 0.24 6.45 3 PG 2 31.50 1.14 0.27 5.53 3 Sandstone Control All 8 49.93 (±7.84) 1.23 (±0.08) 0.22 (±0.16) 7.07 (±2.39) 2 U 2 45.31 1.25 0.25 6.69 2 P 2 52.51 1.16 0.21 6.25 2 G 2 51.51 1.28 0.14 10.47 2 PG 2 50.41 1.21 0.27 4.89 2 Stress ed All 8 44.53 (±14.16) 1.23 (±0.19) 0.17 (±0.14) 6.90 (±0.86) 2 U 2 43.68 1.14 0.04 7.40 2 P 2 31.99 1.33 0.17 6.01 3 G 2 42.44 1.21 0.24 6.37 2 PG 2 59.99 1.22 0.23 7.85 1 *No local axial data and associated Young’s modulus values were obtained. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. **Calculated from local strain data *Difference from mean baseline values. alculated from local strain data This article is protected by copyright. All rights reserved. Table 5: Strength properties of samples weathered in each environmental setting and test conditions. Absolute and percentage differences from equivalent baseline samples are displayed. Negative values are weaker than corresponding baseline values, and positive values are stronger. UCS Young 's Modul us perce nt differe nce (%)** Perce nt differe nce in net axial strain at failure (%) Mean (MPa) Standard deviation (MPa) Absol ute differe nce (MPa) * Perce nt differe nce (%)* Statisti cal signific ance tests (p values) Siltstone All sample s 27.43 13.76 -5.25 -18.10 <0.001 -61.43 -16.62 All field 37.10 13.57 2.21 5.23 0.583 -54.54 -12.55 All lab. 17.81 3.10 -12.70 -41.36 <0.001 -69.70 -20.69 Stress ed 28.80 13.86 -4.33 -14.52 0.043 -66.82 -18.57 Control 25.81 14.13 -6.34 -22.26 0.035 -54.96 -14.31 U 19.17 3.06 -11.79 -38.28 0.011 -59.90 -17.45 P 27.86 14.94 -5.95 -20.38 0.099 -74.10 -10.94 G 29.6 14.88 -3.64 -12.70 0.734 -59.87 -17.52 PG 27.85 16.01 -6.94 -22.84 0.898 -57.03 -19.78 Sandstone All sample s 42.24 10.70 -13.45 -24.15 <0.001 14.43 -1.10 All field 47.23 11.40 -8.46 -15.19 0.042 22.80 -1.81 All lab. 37.24 7.32 -18.45 -33.12 <0.001 6.06 -0.38 Stress ed 41.63 11.29 -14.06 -25.25 0.001 8.88 -0.66 Control 42.85 10.41 -12.84 -23.06 0.001 19.98 -1.53 U 42.00 5.49 -13.69 -24.60 <0.001 25.01 -3.24 P 42.92 13.15 -12.77 -22.93 0.001 7.18 0.23 G 44.11 11.77 -11.58 -20.80 0.002 24.88 0.11 PG 47.10 13.12 -8.59 -15.42 0.089 0.66 -1.49 *Difference from mean baseline values. **Calculated from local strain data **Calculated from local strain data
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Corrosion in reinforced concrete: Diagnostics through Potential Corrosion Technique
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Corrosion in reinforced concrete: Diagnostics through Potential Corrosion Technique Lucas Alexandre Reginato  Federal University of Rio Grande do Sul Alexandre Lorenzi  (  alexandre.lorenzi@gmail.com ) Federal University of Rio Grande do Sul Leonardo Covatti de Oliveira  Federal University of Rio Grande do Sul Luciani Somensi Lorenzi  Federal University of Rio Grande do Sul Luiz Carlos Pinto da Silva Filho  Federal University of Rio Grande do Sul Abstract Among the processes that accelerate the loss performance of reinforced concrete structures, considering their frequent occurrence and severity, reinforcement corrosion can be seen as one of the most important. A worrying factor is that most buildings in the Brazilian urban centers are between 40 and 50 years old, a time when repairs and maintenance become more routine. Therefore, the problems related to the corrosion of the reinforcements tend to accentuate in the coming years, and the identification of corrosion will play an important role. Thus, this research has the purpose to analyze the application of the diagnosis through potential corrosion techniques. For this, full-scale reinforced concrete beams were corrosion analyzed. Corrosion was stimulated by the Modified Immersion Accelerated Corrosion - CAIM method, at three levels of mass loss: 5, 10 and 15%. The diagnostic technique through the corrosion potential was applied, in which it was observed that there is uncertainty in the corrosion evaluation for the mass loss levels of 5 and 10%, resulting in a corrosion uncertainty interval, but at 15%, the potential technique proved to be satisfactory. Research Article Keywords: Corrosion, Diagnosis, Corrosion Potential, Corrosion Acceleration Posted Date: July 24th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3156721/v3 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Keywords: Corrosion, Diagnosis, Corrosion Potential, Corrosion Acceleration Posted Date: July 24th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3156721/v3 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/20 Page 1/20 1 INTRODUCTION The method accelerates the corrosion process, enabling experimental evaluation of corrosion’s effects in reinforced concrete structures. Some parameters are adopted to avoid divergences with the natural phenomenon. Reinforcement corrosion is one of the main challenges Civil Engineering faces. According to El-Reedy [2], this type of pathological manifestation costs billions of dollars around the world. A worrying factor, according to Cascudo [3] and Ribeiro [4], a good part of the buildings in the Brazilian urban center and its infrastructure were built in the ’70s and ’80s, reaching, nowadays, approximately between 40 to 50 years of age, respectively. Structures  built at that time are, thus, reaching a point in which repairs and maintenance become routine therefore, the problems related to reinforcement corrosion tend to accentuate in the coming years. The conservation and maintenance required to prevent the deterioration of the construction heritage entail high costs, given the characteristics of the technologies and processes involved in rehabilitation and repair. Buildings and infrastructure form an essential part of developed societies, and their usability to the highest safety standards is an indisputable priority [5]. Therefore, corrosion evaluation in different levels of degradation, defining a correlation with the diagnostics technique through corrosion potential, and aiding in detecting corrosion in this type of structure are sought after. 1 INTRODUCTION Civil Construction, beginning in the early years of the twentieth century, suffered a paradigm change: the main structures, at the time, were executed with traditional constructive methods based on self-supporting masonry and metallic structures. The paradigm changed as reinforced concrete appeared. The material had innovative potential as it united concrete’s versatility and disponibility with steel’s high strength. This way, there has been an ascension in reinforced concrete technologies starting in the last century’s early decades. Reinforced concrete structures such as buildings, bridges, etc. are designed to last a long time – it is not uncommon for bridge structures to have a design life of 100 years or more. By having the correct concrete cover with an appropriate mix design, the concrete itself generally provides the necessary corrosion protection for the embedded steel reinforcement bars [1]. A great sum of this growth occurred because, initially, it was believed that reinforced concrete structures, in general, had illimited durability thus not requiring maintenance. Therefore, during reinforced concrete’s advent, it was proclaimed as a material with illimited durability. Nonetheless, it is known today that no constructive material has these characteristics, as all wear and decay with time. Truly, nowadays, it is recognized that when adequately projected, executed, and dosed, reinforced concrete presents a considerable lifespan, if properly evaluated, among other factors, the conditions of exposure to the environment, protective measures, and maintenance. The search for an understanding of this material’s behavior, throughout its lifespan, gave birth to various studies that sought to comprehend the causal mechanisms in these structures' degradations. Among the degradation mechanisms that accelerate the loss of reinforced concrete structural performance, considering the frequent occurrence and severity, the reinforcement’s corrosion can be considered the most important. The reinforcement’s corrosion has to be observed cautiously, as it will modify some fundamental parameters of the structural project, generating deleterious effects, among them: reduction of the reinforcement’s transversal section; concrete fissuration due to the corrosion products' tension pressure; displacement of the covering layer; reduction of adherence between the reinforcement and the concrete; and a decrease in steel’s ductile behavior. However, corrosion’s phenomenological mechanism is naturally slow, making the natural evaluation of the process unfeasible, creating a necessity for acceleration methods of the corrosion process for experimental evaluations. Page 2/20 One of the most widespread and utilized methods is Modified Immersion Corrosion. 2 CORROSION IN REINFORCED CONCRETE STRUCTURES: EFFECTS OF CORROSION IN THE STRUCTURAL BEHAVIOR The study of corrosion in reinforced concrete is of the most relevant research lines among the Structural Models Testing Lab Research Group (LEME) and Graduate School Program in Civil Engineering (PPGEC) from Rio Grande do Sul Federal University (UFRGS). The present chapter introduces a short review of corrosion’s effects on reinforced concrete structures related to their structural behavior. Considering PPGEC/UFRGS’ history, the elaborated works that present an in-depth review related to corrosion are registered, in other words, the works of Andrade [6], Andrade [7], Adamatti [8], Caetano [9], Graeff [10], and Stein [11]. Other than these works there is a vast bibliography covering the subject, such as Cascudo [3], Helene [12; 13], Meira [14], Mehta and Monteiro [15], Ribeiro and Cunha [16], Tuutti [17], and others. 2.1 Reinforced Concrete Structures corrosion degree: general considerations Research performed by Apostolopoulos [18], Caprili and Salvatore [19], Fernandez et al. [20], Gehlen and Weirich [21], Kashani et al. [22], and Zhang et al. [23], evaluated the influence of corrosion on reinforced steel structures reinforcement bars, incorporated and not incorporated in concrete. The results converge to the confirmation of corrosion’s effects on steel’s mechanical properties. Steel’s tensile strength is directly affected by corrosion. Yet, simultaneously, a fragmentation of the steel occurs and, as a consequence, a reduction of the ductility. Under structural behavior’s point of view, the structure ductibility is always desirable, as the lack thereof may lead to fragile rupture and, in many times, unpredictability. As ductility is guaranteed by steel in reinforced concrete structures, degrees of corrosion, expressed in mass loss, above 5% there are relative losses in the mentioned characteristics (strength and ductility). However, these adverse effects are intensified in corrosion degrees above 10%. Page 3/20 Page 3/20 Page 3/20 It is emphasized that these effects have been related to research in which only the influence of corrosion on isolated bars of steel was analyzed. On the other hand, the effects of corrosion in reinforced concrete structures tend to be more severe, as there is, also, a reduction of adherence between the reinforcement and the concrete. Almusallam et al. [24], Graeff [10], and Caetano [9] report that the reduction of adherence is intensified in corrosion degrees greater or equal to 10% and that, in mild levels (1.5% to 4.0%), there is added adherence among both materials. As corrosion is analyzed in reinforced concrete structures, in the case of elements subject to bending such as beams, it is observed a behavior similar to the ones described above. However, with a degree of corrosion in the order of 5% there is a reduction in the structural performance of these elements. Nonetheless, this effect of the decrease in the structural performance is intensified in corrosion degrees above 10%. Such a fact is justified by the synergy betwixt the reduction of steel’s mechanical properties and the adherence loss between concrete and steel. 3.1 Corrosion Potential The measurement of the reinforcement corrosion potential consists of recording the voltage difference between the reinforcement and a reference electrode, which is placed in contact with the concrete surface. It is, basically, a qualitative technique that offers data about the probability for corrosion to occur in the analyzed reinforcement. The testing procedure follows ASTM C-876 - Standard Test Method for Corrosion Potentials of Uncoated Reinforcing Steel in Concrete [28]. This norm establishes a correlation between the intervals of potential difference, in relation to the reference electrode of Cu/CuSO4 (Copper/Copper Sulfate), in terms of corrosion probability, as can be observed in Table 1. Table 1. Correlation between the values of p.d. and the probability of corrosion (Font: adapted from [28]). Potential Difference (p.d.) Corrosion Probability p.d. > -200 mV Corrosion probability smaller than 10% -200 mV ≤ p.d. ≤ -350 mV Uncertain corrosion probability 50% p.d. < -350 mV Corrosion probability greater than 90% . Correlation between the values of p.d. and the probability of corrosion (F d d f [28]) Table 1. Correlation between the values of p.d. and the probability of 2.2 Corrosion mechanism in reinforced concrete structures Tuutti [17] proposed a phenomenological model of the corrosion mechanism present in reinforced concrete reinforcements, dividing it into two steps: start and propagation. Under Cascudo [3], for the development of corrosion, a rupture in steel’s passive layer is required. Andrade [7] defines the start process as the time interval necessary for aggressive agents (usually carbon dioxide or chlorides) to penetrate through the concrete’s covering layer and break the passive layer of the reinforcement. The passive layer, once broken, leaves the reinforcement open to corrosion, beginning the step of propagation [3]. Referring to this period, Meira [14] highlights that this phase of corrosion is related to the process kinetics, i.e., to the advancement of the corrosion process. This step of the mechanism is where the deleterious effects of corrosion occur. Because of the studies developed in this research, based on the propagation step of corrosion, the main deleterious effects of corrosion in reinforced concrete structures are introduced and discussed. The effects of corrosion, in reinforced concrete structures, are divided into three main consequences, i.e.: the mechanical properties of the reinforcements; the adherence between the reinforcements and the concrete; and the concrete’s fissuration. It stands out that the main reports found in the bibliography, encompassing corrosion in reinforced concrete structures, are about the structure’s loss of monolithic, reducing, thus, its resistant capacity. Another point that is talked about in the bibliography is steel’s weakening when suffering a corrosive process, which reduces its deformation capabilities when under load. In light of this, the relative contributions of corrosion’s effects on reinforced concrete structural behavior, find themselves developed along the themes of mechanical properties of the reinforcements and loss of adherence between the concrete and the reinforcement [25]. In reinforced concrete structures, reinforcement is presented as one of the main elements. The quantity is the result of sizing, many times complex, and, generally, it is expressed in terms of the necessary area of reinforcement. Thus, the area of reinforcement is understood as one of the major parameters in reinforced concrete structures [10]. [10]. Page 4/20 Page 4/20 In the corrosion process, during the propagation step, there is the formation of corrosion products (oxides and iron hydroxides) and, as a consequence, the consumption of the reinforcement’s mass. Therefore, there is material loss and, due to this, a reduction in the transversal section of the reinforcement [3]; [4]; [12]; [14]; [15]; [16]. 3 NONDESTRUCTIVE METHODS FOR THE EVALUATION OF CORROSION Throughout the last decades, the application of nondestructive testing (NDT), in Civil Engineering, became a topic of interest in numerous countries [26]. NDT usage in this area is directly related to the reliability of the application methods, to the knowledge about the application, and to its economical aspect [27]. Given that corrosion in reinforced concrete structures is one of the most frequent degradation mechanisms, different NDT techniques intended for the evaluation of corrosion can be used. Among them are corrosion potential technique, resistivity, and corrosion rate. 2.2 Corrosion mechanism in reinforced concrete structures Corrosion can affect the tensile strength and ductility of the reinforcements. Both are major properties, having an effect on the structural performance, altering from loading capacity to rupture form [14]. 3.2 Resistivity Concrete resistivity is a major parameter in the corrosion of reinforced concrete structures. High resistivity concrete has a low possibility to have its reinforcements suffer a corrosive process. In sum, electrical resistivity is determined by measuring the differences in potential, on the concrete’s surface, caused by the application of a small current on the surface [15]. Table 2 shows the recommendations of the Comité Euro-International du Béton at CEB 192 [29], relating concrete’s resistivity to a probable corrosion rate. Table 2. Correlation between the resistivity values and the probability of corrosion rrelation between the resistivity values and the probability of corrosion Page 5/20 (Font: adapted from [29]) Concrete’s Resistivity (Ω.m) Probable Corrosion Rate > 200  Negligible corrosion probability 100 a 200 Low corrosion probability 50 a 100 High corrosion probability < 50 Very high corrosion probability (Font: adapted from [29]) There are many techniques for resistivity evaluation. One of them is the external electrode method. This method consists of a metallic electrode, disc-shaped, placed on the surface of the concrete, and connected to the reinforcement bar: which determines the resistivity between the disc and the steel bar. The method follows RILEM TC 154 recommendations [30]. 3.3 Corrosion rate The quantitative information regarding steel corrosion rate is of sum importance to the evaluation of the repair methods, to the lifespan prevision, and to the structural evaluation of the elements with corrosion. One of the few available techniques is the resistance to polarization method [31]. The recommendation RILEM TC 154-EMC describes the testing method for the determination of the reinforcement corrosion rate on reinforced concrete structures, through polarization resistance [32]. Through the method, two characteristics related to corrosion can be obtained: instantaneous corrosion current density (Icorr), and the corrosion rate (Vcorr). Both can be used to evaluate the risk of corrosion. Current density (Icorr) is expressed, usually, in μA/cm²: values under 0.1 represent a negligible degree of corrosion, in contrast, the ones greater than 1.0 present high corrosion risk. The speed of corrosion (Vcorr) represents the volumetric loss of steel by area and time units, generally expressed in mm/year. This value is obtained as a function of current density (Icorr), through Faraday's Law. For Icorr equal to 1 μA/cm², the speed of corrosion is equivalent to 0.0116 mm/year considering uniform corrosion. This, can, too, be expressed as corresponding to a degree of corrosion [31]. In Table 3, the degree of corrosion in relation to current density is related to corrosion speed, according to Andrade et al. (2004). Page 6/20 Table 3. Correlation Ranges of current density and corrosion rate values related to significance i terms of armature service life (Font: adapted from [28]) Current’s density - Icorr  (μA/cm²) Corrosion’s speed - Vcorr (mm/year) Degree of corrosion  ≤ 0,1  ≤ 0,001 negligible 0,1 a 0,5 0,001 a 0,005 low 0,5 a 1,0 0,005 a 0,010 moderate > 1,0 > 0,010 high Table 3. Correlation Ranges of current density and corrosion rate values related to significance in terms of armature service life (Font: adapted from [28]) Table 3. Correlation Ranges of current density and corrosion rate values related to significance in terms of armature service life (Font: adapted from [28]) Current’s density - Icorr  (μA/cm²) Corrosion’s speed - Vcorr (mm/year) Degree of corrosion  ≤ 0,1  ≤ 0,001 negligible 0,1 a 0,5 0,001 a 0,005 low 0,5 a 1,0 0,005 a 0,010 moderate > 1,0 > 0,010 high Page 6/20 4.1 Degree of Corrosion Corrosion Degrees have been adopted, in relation to the mass loss of the reinforcements, of 05 (reference), 5%, 10%, and 15%. Therefore, there is the objective of evaluating corrosion’s influence on reinforced concrete beams. Having the finality of comprehending with greater accuracy the laboratory results, in relation to real-scale structures, beam prototypes with representative dimensions have been utilized: a transversal section of 15 cm in base, 30cm in height, and 300 cm in length. The detailing of the beams is described in item 4.2. 4 EXPERIMENTAL PROGRAM The experimental program is divided into three steps: molding, corrosion acceleration, and evaluation through non- destructive testing. The study has been structured on a comparative analysis of corrosion’s effect concerning whole elements, i.e., that did not suffer deleterious effects of corrosion. The experimental program’s detailing is represented in Figure 1, relating the different steps developed in the study. Establishing a relationship with the experimental study development steps, it is presented in Table 4 the nomenclature of the beams utilized throughout its development. It is highlighted that, for each combination, two beams were used. Table 4. Combinations and beam nomenclature Degree of Corrosion Nomenclature* 0% (Reference) COR-0-V1 COR-0-V2 5% COR-5-V1 COR-5-V2 10% COR-10-V1 COR-10-V2 15% COR-15-V1 COR-15-V2 * V1 and V2 refer to samples 1 and 2 for each nomenclature COR 15 V2 * V1 and V2 refer to samples 1 and 2 for each nomenclature 4.2 Reinforced Concrete Beams Structural Project With the purpose of approximation of the studied situation to actual urban constructions and in coastal ambients, the utilization of concrete with a characteristic compression strength of 30 MPa has been opted for, i.e., the minimum tolerated by NBR 6118 [32] to the Ambiental Aggressivity Class III (CAAIII). This choice is due to the finding that concrete with strength between 20 and 30 MPa, is used in buildings in a general manner. In addition, concrete, with greater resistance to compression, is less prone to reinforcement corrosion. Such a fact is due to low porosity in which oxygen and humidity transports are hampered, and, as a consequence, the passage of electrical current and accelerated corrosion is reduced [9], [10], and [11]. Page 7/20 As for its geometry, important aspects have conditioned the dimensions, a discussion regarding the subject can be found in [24]. All framed beams have a transversal section of 15 cm (base) x 30 cm (height) and a length of 300 cm. The lower longitudinal reinforcement is composed of two steel CA-50 bars, with 12.5 mm of diameter anchored with hooks in the extremities. The upper reinforcement is composed of, also, steel CA-50, and is 6.3 mm in diameter. The transversal reinforcement is of stirrups, with a diameter of 6.3 mm and uniform spacing of 7 cm. The reinforcement has a covering of 1.5 cm. In Figure 2, the detailing of the reinforced concrete beams is presented. cm. The lower longitudinal reinforcement is composed of two steel CA-50 bars, with 12.5 mm of diameter anchored with hooks in the extremities. The upper reinforcement is composed of, also, steel CA-50, and is 6.3 mm in diameter. The transversal reinforcement is of stirrups, with a diameter of 6.3 mm and uniform spacing of 7 cm. The reinforcement has a covering of 1.5 cm. In Figure 2, the detailing of the reinforced concrete beams is presented. 4.3 Materials The materials used in this research were provided by the company responsible for the reinforced concrete beam’s production. Were employed: Portland cement CPV-ARI, natural sand, thin and medium, basaltic gravel types 1 and 0, and superplasticizer additive. The concreting of the beams was executed by a precast factory, located in Porto Alegre -RS. A computerized concrete plant with mass dosing was used. All beams were cast with concrete in a single dosage, in addition, the same concrete mix composition was used. The composition data of the concrete mix used are shown in Table 5. Table 5. Description of the mix used in the execution of the reinforced concrete beams Cement (kg/m³) Thin sand (kg/m³) Medium sand (kg/m³) Gravel 0 (kg/m³) Gravel 1 (kg/m³) Water(kg/m³) Additive (l/m³) RelationA/C 271 280 654 231 692 185 0.8 0.68 The beams were analyzed at an age of 502 days, presenting a medium compression strength of 32.4 MPa, and an elasticity module of 34.8 GPa. Table 5. Description of the mix used in the execution of the reinforced concrete beams Cement (kg/m³) Thin sand (kg/m³) Medium sand (kg/m³) Gravel 0 (kg/m³) Gravel 1 (kg/m³) Water(kg/m³) Additive (l/m³) RelationA/C 271 280 654 231 692 185 0.8 0.68 Table 5. Description of the mix used in the execution of the reinforce The beams were analyzed at an age of 502 days, presenting a medium compression strength of 32.4 MPa, and an elasticity module of 34.8 GPa. The steel was CA-50 for both positive and negative reinforcements. For the positive reinforcement, with 12.5 mm in diameter, an average outflow stress of 670 MPa and rupture of 770 MPa was obtained. The negative reinforcement, having 6.3 mm in diameter, showed the following tensions: mean outflow of 650 MPa, and rupture at 765MPa. 4.4 Accelerated Corrosion The adopted corrosion acceleration method, as mentioned, was the CAIM testing. The CAIM testing was conceived by Lima [34], based on Varela and Espinosa's [35] studies. The method has already been employed, with success, by many of LEME’s researchers [8]; [9]; [10]; [11]; [36]; [37]; [38]; [39]. The method characterizes itself by stimulating corrosion through electrochemical induction, by applying a potential (p.d.) or current (i) difference, in a chloride-rich environment. In relation to the electrochemical induction, it was decided that a stimulus through constant current would be adopted, based on Graeff’s [10] studies and in the discussion brought forth by Caetano [9]. Page 8/20 Page 8/20 One of the main parameters that influence the method is the density of the current used for the corrosion acceleration. Regarding this, El Maaddawy and Soudki [40] report that there are more appropriate current ranges to accelerate corrosion in reinforced concrete reinforcements. High values in corrosion current density (above 500 μA/cm²) tend to distort the effective mechanism of corrosion [10]. When the density is too high, the corrosion products do not have enough time to fix themselves into the concrete’s pores, having an increment in tensions which provokes fissuration levels that do not correspond to the naturally occurring phenomena in structures. In CAIM’s utilization, the incorporation of chlorides in the immersion water occurs. To minimize this effect chloride was used in the immersion water, through the dissolution of sodium chloride in content (35 g/l), producing a saline solution with a chloride concentration close to the one encountered in the Atlantic Ocean, which bathes the Brazilian coast [8]; [9]; [10]; [11]. To enable the presence of oxygen and humidity, so that the corrosive process can develop, the reinforced concrete beam is submerged until the longitudinal reinforcement’s lower face, as presented in Figure 3. In the solution, a negative and a positive electrode both made out of hard copper wire, 2.5 mm in diameter. The positive electrode was positioned 3 mm above the longitudinal reinforcement. Butyl rubber was used, in a “U” shape, enveloping the electrode, and avoiding contact with the reinforcement, as can be observed in Figure 3. As the current rate is proportional to the reinforcement’s area (surface area) that is corroded, the longitudinal reinforcements and a portion of the transversal were considered for this. 4.5  Corrosion Potential To determine the corrosion potential, it is necessary to detect the location of the reinforcement bars. For this, cover meter detection was used, employing a cover meter model PROFOMETER 600, as shown in Figure 4. The corrosion potential testing procedure follows ASTM C-876 [28] which establishes a correlation between the intervals in potential difference, in relation to the reference electrode, made out of Cu/CuSO4 (Copper, Copper Sulfate), in terms of corrosion probability, as established in chapter 3. For the carrying of the test, the equipment GECOR 8 was used, with a reference electrode, which in this case was made out of Cu/CuSO4. The negative pole is connected to the electrode; meanwhile, the positive is connected to the reinforcement bar through the copper wire that was connected to the reinforcement before concreting. The electrode is positioned on the concrete’s surface in a way that an electrical connection with the reinforcement bar is present. The application of the method and the equipment are presented in Figure 5. The evaluation was carried out through a mapping of the corrosion potential for each analyzed beam. The measurement of the potentials was made over each of the longitudinal bars, with a 25 cm spacing between each reading point. 4.4 Accelerated Corrosion Where Δm = mass of consumed steel (g); M = metal’s atomic weight (56 Fe); I = current applied (A); t = corrosion acceleration time (s); z = ionic charge (= 2); F = Faraday’s constant (=96500A/s). As described by the researchers, enlarged test times are required, about the ones predicted through the simple application of Faraday’s Law, to achieve the desired corrosion degrees [8]; [9]; [10]; [11]. Because of this, the methodology proposed by Graeff [10] is applied, for the calibration of the corrosion degrees, and the values presented by Stein [11] are utilized. Thus, by equation (2), the predicted time for corrosion is determined, according to the mass loss required to reach the intended degree of corrosion: Where tc = time of corrosion (s). 4.4 Accelerated Corrosion The longitudinal reinforcement was considered in its entirety (297 cm in length), adding 2.5 cm of length for each anchoring hook. This value is equivalent to twice the reinforcement’s diameter. The transversal reinforcement was considered in two portions. One below the longitudinal reinforcement, and another equivalent to a height of 2.5 cm above the longitudinal reinforcement. This value is, also, equivalent to twice the longitudinal reinforcement’s diameter. Finally, the reinforcement to be corroded possessed a mass of 7,800 g and a surface area, subjected to the corrosive process, of 3,878 cm2, considering the longitudinal reinforcement and the portion of the transversal reinforcement. Having the current rate of 500 µA/cm², for each beam a current of 1.94 A was applied with power supplies. Each one of them has two outputs, capable of providing constant current or tension, in ranges from 0 to 3 A, for current, and 0 to 30 V for tension. Each one of them has two outputs, capable of providing constant current or tension, in ranges from 0 to 3 A, for current, and 0 to 30 V for tension. To obtain the intended corrosion degrees, a theory based on Faraday’s Laws is used to predict the mass loss and, consequently, the degree of corrosion. This theory has already been employed by various authors [8]; [9]; [10]; [11]; [12]. The degree of corrosion, predicted by the mentioned law, corresponds to the area below the curve of current as a function of time. For the cases in which the corrosion is accelerated through the application of constant current, this area can be approximated to a rectangle. Therefore, the degree of corrosion is obtained by the mass loss determined by Faraday’s Law in accordance with the equation (1): Page 9/20 Page 9/20 Where Δm = mass of consumed steel (g); M = metal’s atomic weight (56 Fe); I = current applied (A); t = corrosion acceleration time (s); z = ionic charge (= 2); F = Faraday’s constant (=96500A/s). Where Δm = mass of consumed steel (g); M = metal’s atomic weight (56 Fe); I = current applied (A); t = corrosion acceleration time (s); z = ionic charge (= 2); F = Faraday’s constant (=96500A/s). 4.6 Evaluation of the Degree of Corrosion The beam reinforcements were retrieved, as illustrated in Figure 6. It was opted for the region close to the support. The reinforcements were retrieved in every beam, even the ones that did not suffer a corrosive process, which Page 10/20 Page 10/20 would serve as a control group for determining the degree of corrosion. would serve as a control group for determining the degree of corrosion. Retrieved the beam reinforcements, the concrete adhered to the bars was removed. Following, the reinforcements were immersed in a 3.5 g hexamethylenetetramine solution, diluted in a hydrochloric acid (500 ml), and distilled water (500 ml) solution. This procedure is standardized by ASTM G1-03 [40]. The bars were kept in the solution for 40 minutes in order to remove the products of corrosion. After, they were cleaned in running water to remove the hydrochloric acid. Subsequently, to the cleaning, the bars were transferred to a greenhouse. Kept at a 30oC temperature, there they remained until total humidity removal. In sequence, the mass and length of each one were measured, determining the linear mass. The degree of corrosion, expressed in the mass loss was determined according to Equation (3). The reference linear mass was considered as the average of the reinforcements without corrosion. 5 RESULTS AND DISCUSSIONS The results obtained throughout this experimental program are presented. The values are discussed to subsidize the conclusions of the research. 5.1 Accelerated corrosion Real corrosion degree obtained empirically in comparison to the degree obtained empirically in comparison to the theoric values 5.2 Corrosion diagnosis through non-destructive methods: corrosion potential 5.1 Accelerated corrosion The accelerated corrosion was realized following the parameters and methodology introduced in item 0. During the acceleration, it was observed, all beams that went through the corrosion process presented the following behavior. By accompanying the corrosion process, which used the formation of an electrical circuit between the solution and the corrosion electrode, fed by a constant current, it was verified that the latter did not remain constant. The current decreased right at the beginning of the acceleration process (between 3 to 5 days) and, after a period (1 to 2 days), the current went back to its initial level and kept constant throughout the rest of the tests. This behavior is related to the kinematics of the corrosive process, a discussion in regards to this behavior, denominated concrete pore clogging is addressed by Reginato [24] and Stein [10]. After carrying out the tests, the longitudinal reinforcement bars were extracted from each beam in order to determine the real degree of corrosion. The procedure followed ASTM     G1-03 [40]. Because of the possible differences between theoretical and effective corrosion, in the result evaluations throughout the analysis, the corrosion values were adopted in a variation range. The ranges adopted refer to the effective evaluation of corrosion. The mass loss percentage was determined with average values, being that, for Page 11/20 Page 11/20 each beam, two reinforcement bars were analyzed. It is shown, in Table 6, the real degree of corrosion for one of the beams. According to Table 6, it was observed that the real values of corrosion were approximate to the ones predicted in the experimental program definition when compared with the average values obtained for each group of corroded beams. The exception occurred in the 5% level, resulting in actual corrosion 1.27% below the predicted outcome. This behavior can be the influence of concrete pore clogging, which happens right at the beginning of corrosion acceleration, and, thus, there is a required adaptation time foreseen so that the corrosion level can be reached. Table 6. Real corrosion degree obtained empirically in comparison to the theoric values Beam Theoric corrosion degree    Real corrosion degree Average corrosion degree  Intended corrosion degree COR-5- V1 5,03% 3,49% 3,73 % 5 % COR-5- V2 5,09% 3,97% COR-10- V1 10,17% 9,97% 10,05 % 10 % COR-10- V2 10,14% 10,13% COR-15- V1 15,65% 14,20% 13,87 % 15 % COR-15- V2 15,46% 13,55% able 6. 5.2 Corrosion diagnosis through non-destructive methods: corrosion potential The measurement of the corrosion potential is a qualitative technique, widely used, which provides data regarding the probability of corrosion in the analyzed reinforcement. The testing procedure was made respecting ASTM C- 876 [28]. The corrosion potential values, shown in Table 7, are the average for each beam and the standard deviation. Indicated, also, these parameters for each level of corrosion (beam group), are related to the corrosion probability. As expected, it was observed that there was a decrease in corrosion potential according to the corrosion level. Comparing the acquired data, with ASTM C-876 [28] value ranges, it was verified that for the beam control group (COR -0), the probability of corrosion was below 10%; for the corroded beams in level 5 and 10% (COR-5 and COR- 10) was uncertain; and for corrosion level 15% (COR-15), it was greater than 90%. It was noted that there existed an uncertainty in the corrosion measurements, utilizing the corrosion potential technique, for levels 5 and 10%. In both levels, the corrosion potential remained in the same reading range and was classified with a 50% chance of occurring corrosion. However, more than visually, it was found, through reinforcement mass loss, the existence of corrosion. Table 7. Average and standard deviation of corrosion potential for the control group and corroded beams Table 7. Average and standard deviation of corrosion potential for the control group and corroded beams Page 12/20 Page 12/20 Beam Group Beam Av. (mV)  SD (mV)     Group Av. (mV) Group SD (mV) Corrosion Probability COR-0 V1 28,84 7,45 -30,78   25,24   Below 10% V2 -90,40 43,03 COR-5 V1 -298,22 21,34 -257,57   22,59   Uncertain 50% V2 -216,92 23,85 COR-10 V1 -240,96 17,95 -252,99   19,71   Uncertain 50% V2 -265,03 21,47 COR-15 V1 -494,11 32,94 -438,42 33,01 Above 90% V2 -382,72 33,08 Therefore, corrosion evaluation, through corrosion potential, showed little efficacy in diagnosing corrosion for values up to 10%, nonetheless, it was adequate for 15% levels. This way, the corrosion potential testing did not present safe results, despite this it can be used for this type of diagnosis when done so with caution, as it can present potentially fragile results. Other than corrosion potential statistical data, shown in Table 2, a mapping of each beam’s potential was realized. It is presented in Figure 7. The mapping was done through hypsometric curve elaboration, utilizing data interpolation with the minimum curve technique. Other than corrosion potential statistical data, shown in Table 2, a mapping of each beam’s potential was realized It is presented in Figure 7. The mapping was done through hypsometric curve elaboration, utilizing data interpolation with the minimum curve technique. Basically, it is an interpolation technique that uses a polynomial to create a surface that reduces its curvature, resulting in a smoothed surface, which passes through sampled points, reproducing the variable values. The interpolation and mapping creation was made with image generation software [42]. Basically, it is an interpolation technique that uses a polynomial to create a surface that reduces its curvature, resulting in a smoothed surface, which passes through sampled points, reproducing the variable values. The interpolation and mapping creation was made with image generation software [42]. The scale used was based on ASTM C-876 [30] parameters, expressing corrosion potential in mV, and relating them with corrosion probability. The corrosion potential mapping - Figure 7 - provides global visualization, instead of a numerical parameter. Hence, it was observed that the potential is uniform, for each of the analyzed beams, and noted that there was also a regularity between the potential and the beam groups. With this mapping it is demonstrated, graphically, the corrosion potential changes according to its level, being that the higher the corrosion level, the lower its corrosion potential. AUTHORS' CONTRIBUTIONS Lucas Alexandre Reginato, Alexandre Lorenzi, Leonardo Covatti de Oliveira, and Luciani Somensi Lorenzi wrote the main manuscript text and Lucas Alexandre Reginato prepared all figures. All authors reviewed the manuscript. COMPETING INTERESTS – Not applicable FUNDING – Not applicable ETHICS APPROVAL AND CONSENT TO PARTICIPATE ETHICS APPROVAL AND CONSENT TO PARTICIPATE ETHICS APPROVAL AND CONSENT TO PARTICIPATE The authors approve the manuscript and give consent to participate. CONSENT FOR PUBLICATION All authors consent for publication. All authors consent for publication. 6 CONCLUSIONS This research evaluated corrosion potential techniques for corrosion diagnosis in reinforced concrete beams. On the whole, it is highlighted, concerning the corrosion acceleration process: a)  the accelerated corrosion technique, in reinforced concrete beams, through CAIM testing proved to be quite effective; b) the average values of real corrosion degree were close to the theoric values and the ones established in the experimental program elaboration; With CAIM testing, it was verified that the accelerated corrosion technique in reinforced concrete beams is quite effective. Corrosion’s time adjustment by Graeff’s [10] proposed methodology in addition to the values presented by Stein [11], provided a relation between the theoretical and real degree of corrosion (empirically obtained), indicating that the corrosion acceleration procedure adopted in this research has shown itself adequate to accelerate the corrosion process. Page 13/20 Page 13/20 As for the corrosion diagnosis technique by NDT through corrosion potential, concludes that there was an uncertainty in corrosion measurement for both 5 and 10% levels. Both had their corrosion potential in the same reading range and were classified as having a 50% probability of occurring corrosion. Thus, only for the 15% corrosion level, the corrosion potential method was efficient. Hence, it was concluded that corrosion potential testing does not present ideal results, however, it can be used for corrosion diagnosis, as long as used carefully, since it can show potentially fragile results. Likewise, it is recommended the application of other corrosion diagnosis techniques simultaneously to ensure higher precision for corrosion levels of 5 to 10% mass loss. ACKNOWLEDGEMENTS REFERENCES Page 14/20 [1] M. Donadio, J. Capacho, L. Santander, “Total Corrosion Management. Documentary analysis.” Revista ALCONPAT, vol. 13, no. 2, pp. 235 – 253, May, 2023. DOI: https://doi.org/10.21041/ra.v13i2.690 [2] M. A. El-reedy, Steel-Reinforced Concrete Structures: Assessment and Repair of Corrosion. 2ª ed. New York, New York, United States: Taylor & Francis Group, 2018. [3] O. Cascudo, O controle da Corrosão de Armaduras de Concreto. Inspeções e técnicas eletroquímicas. 1. ed. São Paulo, Brazil: PINI, 1997. [4] D. V. Ribeiro, Corrosão e Degradação em Estruturas de Concreto. 2nd ed. Elsevier, 2018. [5] C. Andrade, “Propagation of reinforcement corrosion: principles, testing and modelling.” Revista Materials and Structures, vol. 52, no. 2, May, 2019. DOI: https://doi.org/10.1617/s11527-018-1301-1 [6] C. Andrade, Manual para Diagnóstico de Obras Deterioradas por Corrosão de Armaduras. 1st ed. São Paulo, Brazil: PINI, 1992. [7] J. J. De O. Andrade, “Estruturas De Concreto Armado Atacadas Pela Corrosão De Armaduras : Iniciação Por Cloretos,” Ph.D. thesis, PPGEC, UFRGS, Porto Alegre, RS, 2001. [8] D. S. Adamatti, ”Análise da eficiência de espaçadores no concreto armado: impacto da corrosão por íons cloreto em diferentes condições de exposição.” M.S. thesis, PPGEC, UFRGS, Porto Alegre, RS, 2016. 9] L. F. Caetano, “Estudo do comportamento da aderência em elementos de concreto armado submetidos à corrosão e elevadas temperaturas.” M.S. thesis, PPGEC, UFRGS, Porto Alegre, RS, 2008. [10] A. G. Graeff, “Avaliação experimental e modelagem dos efeitos estruturais da propagação da corrosão em elementos de concreto armado.” M.S. thesis, PPGEC, UFRGS, Porto Alegre, RS, 2007. [11] K. J. Stein, “Avaliação experimental de vigas de concreto armado corroídas submetidas a ciclos de carregamentos.” M.S. thesis, PPGEC, UFRGS, Porto Alegre, RS, 2019. [12] P. Helene, “Contribuição ao Estudo da Corrosão em Armaduras de Concreto Armado.” Ph.D. thesis, USP, São Paulo, SP, 1993. [13] P. Helene, Corrosão em Armaduras para Concreto Armado. 1st ed. São Paulo, Brazil: PINI, 1986. [14] G. R. Meira, Corrosão de armaduras em estruturas de concreto armado: ensaios eletroquímicos. 1st ed João Pessoa, Brazil: IFPB, 2017. [15] P. K. Mehta; P. J. M. Monteiro, Concreto: microestrutura, propriedades e materiais. 1st ed. São Paulo, Brazil: IBRACON, 2014. [16] M. P. T. Cunha, “Deterioração das estruturas de concreto armado” in Corrosão em Estruturas de Concreto Armado: Teoria, Controle e Métodos de Análise, D. V. Ribeiro, 1st ed., Rio de Janeiro, Brazil: Elsevier, 2014, pp. 87– 118. [17] K. ACKNOWLEDGEMENTS Tuutti, “Corrosion of Steel in Concrete,” in SCCR, Sweden, 1982, pp. 469. [17] K. Tuutti, “Corrosion of Steel in Concrete,” in SCCR, Sweden, 1982, pp. 469. Page 15/20 Page 15/20 Page 15/20 [18] C. A. Apostolopoulos, ”Mechanical behavior of corroded reinforcing steel bars S500s tempcore under low cycle fatigue.” Constr. and Building Materials, vol. 21, no. 7, pp. 1447-1456, July, 2007. [19] S. Caprili; W. Salvatore, “Cyclic behavior of uncorroded and corroded steel reinforcing bars.” Constr. and Building Materials, vol. 76, no. 1, pp. 168-186, February, 2015. [20] I. Fernandez; J. M. Bairán; A. R. Marí, “Corrosion effects on the mechanical properties of reinforcing steel bars. Fatigue and σ-ε behavior.” Constr. and Building Materials, vol. 101, no. 1, pp. 772-783. December, 2015. [21] C. Gehlen; T. Weirich, “High-cycle fatigue behavior of reinforcing steel under the effect of ongoing corrosion.” Struct. Concrete, vol. 17, no. 3, pp. 329-337. February, 2016. [22] M. M. Kashani; P. Alagheband; R. Khan et al, “Impact of corrosion on low-cycle fatigue degradation of reinforcing bars with the effect of inelastic buckling.” Intern. Journal of Fatigue, vol. 77, pp. 174-185, August, 2015. [23] ZHANG, W. et al. Tensile and fatigue behavior of corroded rebars. Construction and Building Materials. Vol 34, pp 409-417, September 2012. [24] A. A. Almusallam, ”Effect of degree of corrosion on the properties of reinforcing steel bars.” Constr. and Building Materials, vol. 15, no. 8, pp. 361-368. December, 2001. [25] L. A. Reginato, “Avaliação experimental dos efeitos da corrosão em concreto armado: diagnóstico e reabilitação com UHPFRC e Concreto Têxtil.” Ph.D. thesis, PPGEC, UFRGS, Porto Alegre, RS, 2020. [26] A. Lorenzi; L. A. Reginato; L. S. Lorenzi; F. Pedron; L. C. P. Silva Filho, “Utilização de Tomografia Ultrassônica para Avaliação de Postes de Concreto após Sinistro.” in XIII CONPAT, Lisboa, 2015. [27] R. Beutel; H. Reinhardt; C. U. Grosse; et. al, “Performance Demonstration of Non-Destructive Testing Methods.” in ECNDT, Berlin, 2006. [28] AMERICAN SOCIETY FOR TESTING AND MATERIALS, C876-15, Standard Test Method for Corrosion Potentials of Uncoated Reinforcing Steel in Concrete, ASTM International, West Conshohocken, PA, 2015. [29] COMITE EURO-INTERNATIONAL DU BETON, CEB 192: Diagnosis and Assessment of Concrete Structures - State-of-Art Report, Bulletin d’ Information, Suecis, n. 192, Jan 1988. [30] B. Elsener, C. Andrade, J. Gulikers, et al. “Half-cell potential measurements - Potential mapping on reinforced concrete structures,” Mat. Struct., vol. 36, pp. 461–471,  2003. [31] L. Bertolini; B. Elsener; P. Pedeferri; R. Polder, Corrosion of Steel in Concrete Prevention, Diagnosis, Repair. 1st ed. Weinheim, Germany: WILEY-VCH Verlag, 2004. [32] J. J. De O. Page 15/20 Andrade, “Estruturas De Concreto Armado Atacadas Pela Corrosão De Armaduras : Iniciação Por Cloretos.” Ph.D. thesis,  PPGEC, UFRGS, Porto Alegre, RS, 2001. [33] ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS, NBR 6118: Projeto de estruturas de concreto - Procedimento, Rio de Janeiro, 2014. Page 16/20 Page 16/20 Page 16/20 Page 16/20 [34] M. G. Lima, “Influência dos componentes do concreto na corrosão de armaduras.” M.S. thesis, PPGEC, UFRGS, Porto Alegrem RS, 1990. [35] H. Varela; L. V. Espinoza “Penetrabilidad De Iones Cloruors En Morteros Con Y Sin Revestimiento,” in VII Jornadas Chilenas Del Hormigón E I Jornada Latinoamericana Del Cimento Y Hormigón, Santiago De Chile, 1988. [35] H. Varela; L. V. Espinoza “Penetrabilidad De Iones Cloruors En Morteros Con Y Sin Revestimiento,” in VII Jornadas Chilenas Del Hormigón E I Jornada Latinoamericana Del Cimento Y Hormigón, Santiago De Chile, 1988. [36] A. P. Kirchheim; V. Pasa; D. Dal Molin; L. C. P. Silva Filho, Análise comparativa da utilização de diferentes sistemas de proteção de corrosão de armaduras na aderência entre concreto branco e barras de aço,” in 47º CONGRESSO BRASILEIRO DO CONCRETO, Recife, 2005. [37] P. R. C. Marchesan; D. Santarosa; J. L. Campagnolo; L. C. P. Silva Filho; A. R. Pacheco “Estudo da influência da aplicação de revestimentos no controle da corrosão,” in IV Congresso Iberoamericano De Patologia Das Construções e VI Congresso De Controle Da Qualidade, Porto Alegre, 1997. [38] S. C. Selistre; F. P. S. L. Gastal; J. L. Campagnolo “Estudo de técnicas para reversão do processo corrosivo em estruturas de concreto contaminadas com cloretos.” in VI Salão de Iniciação Científica e III Feira de Iniciação Científica (SIC), Porto Alegre, 1994. [39] A. S. Torres, “Avaliação da sensibilidade do ensaio CAIM – corrosão acelerada por imersão modificada – frente ao processo de corrosão de armaduras de concreto armado.” M.S. thesis, PPGEC, UFRGS, Porto Alegre, RS, 2006. [40] T. A. El Maaddawy; K. A. Soudki, “Effectiveness of Impressed Current Technique to Simulate Corrosion of Steel Reinforcement in Concrete,” Journal of Materials in Civil Engineering, vol. 15, no. 1, pp. 41–47. January, 2003. [41] AMERICAN SOCIETY FOR TESTING AND MATERIALS, ASTM G1-03 Standard practice for preparing, cleaning, and evaluating corrosion test specimens, Philadelphia, 2017. [42] J. L. S. Andriotti, Fundamentos de Estatística e Geoestatística. 2nd ed. São Leopoldo, Brazil: UNISINOS, 2009. Figure 1 Experimental program’s detailing. Figure 2 Page 18/20 Figure 2 Reinforced concrete beams’ reinforcement detailing Figure 3 Layout adapted for the corrosion acceleration einforced concrete beams’ reinforcement detailing Reinforced concrete beams’ reinforcement detailing Page 18/20 g Figure 3 Layout adapted for the corrosion acceleration Figures Page 17/20 Figures Page 17/20 Figures Page 17/20 Page 17/20 Figure 1 Experimental program’s detailing. Figure 2 Reinforced concrete beams’ reinforcement detailing Figure 3 Figure 3 Layout adapted for the corrosion acceleration Page 18/20 Figure 4 Locating the reinforcements through a cover meter and marking the reading grid for the NDT evaluation Figure 5 Determining the corrosion potential with the GECOR 8 equipment Figure 4 Locating the reinforcements through a cover meter and marking the reading grid for the NDT evaluation Figure 4 Locating the reinforcements through a cover meter and marking the reading grid for the NDT evaluation Figure 5 Figure 4 Locating the reinforcements through a cover meter and marking the reading grid for the NDT evaluation Locating the reinforcements through a cover meter and marking the reading grid for the NDT evaluation Locating the reinforcements through a cover meter and marking the reading grid Page 19/20 Locating the reinforcements through a cover meter and marking the reading grid for the NDT evaluation Figure 5 Determining the corrosion potential with the GECOR 8 equipment Figure 5 Determining the corrosion potential with the GECOR 8 equipment etermining the corrosion potential with the GECOR 8 equipment Page 19/20 Figure 6 Region of beam reinforcement retrieval, distances in centimeters Region of beam reinforcement retrieval, distances in centimeters Region of beam reinforcement retrieval, distances in centimeters Figure 7 Corrosion potential mapping, distance in centimeters Figure 7 Corrosion potential mapping, distance in centimeters Figure 7 Corrosion potential mapping, distance in centimeters Corrosion potential mapping, distance in centimeters Page 20/20
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Spatial and temporal variability of biophysical variables in southwestern France from airborne L-band radiometry
Hydrology and earth system sciences
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Spatial and temporal variability of biophysical variables in Southwestern France from airborne L-band radiometry Elena Zakharova, Jean-Christophe Calvet, Sébastien Lafont, Clément Albergel, Jean-Pierre Wigneron, Mickaël Pardé, Yann H. Kerr, Mehrez Zribi To cite this version: Elena Zakharova, Jean-Christophe Calvet, Sébastien Lafont, Clément Albergel, Jean-Pierre Wigneron, et al.. Spatial and temporal variability of biophysical variables in Southwestern France from airborne L-band radiometry. Hydrology and Earth System Sciences, 2012, 16 (6), pp.1725-1743. ￿10.5194/hess- 16-1725-2012￿. ￿hal-00661546￿ Distributed under a Creative Commons Attribution 4.0 International License Correspondence to: J.-C. Calvet (jean-christophe.calvet@meteo.fr) The presence of small wa- ter bodies within the ISBA-A-gs grid cells tended to increase the CAROLS SSM spatial variability, up to 0.10 m3 m−3. Also, the grid cells characterised by a high vegetation cover heterogeneity presented higher standard deviation values, for both SSM and VOD. Correspondence to: J.-C. Calvet (jean-christophe.calvet@meteo.fr) Correspondence to: J.-C. Calvet (jean-christophe.calvet@meteo.fr) Received: 5 January 2012 – Published in Hydrol. Earth Syst. Sci. Discuss.: 17 January 2012 Revised: 30 May 2012 – Accepted: 31 May 2012 – Published: 25 June 2012 Received: 5 January 2012 – Published in Hydrol. Earth Syst. Sci. Discuss.: 17 January 2012 Revised: 30 May 2012 – Accepted: 31 May 2012 – Published: 25 June 2012 Received: 5 January 2012 – Published in Hydrol. Earth Syst. Sci. Discuss.: 17 January 2012 Revised: 30 May 2012 – Accepted: 31 May 2012 – Published: 25 June 2012 Abstract. In 2009 and 2010 the L-band microwave Coopera- tive Airborne Radiometer for Ocean and Land Studies (CAR- OLS) campaign was performed in southwestern France to support the calibration and validation of the new Soil Mois- ture and Ocean Salinity (SMOS) satellite mission. The L- band Microwave Emission of the Biosphere (L-MEB) model was used to retrieve surface soil moisture (SSM) and the veg- etation optical depth (VOD) from the CAROLS brightness temperature measurements. The CAROLS SSM was com- pared with in situ observations at 11 sites of the SMOSMA- NIA (Soil Moisture Observing System-Meteorological Au- tomatic Network Integrated Application) network of M´et´eo- France. For eight of them, significant correlations were ob- served (0.51 ≤r ≤0.82), with standard deviation of differ- ences ranging from 0.039 m3 m−3 to 0.141 m3 m−3. Also, the CAROLS SSM was compared with SSM values simulated by the A-gs version of the Interactions between Soil, Biosphere and Atmosphere (ISBA-A-gs) model along 20 flight lines, at a resolution of 8 km × 8 km. A significant spatial corre- lation between these two datasets was observed for all the flights (0.36 ≤r ≤0.85). The CAROLS VOD presented sig- nificant spatial correlations with the vegetation water content (VWC) derived from the spatial distribution of vegetation types used in ISBA-A-gs and from the Leaf Area Index (LAI) simulated for low vegetation. On the other hand, the CAR- OLS VOD presented little temporal changes, and no tempo- ral correlation was observed with the simulated LAI. For low vegetation, the ratio of VOD to VWC tended to decrease, from springtime to summertime. The ISBA-A-gs grid cells (8 km × 8 km) were sampled every 5 m by CAROLS obser- vations, at a spatial resolution of about 2 km. For 83 % of the grid cells, the standard deviation of the sub-grid CAROLS SSM was lower than 0.05 m3 m−3. 1 Introduction Biophysical variables, such as soil moisture, Leaf Area In- dex (LAI), and vegetation biomass, need to be monitored for applications in ecohydrology, hydrometeorology and agrocli- matology, at global and regional scales. Soil moisture plays an important role in hydrological models, controlling the soil drainage and the surface runoff. It is also a crucial variable for land surface models (LSM), as it regulates the water and energy surface fluxes (e.g. Mohr et al., 2000). Finally, the seasonal dynamics of vegetation properties, such as LAI, is connected to soil moisture dynamics (Kochendorfer and Ramirez, 2010). This is particularly true in regions affected by droughts, where water is a limiting factor of plant growth (Porporato and Rodriguez-Iturbe, 2002). Developing obser- vation capacities able to monitor the spatial and temporal variability of vegetation and soil moisture characteristics at HAL Id: hal-00661546 https://hal.science/hal-00661546v1 Submitted on 28 Dec 2015 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Hydrology and Earth System Sciences Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 www.hydrol-earth-syst-sci.net/16/1725/2012/ doi:10.5194/hess-16-1725-2012 © Author(s) 2012. CC Attribution 3.0 License. Hydrology and Earth System Sciences E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 1726 egional scale, is needed for ecohydrology research (Wang al., 2012). parameters of the L-band Microwave Emission of the Bio- sphere model (L-MEB) (Bircher et al., 2012). The L-MEB a regional scale, is needed for ecohydrology research (Wang et al., 2012). parameters of the L-band Microwave Emission of the Bio- sphere model (L-MEB) (Bircher et al., 2012). The L-MEB model simulates the L-band emission of the soil-plant sys- tem (Wigneron et al., 2007), and the SMOS SSM retrieval algorithm is based on the inversion of L-MEB. The L-MEB SSM retrieval capability was extensively tested in homoge- neous vegetation cover conditions (Grant et al., 2008; Saleh et al., 2007; Wigneron et al., 2007; Guglielmetti et al., 2008). Panciera et al. (2008, 2009) found that after site-specific cali- bration of the vegetation and roughness parameters, the SSM retrieval accuracy can be better than 0.048 m3 m−3 for crops and grasslands. After the launch of SMOS, there is now a need to assess the accuracy of SSM retrievals for various ground properties and vegetation types in the framework of the calibration/validation of the instrument. This remote sensing study investigates the joint soil mois- ture and vegetation growth dynamics using the L-band Co- operative Airborne Radiometer for Ocean and Land Stud- ies (CAROLS, Zribi et al., 2011). The analysis is based on a very high number of flights (20 flights covering the same transect), in situ soil moisture observations, and sim- ulated biophysical variables. The CAROLS campaign was performed in the framework of the calibration/validation of the SMOS (Soil Moisture and Ocean Salinity) spaceborne ra- diometer operating at L-band (Kerr et al., 2001). Over land, the main product of SMOS is surface soil moisture (SSM). The multi-angular bipolarized observations of SMOS permit the retrieval of the vegetation optical depth (VOD), in addi- tion to SSM (Wigneron et al., 1995; Lee et al., 2002; Pel- larin et al., 2003a). The satellite-derived SSM or soil wet- ness index (SWI) products from passive (e.g. E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France the Advanced Microwave Scanning Radiometer on EOS – AMSR-E) and active (the Advanced Scatterometer – ASCAT, and the Euro- pean Remote Sensing Satellite Scatterometer – ERS-Scat) C- band microwave sensors were recently validated over south- western France (Albergel et al., 2009; Pellarin et al., 2006; R¨udiger et al., 2009), Western Africa (Gruhier et al., 2010; de Rosnay et al., 2009), Canada and Ukraine (Wagner et al., 1999a,b), China (Zhao et al., 2008), the United States of America (Drusch et al., 2004) and Australia (Draper et al., 2007). Microwave sensors operating at low frequencies (1– 10 GHz) are particularly useful for SSM monitoring. Com- pared to higher frequencies, low frequencies are able to sample thicker surface soil layers. Moreover, the vegetation masking effect are less pronounced, and atmospheric effects are weaker (Wagner et al., 2007). Besides SSM, the retrieval algorithm based on L-MEB produces a L-band VOD. In this study, VOD is defined as the effective zenith (i.e. nadir) opacity of the vegetation (“τ”, dimensionless) in the microwave domain. A number of stud- ies have shown the usefulness of VOD values retrieved from C-band or X-band satellite microwave brightness tempera- tures (Liu et al., 2007, 2011; Jones et al., 2011; Miralles et al., 2011). At L-band, this quantity can be produced by the inversion of the simplified “τ −ω” approach used in the L- MEB model (Wigneron et al., 2007). When VOD = 0, there is no vegetation attenuation of the soil microwave emission. The VOD value tends to increase with the vegetation water content (VWC, in kg m−2). From L-band to X-band, VOD is proportional to VWC and to frequency (f ) (Jackson and Schmugge, 1991; Schmugge and Jackson, 1992; Kerr and Wigneron, 1995; Njoku and Chan, 2006). The VOD value is often expressed as VOD = b VWC, with b = A ε′′ S f (Kirdya- shev et al., 1979), where the value of A is related to the canopy structure, and ε′′ S is the imaginary part of the dielec- tric constant of saline water in the vegetation. The latter de- pends only slightly on temperature, at the low salinity lev- els generally observed in plants. At low frequencies, it is often assumed that the A × ε′′ S product does not vary much from one vegetation type to another and across frequency values, and that b is proportional to f . Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 1727 Fig. 1. Atlantic-Mediterranean transect and location of SMOSMA- NIA stations (background is a Landsat GeoCover Mosaics). southwestern France and in Spain (Zribi et al., 2011; Pard´e et al., 2011a). Albergel et al. (2011) showed the good sensitiv- ity of the CAROLS L-band brightness temperatures (Tb) to the SSM variability at 11 stations of the SMOSMANIA soil moisture network (Calvet et al., 2007; Albergel et al., 2008), with an average Pearson correlation coefficient of −0.76 at nadir. However, radio frequency interferences (RFI) affected the CAROLS observations (Albergel et al., 2011; Pard´e et al., 2011a; Zribi et al., 2011). southwestern France and in Spain (Zribi et al., 2011; Pard´e et al., 2011a). Albergel et al. (2011) showed the good sensitiv- ity of the CAROLS L-band brightness temperatures (Tb) to the SSM variability at 11 stations of the SMOSMANIA soil moisture network (Calvet et al., 2007; Albergel et al., 2008), with an average Pearson correlation coefficient of −0.76 at nadir. However, radio frequency interferences (RFI) affected the CAROLS observations (Albergel et al., 2011; Pard´e et al., 2011a; Zribi et al., 2011). , ; , ) In this study, the temporal and spatial variability of the SSM and VOD values retrieved by L-MEB from 2009 and 2010 springtime CAROLS Tb observations in southwest- ern France is investigated. Details on the parameterisation of the L-MEB model are given in Pard´e et al. (2011b). In a first stage, post-processing techniques are used to limit the detrimental impact on the retrievals of RFI and open water surfaces. Then, the accuracy of the SSM retrievals is assessed using in situ SSM observations at 11 instru- mented sites. The CAROLS SSM and VOD transects are compared with SSM and Leaf Area Index (LAI) simulations of the CO2-responsive version of the Interactions between Soil, Biosphere and Atmosphere (ISBA-A-gs) model, over 8 km × 8 km grid cells. Fig. 1. Atlantic-Mediterranean transect and location of SMOSMA- NIA stations (background is a Landsat GeoCover Mosaics). instrument allowed oversampling the swath, with an along- track spatial resolution of about 5 m. The transect between the Bay of Biscay and the Mediterranean Sea was about 385 km, and the complete flights were covered in 3 h. 2.1.1 The CAROLS instrument 2.1.1 The CAROLS instrument Four flights (18 and 27 May 2009, 4 and 22 June 2010) were performed in the late afternoon (between 18:00 UTC and 20:00 UTC). All the other flights were performed in the morning (between 05:00 UTC and 08:00 UTC). The flights did not cover mountainous areas, and the surface altitude var- ied between 0 m a.s.l. and 400 m a.s.l. The CAROLS L-band microwave radiometer is fully polari- metric and has two antennas: one looking at nadir and a side- looking antenna slanting with an incidence angle of 33.5◦. The sampling frequency range is 1.401–1.426 GHz, and an advanced analogue filter permits the suppression of the main RFI affecting the Tb. The instrument sensitivity is 0.1 K for 1s integration time, and the stability is better than 0.1 K over 15 min. The internal calibration is achieved by load and noise diodes. The radiometer was calibrated during test flights per- formed in November 2008 (Zribi et al., 2011). The transect can be divided into three main areas: – the Atlantic lowland plain, between 1◦W and 0.2◦W, corresponding to Les Landes pine forest, on sandy soils (with sand fractions of about 90 %). E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France In 2009, six CAROLS flights were conducted over south- western France between 28 April and 27 May, and all of them are used in this study. While half of them (28 April, 15 and 27 May) covered the whole transect, the others (18, 20 and 26 May) only covered the Toulouse-Atlantic coast transect (225 km long). Moreover, 14 flights performed in 2010, cov- ering the full transect between 15 April and 1 July, are used in this study. 2.1.2 The 2009 and 2010 flights in southwestern France – the Garonne plain, between 0.2◦W and 2.1◦E, cor- responding to hilly Armagnac, Garonne terraces and Lauragais regions: It is characterized by undulating ter- rain, intersected by numerous rivers, and mainly cov- ered by croplands (e.g. wheat and irrigated maize), by grassland and forest patches over the steepest slopes, and with areas covered by vineyards. This part of the transect presents the highest altitudes (from 150 m to 380 m). The loamy soils are characterised by a higher clay content (20–30 %), in comparison with the sur- rounding regions, and a sand fraction ranging between 20 % and 30 %. The CAROLS microwave Tb airborne measurements were carried out from the French ATR-42 research aircraft, to- gether with infrared temperature (TIR) observations per- formed by a CIMEL radiometer. The flights consisted of straight lines between Toulouse and the Atlantic coast (Bay of Biscay), then to the Mediterranean coast, and back to Toulouse (Fig. 1). The observations were performed at an al- titude of 2000 m a.s.l. (above sea level), and the ground foot- print sizes of the nadir and side-looking antennas were about 1.4 km and 2.1 km, respectively (Albergel et al., 2011). The areas observed by the two antennas formed a swath with a mean width of about 3 km. The high acquisition rate of the Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France For example, the fre- quency used by the SMOS radiometer is 1.42 GHz (L-band) and the C-band and X-band channels of AMSR-E correspond to 6.925 GHZ and 10.65 GHz, respectively. Therefore, if one assumes that f dominates the VOD response to VWC, VOD is about 5 times more sensitive to VWC at C-band than at L- band, even more at X-band. Using ground multi-frequency passive microwave observations, Calvet et al. (2011) have shown that C-band and X-band are more appropriate than L- band to monitor the VWC of a wheat field, with VWC values up to 3 kg m−2 at the end of May. This is consistent with the lower sensitivity of VOD to changes in VWC (i.e. the lower b value) at L-band. The high capability of SSM retrieval at L-band (1–2 GHz) was demonstrated by several studies (e.g. Wigneron et al., 2002; Kerr et al., 2001; de Rosnay et al., 2006; Calvet et al., 2011). The first ground-based studies dedicated to soil moisture measurement at L-band started in the 1970s (Njoku and Kong, 1977). Later, they were complemented by airborne measurements (Jackson et al., 1986; Schmugge et al., 1992; Chanzy et al., 1997), which finally led to the development the SMOS (Soil Moisture and Ocean Salinity) spaceborne instrument operating at L-band (Kerr et al., 2001). p g A number of field experiments (CoSMOS, SMOSREX, VAS, MELBEX-1, ELBARA-ETH) (Saleh et al., 2007, 2009; Cano et al., 2010) were carried out in order to pre- pare the SMOS mission. In addition, several airborne cam- paigns were performed to assess SSM retrieval over large ar- eas: EuroSTARRS in France and Spain (Saleh et al., 2004), NAFE/CoSMOS in Australia (Panciera et al., 2008), EA- GLE2006 in Germany (Su et al., 2009), HOBE in Den- mark (Bircher et al., 2012). These studies showed that the response of L-band brightness temperatures to SSM is af- fected by biomass conditions (Saleh et al., 2004). They also showed the importance of specific biome calibration of the One of the more recent airborne campaigns dedicated to SMOS is the CAROLS experiment, co-funded by CNES and ESA. CAROLS was performed in 2009 and 2010 in www.hydrol-earth-syst-sci.net/16/1725/2012/ www.hydrol-earth-syst-sci.net/16/1725/2012/ Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 2.1.3 SSM and VOD retrievals As illustrated by Fig. 2, a number of unrealistic SSM val- ues, close to 1 m3 m−3 or higher than 1 m3 m−3, as well as a lot of smaller peaks are obtained. The spatial analysis of the successful SSM retrievals shows that the perturbation of SSM values are obtained over areas where (1) very low Tb values and/or (2) significant differences between nadir V- and H-polarized Tb are observed (TbV and TbH, respectively). In order to improve the retrievals, a post-processing filter- ing technique was applied to the data set. Two methods were used: (1) a physical method based on the fact that TbH and TbV should be close at nadir and (2) a statistical method us- ing 2-D histograms. The SSM and VOD values were retrieved from the biangular/dual-polarized L-band CAROLS Tb measure- ments, inverting the most recent version of the L-MEB model (Panciera et al., 2008; Pard´e et al., 2011a,b). According to the Fresnel law, TbV and TbH values are confounded at nadir, and only three independent Tb values are used in the inver- sion: Tb at nadir, and slant (33.5◦) TbV and TbH. The present version uses the Dobson model (Dobson et al., 1985) of the soil microwave dielectric properties. The soil roughness is accounted for using the Wang and Choudhury (1981) model, based on two parameters: h and Q. The Q parameter is set to zero, and the h parameter is related to SSM as proposed by Saleh et al. (2007) for a grassland site in southwestern France, and applied by Pard´e et al. (2011b) to the CAROLS data: The physical method was based on the paired view analy- sis of TbV and TbH at nadir. According to the Fresnel law, the surface emissivities at vertical and horizontal polariza- tions are equal at nadir. Therefore, the difference between nadir TbH and nadir TbV (1Tb) should not deviate much from zero. It was assumed that all the retrievals correspond- ing to 1Tb values higher or lower than ±2 standard devia- tions (calculated using all 20 flights) were affected by RFI. Filtering the data using this criterion removed many peaks around the largest urban areas. h = 1.3 −1.13 × SSM (1) (1) h = 1.3 −1.13 × SSM (1) h = 1.3 −1.13 × SSM with SSM in units of m3 m−3. with SSM in units of m3 m−3. E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 1728 – the Mediterranean plain, between 2.1◦E and 3.3◦E is flat and the vegetation cover is contrasted, with forest patches alternating with sparse vegetation, dry shrubs, and vineyards. In the easternmost part the soils are more sandy, with sand fractions ranging from 40 % to 50 %. and their detection, were described in detail by Pard´e et al. (2011a) and Zribi et al. (2011). Before applying the SSM and VOD retrieval algorithm, a method of detection and mit- igation of the undesirable effects of RFI was applied to the CAROLS Tb dataset by Pard´e et al. (2011a). The analysis of the SSM and VOD products performed in this study showed that further RFI filtering is needed. 2.1.3 SSM and VOD retrievals The vegetation contribution is computed using the τ −ω approach, where τ represents VOD, and ω represents the sin- gle scattering albedo of the vegetation, assumed to be close to zero (Wigneron et al., 2007). The effective temperature (Teff) contribution is approximated as The statistical method was based on the analysis of TbH vs. TbV at 33.5◦for the 2009 flights (Fig. 3). A density-based clustering of the data (Jain et al., 1999) was applied to con- tour a main cluster A (Fig. 3), and remove the data outside this cluster. The detection of the cluster borders depends on a density threshold, which was determined using the 2009 data, in order to optimize the spatial correlation between the retrieved and the modelled SSM values. The same threshold was used for the 2010 data. Teff = Tdepth + TIR −Tdepth  × (SSM/w)β (2) (2) where Tdepth is the ground temperature at a depth of 30 cm measured at the meteorological stations of the SMOSMA- NIA network (see Sect. 2.2) and then interpolated along the aircraft transect, and TIR is the infrared temperature, remotely sensed from the aircraft. The w and β parame- ters have constant values: 0.30 m3 m−3 and 0.3, respectively (Grant et al., 2008; Wigneron et al., 2007, 2008). The soil properties like the sand and clay contents used in the model are derived from the SMOSMANIA auxiliary dataset (Al- bergel et al., 2008) linearly interpolated along the CAROLS flight transect. The spatial analysis of the outliers corresponding to high TbH and to high TbV values (B and C subsets of Fig. 3, re- spectively) indicates that they are caused by unmitigated RFI perturbations close to urban areas (not shown). The D cluster corresponds to low values of both TbH and TbV, observed in regions presenting a high density of water bodies (not shown). In order to further reduce the impact of open water surfaces, we have arbitrarily excluded the cluster A measure- ments corresponding to TbH values less than 220 K. It re- moved all the unrealistic SSM higher than 1 m3 m−3 (Fig. 2). The application of the above-mentioned post-processing fil- tering techniques improved significantly the overall quality of the SSM and VOD retrieval data set. www.hydrol-earth-syst-sci.net/16/1725/2012/ www.hydrol-earth-syst-sci.net/16/1725/2012/ E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 2.2 SMOSMANIA stations At each station, the soil moisture acquisition is done ev- ery 12 min by four ThetaProbe ML2X instruments at 5 cm, 10 cm, 20 cm and 30 cm depths. Soil temperature is measured at the same depths. The soil moisture dataset is available on- line at http://www.ipf.tuwien.ac.at/insitu/. The observations from this network were extensively used for the validation of modelled and satellite-derived soil moisture (Albergel et al., 2008, 2009, 2010, 2011; Parrens et al., 2012). The SSM retrievals were validated using in situ observa- tions of the Soil Moisture Observing System – Meteorolog- ical Automatic Network Integrated Application (SMOSMA- NIA) network (Calvet et al., 2007). This network consists of 21 stations in southern France, of which 12 have been operating in southwestern France since January 2007. It is based on the existing automatic weather station network of M´et´eo-France, and soil moisture profiles are monitored, in addition to standard hydrometeorological observations (pre- cipitation, air temperature, air humidity, wind speed). The stations form a transect between the Atlantic coast and the Mediterranean sea (Fig. 1) and sample soil moisture un- der contrasting climatic conditions (Albergel et al., 2008). 2.1.4 Mitigation of perturbing factors In southwestern France the CAROLS microwave measure- ments are affected by RFI and by the presence of water bodies. Albergel et al. (2011) had to filter out about half of CAROLS Tb observations affected by RFI, over 11 SMOS- MANIA sites. The RFI tend to increase the Tb values and undetected RFI may result in the underestimation of SSM and/or in the overestimation of VOD. The structure and qual- ity of the CAROLS observations, as well as the RFI sources www.hydrol-earth-syst-sci.net/16/1725/2012/ www.hydrol-earth-syst-sci.net/16/1725/2012/ Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France va et al.: Spatial and temporal variability of biophysical variables in southwestern France 1729 E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 1729 Fig. 2. Spatial variability of (A) CAROLS TbH (green) and TbV (black) at nadir, (B) CAROLS brightness temperature difference (TbV– TbH) at nadir, and (C) CAROLS surface soil moisture (SSM), for 28 April 2009. Red dots represent the SSM data after the post-processing filtering. Fig. 2. Spatial variability of (A) CAROLS TbH (green) and TbV (black) at nadir, (B) CAROLS brightness temperature difference (TbV– TbH) at nadir, and (C) CAROLS surface soil moisture (SSM), for 28 April 2009. Red dots represent the SSM data after the post-processing filtering. 2.3 ISBA-A-gs surface soil moisture and LAI simulations In order to assess the ability of the CAROLS measurements to represent the spatial and temporal variability of SSM and VOD, the retrievals were compared with SSM and LAI Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 www.hydrol-earth-syst-sci.net/16/1725/2012/ www.hydrol-earth-syst-sci.net/16/1725/2012/ www.hydrol-earth-syst-sci.net/16/1725/2012/ (4) for the flights presenting a significant spatial correlation between VWC and VOD, with VWC and VOD estimates derived from ISBA-A-gs and from CAROLS, respectively, for grid cells with a fraction of woody vege- tation higher than 70 %, and with a fraction of low vegeta- tion higher than 80 %, respectively (see Sect. 3.2.2). Using these average values of bhigh and blow, aggregated bottom-up VOD values can be calculated from the ISBA-A-gs model for each flight: the VWC and VOD values derived from Eqs. (3)– (4) for low and for high vegetation are aggregated over each ISBA-A-gs grid cell using a linear mixing equation based on the ECOCLIMAP-II vegetation fractions. The SSM and LAI simulations were carried out using the SURFEX (SURFace Externalis´ee) platform developed at M´et´eo-France. ISBA-A-gs is a version of the ISBA LSM that includes a photosynthesis and a plant growth model (Cal- vet et al., 1998, 2004; Calvet, 2000; Calvet and Soussana, 2001). The simulations were driven by the SAFRAN analy- sis of surface meteorological variables (Durand et al., 1993; Quintana-Segui et al., 2008), and the spatial distribution of the model parameters was based on ECOCLIMAP-II (Mas- son et al., 2003; Faroux et al., 2009). The ISBA-A-gs version used in this study simulates three soil layers. The simulated SSM corresponds to the modelled surface soil layer, about 1 cm thick. The sub-grid heterogene- ity is represented by aggregating the simulations performed for the various surface types found in the grid cell, according to the surface type fractions provided by ECOCLIMAP-II (Faroux et al., 2009; Brut et al., 2009). Also, this allows to provide separate estimates of SSM and LAI for high (trees) and low (grasslands and crops) vegetation types. www.hydrol-earth-syst-sci.net/16/1725/2012/ Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 1730 Fig. 3. Side-looking antenna TbV vs. TbH for the six CAROLS flights in 2009. The measurements outside of the red contour were removed from the analysis. The A–D letters correspond to data clusters in the TbH–TbV space. The main cluster A (except for TbH < 220 K) corresponds to the data analyzed in Sects. 3–4. Clus- ters B–D correspond to observations excluded from the dataset: B and C are related to unmitigated RFI perturbations close to ur- ban areas; D is related to a high density of water bodies. crops) could be estimated as VWClow = 0.5 × LAI, in units of kg m−2. For high (forest) canopies they set constant val- ues of VWChigh, as at L-band this quantity depends mostly on the water content of the branches. The latter was estimated by Pellarin et al. (2003a) as 3 kg m−2 and 4 kg m−2 for conif- erous and deciduous trees, respectively. As lower branch wa- ter content values were reported by Grant et al. (2008) for European forests, the 1.5 kg m−2 and 3 kg m−2 VWC values are used in this study for coniferous and deciduous trees, re- spectively. Using the LAI simulated by ISBA-A-gs for low vegetation, and the ECOCLIMAP-II forest fraction, the veg- etation water content affecting the L-band land emission for low and high vegetation (in kg m−2) is estimated along the aircraft transect as Fig. 3. Side-looking antenna TbV vs. TbH for the six CAROLS flights in 2009. The measurements outside of the red contour were removed from the analysis. The A–D letters correspond to data clusters in the TbH–TbV space. The main cluster A (except for TbH < 220 K) corresponds to the data analyzed in Sects. 3–4. Clus- ters B–D correspond to observations excluded from the dataset: B and C are related to unmitigated RFI perturbations close to ur- ban areas; D is related to a high density of water bodies. VWClow = 0.5 × LAIlow (3a) VWChigh = 1.5 × αconif + 3 × αdecid (3b) (3a) (3b) where LAIlow is the LAI of low vegetation in a given grid cell, and αconif and αdecid are coniferous and deciduous for- est fractions, respectively. www.hydrol-earth-syst-sci.net/16/1725/2012/ As shown by Eq. (3b), VWChigh does not depend on LAI and does not present seasonal vari- ations. This is coherent with ground observations of the L- band microwave emission of forest canopies showing little or no change across seasons (e.g. Guglielmetti et al., 2008; Grant et al., 2008). Finally, VOD can be derived from VWC as simulations produced by the ISBA-A-gs LSM, at a spatial resolution of 8 km × 8 km. as Prior to the comparison, the airborne observations were averaged within each ISBA-A-gs grid cell crossed by the flight line. As Tb values are very sensitive to open water surfaces, the grid cells with water fraction more than 0.8 % (corresponding to a total open water area of 0.5 km2) were removed. VODlow = blow VWClow (4a) VODhigh = bhigh VWChigh (4b) (4a) VODlow = blow VWClow (4a) VODhigh = bhigh VWChigh (4b) (4b) where blow and bhigh are constant values, for a given vegeta- tion type. Pellarin et al. (2003a) proposed blow values rang- ing from 0.15 to 0.20 m2 kg−1 for low vegetation (crops and grasslands, respectively), and bhigh = 0.33 m2 kg−1. Grant et al. (2008) found a bhigh value of 0.4 m2 kg−1 for 26-yr-old pine trees. Also, values of bhigh and blow can be obtained by inverting Eq. (4) for the flights presenting a significant spatial correlation between VWC and VOD, with VWC and VOD estimates derived from ISBA-A-gs and from CAROLS, respectively, for grid cells with a fraction of woody vege- tation higher than 70 %, and with a fraction of low vegeta- tion higher than 80 %, respectively (see Sect. 3.2.2). Using these average values of bhigh and blow, aggregated bottom-up VOD values can be calculated from the ISBA-A-gs model for each flight: the VWC and VOD values derived from Eqs. (3)– (4) for low and for high vegetation are aggregated over each ISBA-A-gs grid cell using a linear mixing equation based on the ECOCLIMAP-II vegetation fractions. where blow and bhigh are constant values, for a given vegeta- tion type. Pellarin et al. (2003a) proposed blow values rang- ing from 0.15 to 0.20 m2 kg−1 for low vegetation (crops and grasslands, respectively), and bhigh = 0.33 m2 kg−1. Grant et al. (2008) found a bhigh value of 0.4 m2 kg−1 for 26-yr-old pine trees. Also, values of bhigh and blow can be obtained by inverting Eq. 3.1 CAROLS SSM validation at the SMOSMANIA stations In this study, the CAROLS-derived VOD was compared with the version 1 of the GEOLAND2 LAI product (Baret et al., 2012). This product is based on an algorithm trained with two satellite data streams: MODIS, and SPOT/VEGETATION. The fused algorithm uses the SPOT/VEGETATION data to produce the final LAI values. The initial 10-day, 1 × 1 km satellite-derived LAI product was resampled to the SAFRAN 8 × 8 km spatial resolution for the 2009 and 2010 years. In order to evaluate the accuracy of the airborne L-band SSM retrievals, the latter were compared with in situ soil moisture observations performed at a depth of 5 cm at the 12 SMOS- MANIA stations located in southwestern France (Fig. 4 and Table 1). It must be noted that the L´ezignan-Corbi`eres sta- tion (LZC) did not provide SSM observations during the air- borne campaign of 2010. Therefore, only 11 stations are con- sidered. For this comparison, the SSM retrievals were aver- aged over a 10 km radius zone around each station. Signif- icant correlations (p-value < 0.05) between airborne and in situ observations are found for 8 out of 11 stations. For the three other stations, Sabres (SBR), Peyrusse-Grande (PRG) and Condom (CDM), discrepancies are observed and the cor- relation of the CAROLS SSM retrievals with ground obser- vations is not significant (Table 1). More often than not, at PRG and CDM stations, the CAROLS SSM retrievals are lower than the in situ data (Fig. 4). However, PRG presents one SSM retrieval much higher than the in situ observations, and the mean bias is slightly negative, in conjunction with a high SDD value of 0.18 m3 m−3. E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France va et al.: Spatial and temporal variability of biophysical variables in southwestern France E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 1731 Table 1. Comparison of SSM time series for (from left to right) SMOSMANIA vs. CAROLS, CAROLS vs. ISBA-A-gs, and ISBA-A-gs vs. SMOSMANIA. The Pearson correlation coefficient (r), p-value, RMSD, bias (SMOSMANIA minus CAROLS, CAROLS minus ISBA- A-gs, ISBA-A-gs minus SMOSMANIA, respectively) and the standard deviation of differences (SDD) are given for the pooled 2009 and 2010 CAROLS flights. Table 1. Comparison of SSM time series for (from left to right) SMOSMANIA vs. CAROLS, CAROLS vs. ISBA-A-gs, and ISBA-A-gs vs. SMOSMANIA. The Pearson correlation coefficient (r), p-value, RMSD, bias (SMOSMANIA minus CAROLS, CAROLS minus ISBA- A-gs, ISBA-A-gs minus SMOSMANIA, respectively) and the standard deviation of differences (SDD) are given for the pooled 2009 and 2010 CAROLS flights. Station r p-value RMSD Mean bias SDD Nobs (m3 m−3) (m3 m−3) (m3 m−3) SBR 0.36/0.54/0.69 NS/∗/∗∗∗ 0.069/0.064/0.069 −0.024/−0.034/0.058 0.065/0.056/0.037 20 URG 0.56/0.50/0.64 ∗/∗/∗∗ 0.212/0.077/0.168 0.195/−0.046/−0.149 0.085/0.063/0.080 20 CRD 0.66/0.55/0.68 ∗∗/∗/∗∗∗ 0.102/0.069/0.092 −0.080/−0.005/0.085 0.063/0.069/0.036 20 PRG 0.23/0.58/0.67 NS/∗∗/∗∗ 0.175/0.157/0.067 −0.012/−0.028/0.040 0.175/0.157/0.055 17 CDM 0.41/0.61/0.61 NS/∗∗/∗∗ 0.134/0.090/0.085 0.088/−0.018/−0.070 0.101/0.090/0.050 17 LHS 0.62/0.73/0.59 ∗∗/∗∗∗/∗∗ 0.108/0.101/0.064 0.030/−0.009/−0.021 0.105/0.101/0.062 20 SVN 0.51/0.46/0.58 ∗/∗/∗∗ 0.212/0.213/0.068 −0.159/0.154/0.005 0.141/0.150/0.068 20 MNT 0.82/0.63/0.64 ∗∗∗/∗∗/∗∗ 0.121/0.067/0.150 0.113/0.032/−0.144 0.044/0.060/0.044 17 SFL 0.67/0.74/0.53 ∗∗/∗∗∗/∗ 0.073/0.066/0.074 −0.017/−0.018/0.029 0.071/0.065/0.069 17 MTM 0.80/0.72/0.54 ∗∗∗/∗∗/∗ 0.066/0.064/0.039 0.038/−0.031/−0.012 0.054/0.057/0.038 17 LZC – – – – – 2 NBN 0.67/0.60/0.66 ∗∗/∗/∗∗ 0.039/0.044/0.043 −0.006/−0.020/0.023 0.039/0.040/0.036 17 Average 0.57/0.61/0.62 – 0.119/0.097/0.084 0.015/−0.002/−0.014 0.086/0.083/0.052 – NS – non significant; ∗, ∗∗, ∗∗∗stand for p-value greater than 0.05, between 0.05 and 0.01, between 0.01 and 0.001, and below 0.001, respectively. (8 km × 8 km) may contain up to about 500 CAROLS re- trievals. After the pre- and post-processing filtering, about 60 valid SSM and VOD retrievals are obtained, on average. This oversampling capability was used to investigate the sub- grid variability of the model’s parameters in grid cells. following thresholds on p-values are used: (i) NS (non sig- nificant) for p-value greater than 0.05, (ii) ∗between 0.05 and 0.01, (iii) ∗∗between 0.01 and 0.001, (iv) ∗∗∗be- tween 0.001 and 0.0001 and (v) ∗∗∗∗below a value of 0.0001. 2.6 Metrics used for the comparison of the various data sets In order to quantify the differences between two independent estimates of the same quantity (SSM or VOD, either through time at given locations, or throughout the CAROLS tran- sect at given days), four scores are considered in this study: the Pearson correlation coefficient (r), the root mean square difference (RMSD), the standard deviation of differences (SDD) and the Fisher’s F-test p-value. If the two estimates to be compared present the same mean value, then SDD is equal to RMSD. Otherwise, SDD is lower than RMSD as the latter is impacted by the bias. The p-value indicates the sig- nificance of the test: if it is small (e.g. below 0.05), it means that the correlation is not a coincidence. In this study, the Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 www.hydrol-earth-syst-sci.net/16/1725/2012/ www.hydrol-earth-syst-sci.net/16/1725/2012/ 2.4 Investigating the ISBA-A-gs sub-grid variability using the CAROLS retrievals The LAI simulations of ISBA-A-gs can be used, to some extent, to estimate the VWC affecting the L-band land emis- sion. In a study at a global scale, Pellarin et al. (2003a) assumed that the VWC of low vegetation (grasslands and While the ground footprint of the CAROLS radiometer is about 1.4 km at nadir, its along-track spatial resolution al- lows sampling Tb every 5 m. Each ISBA-A-gs grid cell Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 www.hydrol-earth-syst-sci.net/16/1725/2012/ E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 1731 Table 1. Comparison of SSM time series for (from left to right) SMOSMANIA vs. CAROLS, CAROLS vs. ISBA-A-gs, and ISBA-A-gs vs. SMOSMANIA. The Pearson correlation coefficient (r), p-value, RMSD, bias (SMOSMANIA minus CAROLS, CAROLS minus ISBA- A-gs, ISBA-A-gs minus SMOSMANIA, respectively) and the standard deviation of differences (SDD) are given for the pooled 2009 and 2010 CAROLS flights. Station r p-value RMSD Mean bias SDD Nobs (m3 m−3) (m3 m−3) (m3 m−3) SBR 0.36/0.54/0.69 NS/∗/∗∗∗ 0.069/0.064/0.069 −0.024/−0.034/0.058 0.065/0.056/0.037 20 URG 0.56/0.50/0.64 ∗/∗/∗∗ 0.212/0.077/0.168 0.195/−0.046/−0.149 0.085/0.063/0.080 20 CRD 0.66/0.55/0.68 ∗∗/∗/∗∗∗ 0.102/0.069/0.092 −0.080/−0.005/0.085 0.063/0.069/0.036 20 PRG 0.23/0.58/0.67 NS/∗∗/∗∗ 0.175/0.157/0.067 −0.012/−0.028/0.040 0.175/0.157/0.055 17 CDM 0.41/0.61/0.61 NS/∗∗/∗∗ 0.134/0.090/0.085 0.088/−0.018/−0.070 0.101/0.090/0.050 17 LHS 0.62/0.73/0.59 ∗∗/∗∗∗/∗∗ 0.108/0.101/0.064 0.030/−0.009/−0.021 0.105/0.101/0.062 20 SVN 0.51/0.46/0.58 ∗/∗/∗∗ 0.212/0.213/0.068 −0.159/0.154/0.005 0.141/0.150/0.068 20 MNT 0.82/0.63/0.64 ∗∗∗/∗∗/∗∗ 0.121/0.067/0.150 0.113/0.032/−0.144 0.044/0.060/0.044 17 SFL 0.67/0.74/0.53 ∗∗/∗∗∗/∗ 0.073/0.066/0.074 −0.017/−0.018/0.029 0.071/0.065/0.069 17 MTM 0.80/0.72/0.54 ∗∗∗/∗∗/∗ 0.066/0.064/0.039 0.038/−0.031/−0.012 0.054/0.057/0.038 17 LZC – – – – – 2 NBN 0.67/0.60/0.66 ∗∗/∗/∗∗ 0.039/0.044/0.043 −0.006/−0.020/0.023 0.039/0.040/0.036 17 Average 0.57/0.61/0.62 – 0.119/0.097/0.084 0.015/−0.002/−0.014 0.086/0.083/0.052 – NS – non significant; ∗, ∗∗, ∗∗∗stand for p-value greater than 0.05, between 0.05 and 0.01, between 0.01 and 0.001, and below 0.001, respectively. www.hydrol-earth-syst-sci.net/16/1725/2012/ E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 3.2 Observed and modelled spatial and temporal variability of biophysical variables along the CAROLS transect The ISBA-A-gs LSM simulates SSM, together with the root- zone soil moisture, as prognostic variables. Also, the vege- tation growth component of the model provides LAI simu- lations. The simulated SSM and LAI are expected to corre- late with the SSM and VOD values retrieved from the CAR- OLS Tb, both temporally and spatially. Figure 5 presents the SSM and LAI time series simulated by ISBA-A-gs, to- gether with the precipitation provided by SAFRAN, over the Atlantic-Mediterranean transect (Fig. 1), for the 2009– 2010 period. The three regions described in Sect. 2.1.2 can be distinguished in both SSM and LAI time series. Higher SSM values are simulated for the Garonne central part of the transect, in relation to a higher soil water holding capacity. Higher LAI values are simulated over the Atlantic evergreen Les Landes pine forest, at the western part of the transect (3.5 m2 m−2 in April, at the beginning of the 2009 and 2010 CAROLS campaigns, and close to 5 m2 m−2 at the end of May 2009 and June 2010). In the central and eastern parts of the transect, the simulated LAI varies between 1.1 m2 m−2 and 4 m2 m−2, from the beginning to the end of the 2009 and 2010 CAROLS campaigns. The simulated leaf onset varies from the end of February in Les Landes forest to the end of March in other areas. Figure 5 also shows the satellite- derived LAI values. The latter present lower and smoother annual peak values than the simulated LAI. Fig. 5. Spatial and temporal variability of (A) precipitation (SAFRAN reanalysis), (B) ISBA-A-gs surface soil moisture and (C) ISBA-A-gs and satellite-derived LAI (solid and dashed lines, respectively), during the 2009–2010 period, over the three regions covered by the CAROLS Atlantic-Mediterranean transect (Atlantic, Garonne, Mediterranean). Monthly values are plotted. more local precipitation events took place in different parts of the transect. Figure 6 presents the retrieved CAROLS SSM along the Atlantic-Mediterranean transect in contrasting 2009 and 2010 conditions (end of April and end of May), together with the SSM simulated by ISBA-A-gs. At the end of April, the CAROLS SSM values observed in 2009 are systematically higher than the 2010 values. At the end of May, the CAR- OLS SSM is higher in 2009 close to the Atlantic coast and for the eastern part of the transect, and lower at the central part of the transect. E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 1733 Fig. 5. Spatial and temporal variability of (A) precipitation (SAFRAN reanalysis), (B) ISBA-A-gs surface soil moisture and (C) ISBA-A-gs and satellite-derived LAI (solid and dashed lines, respectively), during the 2009–2010 period, over the three regions covered by the CAROLS Atlantic-Mediterranean transect (Atlantic, Garonne, Mediterranean). Monthly values are plotted. situ observations. CAROLS presents fair correlations with ISBA-A-gs for all the stations. While, for the eastern sta- tions (MNT, SFL, MTM, NBN), the CAROLS SSM corre- lates better with the in situ observations than ISBA-A-gs, systematically higher SDD values are obtained with CAR- OLS. On average, the CAROLS SDD scores are higher than 0.08 m3 m−3. The ISBA-A-gs SSM is closer to the in situ ob- servations, with SDD = 0.052 m3 m−3, on average. The lower average SDD between the model and in situ observations is associated with a better average correlation (0.62, against 0.57 for CAROLS). 3.2 Observed and modelled spatial and temporal variability of biophysical variables along the CAROLS transect The same differences between 2010 and 2009 can be observed in Fig. 6 for the simulated SSM. Also, the spatial distribution of the CAROLS SSM measurements is in good agreement with that of the modelled SSM. The lowest SSM values are observed in the forested Atlantic part of the transect and close to the Mediterranean coast. Table 2 shows that the spatial correlation between the two datasets is significant (p-value < 0.05) for all 20 flights, with correlation coefficients ranging from 0.36 to 0.85 and root mean square differences (RMSD) ranging from 0.05 to 0.20 m3 m−3. On average, RMSD = 0.083 m3 m−3 and SDD = 0.066 m3 m−3. For 14 flights out of 20, there is a very good agreement (p- value < 0.001) between the CAROLS and ISBA-A-gs SSM E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France kharova et al.: Spatial and temporal variability of biophysical variables in southwestern Franc 1732 ral variability of the SSM measured by the SMOSMANIA stations (black line) and of the CAROLS SSM (red dots with std BR – Sabres, URG – Urgons, CRD – Cr´eon d’Armagnac, PRG – Peyrusse-Grande, CDM – Condom, LHS – Lahas, SVN – – Montaut, SFL – Saint-F´elix de Lauragais, MTM – Mouthoumet, LZC – L´ezignan-Corbi`eres, NBN – Narbonne. Fig. 4. Temporal variability of the SSM measured by the SMOSMANIA stations (black line) and of the CAROLS SSM (red dots with std error bars): SBR – Sabres, URG – Urgons, CRD – Cr´eon d’Armagnac, PRG – Peyrusse-Grande, CDM – Condom, LHS – Lahas, SVN – Savenes, MNT – Montaut, SFL – Saint-F´elix de Lauragais, MTM – Mouthoumet, LZC – L´ezignan-Corbi`eres, NBN – Narbonne. The most marked CAROLS biases (0.19 m3 m−3 and −0.16 m3 m−3) vs. in situ observations are observed for the Urgons (URG) and Savenes (SVN) stations. As both sta- tions are far from the CAROLS flight transect, it is likely that the station soil properties differ from those seen by the L-band radiometer. This explanation does not hold for the Montaut (MNT) station, which presents a marked bias (0.12 m3 m−3) while lying very close to the CAROLS tran- sect. Moreover, the highest Pearson correlation coefficient (r = 0.82) is obtained for this station. The MNT station is lo- cated in a hilly area, and while the SSM in situ measurements at this station represent well the SSM temporal variability of the area, topographical effects impact the spatial variability of SSM. In Table 1, the CAROLS SSM retrievals are com- pared, also, with the ISBA-A-gs SSM simulations at the 8 km × 8 km grid cells covering the SMOSMANIA stations. For the sake of comparison, they are compared with the in www.hydrol-earth-syst-sci.net/16/1725/2012/ Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 www.hydrol-earth-syst-sci.net/16/1725/2012/ E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 3.2.1 Surface soil moisture The various CAROLS flights permitted the sampling of a large range of SSM conditions, as illustrated by the SSM values simulated by ISBA-A-gs in Fig. 5. The first CAROLS flight on 28 April 2009 took place at the end of a marked rainfall episode. For this date, the retrieved SSM was high and exceeded 0.40 m3 m−3 at the central part of the transect. The end of the 2009 campaign was relatively dry, and the last rainfall events (24–26 May 2009) triggered an increase of the retrieved SSM in the central part of transect only. In 2010 two marked rainfall episodes occurred from 29 April to 14 May and from 6 June to 19 June. At the end of May 2010, www.hydrol-earth-syst-sci.net/16/1725/2012/ Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 www.hydrol-earth-syst-sci.net/16/1725/2012/ SSM VOD Date R p-value RMSD SDD R p-value RMSD SDD Nobs (m3 m−3) (m3 m−3) (−) (−) 28 Apr 2009 0.37 ∗∗ 0.084 0.083 0.57 ∗∗∗∗ 0.056 0.055 67 15 May 2009 0.61 ∗∗∗∗ 0.098 0.073 0.48 ∗∗∗∗ 0.079 0.053 68 18 May 2009 0.61 ∗∗∗ 0.066 0.065 0.57 ∗∗∗ 0.063 0.058 40 20 May 2009 0.56 ∗∗ 0.056 0.052 0.55 ∗∗ 0.080 0.064 33 26 May 2009 0.37 ∗ 0.092 0.067 0.48 ∗∗∗ 0.077 0.066 48 27 May 2009 0.57 ∗∗∗∗ 0.070 0.063 0.49 ∗∗∗∗ 0.075 0.058 67 15 Apr 2010 0.41 ∗∗ 0.077 0.067 0.56 ∗∗∗∗ 0.052 0.051 68 28 Apr 2010 0.36 ∗∗ 0.059 0.050 0.61 ∗∗∗∗ 0.055 0.055 68 3 May 2010 0.38 ∗ 0.197 0.137 0.01 NS 0.218 0.126 60 9 May 2010 0.61 ∗∗∗∗ 0.088 0.068 0.43 ∗∗∗ 0.081 0.081 66 21 May 2010 0.70 ∗∗∗∗ 0.061 0.059 0.38 ∗∗∗ 0.064 0.060 68 26 May 2010 0.85 ∗∗∗∗ 0.074 0.062 0.32 ∗ 0.082 0.080 57 31 May 2010 0.43 ∗∗∗ 0.084 0.076 0.51 ∗∗∗∗ 0.077 0.052 71 4 Jun 2010 0.74 ∗∗∗∗ 0.061 0.042 0.61 ∗∗∗∗ 0.083 0.048 66 8 Jun 2010 0.52 ∗∗∗ 0.101 0.063 0.18 NS 0.120 0.094 70 13 Jun 2010 0.80 ∗∗∗∗ 0.088 0.079 0.37 ∗∗ 0.074 0.055 73 18 Jun 2010 0.65 ∗∗∗∗ 0.086 0.085 0.31 ∗∗ 0.089 0.066 69 22 Jun 2010 0.78 ∗∗∗∗ 0.106 0.060 0.48 ∗∗∗∗ 0.066 0.056 76 26 Jun 2010 0.70 ∗∗∗∗ 0.066 0.028 −0.17 NS 0.154 0.037 62/22 1 Jul 2010 0.80 ∗∗∗∗ 0.049 0.040 0.57 ∗∗∗∗ 0.082 0.046 65 Average 0.59 – 0.083 0.066 0.49 – 0.073 0.059 – ∗, ∗∗, ∗∗∗, ∗∗∗∗stand for p-value between 0.05 and 0.01, between 0.01 and 0.001, between 0.001 and 0.0001 and below 0.0001, respectively. Note that only 22 ISBA-A-gs grid cells presented successful VOD retrievals on 26 June 2010. The average significant score values are indicated. ∗stand for p-value between 0.05 and 0.01, between 0.01 and 0.001, between 0.001 and 0.0001 and below 0.0001, respectively. Note BA-A-gs grid cells presented successful VOD retrievals on 26 June 2010. The average significant score values are indicated. ∗, ∗∗, ∗∗∗, ∗∗∗∗stand for p-value between 0.05 and 0.01, between 0.01 and 0.001, between 0.001 and 0.0001 and below 0.0001, respectively. hat only 22 ISBA-A-gs grid cells presented successful VOD retrievals on 26 June 2010. The average significant score values are indicated. Fig. 6. www.hydrol-earth-syst-sci.net/16/1725/2012/ E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 3 3 rova et al.: Spatial and temporal variability of biophysical variables in southwestern France E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 1734 fficient (R), p-value, RMSD and SDD (m3 m−3 for SSM) and number of grid cells taken for score calculation CAROLS SSM and VOD for 20 flights in 2009 and 2010. Table 2. Spatial correlation coefficient (R), p-value, RMSD and SDD (m3 m−3 for SSM) and number of grid cells taken for score calculation (Nobs) between ISBA-A-gs and CAROLS SSM and VOD for 20 flights in 2009 and 2010. Table 2. Spatial correlation coefficient (R), p-value, RMSD and SDD (m3 m−3 for SSM) and number of grid cells taken for score calculation (Nobs) between ISBA-A-gs and CAROLS SSM and VOD for 20 flights in 2009 and 2010. E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France va et al.: Spatial and temporal variability of biophysical variables in southwestern France 1735 Fig. 7. Flight transect of (A, B) the vegetation optical thickness (VOD) retrieved from CAROLS Tb and of (C, D) the ISBA-A-gs LAI simulations, at (A, C) the end of April 2010, and (B, D) the end of May 2010. variability of the retrieved VOD, either seasonal or interan- nual, is very low and not significant (Fig. 7). spatial distribution, with r ranging from 0.43 to 0.85. For these flights, the mean SDD value is 0.062 m3 m−3. The six remaining CAROLS SSM transects do not correlate very well with the simulated SSM and present higher p-values: 28 April 2009, 20 May 2009, 26 May 2009, 15 April 2010, 28 April 2010, and 3 May 2010. Figure 6 illustrates the dis- agreement between the simulated and observed SSM spa- tial patterns on 28 April 2009 and 28 April 2010. For these dates, the CAROLS retrievals present a more pronounced spatial variability than the simulations. This discrepancy can be caused by uncertainties in both retrievals and simulations. In spite of the lack of temporal correlation between the CAROLS VOD and the ISBA-A-gs LAI, the spatial correla- tion between the two quantities is significant for 19 flights, with r ranging from 0.34 to 0.85 (not shown). The slope of this relationship changes with time (Fig. 8), decreasing from April to July (Table 3). The comparison of the VOD spatial distribution with the 10-day satellite-derived LAI confirms this tendency (Fig. 8). However, in this case the spatial cor- relation is significant at the end of the 2010 campaign only. For the same flights, a significant correlation is found be- tween the VWC derived from the ISBA-A-gs simulations (Eq. 3) and the VOD derived from the CAROLS observa- tions. Inverting Eq. (4) for these flights, mean values of bhigh = 0.16 ± 0.03 m2 kg−1 and blow = 0.09 ± 0.07 m2 kg−1 are obtained. The spatial correlation between the CAR- OLS VOD and the aggregated model-derived VOD is sig- nificant for 17 flights, with correlation coefficients vary- ing from 0.31 to 0.61 (Fig. 8 and Table 2). The mean SDD value is 0.059. Highly significant correlations (p- value < 0.001) are found for 13 flights. For these flights, the mean SDD value is 0.057. E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France Seven CAROLS VOD tran- sects do not correlate very well with the simulated VOD and present higher p-values: 20 May 2009, 3 May 2010, 26 May 2010, 8 June 2010, 13 June 2010, 18 June 2010, 26 June 2010. Among them, only two are associated with rather poor SSM spatial correlations: 20 May 2009 and 3 May 2010. For these two flights, the CAROLS observations www.hydrol-earth-syst-sci.net/16/1725/2012/ Flight transect of the surface soil moisture from (A, B) CAROLS retrievals and from (C, D) ISBA-A-gs simulations, at (A, C) the end of April 2010, and (B, D) the end of May 2010. Fig. 6. Flight transect of the surface soil moisture from (A, B) CAROLS retrievals and from (C, D) ISBA-A-gs simulations, at (A, C) the end of April 2010, and (B, D) the end of May 2010. www.hydrol-earth-syst-sci.net/16/1725/2012/ www.hydrol-earth-syst-sci.net/16/1725/2012/ Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 1735 Fig. 7. Flight transect of (A, B) the vegetation optical thickness (VOD) retrieved from CAROLS Tb and of (C, D) the ISBA-A-gs LAI simulations, at (A, C) the end of April 2010, and (B, D) the end of May 2010. E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 1736 1736 E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France Fig. 8. Spatial correlation between the CAROLS VOD and (A) the ISBA-A-gs LAI, (B) the satellite-derived LAI, and (C) the VOD estimated from the ISBA-A-gs LAI and the forest fraction from ECOCLIMAP-II using the same blow parameter 0.09 for three dates in 2010 (from left to right): 15 April, 4 June, 1 July. The solid lines on plots (A) and (B) represent linear regression lines with forced zero intercept. Fig. 8. Spatial correlation between the CAROLS VOD and (A) the ISBA-A-gs LAI, (B) the satellite-derived LAI, and (C) the VOD estimated from the ISBA-A-gs LAI and the forest fraction from ECOCLIMAP-II using the same blow parameter 0.09 for three dates in 2010 (from left to right): 15 April, 4 June, 1 July. The solid lines on plots (A) and (B) represent linear regression lines with forced zero intercept. Table 3. Spatial correlation coefficient (R), p-value, RMSD, and the slopes of the regression lines (Fig. 8) between ISBA-A-gs/satellite derived LAI and the CAROLS VOD. the retrieval accuracy, especially for VOD. On 26 June 2010, only 22 CAROLS VOD retrievals were successful, mainly at the westernmost and easternmost parts of the transect. Fig- ure 9 shows that the spatial distribution of the CAROLS VOD retrievals aggregated at the grid-cell level is consistent with the simulated VOD for high vegetation. From 28 April 2010 to 4 June 2010, the simulated VOD for low vegetation in- creases significantly and this increase does not appear in the CAROLS VOD retrievals. Date R p-value RMSD slope ISBA-A-gs LAI 15 Apr 2010 0.48 ∗∗∗∗ 1.48 0.096 4 Jun 2010 0.48 ∗∗∗∗ 3.44 0.039 1 Jul 2010 0.44 ∗∗∗ 4.03 0.037 Satellite-derived LAI 15 Apr 2010 – NS 1.46 0.091 4 Jun 2010 0.02 NS 2.23 0.056 1 Jul 2010 0.70 ∗∗∗∗ 2.32 0.063 ∗∗, ∗∗∗, ∗∗∗∗stand for p-value between 0.01 and 0.001, between 0.001 and 0.0001 and below 0.0001, respectively. The linear regression, with forced zero intercept (Fig. 8), fails for the flight of 15 April 2010. 3.2.2 LAI and VOD The VOD values retrieved from CAROLS Tb range from 0.05 to 0.35. The highest VOD values are found in forested areas, presenting an average VOD value of 0.2. The arable lands are characterised by relatively low VOD val- ues varying between 0.05 and 0.15. Figure 7 shows that the ISBA-A-gs simulated LAI increases sharply in 2009 and in 2010, from the end of April to the end of May, from 0.8 m2 m−2 in Les Landes forest to 1.2 m2 m−2 in the other parts of the transect. In 2010, the airborne cam- paign lasted 2.5 months and the simulated LAI increases, on average, from about 2 m2 m−2 in Les Landes forest, to 2.6 m2 m−2 in the central agricultural part of the transect, and to 1.8 m2 m−2 in the eastern Mediterranean part. However, the CAROLS VOD retrievals do not show marked changes during these periods. For most grid cells, the temporal Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 www.hydrol-earth-syst-sci.net/16/1725/2012/ E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France va et al.: Spatial and temporal variability of biophysical variables in southwestern France E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 1737 E. Zakharova et al.: Spatial and temporal variability of b Fig. 9. Along-track variability of the CAROLS VOD and the VOD estimated using the ISBA-A-gs LAI of low vegetation, and the for- est fraction from ECOCLIMAP-II and b parameter = 0.09 for low vegetation and 0.16 for high vegetation, for (A) 28 April 2010 and (B) 4 June 2010. close to the Lahas SMOSMANIA station, for nine CAROLS flights (3 in 2009 and 6 in 2010). These data were compared with the CAROLS Tb by Albergel et al. (2011). They found that the average SSM at each site correlated well with CAR- OLS retrievals based on semi-empirical statistical relation- ships, except for the Berat agricultural site. Indeed, this site was less representative of the area where distributed in situ measurements were taken, as it consisted of large flat maize fields with mainly bare soil in April and May, and rapidly growing maize in June. More research is needed to analyse the CAROLS observations over these sites. In particular, the SSM observations made over hilly terrain at the Le Mona site could be used to investigate the impact of topography on the spatial variation of SSM (Yang et al., 2012). p ( g ) Along the Atlantic-Mediterranean CAROLS transect, the SSM difference between CAROLS and the ISBA-A-gs model is consistent with the CAROLS SSM departure from the in situ observations. This indicates that the CAROLS SSM retrievals behave differently from the other two SSM estimates. Although the mean bias (of 20 flights) between the CAROLS and ISBA-A-gs SSM is low and equal to −0.02 m3 m−3, it presents a high spatial variability with a standard deviation of 0.07 m3 m−3. The same spatial dis- tribution of the bias is observed for all the flights (not shown). The best agreement between airborne measure- ments and model simulations is found in the Mediterranean part of the transect with an average bias between these datasets less than 0.01 m3 m−3. In the Armagnac region at the western part of the transect, between 0.5◦W and 1◦E, the CAROLS SSM retrievals are constantly lower, of about −0.06 m3 m−3, on average. E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France On the other hand, a positive bias of about 0.05 m3 m−3 is observed over the Garonne River valley at the centre of the transect, between 1◦E and 1.5◦E. This area is characterised by the highest discrepancy between the CAROLS and ISBA-A-gs SSM estimates, and the maxi- mal difference can reach 0.38 m3 m−3. It must be noted that, although physical and statistical filters were applied to the CAROLS dataset in order to mitigate the perturbing factors (see Sects. 2.1.4 and 2.3), the influence on SSM of open wa- ters like river channels or small ponds has not been fully eliminated (Sect. 3.3). Using high-resolution Landsat Geo- Cover Mosaic images, it was checked that areas with indi- vidual SSM reaching 0.5 m3 m−3 in Fig. 2 correspond to re- gions presenting a high density of small water bodies. Also, the lower CAROLS SSM values in the western part of the transect may be the signature of possible unfiltered low-level RFI. These perturbing factors can also be an issue for SMOS. The physical filter used in this study cannot be applied to SMOS since valid SMOS observations close to nadir are scarce. The statistical approach could be used for SMOS Tb filtering, provided specific threshold curves are developed. Indeed, the threshold curve of Fig. 3 is valid for an incidence angle of 33.5◦and cannot be used at other incidence angles. Also, the threshold curve is valid for southwestern France Fig. 9. Along-track variability of the CAROLS VOD and the VOD estimated using the ISBA-A-gs LAI of low vegetation, and the for- est fraction from ECOCLIMAP-II and b parameter = 0.09 for low vegetation and 0.16 for high vegetation, for (A) 28 April 2010 and (B) 4 June 2010. close to the Garonne River valley. In the eastern part of the transect, they are found in the hilly areas with a high fraction of bare soil and deciduous forests. 4.1 Causes of mismatch between SSM estimates The CAROLS SSM retrievals may present significant biases with respect to the in situ SMOSMANIA observations (Ta- ble 1 and Fig. 4), and to the ISBA-A-gs simulations (Fig. 6). Part of these differences may be caused by the spatial het- erogeneity of SSM, which is not fully represented in any of the SSM estimates considered in this study (Sect. 3.3). Also, as shown by Fig. 10, large-scale geographic patterns, such as soil texture and vegetation types, may trigger systematic differences in the representation of soil moisture. The CAROLS transect is very long (385 km), and sam- pling soil moisture using ground observations over the whole transect was not feasible. This is why sparse automatic ground observations were associated with simulated SSM values in this study. However, during the campaign, an ef- fort was made to perform additional manual SSM observa- tions at three contrasting sites (Le Mona, Lahage and Berat) Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 www.hydrol-earth-syst-sci.net/16/1725/2012/ 3.3 Small-scale spatial heterogeneity of SSM and VOD In order to assess the local heterogeneity of SSM and VOD, we calculated and analysed the standard deviation (std) of the CAROLS retrievals within each ISBA-A-gs grid cell, using the oversampling capability of the in- strument (see Sect. 2.4). For 83 % of the model’s grid cells std(SSM) and std(VOD) present relatively low values: std(SSM) ≤0.05 m3 m−3 and std(VOD) ≤0.05. High values of std(SSM) and std(VOD) are found for 5 % of the model’s grid cells: std(SSM) > 0.08 m3 m−3 and std(VOD) > 0.08. The grid cells presenting the highest sub-grid variability are found on the western part of the transect, in Les Landes forest area. In the central part of the transect, they are found close to the Auch city (between Peyrusse and Lahas in Fig. 1), and were markedly affected by RFI along the whole transect, and it is likely that the filtering technique described in Sect. 2.1.4 was not able to screen out all the perturbations. On 8 June 2010, the RFI affected two zones in the middle of the transect. They were not that strong and did not stop the retrieval of either SSM or VOD values, but they affected Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 www.hydrol-earth-syst-sci.net/16/1725/2012/ E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 1739 E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France While the flight to flight variability of bhigh is small, the blow values present a significant seasonal trend (Fig. 11) and range from values close to 0.15 m2 kg−1 in April, to values close to 0.05 m2 kg−1 in summertime. The blow trend is signifi- cant and equal to −0.0012 m2 kg−1 day−1. The decrease of blow during the vegetation period compensates for the rise in VWC, and this may explain the absence of significant tempo- ral variability in the CAROLS VOD retrievals (Figs. 7 and 9). It must be noted that the ISBA-A-gs model simulates the green LAI, and that Eq. (3a) was derived by Pellarin et al. (2003b) from field data over a variety of crops, with a VWC to LAI ratio of 0.5 kg m−2. For the SMOSREX grass- land, de Rosnay et al. (2006) and Saleh et al. (2006b) found very good correlations between the green vegetation VWC and the green LAI (r2 close to 0.9), with VWC to LAI ra- tio values ranging between 0.3 and 0.4 LAI kg m−2. An at- tempt was made (not shown) to use such values for grass- lands in deriving VOD estimates from ISBA-A-gs simula- tions. This tended to degrade the scores presented in Table 2 for the VOD spatial correlation. Therefore, the same value of 0.5 kg m−2 (Eq. 3a) was used for both grasslands and crops. The presence of dead vegetation residues may affect VWC and tends to reduce the correlation of VWC with LAI (de Rosnay et al., 2006). In this study, we focused on the plant growing period, and it was assumed that most low vegetation covers consisted of green vegetation. Fig. 11. Temporal variation of the b parameter (in units of m2 kg−1) for high and low vegetation. Time is in day of year (DOY) 2010. For bhigh the correlation is insignificant with r = −0.37; for blow the correlation is significant (p-value = 0.0003 and r = −0.85); the trend in blow is significant at the 0.1 % level and equal to −0.0012 m2 kg−1 day−1. The same kind of problem may exist in the ISBA-A-gs simulations. However, the main factors affecting SSM are accounted for by the model. www.hydrol-earth-syst-sci.net/16/1725/2012/ www.hydrol-earth-syst-sci.net/16/1725/2012/ rova et al.: Spatial and temporal variability of biophysical variables in southwestern France 1738 738 E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France g. 10. Spatial variability (A) of the CAROLS minus ISBA-A-gs (crosses) and CAROLS minus SMOSMANIA (dots) bias averaged for 20 ghts; (B)sand (black) and clay (red) fraction used for ISBA-A-gs simulations (line) and CAROLS retrievals (dots); (C) fraction of surface pes (bare soil, broadleaf deciduous forest, coniferous evergreen forest, C3 crops, rainfed maize, irrigated maize, grasslands, wetlands) from COCLIMAP-II. E. Zakharova et al.: Spatial and temporal variability of biophysical variables in s Fig. 10. Spatial variability (A) of the CAROLS minus ISBA-A-gs (crosses) and CAROLS minus SMOSMANIA (dots) bias averaged for 20 flights; (B)sand (black) and clay (red) fraction used for ISBA-A-gs simulations (line) and CAROLS retrievals (dots); (C) fraction of surface types (bare soil, broadleaf deciduous forest, coniferous evergreen forest, C3 crops, rainfed maize, irrigated maize, grasslands, wetlands) from ECOCLIMAP-II. and it is likely that performing the same exercise in other re- gions of the globe would give different results. litter. Finally, soil roughness may vary from one vegetation type to another, and also within a given vegetation type. Fig- ure 10 suggests that high values of either the sand propor- tion of the soil or the fraction of forest tend to match the areas where the CAROLS SSM values are lower than the simulated ones. This may denote the limit of using the single Eq. (1) to represent soil roughness over all the CAROLS tran- sect. Pard´e et al. (2011b) have shown that the accuracy of the soil moisture retrieval from CAROLS Tb observations can be improved, up to 0.053 m3 m−3, through local soil roughness calibration. To some extent, the large-scale spatial patterns in the CAROLS/ISBA-A-gs SSM bias (Fig. 10) can be explained by uncertainties related to the L-MEB inversion process. In- deed, key processes affecting the microwave emission are not represented, such as the topography and its impact on the an- gular signature of Tb and on the spatial distribution of soil moisture. Also, a number of studies have shown that it is difficult to represent litters in L-MEB (Saleh et al., 2006a, 2007). The latter factor may affect all the vegetation types, but grasslands and forests tend to favour the formation of a www.hydrol-earth-syst-sci.net/16/1725/2012/ www.hydrol-earth-syst-sci.net/16/1725/2012/ Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 E. 4.2 L-band b values through time and vegetation covers The b parameter for low vegetation obtained in this study (blow = 0.09 ± 0.07 m2 kg−1) is close to the value 0.12 ± 0.03 m2 kg−1 that was found to be representative of most agricultural crops by Wigneron et al. (2007). On the other hand, the estimated b parameter for high vegeta- tion (bhigh = 0.16 ± 0.03 m2 kg−1) is lower than the value of 0.33 m2 kg−1 reported in Pellarin et al. (2003a), or than the range of values (0.26 to 0.34 m2 kg−1) used to retrieve VOD of high vegetation from SMOS measurements (Kerr et al., 2001). Moreover, Fig. 8 and Table 3 show that the CAROLS VOD retrievals correlate with both LAI observations and ag- gregated LAI simulations, and that the VOD sensitivity to LAI (either observed or modelled) tends to decrease along the growing season. E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France The maximum SSM and the rate at which SSM varies after a rain, in relation to the root- zone soil moisture value, depend on pedotransfer functions driven by soil characteristics such as soil texture (Noilhan and Lacarr`ere, 1995). The model uses a map of soil proper- ties and represents the interception of rain by the vegetation. Overall, this study tends to confirm that the L-band VOD relationship with vegetation characteristics is less straight- forward than at C-band or X-band. In addition to the reduced sensitivity to VWC, the L-band b value is found to present a seasonal variability for low vegetation canopies. This finding is consistent with the microwave observations over a wheat field analysed by Wigneron et al. (1996). The L-band b val- ues given by Wigneron et al. (1996) for a wheat field range from 0.125 m2 kg−1 for green vegetation at the start of the growing season, to 0.040 m2 kg−1 at the end of June. Ex- pressing b as b = Aε′′ S f (see Sect. 1), this could be explained by changes in the value of the A coefficient, related to the canopy structure, and to leaf microwave properties, from the growing phase to the senescence. 4.3 Impact of surface conditions on the retrievals Overall, SSM SDD values and to a lesser extent VOD SDD values tend to increase in wet conditions. Figure 12 shows that the SSM SDD is significantly correlated with the CAR- OLS SSM (r2 = 0.44, p-value < 0.01). For VOD, no signif- icant trend is observed (r2 = 0.10). This is consistent with the findings of Pellarin et al. (2003a) regarding the L-MEB retrievals. They showed that (1) for VOD values higher than 0.1, the value of SSM impacts the success of the SSM retrievals, and (2) the retrieval performance is lower in wet conditions. Indeed, the weaker L-band emission of a wet soil is more easily attenuated by the vegetation. This study confirms that surface conditions have to be accounted for in The model simulations permit the analysis of the sub-grid variability of the b parameter and to distinguish the temporal evolution of blow and bhigh (Fig. 11). Indeed, forest VOD val- ues are generally not correlated with LAI (e.g. Grant et al., 2008). In particular, the simulated forest VOD does not de- pend on LAI (Eqs. 3b, and 4b) and does not present seasonal variations. The use of the ECOCLIMAP-II land cover infor- mation permits the simulation of separate contributions of low vegetation and forests to the simulated VOD (Figs. 8–9). www.hydrol-earth-syst-sci.net/16/1725/2012/ www.hydrol-earth-syst-sci.net/16/1725/2012/ Zakharova et al.: Spatial and temporal variability of b Fig. 11. Temporal variation of the b parameter (in units of m2 kg−1) for high and low vegetation. Time is in day of year (DOY) 2010. For bhigh the correlation is insignificant with r = −0.37; for blow the correlation is significant (p-value = 0.0003 and r = −0.85); the trend in blow is significant at the 0.1 % level and equal to −0.0012 m2 kg−1 day−1. arova et al.: Spatial and temporal variability of biophysical variables in southwestern France The publication of this article is financed by CNRS-INSU. The publication of this article is financed by CNRS-INSU. www.hydrol-earth-syst-sci.net/16/1725/2012/ Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 5 Conclusions In this paper we have presented an analysis of the data ac- quired during the CAROLS flights that took place in south- western France between the Atlantic and the Mediterranean coasts, in April–May 2009 and in April–July 2010. These data were acquired to validate the retrieval algorithm used for the L2 SMOS SSM processing, and to investigate the ac- curacy of L-band soil moisture sensing over a large area pre- senting variable soil and vegetation conditions. Edited by: L. Wang E. Zakharova et al.: Spatial and temporal variability of biophysical variables in southwestern France 1740 Fig. 12. Impact of SSM conditions (as observed by CAROLS) on the spatial standard deviation of differences (SDD) between the CAROLS SSM and VOD and the simulated values (Table 2, only for p-value < 0.05). L-MEB optimisation technique depends on hypotheses, par- ticularly on soil roughness, and this may affect the quality of the SSM retrievals. Also, it is shown that deriving VWC estimates from VOD retrievals is difficult, because forest and herbaceous vegetation present contrasting VWC responses to VOD. Finally, it was found that the b parameter for low veg- etation may present a seasonal variation from springtime to summertime and this effect should be investigated further as it is currently not accounted for in L-band microwave emis- sion models. Fig. 12. Impact of SSM conditions (as observed by CAROLS) on the spatial standard deviation of differences (SDD) between the CAROLS SSM and VOD and the simulated values (Table 2, only for p-value < 0.05). Acknowledgements. This work was supported by the STAE (Sci- ences et Technologies pour l’A´eronautique et l’Espace) foundation, in the framework of the CYMENT project, as well as by Centre National d’Etudes Spatiales (CNES) and M´et´eo-France. The CAROLS project was funded by the “Programme Terre Oc´ean Surface Continentales et Atmosph`ere” (TOSCA, CNES). The ATR-42 aircraft was operated by the SAFIRE UMS 2859. S. Lafont was supported by the GEOLAND2 project, co-funded by European Commission within the GMES initiative in FP7. The authors would like to thank Richard de Jeu (VUA) and one anonymous referee for their fruitful comments. assessing the uncertainty of the SMOS SSM and VOD re- trievals (Wigneron et al., 2000). The publication of this article is financed by CNRS-INSU. The microwave L-band Tb were affected by RFI. A post- processing filtering, based on the TbH vs. TbV analysis at nadir and slant-looking angles, improved the SSM and VOD retrievals. After applying this filtering, a significant (at the 1 % level) temporal correlation between the airborne SSM and in situ SSM observations was achieved at 6 locations (out of 11), with r values ranging from 0.62 to 0.82, and SDD values ranging from 0.039 to 0.105 m3 m−3. Moreover, the spatial variability of the airborne SSM and VOD retrievals was consistent with the ISBA-A-gs simulations. 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Along the Atlantic-Mediterranean transect, 83 % of ISBA grid cells presented a spatial variability of CAROLS SSM retrievals lower than 0.05 m3 m−3, a figure comparable with the accu- racy acceptable for remotely sensed SSM estimates (Walker and Houser, 2004). It was found that small water bodies (100–300 m in length) had a significant impact on the CAR- OLS measurements. Albergel, C., Calvet, J.-C., de Rosnay, P., Balsamo, G., Wagner, W., Hasenauer, S., Naeimi, V., Martin, E., Bazile, E., Bouys- sel, F., and Mahfouf, J.-F.: Cross-evaluation of modelled and remotely sensed surface soil moisture with in situ data in southwestern France, Hydrol. Earth Syst. Sci., 14, 2177–2191, doi:10.5194/hess-14-2177-2010, 2010. 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Wigneron, J.-P., Calvet, J.-C., and Kerr, Y.: Monitoring water inter- ception by crop fields from passive microwave observations, Agr. Forest Meteorol., 80, 177–194, 1996. Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 Hydrol. Earth Syst. Sci., 16, 1725–1743, 2012 www.hydrol-earth-syst-sci.net/16/1725/2012/ www.hydrol-earth-syst-sci.net/16/1725/2012/
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Application of machine learning and genetic optimization algorithms for modeling and optimizing soybean yield using its component traits
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PLOS ONE PLOS ONE RESEARCH ARTICLE Editor: Qiang Zeng, South China University of Technology, CHINA Copyright: © 2021 Yoosefzadeh-Najafabadi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The data are uploaded in an open access file in Github, and can be accessed through the following link: https:// github.com/Mohsen1080/Available-Datasets. Funding: This project was funded in part by Grain Farmers of Ontario (GFO) and SeCan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Application of machine learning and genetic optimization algorithms for modeling and optimizing soybean yield using its component traits Mohsen Yoosefzadeh-NajafabadiID1, Dan TulpanID2, Milad EskandariID1* Mohsen Yoosefzadeh-NajafabadiID1, Dan TulpanID2, Milad EskandariID1* 1 Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada, 2 Department of Animal Biosciences, University of Guelph, Guelph, Ontario, Canada 1 Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada, 2 Department of Animal Biosciences, University of Guelph, Guelph, Ontario, Canada a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * meskanda@uoguelph.ca Abstract Improving genetic yield potential in major food grade crops such as soybean (Glycine max L.) is the most sustainable way to address the growing global food demand and its security concerns. Yield is a complex trait and reliant on various related variables called yield compo- nents. In this study, the five most important yield component traits in soybean were mea- sured using a panel of 250 genotypes grown in four environments. These traits were the number of nodes per plant (NP), number of non-reproductive nodes per plant (NRNP), num- ber of reproductive nodes per plant (RNP), number of pods per plant (PP), and the ratio of number of pods to number of nodes per plant (P/N). These data were used for predicting the total soybean seed yield using the Multilayer Perceptron (MLP), Radial Basis Function (RBF), and Random Forest (RF), machine learning (ML) algorithms, individually and collec- tively through an ensemble method based on bagging strategy (E-B). The RBF algorithm with highest Coefficient of Determination (R2) value of 0.81 and the lowest Mean Absolute Errors (MAE) and Root Mean Square Error (RMSE) values of 148.61 kg.ha-1, and 185.31 kg.ha-1, respectively, was the most accurate algorithm and, therefore, selected as the meta- Classifier for the E-B algorithm. Using the E-B algorithm, we were able to increase the pre- diction accuracy by improving the values of R2, MAE, and RMSE by 0.1, 0.24 kg.ha-1, and 0.96 kg.ha-1, respectively. Furthermore, for the first time in this study, we allied the E-B with the genetic algorithm (GA) to model the optimum values of yield components in an ideotype genotype in which the yield is maximized. The results revealed a better understanding of the relationships between soybean yield and its components, which can be used for selecting parental lines and designing promising crosses for developing cultivars with improved genetic yield potential. OPEN ACCESS Citation: Yoosefzadeh-Najafabadi M, Tulpan D, Eskandari M (2021) Application of machine learning and genetic optimization algorithms for modeling and optimizing soybean yield using its component traits. PLoS ONE 16(4): e0250665. https://doi.org/10.1371/journal.pone.0250665 Editor: Qiang Zeng, South China University of Technology, CHINA Introduction Soybean (Glycine max L. Merrill) is the world’s most widely grown leguminous crop and an important oil and protein source for food and feed [1]. Because of its nutritional and 1 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 PLOS ONE Using machine learning algorithms for estimating soybean yield from yield component traits Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. pharmaceutical properties, soybean is also considered as an important source of healthy plant- based food products in the human diet. While the global demand for soybean is increasing sig- nificantly [2], the current average annual genetic gain for yield seem not to be able to cope with the growing demand [3]. One of the probable main reasons for this low genetic gain is the inefficient selection criteria that are currently used in breeding programs for selecting geno- types with desirable genetic yield potentials [4]. Despite the major advances in molecular technologies and their potential implications in breeding programs, generating reliable phenotypic data and analyzing big datasets have been remained the major bottlenecks [5]. Plant breeding programs, including soybeans, are contin- uously relying on the evaluation of yield and important agronomic traits for making selections and defining commercial products [6]. If a trait is under controlled by a few major genes, and so with limited environmental effects, designing molecular marker tools would be sufficient for selecting desirable genotypes in a given breeding population [7]. However, for complex traits such as yield, which are highly influenced by the environment and controlled by numer- ous genes with minor effects, the dissection of traits underlying the yield can be beneficial for selecting genotypes with improved genetic potentials [8]. In general, soybean breeders have made extensive use of the classical phenotypic selection approaches to evaluate and exploit total seed yield as the main selection criterion in their cultivar development programs [8, 9]. However, genetic gains for yield have generally been low and inconsistent across different environments [8]. The general low genetic gains for yield can be to a large extent because of the nature of this trait, in which several secondary traits drive the final production directly or indirectly [3]. Thus, one possible way to increase the yield genetic gains in new cultivars is to improve their yield component traits [5, 7, 8]. Competing interests: The authors have declared that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 Introduction Yield formation in plant species is mostly governed by yield component traits [10–12]. Traits such as the number of nodes per plant (NP), number of non-reproductive nodes per plant (NRNP), number of reproductive nodes per plant (RNP), and number of pods per plant (PP) are considered as the major yield components in soybean [13]. Therefore, the increase in yield production can be regulated by selecting the soybeans with greater performance in their yield components [13, 14]. Several studies have been conducted to describe yield improvement in plants via improving yield components [15–18]. For example, positive correlations between soybean yield and the number of pods [19] were reported to be the most significant contribu- tor to yield gain in Northeast China [20]. By considering yield components, soybean breeders can model and predict optimum conditions in which the highest yield production can achieve [21]. However, developing reliable prediction models built upon several yield component traits requires dealing with large datasets that are generated from the evaluation of large breeding populations across multi-environments. Due to the essential need for advanced skills in computational and mathematical analyses, exploiting large datasets in many public breeding programs is still a bottleneck. Machine Learning (ML) algorithms, as one of the reliable and efficient computational approaches, were successfully implemented in different fields of study, such as traffic crash frequency modeling [22, 23], environmental science [24], engineering [25], and medicine [26]. Previously, Zeng, Huang (22) developed neural network, as one of the ML algorithms for exploring the non-lin- ear relationship between risk factors and crash frequency. They reported the successfulness of using neural network algorithms to eliminate the overfitting and deal with lack-box character- istic [22]. Also, several studies reported the efficiency of ML algorithms in better detection of genomic regions associated with a trait of interest [26–28]. One of the recent agriculture trends is the use of ML algorithms for analyzing big data [29, 30]. Emerging ML algorithms in agriculture have created new opportunities to quantify and understand the intensive data process in agriculture [29, 31]. In a simple form, ML algorithms PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 2 / 18 PLOS ONE Using machine learning algorithms for estimating soybean yield from yield component traits can be defined as machines with the ability to learn without explicitly programmed [29, 32]. Introduction Theoretically, each algorithm is involved in a specific learning process from training data to per- form a task of clustering, predicting, and classifying new datasets using the knowledge attained during the learning process [31]. Various ML algorithms have been developed and can be implemented for complex interactions between features [8, 29, 32, 33]. Multilayer Perceptron (MLP), for example, is known as the common neural network algorithm that is widely used in different areas such as plant sciences [30], remote sensing [34], environmental sciences [35], and engineering [36]. Like other neural network algorithms, MLP is built upon many neurons in which each neuron has its own specific weight [37]. In any case that one neuron is insuffi- cient to explain the algorithm, MLP will be useful by providing multi-neurons [38, 39]. Radial Basis Function (RBF) is another ML algorithm commonly used in plant sciences [40–42]. RBF is reported to be successful for predictions wherever relevant features are used [40]. However, its performance for predicting soybean yield from its components is still unknown. Random Forest (RF) is another algorithm that its performance was evaluated in this study. RF has drawn many researchers’ attention because of adequate performances in various fields, including plant science [30, 43], animal science [44, 45], human science [46], and remote sensing [47]. One of the major impediments of using individual ML algorithms is the high probability of overfitting in single predictive algorithms [48]. To overcome this obstacle, the ensemble tech- niques can be employed [49]. Ensemble techniques are known as the most influential develop- ment in the application of ML algorithms [50], in which combined algorithms are exploited to improve prediction accuracies by reducing overfitting rates [48–50]. Three commonly used ensemble algorithms are stacking, boosting, and bagging methods that are used according to the nature of the dataset and the individual ML algorithms that are used [50]. The success of using ensemble techniques was reported in different areas such as plant science [30], engineer- ing [51], and computer sciences [52]. In soybean cultivar development programs, an optimum selection among important yield components can significantly improve the yield genetic gain. Therefore, the implementation of optimization methods in this field is of particular interest. Optimization algorithms for improving important traits are becoming more and more attractive in plant science [40, 53]. Introduction Genetic Algorithm (GA) is known as one of the most well-known single objective optimization methods designed and developed by Holland [54], as a searching algorithm based on natural selection. GA searching algorithm is based on Darwin’s notion that more stable organisms across different environments survive better than the others [55]. Although the successful uses of ML ensemble methods have been reported in different agri- culture-related fields [30, 48, 53, 56, 57], the potential use of these algorithms to predict soy- bean yield using yield components remains unknown. Therefore, this study aimed to investigate the potential use of soybean yield components for predicting the final seed yield using individual ML algorithms as well as ensemble learning methods. In addition, linking the best machine learning algorithm with GA for estimating the optimized values of the yield com- ponents for maximizing the soybean yield was investigated. The outcomes of this study can pave the way to understand the importance of soybean yield components in determining the total seed yield and implement the proposed pipeline for making the genotypic selection more accurate. Phenotypic evaluations Seed yield (ton ha-1) of each plot was measured by harvesting three middle rows and adjusting to a 13% moisture level. Soybean yield components, including NP, NRNP, RNP, and PP, were hand-measured by randomly selected ten plants from each phenotypic plot for each genotype. Also, the PP to NP ratio (P/N) for each genotype was calculated using the following equation: P=N ¼ PP NP ð1Þ ð1Þ where PP indicates the number of pods per plant, and NP indicates the total number of nodes per plant. Data pre-processing, correlation coefficient, and statistical analyses All the phenotypic plot-based data were adjusted for spatial variations within the fields using nearest neighbor analysis (NNA) as one of the spatial error control methods to reduce and minimize the possible error in the field data [58, 59]. The yield components were collected for 250 soybean genotypes from 2000 plots across four environments. The average value of each yield component for each genotype was estimated through the best linear unbiased prediction (BLUP) as a mixed model [60]. For BLUP analysis, the environment factor was selected as a fixed effect, and the genotype factor was considered as a random effect. Afterward, all 250 data-points were used for constructing training and testing datasets. In this study, all the yield component traits such as NP, NRNP, RNP, PP, and P/N were considered as input variables for predicting the soybean yield as the output variable. In order to improve the prediction accu- racy of input variables, data scaling and centering were applied for the pre-processing and pre- treatment steps [61]. Before performing ML algorithms, the principal component (PC) analy- sis was applied to identify outliers; however, no outlier was detected. The Pearson coefficient of correlations between seed yield and yield components were estimated using the R software version 3.6.1. Plant material and experimental design Two hundred and fifty soybean genotypes were grown under field conditions at two locations: Palmyra (42˚25’50.1"N 81˚45’06.9"W, 195 m above sea level) and Ridgetown (42˚27’14.8"N 3 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 PLOS ONE Using machine learning algorithms for estimating soybean yield from yield component traits 81˚52’48.0"W, 200m above sea level), in Ontario, Canada in 2018–2019. The population used in this study was selected from the core germplasms of soybean breeding programs at the Uni- versity of Guelph, Ridgetown campus that have been created over 35 years and used for culti- var development and genetic studies. The experiment was conducted using randomized complete block designs (RCBD) with two replications in four environments (two locations × two years), consisting of 2000 phenotypic plots in total. Each plot consisted of five rows, each 4.2 m long and 40 cm spacing between each row. The seeding rates used in this study were 50–57 seeds per m2. Optimization process via GA For obtimizing the values of NP, PP, RNP, NRNP, and P/N in a theoretical maximized yield ideotype genotype, the best ML algorithm was linked to the genetic algorithm (GA). In order to have the best implement of GA, some parameters such as mutation rate, crossover fraction, and the number of chromosomes should be determined. For that, optimum values of the men- tioned parameters are estimated by the trial and error methods. In brief, a chromosome is known as a set of variables that define as a possible solution to the problem. In this study, all the possible combinations of the yield component traits were considered as different chromo- somes to form the maximum soybean yield production. All proposed chromosomes are con- structed from the initial population, which is the first step in the GA optimization process [53]. Crossover in GA is the process of creating a new generation of chromosomes by mixing the existing chromosomes [69]. In this study, the possible crossover between two chromo- somes, each containing a combination pattern of the yield components, was evaluated by using a two-point crossover, which is known as one of the common crossover fractions. Muta- tion is another parameter in GA, which is used to control the local minima in the population [69]. By using a mutation rate, the possibility of having similar chromosomes will be decreased, and therefore, the possible local minima are decreased [53]. In this study, the mutation and crossover rates as well as the number of chromosomes were set to 0.1, 0.7, and 100, respectively (Fig 1). Also, the Roulette wheel was used for selecting elite populations for crossover to obtain the appropriate fitness. In order to achieve the generation number, the generational practice was iteratively implemented. The lower and upper bounds of the dataset were considered as the constraints in the optimization process (Fig 1). Data-driven modeling MLP, RBF, and RF are the most commonly used ML algorithms with distinct functions and abilities that are used for predicting yield in plant crop species [37, 48, 62]. The MLP, as one of the most common feed-forward neural networks, consists of input, hidden, and output layers of interconnected neurons [63]. RBF is another type of neural network that used approximate multivariate functions [64]. RBF has the same functionality as that of MLP but in an effective way for using in more than one dimension [65]. RF is also another commonly used ML algo- rithm that generates combined trees representing n number of independent observations [66]. The final prediction in RF is determined based on the average predictions of all possible inde- pendent trees [48]. In order to improve the prediction performance of individual ML 4 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 PLOS ONE Using machine learning algorithms for estimating soybean yield from yield component traits algorithm, an ensemble method was proposed based on the bagging strategy (E-B). The steps for the E-B analyses are as follows: (1) applying and training MLP, RBF, and RF, indepen- dently; (2) selecting the best ML algorithm, based on the validation set, as the metaClassifier for the E-B; and (3) combining the prediction results in order to improve the prediction accu- racy of the soybean yield [67]. All the used parameters in each algorithm were optimized based on the training dataset. The Weka software version 3.9.4 [68] was used to run all the tested ML algorithms and the E-B method for analyzing training and validation sets. PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 Quantification of model performance and error estimations The original dataset consists of 250 observations was randomly divided into training and vali- dation sets based on the five k-fold cross-validation method [70] with ten repetitions (Fig 2). To quantify the performance of the ML algorithms for predicting soybean seed yield from the yield components, the following statistical measurements between independent reference val- ues (Y) and estimated values (Y0) were applied: The Root Mean Square Error (RMSE, Eq 2) and the Mean Absolute Errors (MAE, Eq 3) of the validation set.fififififififififififififififififififififififififi RMSE ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi P ðY0 YÞ 2 n s ð2Þ MAE ¼ Pn i¼1 jY0 i YIj n ð3Þ ð2Þ ð3Þ where Y0 stands for predicted value, Y is the field measured value, and n is the number of observations for a given genotype. where Y0 stands for predicted value, Y is the field measured value, and n is the number of observations for a given genotype. 5 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 PLOS ONE Using machine learning algorithms for estimating soybean yield from yield component traits Fig 1. The schematic diagram of the genetic algorithm as the single objective evolutionary optimization algorithm. https://doi org/10 1371/journal pone 0250665 g001 Fig 1. The schematic diagram of the genetic algorithm as the single objective evolutionary optimization algorithm. https://doi.org/10.1371/journal.pone.0250665.g001 Fig 1. The schematic diagram of the genetic algorithm as the single objective evolutionary optimization algorithm. Fig 1. The schematic diagram of the genetic algorithm as the single objective evolutionary optimization algorithm. The analyses of the goodness of fit between observed and the predicted values were per- formed using the coefficient of determination (R2, Eq 4). While we provide their definitions below, more comprehensive descriptions and definitions can be found in [71–73]: R2 ¼ SST SSE SST ð4Þ ð4Þ where SST stands for the sum of squares for total, and SSE stands for the sum of the squares for error. where SST stands for the sum of squares for total, and SSE stands for the sum of the squares for error. PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 Pearson correlation analyses and individual ML evaluations Based on the field data analyses, the average yield of 250 soybean genotypes was estimated to be between 2.58 to 5.71 ton ha-1 with a mean and standard deviation of 4.22 and 0.57 ton ha-1, respectively. The potential benefits of using each soybean yield component for predicting the soybean seed yield was quantified using the Pearson coefficients of correlation among all the measured traits. Based on the correlation coefficients, all the yield components, except NRNP, were posi- tively correlated with soybean seed yield. The linear correlation between soybean seed yield and PP (r = 0.71) was found to be the strongest followed by its correlation with NP (r = 0.68), RNP (r = 0.67), and P/N (r = 0.64). The negative correlation between soybean seed yield and NRNP was estimated as r = -0.29 (Fig 3). Based on the results of correlation analyses, the top correlated variables were iteratively added to the algorithms and updated the algorithm training performances until all variables were included. The R2 values of each model were then calculated (Fig 4). Among all the tested ML algorithms, the R2 reached its maximum value of 0.81 in RBF taking into account PP, NP, and RNP. No changes in R2 value were detected after adding P/N and NRNP in the RBF algo- rithm. The maximum R2 value of 0.80 was obtained for both RF and MLP when all the yield components are added into the algorithms (Fig 4). Visualizing and statistical analyzing The results were visualized using the Microsoft Excel software (2019), ggvis [74], and ggplot2 [75] packages in the R software version 3.6.1. All statistical computational procedures were also conducted using MASS [76] package in R software. 6 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 PLOS ONE Using machine learning algorithms for estimating soybean yield from yield component traits Fig 2. The experimental workflow of algorithm selection and validation for predicting the soybean seed yield. Th Number of Nodes per plant (NP), the Number of Non-Reproductive Nodes per Plant (NRNP), the Number of Reproductive Nodes per Plant (RNP), and the Number of Pods per Plant (PP), the ratio of number of Pods to number of Nodes per plant (P/N), Genetic Algorithm (GA). https://doi.org/10.1371/journal.pone.0250665.g002 Fig 2. The experimental workflow of algorithm selection and validation for predicting the soybean seed yield. The Number of Nodes per plant (NP), the Number of Non-Reproductive Nodes per Plant (NRNP), the Number of Reproductive Nodes per Plant (RNP), and the Number of Pods per Plant (PP), the ratio of number of Pods to number of Nodes per plant (P/N), Genetic Algorithm (GA). Fig 2. The experimental workflow of algorithm selection and validation for predicting the soybean seed yield. The Number of Nodes per plant (NP), the Number of Non-Reproductive Nodes per Plant (NRNP), the Number of Reproductive Nodes per Plant (RNP), and the Number of Pods per Plant (PP), the ratio of number of Pods to number of Nodes per plant (P/N), Genetic Algorithm (GA). Fig 2. The experimental workflow of algorithm selection and validation for predicting the soybean seed yield. The Number of Nodes per plant (NP), the Number of Non-Reproductive Nodes per Plant (NRNP), the Number of Reproductive Nodes per Plant (RNP), and the Number of Pods per Plant (PP), the ratio of number of Pods to number of Nodes per plant (P/N), Genetic Algorithm (GA). 7 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 PLOS ONE Using machine learning algorithms for estimating soybean yield from yield component traits PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 Model performance and evaluation The full analysis of ML algorithms are presented in the S1 Table. Among the three tested ML algorithms, RBF had the highest value for R2 (0.81) and the lowest values for MAE (148.61 kg. ha-1) and RMSE (185.31 kg.ha-1) (Fig 5A–5C). The R2 values for MLP and RF were the same Fig 3. Pearson correlation analysis of soybean yield component traits. The Number of Nodes per plant (NP), the Number of Non-Reproductive Nodes per Plant (NRNP), the Number of Reproductive Nodes per Plant (RNP), and the Number of Pods per Plant (PP), the ratio of number of Pods to number of Nodes per plant (P.N). https://doi.org/10.1371/journal.pone.0250665.g003 Fig 3. Pearson correlation analysis of soybean yield component traits. The Number of Nodes per plant (NP), the Number of Non-Reproductive Nodes per Plant (NRNP), the Number of Reproductive Nodes per Plant (RNP), and the Number of Pods per Plant (PP), the ratio of number of Pods to number of Nodes per plant (P.N). https://doi.org/10.1371/journal.pone.0250665.g003 8 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 PLOS ONE OS ONE Using machine learning algorithms for estimating soybean yield from yield component traits Using machine learning algorithms for estimating soybean yield from yield component traits Fig 4. Model training accuracy for Random Forest (RF), Multilayer Perceptron (MLP), and Radial Basis Function (RBF) algorithms by adding variables based on the correlation results. The Number of Nodes per plant (NP), the Number of Non-Reproductive Nodes per Plant (NRNP), the Number of Reproductive Nodes per Plant (RNP), and the Number of Pods per Plant (PP), the ratio of number of Pods to number of Nodes per plant (P/N). https://doi.org/10.1371/journal.pone.0250665.g004 Fig 4. Model training accuracy for Random Forest (RF), Multilayer Perceptron (MLP), and Radial Basis Function (RBF) algorithms by adding variables based on the correlation results. The Number of Nodes per plant (NP), the Number of Non-Reproductive Nodes per Plant (NRNP), the Number of Reproductive Nodes per Plant (RNP), and the Number of Pods per Plant (PP), the ratio of number of Pods to number of Nodes per plant (P/N). https://doi.org/10.1371/journal.pone.0250665.g004 https://doi.org/10.1371/journal.pone.0250665.g004 (0.80); however, they had different values for MAE and RMSE. In comparison with the MLP algorithm, RF had the lower MAE and RMSE with 156.28 kg.ha-1 and 194.75 kg.ha-1, respec- tively (Fig 5A–5C). MLP had the highest MAE (172.98 kg.ha-1), and RMSE (211.57 kg.ha-1) among the ML algorithms. Model performance and evaluation In addition to individual evaluations of the three ML algorithms, an ensemble learning was also developed, which outperformed all the individual machine learning algorithms, attaining an R2 value of 0.82. The E-B method had the acceptable RMSE and MAE with a value of 184.35 kg.ha-1 and 148.37 kg.ha-1, respectively (Fig 5A–5C). In gen- eral, the R2 value of E-B increased by 0.1, while the values of MAE and RMAS decreased by 0.24 kg.ha-1 and 0.96 kg.ha-1, respectively, in comparison with RBF as the most accurate indi- vidual ML algorithm identified in this study. Optimization of the soybean seed yield using E-B-GA The aim of the current study, not only was to predict soybean seed yield using yield compo- nents, but also to estimate the optimum values of these traits, i.e., NP, PP, RNP, NRNP, and P/ N, to maximize the final yield production in a given genotype. The results of the optimization process using GA, as the single objective evolutionary optimization algorithm, are presented in Table 1. Theoretically, the highest soybean seed yield production of 5.64 ton ha-1 can be achieved in an ideotype soybean genotype in which the values of NP, NRNP, RNP, PP, and P/ N are 17.32, 3.07, 14.25, 48.98, and 2.83, respectively. Discussion One of the objectives of this study was to investigate the potential use of soybean yield compo- nents such as NP, PP, RNP, NRNP, and P/N for predicting the final seed yield production. Many studies reported the reliance of the final yield production on the performance of several yield-related traits [15, 77–80]. In soybean, PP and NP are considered as major players in 9 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 PLOS ONE Using machine learning algorithms for estimating soybean yield from yield component traits Fig 5. (A) coefficient of determination (R2), (B) the Root Mean Square Error (RMSE) and (C) the Mean Absolute Errors (MAE) performance of Random Forest (RF), Multilayer Perceptron (MLP), and Radial Basis Function (RBF) algorithms, and the Ensemble-Bagging (E-B) strategy for soybean yield prediction using yield component traits. The mean performance is indicated with an + sign in each Figure. https://doi.org/10.1371/journal.pone.0250665.g005 Fig 5. (A) coefficient of determination (R2), (B) the Root Mean Square Error (RMSE) and (C) the Mean Absolute Errors (MAE) performance of Rando Forest (RF), Multilayer Perceptron (MLP), and Radial Basis Function (RBF) algorithms, and the Ensemble-Bagging (E-B) strategy for soybean yield prediction using yield component traits. The mean performance is indicated with an + sign in each Figure. Fig 5. (A) coefficient of determination (R2), (B) the Root Mean Square Error (RMSE) and (C) the Mean Absolute Errors (MAE) performance of Random Forest (RF), Multilayer Perceptron (MLP), and Radial Basis Function (RBF) algorithms, and the Ensemble-Bagging (E-B) strategy for soybean yield prediction using yield component traits. The mean performance is indicated with an + sign in each Figure. https://doi.org/10.1371/journal.pone.0250665.g005 https://doi.org/10.1371/journal.pone.0250665.g005 Table 1. Optimizing the number of nodes per plant (NP), the number of non-reproductive nodes per plant (NRNP), the number of reproductive nodes per plant (RNP), and the number of pods per plant (PP), the ratio of number of pods to number of nodes per plant (P/N) according to the E-B-GA for maximizing soybean seed yield. Input variables Predicted Yield (ton ha-1) NP NRNP RNP PP P/N 17.32 3.07 14.25 48.98 2.83 5.64 https://doi.org/10.1371/journal.pone.0250665.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 10 / 18 Table 1. Discussion Optimizing the number of nodes per plant (NP), the number of non-reproductive nodes per plant (NRNP), the number of reproductive nodes per plant (RNP), and the number of pods per plant (PP), the ratio of number of pods to number of nodes per plant (P/N) according to the E-B-GA for maximizing soybean seed yield. Input variables Predicted Yield (ton ha-1) NP NRNP RNP PP P/N 17.32 3.07 14.25 48.98 2.83 5.64 https://doi.org/10.1371/journal.pone.0250665.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 10 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 10 / 18 PLOS ONE Using machine learning algorithms for estimating soybean yield from yield component traits determining the final seed yield [15, 81, 82]. In the current study, PP showed the highest linear correlation with the total seed yield. The direct impact of the number of pods per plant on the final soybean yield is also reported by Bastidas, Setiyono (83)]. Among all the tested yield com- ponent traits in this study, NP showed the second-highest linear correlation with total seed yield and showed a positive correlation with PP. Many studies reported that the variations in the number of nodes per plant is usually accounted for the changes in the number of pods per plant [81–84]. A negative correlation between the total soybean seed yield and NRNP was found in this study. It is in agreement with previous studies claimed that increasing the num- ber of non-reproductive nodes decreases the reproductive potential of soybean seed yield [81, 85]. The results of linear correlation analyses in this study illustrated the importance of indi- vidual yield component traits in determining the total soybean seed yield. Conventional statistical methods such as ANOVA and simple regression methods are typi- cally recommended for small datasets with limited dimensions [86, 87]. However, soybean yield is a complex trait under controlled by different continuous variables called yield compo- nent traits. Therefore, more sophisticated methods are required for analyzing large data sets with high dimensions [88]. The successful uses of ML algorithms for analyzing big data with multi-collinearity among the variables have recently been reported in many plant species such as soybean [30], alfalfa [48], chrysanthemum [89], wheat [90], and lime [91]. PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 Discussion Although the difference between RF and RBF in terms of R2 values was not statistically significant (data are not shown), they were statistically different for their MAE and RMSE values. The performance of the RF algo- rithm relies on processing large dimensional data based on generalized error estimation [100, 101]. Also, there is no assumption requirement for RF about the distribution of data [102], and this algorithm can isolate outliers in a small region of the variable space resulted in acceptable performance against nonlinear environmental effects [102, 103]. In addition to individual comparison of the three tested ML algorithms, we developed a bagging ensemble model by combining the results of RBF, MLP, and RF in this study. Since the RBF had the highest performance in predicting the soybean seed yield, this algorithm was chosen as the metaClassifier for developing the E-B algorithm. Using the E-B model, a slight improvement was obtained in predicting the total soybean seed yield from its component traits. Diversity and sufficiency are two of the most important principles in selecting base learners for an ensemble model [67]. It means that the dependency among the used ML algo- rithms in the ensemble model should be minimized, while each based learner should have an acceptable predicting capability as well [104, 105]. Therefore, we selected different ML algo- rithms as the base learners for the E-B with different training data mechanisms. Also, the per- formance of the E-B was optimized by implementing RBF as the metaClassifier for this model. The effectiveness of using ensemble models was reported in different plant species such as chrysanthemum [106], sorghum [107], wheat [108], alfalfa [67], and brassicas [109]. This study demonstrates the benefit of using the RBF-based E-B approach to improve the soybean yield prediction accuracy using yield components. Selecting high-yielding lines has always been one of the major goals in plant breeding pro- grams that can be performed using either direct or indirect selection approaches [110]. Select- ing superior genotypes based on the yield component traits can be considered as an indirect method. An analytical breeding strategy is an alternate breeding approach that is focused on the improvement of components of complex traits such as plant growth and development rates or yield components [111] rather than the traits per se. Discussion The prediction performance of a given machine learning algorithm refers to the power of the model in pre- dicting the values of a dependent variable when non-representative samples, or samples from a different population, are used as the test population [92]. The prediction performance of an ML algorithm is estimated using its R2, RMSE, and MAE values [92–94]. In this study, the three common ML algorithms, RBF, MLP, and RF, were used to predict the soybean seed yield using its components and their prediction performance were estimated. RBF was found to be the most accurate ML algorithm for predicting the soybean seed yield from its component traits. In general, yield components in soybean are traits with low heritability that are influ- enced by environmental factors. The environmental factors can bring some levels of instabil- ity/noise in the results of all the ML analyses [95]. However, the structure of RBF (Fig 6) gives Fig 6. The schematic view of the Radial Basis Function (RBF) algorithm. https://doi.org/10.1371/journal.pone.0250665.g006 Fig 6. The schematic view of the Radial Basis Function (RBF) algorithm. https://doi.org/10.1371/journal.pone.0250665.g006 11 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 PLOS ONE Using machine learning algorithms for estimating soybean yield from yield component traits some level of robustness against the adversarial noises, compared to other tested ML algo- rithms [96, 97]. The specific structure of RBF is the use of the transfer function of input vari- ables to hidden layer name radial basis function [64, 98]. This function plays an important role in reducing noises of input variables resulted in more accurate prediction performance [99]. Although RF and MLP had the same R2 values, the MAE and RMSE values were lower in RF. MLP, as one of the neural network algorithms, is highly susceptible to possible instabili- ties/noises caused by non-heritable factors. The MAE and RMSE values of this algorithm were the highest among all the tested ML algorithms that may indicate the sensitivity of the algo- rithm to noises. As a result, using this algorithm may not recommend for analyzing pheno- typic data that are largely affected by environmental factors. RF with the R2, MAE, and RMSE values of 0.80, 156.28 kg.ha-1, and 194.75 kg.ha-1, respectively, was selected as the second-best ML algorithm for predicting soybean seed yield in this study. PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 Acknowledgments The authors are grateful to the past and current members of Eskandari laboratory at the Uni- versity of Guelph, Ridgetown Campus, including Dr. Sepideh Torabi, Mr. Bryan Stirling, Mr. John Kobler, and Mr. Robert Brandt for their technical support. We would also like to thank Mrs. Maryam Vazin for her assistance with the field data collection. Conclusion Efficient breeding approaches for improving the genetic potential of complex traits such as yield in soybean can be developed based upon secondary traits that govern the final perfor- mance of the complex traits. Thus, in order to increase the genetic yield potential in soybean, breeders can select soybean genotypes with improved yield component traits. However, mea- suring yield components in a large soybean breeding program is time-consuming and labor- intensive, which limit the implication of this approach in cultivar development programs. The main objective of this study was to evaluate the potential use of yield component traits for esti- mating final seed yield in soybean using different ML and E-B algorithms, which in turn can be used by breeders for selecting parental lines and designing promising crosses for developing cultivars with improved genetic yield potential. The results of the current study showed that RBF is a reliable solo ML algorithm for predicting the soybean seed yield from its component traits. However, an E-B algorithm that was built by combining the three ML and using RBF as its metaClassifier outperformed all individual ML algorithms and, therefore, it is recom- mended for predicting the soybean seed yield exploiting yield component traits. In the current study for the first time, we coupled E-B algorithm with GA in order to estimate optimum val- ues of yield component traits in a theoretical genotype in which the yield is maximized using the real field data. The results seem to be promising but are recommended to be evaluated in new and independent breeding populations before using in cultivar development programs for selecting high-yielding potential genotypes. This information can be also used, in com- bined with molecular marker technology, for developing genomic-based toolkits that can be used for selecting genotypes with improved genetic yield potential at early generations. Supporting information S1 Table. Analysis performance of Random Forest (RF), Multilayer Perceptron (MLP), and Radial Basis Function (RBF) algorithms, and the Ensemble-Bagging (E-B) strategy for soybean yield prediction using yield component traits. (DOCX) Discussion This strategy can improve genetic yield potential in varieties by setting up selection criteria on yield components and making the selection more efficient [112]. In order to move toward analytical breeding, it would be impor- tant to have the optimized level of each yield component traits in target populations. Genetic algorithm is commonly used in finding optimized solutions by searching problems through biological parameters such as selection, crossover, and mutation [53, 113]. After selecting E-B as the combined algorithm with the highest prediction ability in this study, GA was linked into this algorithm to estimate the optimum values of the yield component traits (Table 1). The suc- cessfulness of using the GA algorithm for estimating optimized solutions was reported previ- ously in plant tissue culture [89], plant physiology [114], and remote sensing [115]. PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 12 / 18 PLOS ONE Using machine learning algorithms for estimating soybean yield from yield component traits PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 References 1. Hashiguchi A, Komatsu S. Chapter 6—Proteomics of Soybean Plants. In: Colgrave ML, editor. Proteo- mics in Food Science: Academic Press; 2017. p. 89–105. 2. Miransari M. 11—Soybean production and N fertilization. In: Miransari M, editor. Abiotic and Biotic Stresses in Soybean Production. San Diego: Academic Press; 2016. p. 241–60. 3. 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Funding acquisition: Milad Eskandari. Funding acquisition: Milad Eskandari. Methodology: Mohsen Yoosefzadeh-Najafabadi. Methodology: Mohsen Yoosefzadeh-Najafabadi. Methodology: Mohsen Yoosefzadeh-Najafabadi. Validation: Dan Tulpan, Milad Eskandari. Writing – original draft: Mohsen Yoosefzadeh-Najafabadi. Writing – original draft: Mohsen Yoosefzadeh-Najafabadi. Writing – review & editing: Dan Tulpan, Milad Eskandari. Writing – review & editing: Dan Tulpan, Milad Eskandari. 13 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 PLOS ONE | https://doi.org/10.1371/journal.pone.0250665 April 30, 2021 PLOS ONE Using machine learning algorithms for estimating soybean yield from yield component traits References Szymczak S, Biernacka JM, Cordell HJ, Gonza´lez-Recio O, Ko¨nig IR, Zhang H, et al. Machine learn- ing in genome-wide association studies. Genetic epidemiology. 2009; 33(S1):S51–S7. https://doi.org/ 10.1002/gepi.20473 PMID: 19924717 29. 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Epilepsy, hippocampal sclerosis and febrile seizures linked by common genetic variation around SCN1A
Brain
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Zurich Open Repository and Archive Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2013 BRAIN A JOURNAL OF NEUROLOGY Received February 14, 2013. Revised June 28, 2013. Accepted July 2, 2013. Advance Access publication September 6, 2013  The Author (2013). Published by Oxford University Press on behalf of the Guarantors of Brain. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Epilepsy, hippocampal sclerosis and febrile seizures linked by common genetic variation around SCN1A Kasperavičiūtė, Dalia ; Catarino, Claudia B ; Matarin, Mar ; Leu, Costin ; Novy, Jan ; Tostevin, Anna ; Leal, Bárbara ; Hessel, Ellen V S ; Hallmann, Kerstin ; Hildebrand, Michael S ; Dahl, Hans-Henrik M ; Ryten, Mina ; Trabzuni, Daniah ; Ramasamy, Adaikalavan ; Alhusaini, Saud ; Doherty, Colin P ; Dorn, Thomas ; Hansen, Jörg ; Krämer, Günter ; Steinhoff, Bernhard J ; Zumsteg, Dominik ; Duncan, Susan ; Kälviäinen, Reetta K ; Eriksson, Kai J ; Kantanen, Anne-Mari ; Pandolfo, Massimo ; Gruber-Sedlmayr, Ursula ; Schlachter, Kurt ; Reinthaler, Eva M ; Stogmann, Elisabeth ; et al DOI: https://doi.org/10.1093/brain/awt233 DOI: https://doi.org/10.1093/brain/awt233 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-154722 Journal Article Published Version Originally published at: Kasperavičiūtė, Dalia; Catarino, Claudia B; Matarin, Mar; Leu, Costin; Novy, Jan; Tostevin, Anna; Leal, Bárbara; Hessel, Ellen V S; Hallmann, Kerstin; Hildebrand, Michael S; Dahl, Hans-Henrik M; Ryten, Mina; Trabzuni, Da- niah; Ramasamy, Adaikalavan; Alhusaini, Saud; Doherty, Colin P; Dorn, Thomas; Hansen, Jörg; Krämer, Günter; Steinhoff, Bernhard J; Zumsteg, Dominik; Duncan, Susan; Kälviäinen, Reetta K; Eriksson, Kai J; Kantanen, Anne- Mari; Pandolfo, Massimo; Gruber-Sedlmayr, Ursula; Schlachter, Kurt; Reinthaler, Eva M; Stogmann, Elisabeth; et al (2013). Epilepsy, hippocampal sclerosis and febrile seizures linked by common genetic variation around SCN1A. Brain : a journal of neurology, 136(10):3140-3150. DOI: https://doi.org/10.1093/brain/awt233 BRAIN A JOURNAL OF NEUROLOGY doi:10.1093/brain/awt233 Brain 2013: 136; 3140–3150 | 3140 Brain 2013: 136; 3140–3150 | 3140 doi:10.1093/brain/awt233 gy q 2 Epilepsy Society, Chalfont-St-Peter, SL9 0RJ, UK p  The Author (2013). Published by Oxford University Press on behalf of the Guarantors of Brain. Received February 14, 2013. Revised June 28, 2013. Accepted July 2, 2013. Advance Access publication September 6, 2013 gy q 2 Epilepsy Society, Chalfont-St-Peter, SL9 0RJ, UK 1 NIHR University College London Hospitals Biomedical Research Centre, Department of Clinical and Experimenta Neurology, Queen Square, London, WC1N 3BG, UK ology, Queen Square, London, WC1N 3BG, UK 1 NIHR University College London Hospitals Biomedical Research Centre, Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK 1 NIHR University College London Hospitals Biomedical Research Centre, Department of Clinical and Experimental Epilepsy, UCL Institute of N l Q S L d WC1N 3BG UK 1 NIHR University College London Hospitals Biomedical Research Centre, Department of Clinical and Experimenta p istribution, and reproduction in any medium, provided the original work is properly cited. Epilepsy, hippocampal sclerosis and febrile seizures linked by common genetic variation around SCN1A Dalia Kasperavicˇiu¯te_,1,* Claudia B. Catarino,1,2,* Mar Matarin,1,* Costin Leu,1,* Jan Novy,1,2 Anna Tostevin,1,2 Ba´rbara Leal,3,4 Ellen V. S. Hessel,5 Kerstin Hallmann,6,7 Michael S. Hildebrand,8 Hans-Henrik M. Dahl,8 Mina Ryten,9,10 Daniah Trabzuni,9,10,11 Adaikalavan Ramasamy,9,10,12 Saud Alhusaini,13,14 Colin P. Doherty,15 Thomas Dorn,16 Jo¨rg Hansen,16 Gu¨nter Kra¨mer,16 Bernhard J. Steinhoff,17 Dominik Zumsteg,18 Susan Duncan,19 Reetta K. Ka¨lvia¨inen,20,21 Kai J. Eriksson,22 Anne-Mari Kantanen,20 Massimo Pandolfo,23 Ursula Gruber-Sedlmayr,24 Kurt Schlachter,25 Eva M. Reinthaler,26 Elisabeth Stogmann,26 Fritz Zimprich,26 Emilie The´aˆtre,27,28 Colin Smith,29 Terence J. O’Brien,30,31 K. Meng Tan,30,31 Slave Petrovski,30,31,32 Angela Robbiano,33 Roberta Paravidino,33 Federico Zara,33 Pasquale Striano,34 Michael R. Sperling,35 Russell J. Buono,36 Hakon Hakonarson,37 Joa˜o Chaves,38 Paulo P. Costa,3,4,39 Berta M. Silva,3,4 Anto´nio M. da Silva,4,38 Pierre N. E. de Graan,5 Bobby P. C. Koeleman,40 Albert Becker,41 Susanne Schoch,41 Marec von Lehe,42 Philipp S. Reif,43 Felix Rosenow,43 Felicitas Becker,44 Yvonne Weber,44 Holger Lerche,44 Karl Ro¨ssler,45 Michael Buchfelder,45 Hajo M. Hamer,46 Katja Kobow,47 Roland Coras,47 Ingmar Blumcke,47 Ingrid E. Scheffer,8,48,49 Samuel F. Berkovic,8 Michael E. Weale,12 UK Brain Expression Consortium9,10,† Norman Delanty,13,50 Chantal Depondt,23 Gianpiero L. Cavalleri,13 Wolfram S. Kunz6,7 and Sanjay M. Epilepsy, hippocampal sclerosis and febrile seizures linked by common genetic variation around SCN1A Ricardo Jorge (INSA), 4049-019 Porto, Portugal 40 Department of Medical Genetics, University Medical Centre Utrecht, 3584 CG Utrec 41 Department of Neuropathology, University of Bonn, 53105 Bonn, Germany of Neuropathology, University of Bonn, 53105 Bonn, Germ 42 Department of Neurosurgery, University of Bochum, 44892 Bochum, Germany Department of Neurology, University Hospitals and Philipps-University Marburg, 35043 Marburg, Germany 43 Epilepsy-Centre Hessen, Department of Neurology, University Hospitals and Philipps-University Marburg, 35 43 Epilepsy-Centre Hessen, Department of Neurology, University Hospitals and Phil 44 Department of Neurology and Epileptology, Hertie Institute for Clinical Brain Research, University of Tu¨ bingen, 72076 Tu¨ bingen, Germany 44 Department of Neurology and Epileptology, Hertie Institute for Clinical Brain Research, University of Tu¨ bingen, 72076 Tu¨ bingen, Germany 44 Department of Neurology and Epileptology, Hertie Institute for Clinical Brain Research, University of Tu¨ bingen, 72076 Tu¨ bingen, Germany 45 Department of Neurosurgery, University Hospital Erlangen, 91054 Erlangen, Germany 44 Department of Neurology and Epileptology, Hertie Institute for Clinical Brain Research, University of Tu¨ binge 46 Department of Neurology, Epilepsy Centre, University Hospital Erlangen, 91054 Erlangen, Ge 46 Department of Neurology, Epilepsy Centre, University Hospital Erlangen, 91054 Erlangen, Germany 47 Department of Neuropathology, University Hospital Erlangen, 91054 Erlangen, Germany 48 Florey Institute of Neuroscience and Mental Health, Melbourne VIC 3010, Australia 49 Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Melbourne VIC 3052, Australia 49 Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Melbourne VIC 3052, Austra 49 Department of Paediatrics, University of Melbourne, Royal Childre 50 Department of Neurology, Beaumont Hospital, Dublin 9, Ireland 50 Department of Neurology, Beaumont Hospital, Dublin *These authors contributed equally to this work. *These authors contributed equally to this work. q The list of members of the UK Brain Expression Consortium is available at http://ukbec.wordpress.com. †The list of members of the UK Brain Expression Consortium is available at http://ukbec.wordpress.com. †The list of members of the UK Brain Expression Consortium is available at http://ukb Correspondence to: Sanjay M. Sisodiya, PhD, FRCP Correspondence to: Sanjay M. Sisodiya, PhD, FRCP Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, 33 Queen Square London, WC1N 3BG, E-mail: s.sisodiya@ucl.ac.uk Epilepsy comprises several syndromes, amongst the most common being mesial temporal lobe epilepsy with hippocampal sclerosis. Epilepsy, hippocampal sclerosis and febrile seizures linked by common genetic variation around SCN1A Gaslini’ Institute, 16147 Genova, Italy 33 Department of Neurosciences, Laboratory of Neurogenetics, University of Genoa, ‘G. Gaslini’ Institute, 16147 Genova, Italy 34 Paediatric Neurology and Muscular Diseases Unit, Department of Neurosciences, Rehabilitation, Ophthalmol Health University of Genoa ‘G Gaslini’ Institute 16147 Genova Italy 34 Paediatric Neurology and Muscular Diseases Unit, Department of Neurosci Health, University of Genoa, ‘G. Gaslini’ Institute, 16147 Genova, Italy y y 35 Department of Neurology, Thomas Jefferson University, Philadelphia, PA 19107, USA 35 Department of Neurology, Thomas Jefferson University, Philadelphia, PA 19107, USA p p 36 Department of Biomedical Science, Cooper Medical School of Rowan University, Camden, NJ 08103, USA 37 Centre for Applied Genomics, The Children’s Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-4318, USA 37 Centre for Applied Genomics, The Children’s Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-4318, USA Neurological Disorders and Senses, Hospital Santo Anto´ nio / Centro Hospitalar do Porto, 4099-001 Porto, Portugal 38 Department of Neurological Disorders and Senses, Hospital Santo Anto´ nio / Centro Hospitalar do Porto, 409 p g p p g 39 Instituto Nacional de Sau´ de Dr. Ricardo Jorge (INSA), 4049-019 Porto, Portugal 39 Instituto Nacional de Sau´ de Dr. Epilepsy, hippocampal sclerosis and febrile seizures linked by common genetic variation around SCN1A Sisodiya1,2 1 NIHR University College London Hospitals Biomedical Research Centre, Department of Clinical and Experimental Epilepsy, UCL Institute of N l Q S L d WC1N 3BG UK p p y y 3 Immunogenetics Laboratory, University of Porto, 4050-313 Porto, Portugal g y, y , , g 4 UMIB - Instituto Cieˆncias Biome´dicas Abel Salazar, University of Porto, 4099-003 Porto, Portugal 5 Rudolf Magnus Institute of Neuroscience, Department of Neuroscience and Pharmacology, University Medical Centre Utrecht, 3584 CG Utrecht, The Netherlands 6 Department of Epileptology, University of Bonn, 53105 Bonn, Germany p p p gy y y 7 Life & Brain Centre, University of Bonn, 53105 Bonn, Germany 7 Life & Brain Centre, University of Bonn, 53105 Bonn, Germany fe & Brain Centre, University of Bonn, 53105 Bonn, Germ y y 8 Epilepsy Research Centre, Austin Health, University of Melbourne, Melbourne VIC 3084, Australia 9 Department of Molecular Neuroscience, UCL Institute of Neurology, London, WC1N 3BG, UK 10 Reta Lila Weston Institute, UCL Institute of Neurology, London, WC1N 3BG, UK 11 Department of Genetics, King Faisal Specialist Hospital and Research Centre, Riyadh, 11211, Saudi Arabia p p p 12 Department of Medical and Molecular Genetics, King’s College London, Guy’s Hospital, London, SE1 9RT, 12 Department of Medical and Molecular Genetics, King’s College London, Guy’s Hospital, London, S Department of Medical and Molecular Genetics, King’s Co 14 Brain Morphometry Laboratory, Neurophysics Department, Beaumont Hospital, Dublin 9, Ireland 15 Department of Neurology, St James’ Hospital, Dublin 8, Ireland 15 Department of Neurology, St James’ Hospital, Dublin 8, Ireland 16 Swiss Epilepsy Centre, 8008 Zurich, Switzerland 16 Swiss Epilepsy Centre, 8008 Zurich, Switzerland 17 Kork Epilepsy Centre, 77694 Kehl-Kork, Germany 18 Department of Neurology, University Hospital Zurich, 8091 Zurich, Switzerland 18 Department of Neurology, University Hospital Zurich, 8091 Zurich, Switzerland 18 Department of Neurology, University Hospital Zuric 18 Department of Neurology, University Hospital Zurich, 8091 Zurich, Switzerland 19 Edinburgh and South East Scotland Epilepsy Service, Western General Hospital Edinburgh, EH4 2XU, Scotland, UK 19 Edinburgh and South East Scotland Epilepsy Service, Western General Hospital Edinburgh, EH4 2XU, Scotla 19 Edinburgh and South East Scotland Epilepsy Service, Western General Hospital E Brain 2013: 136; 3140–3150 | 3141 SCN1A and MTLEHS with FS 0 Kuopio Epilepsy Centre, Kuopio University Hospital, 70211 Kuopio, Finland 20 Kuopio Epilepsy Centre, Kuopio University Hospital, 70211 Kuopio, Finland 20 Kuopio Epilepsy Centre, Kuopio University Hospital, 70211 Kuopio, Finland p p y p y p p ent of Neurology, Institute of Clinical Medicine, University of Eastern Finland, 70211 Kuopio, Finland p p p p p p 21 Department of Neurology, Institute of Clinical Medicine, University of Eastern Finland, 70211 Kuopio, Finlan Department of Neurology, Institute of Clinical Medicine, U 21 Department of Neurology, Institute of Clinical Medicine, University of Eastern F 22 Paediatric Neurology Unit, Tampere University Hospital and Paediatric Research Centre, University of T c Neurology Unit, Tampere University Hospital and Paediatric Research Centre, University of Tampere, 33521 Tam 22 Paediatric Neurology Unit, Tampere University Hospital and Paediatric Research Centre, University of Tampere, 33521 Tampere, Finland 23 Department of Neurology, Hoˆ pital Erasme, Universite´ Libre de Bruxelles, 1070 Brussels, Belgium p p p p 23 Department of Neurology, Hoˆ pital Erasme, Universite´ Libre de Bruxelles, 1070 Brussels, Belgium p gy p 24 Department of Paediatrics, Medical University of Graz, 8036 Graz, Austria 25 Department of Paediatrics, LKH Bregenz, 6900 Bregenz, Austria p g g 26 Department of Clinical Neurology, Medical University of Vienna, 1090 Vienna, Austria Department of Clinical Neurology, Medical University of p 27 Groupe Interdisciplinaire de Ge´noprote´omique Applique´e (GIGA-R) and Faculty of Veterinary Medicin 27 Groupe Interdisciplinaire de Ge´noprote´omique Applique´e (GIGA-R) and Faculty of Veterinary Medicine, University of Lie`ge, 4000 Lie`ge, Belgium 28 Unit of Gastroenterology, Centre Hospitalier Universitaire, University of Lie`ge, 4000 Lie`ge, Belgium 27 Groupe Interdisciplinaire de Genoproteomique Appliquee (GIGA R) and Faculty of Veterinary Medicine, University of Liege, 4000 Liege, Belgium 28 Unit of Gastroenterology, Centre Hospitalier Universitaire, University of Lie`ge, 4000 Lie`ge, Belgium p p p q pp q y y y g g g 28 Unit of Gastroenterology, Centre Hospitalier Universitaire, University of Lie`ge, 4000 Lie`ge, Belgium Unit of Gastroenterology, Centre Hospitalier Universitaire, gy p y g g g 29 Department of Neuropathology, MRC Sudden Death Brain Bank Project, University of Edinburgh, Wilkie Bu of Medicine and Neurology, Royal Melbourne Hospital, University of Melbourne, Melbourne VIC 3050, Australia 30 Departments of Medicine and Neurology, Royal Melbourne Hospital, University of Melbourne, Melbourne V 31 Melbourne Brain Centre, University of Melbourne, Melbourne VIC 3084, Australia 32 Department of Medicine, Austin Health, University of Melbourne, Melbourne VIC 3084, Australia p y 33 Department of Neurosciences, Laboratory of Neurogenetics, University of Genoa, ‘G. Epilepsy, hippocampal sclerosis and febrile seizures linked by common genetic variation around SCN1A Seizures in mesial temporal lobe epilepsy with hippocampal sclerosis are typically drug-resistant, and mesial temporal lobe epilepsy with hippocampal sclerosis is frequently associated with important co-morbidities, mandating the search for better understanding and treatment. The cause of mesial temporal lobe epilepsy with hippocampal sclerosis is unknown, but there is an association with childhood febrile seizures. Several rarer epilepsies featuring febrile seizures are caused by mutations in SCN1A, which encodes a brain-expressed sodium channel subunit targeted by many anti-epileptic drugs. We undertook a genome-wide association study in 1018 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 7552 control subjects, with validation in an independent sample set comprising 959 people with mesial temporal lobe epilepsy with hippo- campal sclerosis and 3591 control subjects. To dissect out variants related to a history of febrile seizures, we tested cases with mesial temporal lobe epilepsy with hippocampal sclerosis with (overall n = 757) and without (overall n = 803) a history of febrile seizures. Meta-analysis revealed a genome-wide significant association for mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures at the sodium channel gene cluster on chromosome 2q24.3 [rs7587026, within an intron of the SCN1A gene, P = 3.36  109, odds ratio (A) = 1.42, 95% confidence interval: 1.26–1.59]. In a cohort of 172 individuals with febrile seizures, who did not develop epilepsy during prospective follow-up to age 13 years, and 6456 controls, no association was found for rs7587026 and febrile seizures. These findings suggest SCN1A involvement in a common epilepsy syndrome, give new direction to biological understanding of mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures, and open avenues for investigation of prognostic factors and possible prevention of epilepsy in some children with febrile seizures. Epilepsy comprises several syndromes, amongst the most common being mesial temporal lobe epilepsy with hippocampal sclerosis. Seizures in mesial temporal lobe epilepsy with hippocampal sclerosis are typically drug-resistant, and mesial temporal lobe epilepsy with hippocampal sclerosis is frequently associated with important co-morbidities, mandating the search for better understanding and treatment. The cause of mesial temporal lobe epilepsy with hippocampal sclerosis is unknown, but there is an association with childhood febrile seizures. Several rarer epilepsies featuring febrile seizures are caused by mutations in SCN1A, which encodes a brain-expressed sodium channel subunit targeted by many anti-epileptic drugs. Introduction considered unknown, and not eligible for analysis. Population-based controls (n = 7552) were included in the discovery stage, and 3591 in the replication (Table 1 and Supplementary Table 1). Mesial temporal lobe epilepsy with hippocampal sclerosis (MTLEHS) is typically a serious epilepsy syndrome and the most common drug-resistant epilepsy (Berg et al., 2010). It is associated with burdensome co-morbidities, such as memory and psychiatric disorders. MTLEHS is the epilepsy most considered for therapeutic neurosurgery. Although surgery is a proven therapy, only 50% of patients have sustained postoperative seizure freedom (de Tisi et al., 2011), and surgery can have important adverse conse- quences. Better treatment options, or even prevention, of MTLEHS are therefore needed, but rational therapy for MTLEHS remains elusive because its causes are obscure (O’Dell et al., 2012). We also studied 542 individuals who had had febrile seizures but by the last follow-up had not had unprovoked seizures. These came from three groups: a German group; an Austrian group and the ALSPAC (Avon Longitudinal Study of Parents and Children) cohort, the latter followed to age 13 years (Supplementary material); MTLEHS after febrile seizures almost always develops by the age of 15 (Neligan et al., 2012). These cases were compared with 7387 control subjects from three relevant populations (Table 1). For the German and Austrian samples, the same controls as in the MTLEHS study were used. To minimize population stratification, only individuals of white European ancestry were included. In the discovery stage, a combin- ation of self-identified ancestry and EIGENSTRAT principal component methods was used to determine European ancestry. In the replication and febrile seizures analyses, only self-reported white individuals of European ancestry were included. More detailed ancestry data were available from all sources except Austria, allowing exclusion of individ- uals self-reported as coming from countries other than those where they were recruited. MTLEHS is associated with a history of febrile seizures in child- hood (Pittau et al., 2009; O’Dell et al., 2012). About 3% of chil- dren have febrile seizures; why only some go on to develop epilepsy, including MTLEHS, is unknown. There are a number of rare, genetically-determined, epilepsy syndromes in which febrile seizures are a prominent feature, such as Dravet syndrome and ‘genetic epilepsy with febrile seizures plus’ (GEFS + ) (Oliva et al., 2012). Genotyping and quality control In the discovery stage, all but the Austrian samples and Belgian con- trols comprised a subset of a previously described data set (Kasperaviciu¯ te et al., 2010), genotyped on Illumina genome-wide genotyping chips, mostly on Illumina Human610-Quadv1/Human1- 2M-DuoCustom. One hundred and fifty-seven Austrian patients and 332 controls were genotyped on Illumina HumanCNV370duo, and 285 Belgian controls were genotyped on Illumina HumanHap300 gen- otyping chips. Gender and relatedness checks were performed on all samples. The cluster plots of the top-associated single nucleotide poly- morphisms were inspected manually. Details are given in Kasperaviciu¯ te et al. (2010) and in the online Supplementary material. For replication analysis, several methods were used for genotyping. We hypothesized that MTLEHS, or MTLEHS with febrile seiz- ures, as common epilepsy syndromes, might be associated with common genetic variation, and tested this ‘common disease- common variant’ hypothesis in a genetic association study. Introduction MTLEHS has rarely been described in families with GEFS + (Abou-Khalil et al., 2001) or familial febrile seizures (Mantegazza et al., 2005) associated with SCN1A mutations. In familial mesial temporal lobe epilepsy, some family members may have hippo- campal sclerosis (Labate et al., 2011). A cluster of families with mesial temporal lobe epilepsy with hippocampal changes has been described in Brazil (Andrade-Valenc¸a et al., 2008). Together, this evidence implies genetic susceptibility to MTLEHS, although its heritability is unknown. Keywords: mesial temporal lobe epilepsy; mesial temporal sclerosis; SCN1A; association; complex genetics Abbreviations: MTLEHS = mesial temporal lobe epilepsy with hippocampal sclerosis; MTLEHS + FS = MTLEHS with febrile seizures; MTLEHS–FS = MTLEHS without febrile seizures Keywords: mesial temporal lobe epilepsy; mesial temporal sclerosis; SCN1A; association; complex genetics Abbreviations: MTLEHS = mesial temporal lobe epilepsy with hippocampal sclerosis; MTLEHS + FS = MTLEHS with febrile seizures; MTLEHS–FS = MTLEHS without febrile seizures Statistical analysis In the discovery stage, genome-wide association analysis was per- formed using PLINK. Only single nucleotide polymorphisms present on both Illumina Human610-Quadv1 and Human1-2M-DuoCustom were analysed. In the discovery stage, we performed logistic regression using an additive model, including all significant EIGENSTRAT axes (assessed using the Tracy-Widom statistic with P 5 0.05) as covariates. Only single nucleotide polymorphisms with minor allele frequency of 5 1% were analysed. Since the replication samples did not have genome-wide data available to calculate EIGENSTRAT axes, we per- formed stratified analysis using the Cochran-Mantel-Haenszel test for 2  2  8 stratified case-control subsamples deriving from eight differ- ent recruitment countries and self-identified ancestry, using R. The Woolf test was used to assess effect heterogeneity. Meta-analysis of discovery and replication studies was performed using the inverse vari- ance-weighted fixed-effects model as implemented in the GWAMA All aspects of the study were approved by the relevant institutional review board. All participants gave written informed consent. Epilepsy, hippocampal sclerosis and febrile seizures linked by common genetic variation around SCN1A We undertook a genome-wide association study in 1018 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 7552 control subjects with validation in an independent sample set comprising 959 people with mesial temporal lobe epilepsy with hippo genome wide association study in 1018 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 7552 control subjects, with validation in an independent sample set comprising 959 people with mesial temporal lobe epilepsy with hippo- campal sclerosis and 3591 control subjects. To dissect out variants related to a history of febrile seizures, we tested cases with mesial temporal lobe epilepsy with hippocampal sclerosis with (overall n = 757) and without (overall n = 803) a history of febrile seizures. Meta-analysis revealed a genome-wide significant association for mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures at the sodium channel gene cluster on chromosome 2q24.3 [rs7587026, within an intron of the SCN1A gene, P = 3.36  109, odds ratio (A) = 1.42, 95% confidence interval: 1.26–1.59]. In a cohort of 172 individuals with febrile seizures, who did not develop epilepsy during prospective follow-up to age 13 years, and 6456 controls, no association was found for rs7587026 and febrile seizures. These findings suggest SCN1A involvement in a common epilepsy syndrome, give new direction to biological understanding of mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures, and open avenues for investigation of prognostic factors and possible prevention of epilepsy in some children with febrile seizures. 3142 | Brain 2013: 136; 3140–3150 D. Kasperavicˇiu¯ t_e et al 3142 | Brain 2013: 136; 3140–3150 D. Kasperavicˇiu¯ t_e et al. Subjects and DQ993523, respectively) (Martin et al., 2007) in the 5’ region of SCN1A, were tested by quantitative RT-PCR as they are not covered by the array. Details are provided in the Supplementary material. Further, we tested whether the associated single nucleotide poly- morphisms have an effect on expression or splicing of any genes in the genome in post-mortem tissue of nine brain regions from 134 control individuals (Supplementary material). and DQ993523, respectively) (Martin et al., 2007) in the 5’ region of SCN1A, were tested by quantitative RT-PCR as they are not covered by the array. Details are provided in the Supplementary material. and DQ993523, respectively) (Martin et al., 2007) in the 5’ region of SCN1A, were tested by quantitative RT-PCR as they are not covered by the array. Details are provided in the Supplementary material. software (Ma¨gi and Morris, 2010). We considered an association to be genome-wide significant at P 5 5  108. To fine map the association signal in the discovery stage, we imputed single nucleotide polymorphisms in the 10 Mb region surrounding rs7587026. Imputation was performed using MINIMAC (Howie et al., 2012), and 1000 Genomes Project data (1000 Genomes Project Consortium et al., 2010) as the reference data set. Subsequent association analysis was performed using MACH2DAT (Li et al., 2010) using significant EIGENSTRAT axes as covariates. Further, we tested whether the associated single nucleotide poly- morphisms have an effect on expression or splicing of any genes in the genome in post-mortem tissue of nine brain regions from 134 control individuals (Supplementary material). Power calculations were performed using Genetic Power Calculator (Purcell et al., 2003). Subjects Patients were recruited during clinical appointments. MTLEHS was defined as in Wieser (2004). The diagnosis was made and/or reviewed by a consultant epileptologist who was part of this study, with access to history and investigation results. Patients with bilateral hippocampal sclerosis or dual pathology were excluded. One thousand and eighteen patients were included in the discovery stage and 959 patients in the replication. The number of patients by country is shown in Table 1, with further details in Supplementary Table 1. A history of presence or absence of febrile seizures was accepted only if contemporary medical records or a parental account was available; otherwise it was Brain 2013: 136; 3140–3150 | 3143 SCN1A and MTLEHS with FS 3143 Table 1 Number of individuals included in the study, after removal of population outliers and individuals of non-European ancestry Population Patients with MTLEHS Individuals with a definite history of febrile seizures Individuals with a definite history of no febrile seizures Controls Discovery Austria 157 45 104 332 Belgium 67 23 20 285 USA 71 23 45 605 Finland 116 18 0* 746 Ireland 148 54 90 209 UK 277 117 101 5116 Switzerland 182 61 0* 259 Total discovery 1018 341 360 7552 Replication Austria 57 18 39 254 Germany 273 112 161 346 Portugal 102 54 48 190 UK 80 42 28 857 Netherlands 164 74 0* 601 Italy 44 18 26 249 Australia 162 83 79 794 USA 77 15 62 300 Total replication 959 416 443 3591 Febrile seizures study Austria NA 158 NA 585** Germany NA 212 NA 346*** UK (ALSPAC) NA 172 NA 6456 Total febrile seizures NA 542 NA 7387 NA = not applicable. *Data were not collected according to the criteria used in the study. **Combined discovery and replication Austrian controls. ***Same as replication German controls. ble 1 Number of individuals included in the study, after removal of population outliers and dividuals of non-European ancestry and DQ993523, respectively) (Martin et al., 2007) in the 5’ region of SCN1A, were tested by quantitative RT-PCR as they are not covered by the array. Details are provided in the Supplementary material. Further, we tested whether the associated single nucleotide poly- morphisms have an effect on expression or splicing of any genes in the genome in post-mortem tissue of nine brain regions from 134 control individuals (Supplementary material). Expression analysis We performed a two-stage study. For discovery, we first investi- gated genome-wide association between all MTLEHS and 531 164 single nucleotide polymorphisms in 1018 MTLEHS cases and 7552 controls from seven populations of European descent (Table 1 and Supplementary Table 1). Using logistic regression analysis and cor- recting for population stratification, suggestive association emerged for three single nucleotide polymorphisms in a region of strong linkage disequilibrium on chromosome 2q24.3 encom- passing SCN1A and other sodium channel genes (Supplementary Fig. 1). The most strongly associated single nucleotide polymorph- ism, rs11692675, is within intron 3 of the SCN1A full-length We tested association between genotypes of the two top single nu- cleotide polymorphisms rs7587026 and rs11692675 and SCN1A exons and gene expression in the middle temporal cortex (Brodmann areas 20 and 21) from 78 patients with MTLEHS who had undergone sur- gical resection, compared with 78 neurologically normal individuals from the MRC Sudden Death Brain and Tissue Bank. We specifically chose not to study the hippocampus to avoid confounding due to tissue changes such as cell loss and gliosis. All samples were randomly hybridized to Affymetrix Human Exon 1.0 ST arrays. Differential expression of SCN1A transcripts incorporating the ‘neonatal’ or ‘adult’ exon 5 form (5N or 5A exon, respectively), and expression of non-coding exons 1a and 1b (GenBank accession numbers DQ993522 3144 | Brain 2013: 136; 3140–3150 D. Kasperavicˇiu¯ t_e et al. Expression analysis Table 2 Genotype counts, allele frequencies and association results for rs7587026 and rs11692675 SNPs in the MTLEHS study SNP n patients n controls Minor allele Genotype count in patients Genotype count in controls Minor allele frequency in patients Minor allele frequency in controls P-value* Odds ratio (95% CI) Discovery MTLEHS versus controls rs7587026 1017 7549 A 99/440/478 536/2895/4118 0.314 0.263 1.19  10–7 1.31 (1.19–1.45) rs11692675 1018 7547 G 147/477/394 794/3352/3401 0.379 0.327 5.26  10–8 1.31 (1.19–1.44) MTLEHS + FS versus controls rs7587026 341 7549 A 43/161/137 536/2895/4118 0.362 0.263 2.64  10–8 1.59 (1.35–1.87) rs11692675 341 7547 G 61/163/117 794/3352/3401 0.418 0.327 1.25  10–6 1.49 (1.27–1.75) MTLEHSFS versus controls rs7587026 359 6544 A 30/143/186 469/2528/2547 0.283 0.265 0.21 1.12 (0.94–1.33) rs11692675 360 6542 G 48/167/145 698/2951/2893 0.365 0.332 0.039 1.19 (1.01–1.40) MTLEHS + FS versus MTLEHSFS rs7587026 341 359 A 43/161/137 30/143/186 0.362 0.283 1.12  10–3 1.48 (1.17–1.87) rs11692675 341 360 G 61/163/117 48/167/145 0.418 0.365 0.030 1.28 (1.03–1.59) Replication MTLEHS versus controls rs7587026 933 3537 A 89/360/484 247/1361/1929 0.288 0.262 0.025 1.15 (1.02–1.29) rs11692675 826 3568 G 108/364/354 394/1615/1559 0.351 0.337 0.19 1.08 (0.96–1.21) MTLEHS + FS versus controls rs7587026 406 3537 A 43/163/200 247/1361/1929 0.307 0.262 5.88  10–3 1.26 (1.07–1.48) rs11692675 371 3568 G 56/156/159 394/1615/1559 0.361 0.337 0.12 1.14 (0.97–1.34) MTLEHSFS versus controls rs7587026 436 2972 A 42/160/234 216/1136/1620 0.280 0.264 0.20 1.11 (0.95–1.31) rs11692675 357 2983 G 42/164/151 336/1332/1315 0.347 0.336 0.35 1.08 (0.92–1.27) MTLEHS + FS versus MTLEHSFS rs7587026 338 436 A 35/137/166 42/160/234 0.306 0.280 0.22 1.16 (0.93–1.44) rs11692675 298 357 G 46/125/127 42/164/151 0.364 0.347 0.50 1.09 (0.87–1.36) Combined (meta-analysis) MTLEHS versus controls rs7587026 1950 11 086 A 188/800/962 783/4256/6047 0.302 0.263 3.78  10–8 1.24 (1.15–1.34) rs11692675 1844 11 115 G 255/841/748 1188/4967/4960 0.366 0.330 4.87  10–7 1.21 (1.12–1.30) MTLEHS + FS versus controls rs7587026 747 11 086 A 86/324/337 783/4256/6047 0.332 0.263 3.36  10–9 1.42 (1.26–1.59) rs11692675 712 11 115 G 117/319/276 1188/4967/4960 0.388 0.330 4.78  10–6 1.30 (1.16–1.46) MTLEHSFS versus controls rs7587026 795 9516 A 72/303/420 685/3664/5167 0.281 0.265 0.067 1.12 (0.99–1.25) rs11692675 717 9525 G 90/331/296 1034/4283/4208 0.356 0.333 0.033 1.13 (1.01–1.27) MTLEHS + FS versus MTLEHSFS rs7587026 679 795 A 78/298/303 72/303/420 0.334 0.281 1.53  10–3 1.30 (1.10–1.52) rs11692675 639 717 G 107/288/244 90/331/296 0.393 0.356 0.039 1.18 (1.01–1.38) *In discovery stage, P-value is logistic regression P-value for additive genetic model; in replication stage, Cochran-Mantel-Haenszel test P-value. Expression analysis This evidence motivated our pre-analysis collection of febrile seizure data, and our previous study of febrile seizures (Petrovski et al., 2009). We performed analysis of patients in the discovery cohort with a known history of presence of childhood febrile seiz- ures (MTLEHS + FS, n = 341) (Table 2 and Fig. 1). The strongest association was for rs7587026, P = 2.64  108 [OR(A) = 1.59, 95% CI: 1.35–1.87] and rs580041, P = 8.91  107 [OR(A) = 1.56, 95% CI: 1.33–1.84], whereas the signal for rs11692675 was slightly weaker, P = 1.25  106 [OR(G) = 1.49, 95% CI: 1.27–1.75]. No association was seen in patients with MTLEHS without febrile seizures (MTLEHSFS), despite similar sample size. Expression analysis Table 2 Genotype counts, allele frequencies and association results for rs7587026 and rs11692675 SNPs in the MTLEHS study l l dd Table 2 Genotype counts, allele frequencies and association results for rs7587026 and rs11692675 SNPs in the MTLEHS t d lele frequencies and association results for rs7587026 and rs11692675 SNPs in the MTLEHS transcript variant (NM_001202435.1) {P = 5.26  108, odds ratio for G allele [OR(G)] 1.31, 95% confidence interval (CI) 1.19–1.44; Table 2}. Two other single nucleotide polymorphisms within SCN1A intron 1, had similarly low P-values: rs7587026 (r2 = 0.806 with rs11692675 in CEU population based on 1000 Genomes data set), P = 1.19  107 [OR(A) = 1.31, 95% CI: 1.19–1.45]; and rs580041 (r2 = 0.806 with rs11692675), P = 5.74  107 [OR(A) = 1.29, 95% CI: 1.17–1.43]. (Oliva et al., 2012). The common SCN1A single nucleotide poly- morphism rs3812718, affecting splicing (Heinzen et al., 2007), has also been associated with febrile seizures (Schlachter et al., 2009), though replication has failed (Petrovski et al., 2009). Retrospective studies show association between MTLE and febrile seizures (Pittau et al., 2009; O’Dell et al., 2012). Whether febrile seizures cause MTLEHS (Koyama et al., 2012) or whether pre-existing hippocampal abnormalities predispose to febrile seizures (Cendes, 2004), which may then also be injurious, is unknown. Clinical differences between patients with and without a history of febrile seizures suggest MTLEHS is heterogeneous (Thom et al., 2010). SCN1A encodes brain-expressed voltage-gated sodium channel type I, alpha subunit. It bears the largest number of known epi- lepsy-related mutations, some associated with febrile seizures Figure 1 The results of genome-wide association analysis in MTLEHS + FS in discovery stage. (A) Manhattan plot, log10 (P-values) of the logistic regression test are plotted against single nucleotide polymorphism positions on each chromosome. (B) Quantile-quantile plot, the grey shaded area represents the 95% confidence interval of expected log10 (P-values). Black dots represent the observed P-values;  = 1.022. (C) Regional association results for the chromosome 2q24.3 locus. The left y-axis represents log10 (P-values) for association with MTLEHS, the right y-axis represents the recombination rate, and the x-axis represents base-pair positions along the chromosome (human genome Build 37). The top single nucleotide polymorphism, rs7587026, is shown in purple, the rest of the single nucleotide polymorphisms are coloured according to their linkage disequilibrium r2 value with rs7587026. Expression analysis SCN1A and MTLEHS with FS Brain 2013: 136; 3140–3150 | 314 Brain 2013: 136; 3140–3150 | 3145 SCN1A and MTLEHS with FS Figure 1 The results of genome-wide association analysis in MTLEHS + FS in discovery stage. (A) Manhattan plot, log10 (P-values) of the logistic regression test are plotted against single nucleotide polymorphism positions on each chromosome. (B) Quantile-quantile plot, the grey shaded area represents the 95% confidence interval of expected log10 (P-values). Black dots represent the observed P-values;  = 1.022. (C) Regional association results for the chromosome 2q24.3 locus. The left y-axis represents log10 (P-values) for association with MTLEHS, the right y-axis represents the recombination rate, and the x-axis represents base-pair positions along the chromosome (human genome Build 37). The top single nucleotide polymorphism, rs7587026, is shown in purple, the rest of the single nucleotide polymorphisms are coloured according to their linkage disequilibrium r2 value with rs7587026. This evidence motivated our pre-analysis collection of febrile seizure data, and our previous study of febrile seizures (Petrovski et al., 2009). We performed analysis of patients in the discovery cohort with a known history of presence of childhood febrile seiz- ures (MTLEHS + FS, n = 341) (Table 2 and Fig. 1). The strongest association was for rs7587026, P = 2.64  108 [OR(A) = 1.59, 95% CI: 1.35–1.87] and rs580041, P = 8.91  107 [OR(A) = 1.56, 95% CI: 1.33–1.84], whereas the signal for rs11692675 was slightly weaker, P = 1.25  106 [OR(G) = 1.49, 95% CI: 1.27–1.75]. No association was seen in patients with MTLEHS without febrile seizures (MTLEHSFS), despite similar sample size. To refine the association signal, we performed regional imput- ation in the discovery data set of a 10 Mb region surrounding rs7587026 using the 1000 Genomes reference panel. Two single nucleotide polymorphisms had slightly lower P-values in the MTLEHS + FS analysis than the original single nucleotide poly- morphisms [rs16851603 (P = 2.23  108) and rs3919196 (P = 2.26  108)], but neither were significantly stronger than the original associations, and these signals reflected regional link- age disequilibrium structure (Supplementary Fig. 2). No known functional variants in SCN1A, nor in other genes in the region, were in high linkage disequilibrium with rs7587026. The associ- ation signal is localized within one linkage disequilibrium block that also spans the promoter and 5’ UTR region of SCN1A (Supplementary Fig. 2). Replication and combined analyses We selected the two top single nucleotide polymorphisms, rs7587026 and rs11692675, for replication in an independent sample of 959 patients with MTLEHS, of whom 416 had MTLEHS + FS, and 3591 population-matched controls of European descent from eight populations (Table 1 and Supplementary Table 1). We did not study rs580041 because of its perfect linkage disequilibrium with rs7587026 in white Europeans (r2 = 1). We detected an association between rs7587026 and MTLEHS + FS, P = 5.88  103 [OR(A) = 1.26, 95% CI: 1.07–1.48; Table 2]; this value remains significant at a revised alpha threshold of 6.3  103 after Bonferroni correction for multiple comparisons in the replication cohort. No association was detected for MTLEHSFS. To refine the association signal, we performed regional imput- ation in the discovery data set of a 10 Mb region surrounding rs7587026 using the 1000 Genomes reference panel. Two single nucleotide polymorphisms had slightly lower P-values in the MTLEHS + FS analysis than the original single nucleotide poly- morphisms [rs16851603 (P = 2.23  108) and rs3919196 (P = 2.26  108)], but neither were significantly stronger than the original associations, and these signals reflected regional link- age disequilibrium structure (Supplementary Fig. 2). No known functional variants in SCN1A, nor in other genes in the region, 3146 | Brain 2013: 136; 3140–3150 D. Kasperavicˇiu¯ t_e et al. Figure 2 Forest plot for association of rs7587026 with MTLEHS + FS. The confidence interval for each study population is given by a horizontal line, and the point estimate is given by a square whose area is inversely proportional to the standard error of the estimate. The combined odds ratio is drawn as a diamond with horizontal limits at the confidence limits and width inversely proportional to its standard error. The study populations are ordered in descending order by the number of MTLEHS + FS cases. followed children to age 13: there was no association of rs7587026 with febrile seizures in 171 individuals who did not go on to develop epilepsy (Table 3) in comparison with 6443 controls from the same cohort. The two other cohorts of children with febrile seizures, from Austria [samples partially overlapping with those reported in Schlachter et al. Replication and combined analyses (2009)] and Germany, were ascertained at a young age (to six years of age only) and had no follow-up to establish whether the children had febrile seizures only, or febrile seizures in the context of subsequent epi- lepsy including MTLEHS, and are therefore not best suited to ad- dress the question, but were examined as few cohorts overall are available. Bearing this key caveat in mind, in these two data sets there was an observed association of febrile seizures with rs7587026 (Table 3). The previously reported association in the Austrian population with an SCN1A functional splice-site single nucleotide polymorphism, rs3812718, was also seen in our Austrian sample [which is not unexpected as there is partial over- lap of cases with those in the original report (Schlachter et al., 2009)] and was present in the German sample. The observed as- sociation of rs7587026 with febrile seizures disappeared in both Austrian and German data sets when analysis was conditioned on rs3812718 (P 4 0.19; Table 3). Moreover, although the associ- ation of febrile seizures with rs3812718 may be thought to be of interest for pure febrile seizures alone, we note there is no association of rs3812718 with febrile seizures in the best charac- terized cohort, from ALSPAC (Table 3), nor in a published sample (Petrovski et al., 2009). Figure 2 Forest plot for association of rs7587026 with Figure 2 Forest plot for association of rs7587026 with g p MTLEHS + FS. The confidence interval for each study population is given by a horizontal line, and the point estimate is given by a square whose area is inversely proportional to the standard error of the estimate. The combined odds ratio is drawn as a diamond with horizontal limits at the confidence limits and width inversely proportional to its standard error. The study populations are ordered in descending order by the number of MTLEHS + FS cases. g p MTLEHS + FS. The confidence interval for each study population is given by a horizontal line, and the point estimate is given by a square whose area is inversely proportional to the standard error of the estimate. The combined odds ratio is drawn as a diamond with horizontal limits at the confidence limits and width inversely proportional to its standard error. The study populations are ordered in descending order by the number of MTLEHS + FS cases. Replication and combined analyses Thus, although other single nucleotide polymorphisms in or near SCN1A may predispose to pure febrile seizures, the signal we observed in MTLEHS + FS is very unlikely to be due to the history of febrile seizures alone. Moreover, no significant association was detected in a group of patients with other partial epilepsies with a history of febrile seizures [data set from Kasperaviciute et al. (2010); rs7587026, P = 0.24, OR(A) = 1.15, 95% CI: 0.91–1.45]. The sample for this analysis was smaller (177 patients; 7552 con- trols), but had 81% power to detect association of OR 5 1.42 (as seen in MTLEHS + FS group combined analysis) under 0.05 signifi- cance level. Collectively, we found no evidence that the MTLEHS + FS association was due to febrile seizures, or that it holds for all partial epilepsies with febrile seizures. Meta-analysis of the discovery and replication samples confirmed the association of the 2q24.3 locus with MTLEHS + FS at genome- wide significant level for rs7587026 [Pmeta = 3.36  109, OR(A) = 1.42, 95% CI: 1.26–1.59]; the signal for rs11692675 did not reach genome-wide significance [Pmeta = 4.78  106, OR(G) = 1.30, 95% CI: 1.16–1.46]. No significant heterogeneity in effect sizes was detected among different populations (Fig. 2, Woolf’s test for heterogeneity P = 0.45, see Supplementary Tables 7–10 for allele frequencies in all populations). Febrile seizure analysis The observed association could act by modulating SCN1A gene expression. The associated region harbours several alternative un- translated SCN1A exons (Martin et al., 2007; Nakayama et al., 2010). We did not detect association between rs7587026 and any protein-coding exon except one (see below) or total SCN1A expression, or with expression of untranslated 5’ exons 1a and 1b (Martin et al., 2007) (data not shown) in 78 patients and 78 control subjects. To explore whether the observed association with rs7587026 is specific for MTLEHS + FS, or is specific for febrile seizures in gen- eral, we examined a total of 7387 controls and three data sets of patients (totalling 542) who had had febrile seizures but had not developed epilepsy by the time of the latest follow-up. It has been shown that almost all children who go on to develop any type of epilepsy after febrile seizures have done so by the age of 15 years (Neligan et al., 2012). Therefore, to address the specificity of the association for MTLEHS + FS rather than febrile seizures alone, the ideal febrile seizures cohort would have been followed to age 15 at least. Of the three data sets available to us, only one met this criterion closely. The ALSPAC prospective cohort, which has the most comprehensive phenotypic data of the three data sets, The presence or absence of transcripts incorporating the ‘neonatal’ SCN1A exon 5 (‘5N’) was significantly different according to genotype of the two top single nucleotide poly- morphisms (rs11692675 and rs7587026, P-values 1.08  109 and 1.17  106, respectively; Supplementary material). Febrile seizure analysis Finally, expression quantitative trait loci analyses for subsets of patients according to a known history of presence (n = 46) or absence (n = 27) of febrile seizures in childhood for rs11692675 or rs7587026 showed significant dif- ferences in the level of expression of 5N exon according to geno- type in both MTLEHS + FS and MTLEHSFS. Including both rs11692675 or rs11692675 and rs922224 in the regression models, only rs922224 remained significant in both MTLEHS + FS and MTLEHSFS groups (Supplementary material). narrowly-defined syndromes. Because the biology of most of the epilepsies is poorly understood, there are few a priori data upon which to base selection of the range of phenotypes to include in studies of possible genetic causation. Our findings suggest that focussing on clinically recognized syndromes or constellations (Berg et al., 2010) may prove fruitful by reducing heterogeneity before genomic analyses. Our association seems to be specific for MTLEHS + FS, with no association for MTLEHSFS, febrile seizures alone or non- MTLEHS partial epilepsies with febrile seizures. Our findings sug- gest that there is genetic susceptibility to MTLEHS, and that it, or hippocampal sclerosis, may not necessarily be only acquired. The results support the concept of heterogeneity in MTLEHS, beyond that already documented clinico-pathologically (Tassi et al., 2009; Thom et al., 2010; Blu¨ mcke et al., 2012). However, further work will be needed to confirm the specificity of our findings, as we did not formally establish a significant difference in odds ratios be- tween MTLEHS + FS and MTLEHSFS. It would also be interesting to explore, in a suitably-powered study, whether there is any association with MTLE without hippocampal sclerosis. We cannot exclude the possibility that rs7587026 (or another single nucleotide polymorphism in the high linkage disequilibrium region) may act as an additional splicing controller to rs3812718, but our data are consistent with rs7587026 having no solo effect on 5N splicing. We also did not detect any correlation using a significance level of P 5 5  105 between rs7587026 and expression/splicing of any other genes across the genome (Supplementary material). Febrile seizure analysis For rs11692675 and rs7587026, respectively, none and 1% of the Brain 2013: 136; 3140–3150 | 3147 SCN1A and MTLEHS with FS Table 3 Genotype counts, allele frequencies and association results for rs7587026, rs3812718 and rs922224 in febrile seizures stage Table 3 Genotype counts, allele frequencies and association results for rs7587026, rs3812718 and rs922224 in febrile seizures stage Table 3 Genotype counts, allele frequencies and association results for rs7587026, rs3812718 and rs922224 in febrile seizures stage Population n patients n controls Minor allele Genotype count in patients Genotype count in controls Minor allele frequency in patients Minor allele frequency in controls P-value in single SNP association (allelic 2 test) P-value in conditional analysis** rs7587026 Austria 158 584 A 19/58/81 31/216/337 0.304 0.238 0.017 0.19 Germany 194 337 A 15/92/87 20/116/201 0.314 0.231 0.003 0.43 UK (ALSPAC) 171 6443 A 23/59/89 498/2550/3395 0.307 0.275 0.194 0.33* rs3812718 Austria 133 209 G 16/65/52 52/100/57 0.365 0.488 0.0015 0.030 Germany 212 344 G 32/98/82 88/166/90 0.382 0.497 0.00018 0.0012 rs922224 (proxy for rs3812718) UK (ALSPAC) 172 6456 G 34/81/57 1371/3144/1941 0.433 0.456 0.40 0.83* *Conditional analysis performed despite a non-significant single SNP association. **In conditional analyses, rs7587026 was conditioned for rs3812718 (or its proxy, rs922224, for the ALSPAC cohort), while rs3812718 and rs922224 were conditioned for rs7587026. allele frequencies and association results for rs7587026, rs3812718 and rs922224 in febrile Table 3 Genotype counts, allele frequencies and association results for rs7587026, rs381271 seizures stage individuals with the GG and AA genotype showed SCN1A transcripts in the neonatal form, compared with 83% and 81% with the genotype AA or CC. This alternative splicing event is influenced by rs3812718 (Heinzen et al., 2007). The association of alternative splicing with rs922224 (r2 = 1 with rs3812718) was stronger, P = 2.33  1031. The level of expression of SCN1A exon 5N was also significantly different according to genotype (P = 1.62  1011 for rs11692675, 2.70  106 for rs7587026, 7.40  1034 for rs922224). In conditional analyses including all three single nucleotide polymorphisms, only rs922224 remained significant (P = 1.08  1025). Febrile seizure analysis The notably weaker association in the replication stage could be due to several factors, the most important of which is the ‘win- ner’s curse’ (Ioannidis et al., 2009); there may be a large number of weak but real associations in the data, some of which achieve genome-wide significance in a particular study through random stochastic chance, but will not do so in another study. The asso- ciation in our discovery cohort was replicated in the second inde- pendent sample, but it is nevertheless important that other studies are undertaken to further replicate our findings. Other limitations of our study are the lack of genome-wide data in the replication sample, preventing direct population stratification assessment, though self-identification closely corresponds to genetically-deter- mined ancestry (Lao et al., 2008; Wang et al., 2010), a phenom- enon we confirmed in the discovery stage, and the small size of some of the replication groups, reducing replication power, and magnifying effects of undetected population admixture. Acknowledgements We would like to thank AROS Applied Biotechnology AS company laboratories and Affymetrix for their valuable input. We are grateful to the Banner Sun Health Research Institute Brain and Body Donation Program of Sun City, Arizona for the provision of human biospeci- mens contributing to gene expression analysis of nine brain re- gions from 134 control individuals. The Brain and Body Donation Program is supported by the National Institute of Neurological Disorders and Stroke (U24 NS072026 National Brain and Tissue Resource for Parkinson’s Disease and Related Disorders), the National Institute on Aging (P30 AG19610 Arizona Alzheimer’s Disease Core Centre), the Arizona Department of Health Services (contract 211002, Arizona Alzheimer’s Research Centre), the Arizona Biomedical Research Commission (contracts 4001, 0011, 05-901 and 1001 to the The location of the associated variants within SCN1A and over- lapping its promoter regions (Long et al., 2008), was suggestive of possible roles in SCN1A expression modulation. In fact, we did not detect a definitive effect on expression of SCN1A or its exons in temporal neocortex. However, this analysis may have been con- founded by many factors: effects may be brain-region or cell- population specific, as in SCN1A-related Dravet syndrome, where consequences are only found in interneurons (Ogiwara et al., 2007); our whole-tissue expression analysis would not detect such subtle signals. Moreover, noting the febrile seizures association, the effects may be temporally or spatially restricted, acting only in childhood or/and in the stress of febrile seizures (Koyama et al., 2012). Further studies will be needed to explore possible functional effects. p The detected association could act in different ways, predispos- ing to MTLEHS + FS as a distinct syndrome, or to the specific de- velopment of MTLEHS in the context of remote febrile seizures. If the association does indeed relate to SCN1A and function of the encoded protein, new lines of investigation may prove possible in the context of the existing deep knowledge of SCN1A, experimen- tal models of MTLE and in vitro study of mechanisms of hippo- campal dysfunction in epilepsy, as well as intriguing reports of the role of SCN1A in many epilepsies, such as the suggestion that mutations in SCN1A in Dravet Syndrome may protect against hippocampal sclerosis (Auvin et al., 2008; Catarino et al., 2011). Acknowledgements The Brain and Body Donation Program is supported by the National Institute of Neurological Disorders and Stroke (U24 NS072026 National Brain and Tissue Resource for Parkinson’s Disease and Related Disorders), the National Institute on Aging (P30 AG19610 Arizona Alzheimer’s Disease Core Centre), the Arizona Department of Health Services (contract 211002, Arizona Alzheimer’s Research Centre), the Arizona Biomedical Research Commission (contracts 4001, 0011, 05-901 and 1001 to the We thank all patients, their families and physicians for participat- ing in this study. We thank Drs. Goldstein, Heinzen and Radtke for use of data from patients at Duke University School of Medicine, and for their comments. We thank Drs. D. Lowenstein and A-E. Lehesjoki, and the ILAE Consortium on Complex Epilepsies (including Carolien de Kovel, Thomas Sander, Dennis Dlugos, Warren Lo, Tom Ferraro, Mike Johnson, Tony Marson, Douglas Speed, Patrick Kwan, Larry Baum, Stacey Cherny, Zhi Wei, Larry Brody, Pak Sham, David Balding and Aarno Palotie) for their sup- port of the work, and for their comments. We thank Prof J. Hardy for his support. We thank the members of the Dutch Collaborative Epilepsy Surgery Program for their cooperation. We acknowledge the Italian League against Epilepsy (LICE) collaborative group for its support. We gratefully acknowledge Professor John Hopper (School of Population Health, The University of Melbourne) for providing the control samples for the Melbourne cohort and Dr. Marian Todaro for processing the DNA samples for the Melbourne cohort; Dr. Liisa Myllykangas (Folkhalsan Institute of Genetics and Department of Pathology, University of Helsinki) and Dr. Pentti Tienari (Molecular Neurology Programme, Biomedicum, University of Helsinki and Department of Neurology, Helsinki University Central Hospital) for providing us the Vantaa85 + Study GWAS genotypes; Professor Edouard Louis (MD, PhD), Head of the Gastroenterology Unit, University Hospital Centre (CHU) of Lie`ge and Professor Michel Georges (DVM, PhD), Head of the Animal Genomics Unit, Faculty of Veterinary Medicine and Groupe Interdisciplinaire de Ge´noprote´omique Applique´e (GIGA-R) for providing us the Belgian control cohort; Drs. P. C. van Rijen and P. H. Gosselaar, Department of Neurosurgery, Rudolf Magnus Institute of Neuroscience, University Medical Centre Utrecht, The Netherlands; Christian Hengsbach for expert assistance with recruitment for the Tu¨ bingen MTLE cohort; Susanne Beyer and Ulrike Strube for the technical assistance in SNP genotyping for the Bonn MTLE cohort; and the whole ALSPAC team, which includes interviewers, computer and labora- tory technicians, clerical workers, research scientists, volunteers, managers, receptionists and nurses. Discussion We show that common variation in and near SCN1A may increase susceptibility to MTLEHS + FS. Our previously published larger genome-wide association study for a broader range of focal epilepsies did not identify any single-single nucleotide polymorph- ism association (Kasperaviciu¯ te et al., 2010), but the findings here demonstrate that associated variants may exist for more 3148 | Brain 2013: 136; 3140–3150 3148 | Brain 2013: 136; 3140–3150 D. Kasperavicˇiu¯ t_e et al. As for many genome-wide association studies, we could not narrow the association to a single gene or functional variant. There are other genes designated ‘SCNxA’ in the vicinity: SCN3A, SCN2A, SCN9A and SCN7A (this last does not show any sodium channel activity in exogenous expression systems) (Meisler et al., 2010). Among these genes, SCN2A has the most published evidence to support its role in the epilepsies. We cannot exclude the possibility that the association is driven by deleterious variants in these or other nearby genes. SCN1A, however, emerges as the most plausible candidate, due both to its proximity to the associated region and its role in other epilepsies with febrile seizures. Notably, our association is with a syndrome involving hippocampal damage, whereas typically no hippocampal damage is observed in patients with Dravet syndrome caused by deleteri- ous changes affecting SCN1A (Catarino et al., 2011), suggesting that SCN1A might influence epileptogenesis through various mechanisms. contribute to understanding the risk of developing MTLEHS after febrile seizures. Acknowledgements g We thank all patients, their families and physicians for participat- ing in this study. We thank Drs. Goldstein, Heinzen and Radtke for use of data from patients at Duke University School of Medicine, and for their comments. We thank Drs. D. Lowenstein and A-E. Lehesjoki, and the ILAE Consortium on Complex Epilepsies (including Carolien de Kovel, Thomas Sander, Dennis Dlugos, Warren Lo, Tom Ferraro, Mike Johnson, Tony Marson, Douglas Speed, Patrick Kwan, Larry Baum, Stacey Cherny, Zhi Wei, Larry Brody, Pak Sham, David Balding and Aarno Palotie) for their sup- port of the work, and for their comments. We thank Prof J. Hardy for his support. We thank the members of the Dutch Collaborative Epilepsy Surgery Program for their cooperation. We acknowledge the Italian League against Epilepsy (LICE) collaborative group for its support. We gratefully acknowledge Professor John Hopper (School of Population Health, The University of Melbourne) for providing the control samples for the Melbourne cohort and Dr. Marian Todaro for processing the DNA samples for the Melbourne cohort; Dr. Liisa Myllykangas (Folkhalsan Institute of Genetics and Department of Pathology, University of Helsinki) and Dr. Pentti Tienari (Molecular Neurology Programme, Biomedicum, University of Helsinki and Department of Neurology, Helsinki University Central Hospital) for providing us the Vantaa85 + Study GWAS genotypes; Professor Edouard Louis (MD, PhD), Head of the Gastroenterology Unit, University Hospital Centre (CHU) of Lie`ge and Professor Michel Georges (DVM, PhD), Head of the Animal Genomics Unit, Faculty of Veterinary Medicine and Groupe Interdisciplinaire de Ge´noprote´omique Applique´e (GIGA-R) for providing us the Belgian control cohort; Drs. P. C. van Rijen and P. H. Gosselaar, Department of Neurosurgery, Rudolf Magnus Institute of Neuroscience, University Medical Centre Utrecht, The Netherlands; Christian Hengsbach for expert assistance with recruitment for the Tu¨ bingen MTLE cohort; Susanne Beyer and Ulrike Strube for the technical assistance in SNP genotyping for the Bonn MTLE cohort; and the whole ALSPAC team, which includes interviewers, computer and labora- tory technicians, clerical workers, research scientists, volunteers, managers, receptionists and nurses. We would like to thank AROS Applied Biotechnology AS company laboratories and Affymetrix for their valuable input. We are grateful to the Banner Sun Health Research Institute Brain and Body Donation Program of Sun City, Arizona for the provision of human biospeci- mens contributing to gene expression analysis of nine brain re- gions from 134 control individuals. Funding This study makes use of data generated by the Wellcome Trust Case-Control Consortium (a full list of the investigators who con- tributed to the generation of the data is available from www. wtccc.org.uk; funding for the project was provided by the Wellcome Trust under award 076113 and 085475) and the Irish Amyotrophic Lateral Sclerosis Study (funding support was pro- vided by Muscular Dystrophy Association, USA, Irish Institute of Clinical Neurosciences Travel Award, and National Institutes of Health, USA; additional genotyping was provided by the National Institute of Neurological Disorders and Stroke (NINDS); the dataset used for the analyses described in this manuscript were obtained from the NINDS Database found at http://www.ncbi. nlm.nih.gov/gap through dbGaP accession number phs000127.v1.p1). Supported by the Wellcome Trust (grant 084730 to S.M.S., N.D, C.D.); the UK Medical Research Council (MRC grants G0400126 to S.M.S., G1100616 to C.S., G0901254 to J.H. and M.E.W., and Training Fellowship G0802462 to M.R.); the King Faisal Specialist Hospital and Research Centre (KFSH & RC grant to D.T.); the University College London Hospitals Charities and the Clinical Research and Development Committee (UCLH/CRDC grant F136 to S.M.S.); the National Institute for Health Research (NIHR grant 08-08-SCC to S.M.S.); the National Society for Epilepsy; the National Institute for Health Research University College London Hospitals Biomedical Research Centre; Marie Curie International Re-integration Grant (FP7-PEOPLE-2009-RG grant No 256545 to M.M. and S.M.S.); the European Commission (FP7 project EpiPGX, grant 279062 to H.L., S.M.S and W.S.K.; FP6 project Epicure, grant LSHM-CT-2006-037315 to H.L., P.S.R. and F.R.); the Robert Bosch Foundation, Stuttgart, and the University of Tu¨ bingen (IZEPHA project 18-0-0, grant to H.L.); the German Federal Ministry of Education and Research, National Genome Research Network (NGFNplus: EMINet grant 01GS08123 to H.L., S.S., A.J.B.); BONFOR (S.S., A.J.B.); the German Federal Ministry of Education and Research (independent research groups in neuroscience); the German Research Foundation (EUROCORES program, EuroEPINOMICS-RES grant RO3396/2-1 to P.S.R. and F.R. and EuroEPINOMICS-RES grant DFG Bl421/3-1 to I.B. and K.K.); the Austrian Science Fund (project FWF I643, grant to F. Zimprich); the National Health and Medical Research Council (NHMRC grant 628952 to S.F.B.); the Royal Melbourne Hospital Neuroscience Foundation (grant to T.J.O’B.); the Foundation of Science and Technology, Lisbon (FCT grant PIC/ IC/83297/2007 to B.M.S.); the National Institutes of Health, USA (NIH grants R01-NS-49306-01 to R.J.B. and R01-NS- 064154-01 to R.J.B. Funding and H.H.); the collection of the Irish patient cohort was supported by the Irish Higher Education Authority Programme for Research in Third Level Institutions (PRTLI3) through a Science Foundation Ireland Research Frontiers Programme award (08/RFP/GEN1538) and a Medical Research Charities Group of Ireland/Health Research Board award (2009/ 001) from Brainwave–the Irish Epilepsy Association. GlaxoSmithKline funded the recruitment and phenotypic data col- lection of the GenEpA Consortium samples used in this study and contributed to the genotyping costs associated with their study. The collection of the Belgian patients was supported by the Fonds National de la Recherche Scientifique, grant n. FC 63574/ 3.4.620.06 F, and the Fonds Erasme, Universite´ Libre de Bruxelles. The collection of the Belgian control cohort was sup- ported by the Walloon Region and the French-Speaking Community of Belgium, the Belgian Science Policy and the University of Lie`ge. J.N. is supported by the Swiss National Science Foundation-Fellowships for prospective researchers and the SICPA Foundation, Prilly, Switzerland. Computing facilities used at King’s College London were supported by the National Acknowledgements Stratifying by febrile seizures type could also prove illuminating, as prolonged, lateralized or repeated febrile seizures within a short interval may have different effects to ‘uncomplicated’ febrile seiz- ures. Our retrospective febrile seizures data were insufficiently resolved to permit such analysis. This is an important avenue for further investigation, because no predictors exist for the develop- ment of epilepsy in the 3% of all the children who have febrile seizures, and because established MTLEHS can have devastating consequences. Eventual reliable prediction of significant risk of MTLEHS after febrile seizures could lead to novel preventative measures in at-risk individuals: here, we note that SCN1A encodes an important anti-epileptic drug target and that it is possible to pharmacologically prevent the development of epilepsy after febrile seizures in an animal model (Koyama et al., 2012). Our findings suggest that further work on SCN1A variation may Brain 2013: 136; 3140–3150 | SCN1A and MTLEHS with FS SCN1A and MTLEHS with FS 3149 3149 Arizona Parkinson’s Disease Consortium) and the Michael J. Fox Foundation for Parkinson’s Research. Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London. Funding support for the ALSPAC was provided by The UK Medical Research Council (grant refs: 74882) the Wellcome Trust (grant refs: 076467) and the University of Bristol. References Ogiwara I, Miyamoto H, Morita N, Atapour N, Mazaki E, Inoue I, et al. Nav1.1 localizes to axons of parvalbumin-positive inhibitory inter- neurons: a circuit basis for epileptic seizures in mice carrying an Scn1a gene mutation. J Neurosci 2007; 27: 5903–14. p Cendes F. Febrile seizures and mesial temporal sclerosis. Curr Opin Neurol 2004; 17: 161–4. Oliva M, Berkovic SF, Petrou S. Sodium channels and the neurobiology of epilepsy. Epilepsia 2012; 53: 1849–59. De Tisi J, Bell GS, Peacock JL, McEvoy AW, Harkness WF, Sander JW, et al. The long-term outcome of adult epilepsy surgery, patterns of seizure remission, and relapse: a cohort study. Lancet 2011; 378: 1388–95. Petrovski S, Scheffer IE, Sisodiya SM, O’Brien TJ, Berkovic SF. Lack of replication of association between scn1a SNP and febrile seizures. Neurology 2009; 73: 1928–30. Heinzen EL, Yoon W, Tate SK, Sen A, Wood NW, Sisodiya SM, et al. Nova2 interacts with a cis-acting polymorphism to influence the pro- portions of drug-responsive splice variants of SCN1A. Am J Hum Genet 2007; 80: 876–83. Pittau F, Bisulli F, Mai R, Fares JE, Vignatelli L, Labate A, et al. Prognostic factors in patients with mesial temporal lobe epilepsy. Epilepsia 2009; 50 (Suppl 1): 41–4. Purcell S, Cherny SS, Sham PC. Genetic power calculator: design of link- age and association genetic mapping studies of complex traits. Bioinformatics 2003; 19: 149–50. Howie B, Fuchsberger C, Stephens M, Marchini J, Abecasis GR. Fast and accurate genotype imputation in genome-wide association studies through pre-phasing. Nat Genet 2012; 44: 955–9. Schlachter K, Gruber-Sedlmayr U, Stogmann E, Lausecker M, Hotzy C, Balzar J, et al. A splice site variant in the sodium channel gene SCN1A confers risk of febrile seizures. Neurology 2009; 72: 974–8. Ioannidis JP, Thomas G, Daly MJ. Validating, augmenting and refining genome-wide association signals. Nat Rev Genet 2009; 10: 318–29. Kasperaviciu¯ te D, Catarino CB, Heinzen EL, Depondt C, Cavalleri GL, Caboclo LO, et al. Common genetic variation and susceptibility to partial epilepsies: a genome-wide association study. Brain 2010; 133: 2136–47. Tassi L, Meroni A, Deleo F, Villani F, Mai R, Russo GL, et al. Temporal lobe epilepsy: neuropathological and clinical correlations in 243 surgi- cally treated patients. Epileptic Disord 2009; 11: 281–92. Thom M, Mathern GW, Cross JH, Bertram EH. Mesial temporal lobe epilepsy: how do we improve surgical outcome? Ann Neurol 2010; 68: 424–34. References Mantegazza M, Gambardella A, Rusconi R, Schiavon E, Annesi F, Cassulini RR, et al. Identification of an Nav1.1 sodium channel (SCN1A) loss-of-function mutation associated with familial simple fe- brile seizures. Proc Natl Acad.Sci USA 2005; 102: 18177–82. 1000 Genomes Project Consortium, Abecasis GR, Altshuler D, Auton A, Brooks L, Durbin R, et al. A map of human genome variation from population-scale sequencing. Nature 2010; 467: 1061–73. Abou-Khalil B, Ge Q, Desai R, Ryther R, Bazyk A, Bailey R, et al. Partial and generalized epilepsy with febrile seizures plus and a novel SCN1A mutation. Neurology 2001; 57: 2265–72. Martin MS, Tang B, Ta N, Escayg A. Characterization of 5’ untranslated regions of the voltage-gated sodium channels SCN1A, SCN2A, and SCN3A and identification of cis-conserved noncoding sequences. Genomics 2007; 90: 225–35. Andrade-Valenc¸a LP, Valenc¸a MM, Velasco TR, Carlotti CG Jr, Assirati JA, Galvis-Alonso OY, et al. Mesial temporal lobe epilepsy: clinical and neuropathologic findings of familial and sporadic forms. Epilepsia 2008; 49: 1046–54. Meisler MH, O’Brien JE, Sharkey LM. Sodium channel gene family: epi- lepsy mutations, gene interactions and modifier effects. J Physiol (Lond.) 2010; 588: 1841–8. p p Auvin S, Dulac O, Valle´e L. Do SCN1A mutations protect from hippo- campal sclerosis? Epilepsia 2008; 49: 1107–8. Nakayama T, Ogiwara I, Ito K, Kaneda M, Mazaki E, Osaka H, et al. Deletions of SCN1A 5’ genomic region with promoter activity in Dravet syndrome. Hum Mutat 2010; 31: 820–9. Berg AT, Berkovic SF, Brodie MJ, Buchhalter J, Cross JH, van Emde Boas W, et al. Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005-2009. Epilepsia 2010; 51: 676–85. Neligan A, Bell GS, Giavasi C, Johnson AL, Goodridge DM, Shorvon SD, et al. Long-term risk of developing epilepsy after febrile seizures: a prospective cohort study. Neurology 2012; 78: 1166–70. O’Dell CM, Das A, Wallace G 4th, Ray SK, Banik NL. Understanding the basic mechanisms underlying seizures in mesial temporal lobe epilepsy and possible therapeutic targets: a review. 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Conflict of interest Y.W. has served on scientific advisory boards for UCB Pharma and has received funding for travel and speaker honoraria from UCB, Desitin Pharmaceuticals, GmbH, and Eisai Inc.; H.L. has served on scientific advisory boards for Eisai Inc., GlaxoSmithKline, Pfizer Inc, UCB, and Valeant Pharmaceuticals International, has received funding for travel from GlaxoSmithKline, Medtronic, Pfizer and UCB and speaker honoraria from Desitin Pharmaceuticals, GmbH, Eisai Inc., GlaxoSmithKline, Pfizer, and UCB, and has received research support from Sanofi-Aventis, UCB, DFG, BMBF, and the EU; J.C. has received funding from the Tecnifar group (BICE Tecnifar Grant 2009); F.R. has received within the last two years honoraria as scientific advisor from GSK, EISAI, UCB and Pfizer, and has received speaker honoraria from UCB, GSK, Eisai, Desitin and Medtronic and educational grants from Nihon- Kohden, UCB, Medtronics, Cyberonics and Cerbomed (F.R. has, however, no conflicts of interest regarding this study); P.S.R. has received travel grants from UCB; G.L.C has received research funding from UCB and speaker honoraria from Eisai; S.M.S has received research funding or personal/institutional honoraria from UCB Pharma, GlaxoSmithKline and Eisai Inc; H.M.H. has served on the scientific advisory board of Eisai, Pfizer, GlaxoSmithKline and UCB Pharma, has served on the speakers’ bureau of Desitin, Eisai, GlaxoSmithKline and UCB Pharma and received research funding from Desitin, Janssen-Cilag, GlaxoSmithKline and UCB Pharma; I.B. received speaker honoraria from Desitin Pharmaceuticals, GmbH, Eisai Inc., and UCB, and has received research support from Boehringer-Ingelheim; R.K.K. has served on scientific advis- ory boards for UCB Pharma, Eisai, Lundbeck, GlaxoSmithKline, and Fennomedical and has received funding for travel and speaker honoraria from UCB Pharma, Eisai, GlaxoSmithKline, Medtronic, Pfizer, Orion, Fennomedical and institutional research funding from UCB Pharma and GlaxoSmithKline; A-M.K has received funding for travel from UCB Pharma, Eisai, Abbott and Biogen; S.F.B. is an inventor on a patent for SCN1A testing held by 3150 | Brain 2013: 136; 3140–3150 D. Kasperavicˇiu¯ t_e et al. Lao O, Lu TT, Nothnagel M, Junge O, Freitag-Wolf S, Caliebe A, et al. Correlation between genetic and geographic structure in Europe. Curr Biol 2008; 18: 1241–8. Bionomics Inc and licensed to various diagnostic companies. All other authors declare that they have no conflicts of interest. Li Y, Willer CJ, Ding J, Scheet P, Abecasis GR. MaCH: using sequence and genotype data to estimate haplotypes and unobserved genotypes. Genet Epidemiol 2010; 34: 816–34. Supplementary material is available at Brain online. Ma¨gi R, Morris AP. GWAMA: software for genome-wide association meta-analysis. BMC Bioinformatics 2010; 11: 288. Supplementary material Long YS, Zhao QH, Su T, Cai YL, Zeng Y, Shi YW, et al. Identification of the promoter region and the 5’-untranslated exons of the human volt- age-gated sodium channel Nav1.1 gene (SCN1A) and enhancement of gene expression by the 5’-untranslated exons. J. Neurosci Res 2008; 86: 3375–81. Supplementary material is available at Brain online. References Koyama R, Tao K, Sasaki T, Ichikawa J, Miyamoto D, Muramatsu R, et al. GABAergic excitation after febrile seizures induces ectopic granule cells and adult epilepsy [Internet]. Nat Med 2012; 18: 1271–8. Available from: http://www.ncbi.nlm.nih.gov/pubmed/ 22797810 (13 September 2012, date last accessed). Wang H, Haiman CA, Kolonel LN, Henderson BE, Wilkens LR, Le Marchand L, et al. Self-reported ethnicity, genetic structure and the impact of population stratification in a multiethnic study. Hum Genet 2010; 128: 165–77. Labate A, Gambardella A, Andermann E, Aguglia U, Cendes F, Berkovic SF, et al. Benign mesial temporal lobe epilepsy. Nat Rev Neurol 2011; 7: 237–40. Wieser HG. ILAE Commission Report. Mesial temporal lobe epilepsy with hippocampal sclerosis. Epilepsia 2004; 45: 695–714.
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https://www.qeios.com/read/WGMVBT/pdf
cy
Brachydactyly-syndactyly, Zhao type
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93
Qeios · Definition, February 10, 2020 Ope n Pe e r Re v ie w on Qe ios Brachydactyly-syndactyly, Zhao type INSERM Source INSERM. (1999). Orphanet: an online rare disease and orphan drug data base. Brachydactyly-syndactyly, Zhao type. ORPHA:93409 Brachydactyly-syndactyly, Zhao type is a recently described syndrome associating a brachydactyly type A4 (short middle phalanges of the 2nd and 5th fingers and absence of middle phalanges of the 2nd to 5th toes) and a syndactyly of the 2nd and 3rd toes. Metacarpals and metatarsals anomalies are common. Qeios ID: WGMVBT · https://doi.org/10.32388/WGMVBT 1/1
https://openalex.org/W2998810908
https://europepmc.org/articles/pmc6988671?pdf=render
English
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The Threshold of the Severity of Diabetic Retinopathy below Which Intensive Glycemic Control Is Beneficial in Diabetic Patients: Estimation Using Data from Large Randomized Clinical Trials
Journal of diabetes research
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Hindawi Journal of Diabetes Research Volume 2020, Article ID 8765139, 6 pages https://doi.org/10.1155/2020/8765139 Hindawi Journal of Diabetes Research Volume 2020, Article ID 8765139, 6 pages https://doi.org/10.1155/2020/8765139 Hindawi Journal of Diabetes Research Volume 2020, Article ID 8765139, 6 pages https://doi.org/10.1155/2020/8765139 Hindawi Journal of Diabetes Research Volume 2020, Article ID 8765139, 6 pages https://doi.org/10.1155/2020/8765139 Review Article The Threshold of the Severity of Diabetic Retinopathy below Which Intensive Glycemic Control Is Beneficial in Diabetic Patients: Estimation Using Data from Large Randomized Clinical Trials Yuqi Liu, Juan Li , Jinfang Ma, and Nanwei Tong Department of Endocrinology and Metabolism, West China Hospital of Sichuan University, Chengdu, 610041 Sichuan, China Department of Endocrinology and Metabolism, West China Hospital of Sichuan University, Chengdu, 610041 Sichuan, China of Endocrinology and Metabolism, West China Hospital of Sichuan University, Chengdu, 610041 Sichuan, China Correspondence should be addressed to Nanwei Tong; tongnw@scu.edu.cn Received 24 July 2019; Revised 13 December 2019; Accepted 3 January 2020; Published 17 January 2020 Academic Editor: Erifili Hatziagelaki Copyright © 2020 Yuqi Liu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Intensive glucose therapy can protect the retina of individuals with diabetes, but it is unknown if it provides the same protection to patients with different severity of diabetic retinopathy (DR). We finally included DR-related studies involving intensive glucose control with large sample size and long follow-up time, including five large and high-quality randomized clinical trials (RCTs): DCCT, UKPDS, ACCORD, AdRem, and VADT. With DCCT as a reference, we supposed a DR severity threshold that is verified by other RCTs then. We found that individuals who have DR lesions that are equivalent to or less severe than moderate NPDR achieve benefits for the retina by intensive glycemic control. However, these are realized only if the HbA1c in type 1 or type 2 diabetic patients is reduced at least by 0.8% versus the control group or it is reduced to <7% and >3 years of intensive glucose control is required. If the severity of DR lesions is worse than moderate NPDR, intensive glycemic control may not bring benefits. 1. Introduction (UKPDS), and their follow-up studies [4, 5], showed a legacy effect of intensive glucose control in terms of macrovascular protection in those patients without established atheroscle- rotic cardiovascular disease (ASCVD). However, a previously published meta-analysis found that intensive glucose control does not have such a legacy effect in diabetic patients with established or very high-risk ASCVD [6]. The current evi- dence suggests that intensive glycemic control can reduce the incidence of diabetic retinopathy and delay the progres- sion of retinopathy in patients with type 1 or type 2 diabetes in the DCCT [3], its follow-up study [7], and UKPDS [4], but there is few focused on whether disparate effects exist in indi- viduals with different DR conditions. Diabetic retinopathy (DR) is a progressive disease that can be divided into two stages: the earlier stage is referred to as “nonproliferative diabetic retinopathy” (NPDR) and the later stage as “proliferative diabetic retinopathy” (PDR). NPDR is characterized by weakness of the capillary wall, microaneur- ysm formation and fluid leakage, and greater adhesion of leu- kocytes and monocytes to the endothelium [1]. Conversely, the proliferative stage is characterized by the development of new retinal blood vessels and fibrous tissue at the optic disc or near venules elsewhere in the retina. The appearance of these vessels is associated with nonperfusion or edema in the macula, vitreous hemorrhage, and distortions or traction retinal detachment, leading to loss of vision [2]. The retinal microvascular protection provided by inten- sive glucose therapy may depend on the severity of the lesions. We hypothesized that there is a particular threshold of lesion severity below which hypoglycemic therapy, espe- cially intensive glycemic control or glycemia-dependent reti- nal microvascular protection, can be effective. Conversely, Studies of intensive glycemic control in patients with newly diagnosed type 1 diabetes and type 2 diabetes, includ- ing the Diabetes Control and Complications Trials (DCCT) [3] and the United Kingdom Prospective Diabetes Study Journal of Diabetes Research 2 Table 1: Characteristics of included studies. Table 1: Characteristics of included studies. Study Author and year Country Age (years) (IG vs. CG) DM type Follow-up (years) HbA1c at baseline (IG vs. CG) Achieved HbA1c (IG vs. CG) DCCT DCCT group, 1993 USA 27 T1DM 10 Primary prevention: 8.8%; secondary prevention: 8.9% 7.2% vs. 8.0% UKPDS UKPDS group, 1998 UK 63 T2DM 15 9.3% vs. 9.4% 7.1% vs. 7.9% VADT Duckworth 2009, Azad 2014 USA 60 T2DM 6 9.3% vs. 2. Search Strategy and Selection Criteria The study was of 1,441 participants and included a primary prevention cohort of 726 patients who had no retinopathy and a secondary intervention cohort of 200 individuals with microangioma or nonproliferative DR in 715 participants [3]. The secondary intervention cohort excluded patients with severe NPDR and those with more severe DR, meaning that the fundic status of all the subjects in this group was at or below the moderate NPDR level of the International Clinical DR Disease Severity Scale. The mean HbA1c at baseline was 8.8%, and the mean follow-up duration was 6.4 years. 3.2. Identification of the Hypothesized DR Severity Threshold. Because DR is a classical and specific complication, the sever- ity of which is closely related to blood glucose concentration in type 1 diabetes, we first decided to identify such a thresh- old using data from the DCCT. The DCCT study, initiated in 1983, was an RCT conducted in patients with type 1 diabetes. The study was of 1,441 participants and included a primary prevention cohort of 726 patients who had no retinopathy and a secondary intervention cohort of 200 individuals with microangioma or nonproliferative DR in 715 participants [3]. 3.2. Identification of the Hypothesized DR Severity Threshold. Because DR is a classical and specific complication, the sever- ity of which is closely related to blood glucose concentration in type 1 diabetes, we first decided to identify such a thresh- old using data from the DCCT. The DCCT study, initiated in 1983, was an RCT conducted in patients with type 1 diabetes. The study was of 1,441 participants and included a primary prevention cohort of 726 patients who had no retinopathy and a secondary intervention cohort of 200 individuals with microangioma or nonproliferative DR in 715 participants [3]. 1. Introduction 9.4% 6.9% vs. 8.4% ACCORD ACCORD group, 2010 USA 61.6 T2DM 4 8.2% vs. 8.2% 7.1% vs. 9.4% AdRem Beulens, 2009 20 countries 66 T2DM 4.1 7.4% vs. 7.4% 6.49% vs. 7.24% DM = diabetes; T1DM = type 1 diabetes; T2DM = type 2 diabetes; IG = intensive glycemia control group; CG = conventional glycemic control group. ype 1 diabetes; T2DM = type 2 diabetes; IG = intensive glycemia control group; CG = conventional glycemic control group. Disease Severity Scale (DR International Classification Standard, Table 2) and the International Clinical Diabetic Macular Edema Disease Severity Scale were developed in Sydney in 2002 by the American Academy of Ophthalmol- ogy (AAO), involving representatives from many countries. The standard scale provides an important basis for standard- izing epidemiological investigations of DR, facilitates com- munication among community doctors, endocrinologists, and ophthalmologists, and is widely applied internationally [11]. The UKPDS [4] made minor adjustments to the ETDRS grading scale (Table 3), but this did not affect the uniformity of the severity scales (Table 2). The methods of fundic assess- ment and grading in studies we included were almost identi- cal to those of the UKPDS. once the lesion reaches a certain degree of severity, intensive glycemic control will not have microvascular benefits. In this review, we aim to infer the threshold of DR lesion severity below which intensive therapy could have protective effects, but above which it would have no benefits for patients. We have pursued this aim by reviewing high-quality published randomized clinical trials (RCTs) of intensive blood glucose control in diabetic patients. Disease Severity Scale (DR International Classification Standard, Table 2) and the International Clinical Diabetic Macular Edema Disease Severity Scale were developed in Sydney in 2002 by the American Academy of Ophthalmol- ogy (AAO), involving representatives from many countries. The standard scale provides an important basis for standard- izing epidemiological investigations of DR, facilitates com- munication among community doctors, endocrinologists, and ophthalmologists, and is widely applied internationally [11]. The UKPDS [4] made minor adjustments to the ETDRS grading scale (Table 3), but this did not affect the uniformity of the severity scales (Table 2). The methods of fundic assess- ment and grading in studies we included were almost identi- cal to those of the UKPDS. 2. Search Strategy and Selection Criteria We screened published literature searched according to the search strategy (Supplement Table 1). Randomized controlled trials were included if they separately assessed the effects of intensive glycemic control in individuals with type 1 or type 2 diabetes and diabetic retinopathy has been reported and if they had at least a 3-year follow-up in both groups and had more than 500 participant-years in each treatment group. The intensive glycemic control has been defined as maintenance of glycated hemoglobin A1c (HbA1c) concentration at ≤7% (53 mmol/mol) in the intervention group or a difference in HbA1c between the intervention and conventional management groups of ≥0.8% [8]. Trials were excluded if they were in a non-English publication or with multifactorial interventions that cannot separate assessment of the effects of glycemic control. In summary, the International Clinical DR Disease Sever- ity Scale can be used to assess the severity of DR in subjects in the studies we included, and the criteria used can be regarded as consistent. In these studies, the progression of DR was defined as the increase in severity of DR from baseline at follow-up of ≥2 or ≥3 steps in the ETDRS grading system, where an increase of 1 step means that the severity of the lesion has progressed from the original level to the next more severe level. Analysis of the ACCORD study showed that the definition of progres- sion used (>1, 2, or 3 steps) does not affect the results of the study, so it can be considered that the definition of DR progression is similar for each study [12]. The primary outcomes were new-onset or any progres- sion in diabetic retinopathy which was defined as a composite of progression of DR by at least two steps on the Early Treat- ment of Diabetic Retinopathy Study (ETDRS) severity scale, development of proliferative retinopathy, or requirement for retinal photocoagulation therapy or vitrectomy. 3.2. Identification of the Hypothesized DR Severity Threshold. Because DR is a classical and specific complication, the sever- ity of which is closely related to blood glucose concentration in type 1 diabetes, we first decided to identify such a thresh- old using data from the DCCT. The DCCT study, initiated in 1983, was an RCT conducted in patients with type 1 diabetes. 3. Determination of the DR Severity Threshold below Which Intensive Glycemic Control Has Benefits for the Retina 3.1. Homogeneity of Assessment of DR Severity in RCTs. Finally, five trials were included in this review (Table 1). Although each study was different, the description and classification of DR were based on the Early Treatment of Diabetic Retinopathy Study (ETDRS) [9]. Based on data from the ETDRS and the Wisconsin Epidemiological Study on Diabetic Retinopathy (WESDR) [10], the Retinopathy The secondary intervention cohort excluded patients with severe NPDR and those with more severe DR, meaning that the fundic status of all the subjects in this group was at or below the moderate NPDR level of the International Clinical DR Disease Severity Scale. The mean HbA1c at baseline was 8.8%, and the mean follow-up duration was 6.4 years. Journal of Diabetes Research 3 he Diabetic Retinopathy Disease Severity and International Clinical Diabetic Retinopathy Disease Severity Scales Table 2: The Diabetic Retinopathy Disease Severity and International Clinical Diabetic Retinopathy Disease Severity Scales. Disease severity level Findings observable upon dilated ophthalmoscopy No apparent retinopathy No abnormalities Mild NPDR Microaneurysms only Moderate NPDR More than just microaneurysms but less than severe NPDR Severe NPDR U.S. definition Any of the following (4-2-1 rule) and no signs of proliferative retinopathy: (i) Severe intraretinal hemorrhages and microaneurysms in each of four quadrants (ii) Definite venous beading in two or more quadrants (iii) Prominent IRMA in one or more quadrants International definition Any of the following and no signs of proliferative retinopathy: (i) More than 20 intraretinal hemorrhages in each of four quadrants (ii) Definite venous beading in two or more quadrants (iii) Prominent IRMA in one or more quadrants PDR One or both of the following: (i) Neovascularization (ii) Vitreous/preretinal hemorrhage IRMA = intraretinal microvascular abnormalities; NPDR = nonproliferative diabetic retinopathy; PDR = proliferative diabetic retinopathy. Note: any patient with two or more of the characteristics of severe NPDR is considered to have very severe NPDR. PDR may be classified as high risk or not high risk. PDR Table 3: The grading system from the Early Treatment of Diabetic Retinopathy Study. Table 3: The grading system from the Early Treatment of Diabetic Retinopathy Study. 3. Determination of the DR Severity Threshold below Which Intensive Glycemic Control Has Benefits for the Retina Level Severity Definitions Scale step 10/10 DR absent All diabetic retinopathy features absent 1 20/<20 MA only Microaneurysm(s) only, other lesions absent, one eye 2 20/20 MA only Microaneurysm(s) only, other lesions absent, both eyes 3 35/<35 Mild NPDR MA plus retinal hemorrhage(s) and/or hard exudates and/or cotton wool spots, one eye 4 35/35 Mild NPDR MA plus retinal hemorrhage(s) and/or hard exudates and/or cotton wool spots, both eyes 5 43/<43 Moderate NPDR Lesions as above + either extensive or severe HMA or IRMA present, one eye 6 43/43 Moderate NPDR Lesions as above + either extensive or severe HMA or IRMA present, both eyes 7 47/<47 Moderately severe NPDR Lesions of 35 + either extensive or severe HMA with IRMA or venous beading, one eye 8 47/47 Moderately severe NPDR Lesions of 35 + either extensive or severe HMA with IRMA or venous beading, both eyes 9 53/<53 Severe NPDR Extensive and severe HMA, IRMA, and/or venous beading, one eye 10 53/53 Severe NPDR Extensive and severe HMA, IRMA, and/or venous beading, both eyes 11 60, 61, 65, 71, 75, 81 Proliferative DR NVD and/or NVE without or with complication 12+ DR = diabetic retinopathy; NPDR = nonprotective diabetic retinopathy; MA = microaneurysm; HMA = hemorrhages and microaneurysms; HE = hard exudates; CWS = cotton wool spots; IRMA = intraretinal microvascular abnormalities; NVD = new vessels on the disc; NVE = new vessels elsewhere. DR = diabetic retinopathy; NPDR = nonprotective diabetic retinopathy; MA = microaneurysm; HMA = hemorrhages and xudates; CWS = cotton wool spots; IRMA = intraretinal microvascular abnormalities; NVD = new vessels on the disc; NVE = n At the end of the study, the mean HbA1c concentrations achieved were 7.2% and 8.0% in the intensive glycemic control and conventional management groups, respectively. The endpoints were the incidence of DR and progression of DR, with the progression of DR being defined as a severity increment of ≥3 steps in the ETDRS grading system between baseline and follow-up. that in the conventional treatment group, and after a mean 6-year follow-up, intensive glucose control reduced the adjusted mean risk of retinopathy by 76%. The reduction in risk increased with time [3]. The patients in the intensive glycemic control group in the secondary intervention cohort had a higher cumulative incidence of progression of DR. 3. Determination of the DR Severity Threshold below Which Intensive Glycemic Control Has Benefits for the Retina Intensive treatment reduced the mean risk of DR progression by 54% over the entire study period (n = 77 events in the intensive group and n = 143 events in the conventional group). It appears that intensive glycemic control can delay the progression of DR in patients whose lesion severity is less than or equal to the moderate NPDR on the International Clinical DR Disease Severity In the primary prevention cohort of the study, there was a significant difference in the cumulative incidence of DR between the two groups at 36 months, which is why we only included RCTs with more than a 3-year follow-up. From 5 years onward, the cumulative incidence of retinopathy in the intensive therapy group was approximately 50% less than Journal of Diabetes Research 4 difference between the intensive glycemic control group and the control group if progression was defined as >4 steps, this may be because few patients progressed this much, meaning that the analysis was insufficiently powered or that most patients whose DR progressed >4 steps had undergone fur- ther photocoagulation. However, the ACCORD trial did show that intensive glycemic control delays the progression of DR, especially in patients with unilateral or bilateral microaneurysms or those with mild NPDR signs in one eye. Scale. Therefore, we contend that the DR severity threshold below which significant retinal benefits of intensive glucose control accrue is no more than that consistent with moderate NPDR, as defined in the International Clinical DR Disease Severity Scale. 3.3. Verification of the Determined DR Severity Threshold. The UKPDS included 3,867 newly diagnosed type 2 diabetes patients with a median age of 54 years, 64% of whom had no NPDR at the time of admission, 24% had mild NPDR, 10% had moderate NPDR, 2% had severe NPDR, and 0.1% had PDR, meaning that >95% of the participants had no DR, or their DR was less severe or equivalent to moderate NPDR at the beginning of the study [4, 13]. After 10 years of fol- low-up, the HbA1c concentration achieved in the intensive glycemic control group was 7.0%, compared with 7.9% in the conventional therapy group. At the commencement of the study and then every 3 years, all the participants under- went a full clinical examination, including tests of visual acu- ity and ophthalmoscopy following pupillary dilation. 3. Determination of the DR Severity Threshold below Which Intensive Glycemic Control Has Benefits for the Retina There was a significant difference in the incidence of progression of DR between the groups from the sixth year onwards. The results suggested that intensive glycemic control slowed the progression of DR, and it is also consistent with intensive blood glucose control having a beneficial effect on individuals with diabetes when their severity of DR does not exceed the moderate NPDR level defined by the International Clinical DR Disease Severity Scale. However, the study did not report the incidence of newly diagnosed DR. y g y These two studies confirmed the beneficial effects of intensive glucose control in the population they enrolled in. The Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation Retinal Measure- ments (AdRem) study is a substudy of ADVANCE. Of these, over 50% of participants had no DR at baseline, more than 30% had moderate NPDR, and 3.7% had severe NPDR or PDR, which means more participants with severe DR com- pared with the population in UKPDS and ACCORD. The results indicated that there was no statistical difference in the incidence or frequency of progression of DR between the intensive glycemic control and conventional management groups. The reason for this negative result is unclear, but the worse DR condition at baseline might partly contribute. Besides, although intensive glucose control achieved a median HbA1c of <7%, the difference in the medians between the groups was <0.8% (6.4% vs. 7.0%), which might be the expla- nation because the protective effect of glucose lowering against macrovascular disease was previously shown to require the difference in achieved HbA1c to be ≥0.8% [15]. y g In the ACCORD study, the effect of intensive glycemic control was evaluated on the incidence of cardiovascular events in type 2 diabetes patients with established cardiovas- cular disease and/or additional cardiovascular risk factors [14]. A total of 2,856 patients were enrolled, and 38% had at least one cardiovascular event or possessed other CVD risk factors. According to the International Clinical DR Disease Severity Scale, less than 2% had severe NPDR or PDR. The median follow-up period for the study was 4 years, during which the mean HbA1c concentration decreased from >8% to 6.3% in the intensive glycemic control group, but only to 7.6% in the conventional therapy group. 3. Determination of the DR Severity Threshold below Which Intensive Glycemic Control Has Benefits for the Retina The cumulative inci- dence of progression of DR over the 4 years differed signifi- cantly between groups (4.8% in the intensive treatment and 13.1% in the conventional management group) [14]. DR pro- gression was significantly reduced by intensive glycemic intervention compared to standard therapy throughout the study. For all the DR severity levels combined, the primary results showed a statistically significant benefit of intensive glycemic control compared with conventional management [14]. Chew et al. considered specific components of the pri- mary eye outcomes from ACCORD and compared the results among subgroups of different DR severity at baseline [12]. The difference was large and statistically significant only for patients with microaneurysms in one or both eyes when compared with those with mild NPDR in only one eye (odds ratio 0.30, 95% confidence interval 0.15–0.59; P = 0:0002), and similar results were obtained for retinopathy progression by 1, 2, and 4 or more steps on the person scale and for ≥2 steps on the eye scale [12]. Although there was no significant The VADT [16, 17] study assessed 858 patients with type 2 diabetes using the ETDRS grading system; using the inter- national clinical DR severity scale, 31% of the patients showed no evidence of fundic lesions at the time of enroll- ment, 21% had mild NPDR, 42% had moderate NPDR, and around 6% had severe NPDR or PDR. Individuals with severe DR were more. The VADT study group defined the progres- sion of DR as a progression of >2 steps between baseline and follow-up. The results of a 5-year follow-up showed that HbA1c in the intensive glycemic control group had decreased from 9.3% to 6.9% on average, while HbA1c in the control group only decreased from 9.4% to 8.4%. Although the two-step progression in the intensive glycemic control group was significantly lower than that in the conventional management group (17.0% vs. 22.1%) [16], the other DR outcomes show no significant difference between the two groups. DR condition at the baseline might be a clue for the negative results. 4. Discussion We reviewed five large-scale RCTs which had shown that a DR severity of no more than moderate NPDR on the Interna- tional Clinical DR Disease Severity Scale is the threshold below which intensive glucose control has beneficial effects on retinal microvessels. When individuals have DR lesions that are equivalent to or less severe than moderate NPDR, >3 years of intensive glucose control is required to achieve benefits for the retina. However, these are realized only if the HbA1c in type 1 or Journal of Diabetes Research 5 type 2 diabetic patients is reduced at least by 0.8% versus the control group or it is reduced to <7%. The same degree of glucose control is associated with less pronounced retinal benefits in type 2 diabetic patients than in those with type 1 diabetes, according to analysis of the DCCT, UKPDS, and ACCORD, which might be the result of the presence of hypertension and/or other risk factors frequently associated with type 2 diabetes, such as hypertriglyceridemia. [3] The Diabetes Control and Complications Trial Research Group, “The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus,” The New England Journal of Medicine, vol. 329, no. 14, pp. 977–986, 1993. [4] UK Prospective Diabetes Study (UKPDS) Group, “Intensive blood-glucose control with sulphonylureas or insulin com- pared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33),” The Lancet, vol. 352, no. 9131, pp. 837–853, 1998. The increase of permeability in retinal vessels is one of the early pathological signs of DR in animal models. Hyper- glycemia induces intracellular reactive oxygen species (ROS) [18] and advanced glycation end product (AGEs) pathway which causes a breakdown of the blood-retina barrier and loss of retinal vascular pericytes [19, 20]. However, overpro- duction of ROS and decreased efficiency of antioxidant defenses eventually worsen over the course of the disease [21]. This might partly explain why glycemic control lost the protective effect for an individual’s retina with severe DR. [5] R. R. Holman, S. K. Paul, M. A. Bethel, D. R. Matthews, and H. A. Neil, “10-year follow-up of intensive glucose control in type 2 diabetes,” The New England Journal of Medicine, vol. 359, no. 15, pp. 1577–1589, 2008. [6] X. Zhang, Y. Liu, F. Zhang, J. Li, and N. Conflicts of Interest The authors declare that they have no conflict of interest. [11] C. P. Wilkinson, F. L. Ferris, R. E. Klein et al., “Proposed inter- national clinical diabetic retinopathy and diabetic macular edema disease severity scales,” Ophthalmology, vol. 110, no. 9, pp. 1677–1682, 2003. 4. Discussion Tong, “Legacy effect of intensive blood glucose control on cardiovascular outcomes in patients with type 2 diabetes and very high risk or secondary prevention of cardiovascular disease: a meta-analysis of randomized controlled trials,” Clinical Therapeutics, vol. 40, no. 5, pp. 776–788.e3, 2018, e773. It is widely accepted that glycemic control does delay the progression of diabetic retinopathy. However, nearly no guideline or review mentioned whether the different severity of diabetic retinopathy conditions affects the beneficial effect of glycemic control. Based on the benefit-risk evaluation, it is vital for clinicians to make their clinical decisions on appro- priate glycemic control goals with their different patients. [7] A. R. Gosmanov and E. O. Gosmanova, “Long-term renal out- comes of patients with type 1 diabetes mellitus and microalbu- minuria: an analysis of the DCCT/EDIC cohort,” Archives of Internal Medicine, vol. 171, no. 17, p. 1596, 2011, author reply 1597. [8] Y. Wu, L. Tang, F. Zhang, Z. Yan, J. Li, and N. Tong, “Evalua- tion of the HbA1c reduction cut point for a nonglycemic effect on cardiovascular benefit of hypoglycemic agents in patients with type 2 diabetes based on endpoint events,” International Journal of Endocrinology, vol. 2018, Article ID 8457538, 7 pages, 2018. We have to admit the inevitable limitation on this review. Because these data have no details in the events in each level of DR severity, a meta-analysis cannot be performed. However, these trials we included can be considered as the “milestones” in diabetes mellitus fields. We can just consider each study as a whole body and infer to find its value. [9] Early Treatment Diabetic Retinopathy Study Research Group, “Grading Diabetic Retinopathy from Stereoscopic Color Fun- dus Photographs–An Extension of the Modified Airlie House Classification: ETDRS Report Number 10,” Ophthalmology, vol. 98, no. 5, pp. 786–806, 1991. In summary, we contend that the concept of a threshold of DR severity can provide clinicians with a reference to judge whether the retinas of individuals with diabetes could be protected by intensive glucose control, and it is also a new point that need more attention for future investigations. [10] R. Klein, B. E. Klein, S. E. Moss, and K. J. Cruickshanks, “The Wisconsin Epidemiologic Study of diabetic retinopathy. XIV. Ten-year incidence and progression of diabetic retinopathy,” Archives of Ophthalmology, vol. 112, no. 9, pp. 1217–1228, 1994. Acknowledgments This work was supported by the Major Project of the Science and Technology Department of Sichuan Province (Grant No. 0040205301D50). [12] E. Y. Chew, M. D. Davis, R. P. Danis et al., “The effects of medical management on the progression of diabetic reti- nopathy in persons with type 2 diabetes: the Action to Control Cardiovascular Risk in Diabetes (ACCORD) Eye Study,” Ophthalmology, vol. 121, no. 12, pp. 2443–2451, 2014. Supplementary Materials Supplement Table 1. Search strategy, Supplement Table 2. The baseline of diabetic retinopathy condition, Supplement Table 3. Diabetic retinopathy outcomes at the end of studies (Supplementary Materials) [13] I. M. Stratton, E. M. Kohner, S. J. Aldington et al., “UKPDS 50: risk factors for incidence and progression of retinopathy in type II diabetes over 6 years from diagnosis,” Diabetologia, vol. 44, no. 2, pp. 156–163, 2001. [14] The ACCORD Study Group and ACCORD Eye Study Group, “Effects of medical therapies on retinopathy progression in type 2 diabetes,” The New England Journal of Medicine, vol. 363, no. 3, pp. 233–244, 2010. References [1] R. N. Frank, “Diabetic retinopathy,” The New England Journal of Medicine, vol. 350, no. 1, pp. 48–58, 2004. [15] J. W. Beulens, A. Patel, J. R. Vingerling et al., “Effects of blood pressure lowering and intensive glucose control on the inci- dence and progression of retinopathy in patients with type 2 [2] “Panel. AAoORV. Preferred Practice Pattern® Guidelines. Diabetic Retinopathy,” 2016, https://www.aao.org/ppp. Journal of Diabetes Research 6 diabetes mellitus: a randomised controlled trial,” Diabetologia, vol. 52, no. 10, pp. 2027–2036, 2009. [16] W. Duckworth, C. Abraira, T. Moritz et al., “Glucose control and vascular complications in veterans with type 2 diabetes,” The New England Journal of Medicine, vol. 360, no. 2, pp. 129–139, 2009. [17] N. Azad, L. Agrawal, N. V. Emanuele et al., “Association of blood glucose control and pancreatic reserve with diabetic retinopathy in the Veterans Affairs Diabetes Trial (VADT),” Diabetologia, vol. 57, no. 6, pp. 1124–1131, 2014. [18] A. S. Al-Kharashi, “Role of oxidative stress, inflammation, hypoxia and angiogenesis in the development of diabetic retinopathy,” Saudi Journal of Ophthalmology, vol. 32, no. 4, pp. 318–323, 2018. [19] R. Milne and S. Brownstein, “Advanced glycation end prod- ucts and diabetic retinopathy,” Amino Acids, vol. 44, no. 6, pp. 1397–1407, 2013. [20] Z. Gurel, B. W. Zaro, M. R. Pratt, and N. Sheibani, “Identifica- tion of O-GlcNAc modification targets in mouse retinal pericytes: implication of p53 in pathogenesis of diabetic reti- nopathy,” PLoS One, vol. 9, no. 5, article e95561, 2014. [21] M. Brownlee, “The pathobiology of diabetic complications: a unifying mechanism,” Diabetes, vol. 54, no. 6, pp. 1615–1625, 2005.
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Une communauté d’artistes et d’artisans français à l’étranger : Le cas des sculpteurs au château royal de Stockholm au XVIIIe siècle
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DOI: http://dx.doi.org/10.7557/4.3528 Une communauté d’artistes et d’artisans français à l’étranger : le cas des sculpteurs au château royal de Stockholm au XVIIIe siècle Une communauté d’artistes et d’artisans français à l’étranger : le cas des sculpteurs au château royal de Stockholm au XVIIIe siècle Anne-Sophie Michel Anne-Sophie Michel Dans le contexte culturel européen du XVIIIe siècle, la France de Louis XV oc- cupe une position éminente. L’art français, dans ses diverses composantes, connaît une diffusion sans précédent imputable au prestige dont il est assorti.1 La Suède, en plein renouveau culturel, se tourne alors vers son allié inaugurant l’âge d’or des relations franco-suédoises. Pour autant, la multiplication de ces échanges est loin d’être un phénomène naturel et résulte, en partie, des choix opérés par une élite francophile soucieuse d’affirmer sa puissance sur la scène européenne.2 C’est ainsi que vont s’épanouir entre les deux pays des relations denses et étroites qui laisseront des traces dans la pierre, sur les murs et les plafonds du château royal de Stockholm. Réalisé selon les plans de l’architecte Nicodème Tes- sin le jeune, le chantier ouvert en 1697 se poursuit jusqu’à la fin du XVIIIe siècle malgré de nombreuses difficultés économiques, politiques et matérielles.3 Très tôt, la France y apparaît comme une source d’inspiration. Son rayonnement se traduit par la mise en place progressive de réseaux artistiques et commerciaux, ainsi que par le recrutement de deux générations successives d’artistes et artisans français.4 Ces derniers seront d’ailleurs les acteurs indispensables de la création d’institutions modernes sur le modèle des Bâtiments du Roi et de l’Académie royale de Peinture et de Sculpture. Dans le sillage des recherches de Linda Hinners sur la première vague de Fran- çais, ce travail se propose d’étudier sous un nouveau jour la seconde vague com- posée principalement de sculpteurs recrutés entre 1728 et 1765 pour participer à la décoration du château royal de Stockholm.5 Il s’agit ici d’abandonner la vision nationaliste des précédents travaux français, selon lesquels le rayonnement général de l’esprit et du goût français se manifeste par l’appel à ses artistes dans toute l’Europe, pour tenter de comprendre à travers ces migrations artistiques le trans- fert culturel opéré.6 Si le recours aux Français apparaît comme un corollaire des liens artistiques noués entre les deux pays, il n’en demeure pas moins un moyen 117 Sjuttonhundratal | 2015 rapide et efficace pour s’approprier le modèle artistique français. Dès lors, qui sont ces sculpteurs et pourquoi sont-ils recrutés ? A quelles conditions ? Quels rôles leurs sont assignés ? Une communauté d’artistes et d’artisans français à l’étranger : le cas des sculpteurs au château royal de Stockholm au XVIIIe siècle Et surtout quels éléments les distinguent de la précé- dente génération de Français ? En répondant à ces questions, cet article invite à relativiser l’idée d’une in- fluence française et à analyser ces migrations au prisme d’une histoire sociale soucieuse de mieux comprendre le rôle joué par ces sculpteurs dans les mutations qu’a connu le milieu artistique suédois au XVIIIe siècle. Le recrutement de sculpteurs français sur le chantier du château royal : raisons et moyens Grâce aux archives du château et à la correspondance diploma- tique, il est possible de retracer l’histoire mouvementée de leur engagement. Dès 1732, Carl Hårleman s’embarque pour la France afin d’engager, avec l’aide de ses amis comme l’ornemaniste parisien Claude III Audran, une première équipe. Si ses relations lui permettent d’entrer en contact avec de nombreux sculpteurs, elles ne l’empêchent pas d’éprouver certaines difficultés. Selon lui, il se trouve bien d’habiles gens ici qui n’ont presque rien à faire, qui meurent de faim qui voudrerent bien changer leur sort mais qui ne sauraient se résoudre à gagner leur pain et à vivre hors de Paris, avec tout cela je n’oserois les presser de peur qu’ils ne se rendent trop cher. Que mon trop peu de sejour dans ce pays cy et le devoir indispensable dans lequel je suis au bon marché s’accordent mal avec une commission tel que la mienne qui ne devrait avoir que l’habileté et le scavoir en vüe.10 Après de longues et laborieuses recherches, il réussit à recruter deux maîtres sculpteurs : Michel Lelièvre et Antoine Belette, ainsi que quatre compagnons : Nicolas Varin, Pierre David, Nicolas Leger, et Charles Ruste.11 L’année suivante, la surintendance des Bâtiments suédois charge le Baron de Gedda épaulé par deux maîtres de l’Académie de Saint Luc liés à Antoine Bellette, Gardy et Montheau, de trouver de nouveaux sculpteurs. La nouvelle équipe compte un maître et dix compagnons : Michel Gardy, André Tiroir, François Ménard, Jean-Baptiste Gues- non, Pierre Hanneguy, Jacques-Gabriel Créssé, Eustache Bouru, Ignace Blaton, Lambert Dequinthe, Robert Lemerle et Louis Roland. Après de longues et laborieuses recherches, il réussit à recruter deux maîtres sculpteurs : Michel Lelièvre et Antoine Belette, ainsi que quatre compagnons : Nicolas Varin, Pierre David, Nicolas Leger, et Charles Ruste.11 L’année suivante, la surintendance des Bâtiments suédois charge le Baron de Gedda épaulé par deux maîtres de l’Académie de Saint Luc liés à Antoine Bellette, Gardy et Montheau, de trouver de nouveaux sculpteurs. La nouvelle équipe compte un maître et dix compagnons : Michel Gardy, André Tiroir, François Ménard, Jean-Baptiste Gues- non, Pierre Hanneguy, Jacques-Gabriel Créssé, Eustache Bouru, Ignace Blaton, Lambert Dequinthe, Robert Lemerle et Louis Roland. Par la suite, le recrutement de Français semble s’essouffler. Les nouvelles re- crues viennent alors simplement remplacer leurs compatriotes ou répondre à des exigences artistiques précises. Le recrutement de sculpteurs français sur le chantier du château royal : raisons et moyens L’âge d’or des relations franco-suédoises se caractérise par la naissance de véritables réseaux artistiques qui résultent de l’action conjointe de l’élite suédoise et de ses agents parisiens. Le rayonnement de l’art français passe en effet par la connaissance des artistes, du goût français, et l’affluence d’objets et d’œuvres d’art.7 Avec les voyages d’étude à Paris des différents surintendants des Bâtiments suédois, Carl Gustaf Tessin, Carl Hårleman, puis Carl Johan Cronstedt, se créent de solides liens d’amitiés entre le milieu artistique français et suédois. Ainsi, entre 1732 et 1754, les échanges avec Paris se multiplient grâce aux actions conjointes du Baron Niklas Peter von Gedda, Ministre plénipotentiaire de 1725 à 1742, du Comte Carl Gustaf Tessin, en mission extraordinaire à la cour entre 1739 et 1742 et du Baron Carl Fredrik Scheffer, ambassadeur de Suède de 1742 à 1752.8 Ces hommes entretien- nent une correspondance régulière avec la surintendance des Bâtiments suédois qui les charge de différentes missions. Ils passent des commandes aux peintres français, font des achats de mobilier, accueillent les Suédois en voyage à Paris, les informent des nouveautés parisiennes et surtout engagent des artistes et artisans pour venir travailler au chantier du château royal. C’est dans ce contexte culturel particulière- ment ouvert sur la France que se poursuit la construction du château. Si l’avènement de l’art rocaille au début du siècle implique de repenser le programme décoratif élaboré par Nicodème Tessin le jeune au XVIIe siècle, le modèle français se maintient et pour cause. Le château construit d’après les plans de l’architecte va se parer d’un décor rocaille suivant la dernière mode parisienne. Carl Gustaf Tessin et Carl Hårleman, tous deux en charge de la reprise des tra- vaux, décident alors de se rendre en France pour se procurer une partie de la main d’œuvre nécessaire à sa réalisation. Notons cependant que leurs recherches ne sont pas circonscrites à la France mais qu’elles seront peu fructueuses, peut être 118 Anne-Sophie Michel | Une communauté d’artistes et d’artisans français à l’étranger en raison de l’absence de réseau, en Italie, en Belgique comme en Flandres.9 La surintendance des Bâtiments suédois semble donc, au départ, moins à la recherche de Français que d’ouvriers spécialisés capables d’exécuter rapidement et à moindre coût un décor de qualité française. Ainsi, arrivent à Stockholm entre 1732 et 1755 une trentaine de sculpteurs français accompagnés de quelques autres artisans spécialisés (peintres, menuisier, fondeur, mouleur). Le recrutement de sculpteurs français sur le chantier du château royal : raisons et moyens C’est le cas, par exemple, de Charles Guillaume Cousin, Jacques Philippe Bouchardon, sculpteurs de figures et d’ornements, de Nicolas Alexandre Vigé, mouleur ou encore de Pierre-Hubert L’Archevêque, aca- démicien. Ainsi, on ne compte pas plus d’une dizaine de nouveaux sculpteurs entre 1735 et 1755.12 Il faut dire que le pays connaît de grosses difficultés finan- cières qui ne tardèrent pas à se répercuter sur le budget dédié au château. Une fois de plus, cette mission échoit aux résidents suédois à Paris qui éprouvent toujours 119 Sjuttonhundratal | 2015 les mêmes difficultés comme le laisse entendre Carl Gustaf Tessin dans sa corres- pondance. Il écrit : « Je suis à Paris depuis six jours, et je chasse actuellement aux sculpteurs ; c’est un gibier plus rare qu’on ne s’imagine d’abord. Les uns ne veu- lent pas aller si loin, les autres mettent leurs marchandises à trop haut prix ».13 Si les Suédois ne peuvent convaincre ni les académiciens ni les sculpteurs ré- putés de la capitale, ils réussissent néanmoins à recruter une trentaine de maîtres et compagnons prêts à s’embarquer pour Stockholm. Ces derniers, plus que les peintres, prennent part aux travaux complémentaires de la construction rendant leur présence sur le chantier indispensable. Anne-Sophie Michel | Une communauté d’artistes et d’artisans français à l’étranger Les contrats stipulent ensuite que les compagnons bénéficient d’un logement commun. Hårleman écrira : J’espere aussy qu’à leur arrivé ils pourront être logé à Rännarebanan,20 une partie dans la maisonnette de bois et l’autre dans la grande de pierre. C’est un article que j’ai eu bien de la peine a leur accorder mais par ou il m’a bien fallu passer comme par le reste vu que de tout tems et partout ce meme avantage est attaché aux ouvriers de toutes espece. Outre que tout bien considéré on les a de cette façon la mieux sous les yeux et que le Roi en est mieux servi.21 Cette prise en charge se présente comme un avantage supplémentaire surtout si le chantier du château de Versailles (1662–1715) est pris comme point de com- paraison.22 Les Français se voient aussi accorder le libre exercice de la religion ca- tholique, d’où leur regroupement autour de la chapelle de l’ambassade française.23 Enfin, tout un chapitre est réservé à la rupture du contrat et à la maladie. En cas de mécontentement, le Roi peut renvoyer les Français en les avertissant à l’avance et en leur accordant une somme pour leur retour. De même, en cas de maladie courte, ils bénéficient du maintien de leur gage jusqu’à leur rétablissement. En France, si les accidents du travail et les décès font l’objet d’une indemnisation, son montant dérisoire ne vise pas à compenser la perte d’une capacité à générer un revenu. En outre, si l’accident ou la maladie intervient en dehors du lieu de travail, des soins seront apportés par les autorités mais aucune indemnisation ne sera versée.24 Ces articles accordent donc de réelles garanties aux Français afin de les rassurer et surtout de les inciter au départ. De leur côté les sculpteurs, dont la plupart sont liés l’Académie de Saint Luc et ont travaillé pour les Bâtiments du Roi, jouissent d’un traitement particulier.25 Leurs contrats reprennent les mêmes clauses que ceux des compagnons mais des assouplissements sont introduits. Les salaires sont compris entre 2000 et 6000 livres par an en fonction de leur statut et de leur talent. La monarchie leur con- cède aussi le droit d’être rémunéré à la pièce et de travailler pour des particuliers, complément de revenu lucratif. Les conditions de vie et de travail offertes aux Français Afin de matérialiser leur engagement, des contrats sont conclus avec la monarchie suédoise au moment du départ.14 Ils nous éclairent sur les conditions de vie et de travail offertes aux Français, tout en faisant ressortir immédiatement une diffé- rence de traitement entre les compagnons et les sculpteurs. Les compagnons ont tous conclu « un contrat pour se transporter à Stockholm et y travailler de leur profession pendant l’espace de trois années ». S’ils bénéficient de la prise en charge des frais, ils voient en contrepartie leur liberté au cours du voyage très encadrée. Une fois sur place, le compagnon « travaillera de sa dite pro- fession sous les ordres des préposés pour les dits bâtiments de sa dite majesté ». Le contrat précise que « les journées dureront de 7h du matin jusqu’à 7h du soir pendant l’espace duquel ledit [compagnon] aura deux heures pour ses repas suivant les usages de cette ville de Paris ». Son activité lui donne droit de percevoir un salaire dont le montant varie entre 700 et 1700 livres. La moyenne étant de 700 livres, leur rémunération reste plus attractive qu’en France où un compagnon perçoit environ 400 livres par an.15 Mais n’oublions pas que leur départ comporte des risques et que les expériences malheureuses sont nombreuses.16 A Hårleman de noter : « à dire le vrai, ce n’est point trop, vu qu’ils perdent ici en attendant leurs pratiques aussi bien que le gout qui dépend tant de la mode et qu’il faut compter ces gens à leur retour au bout d’un certain nombre d’année comme mort pour ce pay ci ».17 De ce fait, la surintendance des Bâtiments devra, malgré ses difficultés financières, faire des concessions comme le paiement de « certaines sommes, à compte sur le premier quartier quelque uns pour payer leur petites dettes à d’autre pour s’équiper, ceux-ci pour laisser à leur femme et enfants à Paris et à ceux là pour s’acheter des estampes, des modèles et des outils ».18 En réalité, c’est surtout la concurrence exercée par la Pologne et la Russie qui les oblige à revoir leur position.19 120 Anne-Sophie Michel | Une communauté d’artistes et d’artisans français à l’étranger Anne-Sophie Michel | Une communauté d’artistes et d’artisans français à l’étranger Malgré ces avantages financiers, les sculpteurs n’auront de cesse de se plaindre de la faiblesse de leur salaire.26 La raison de leur départ est donc à rechercher du côté des opportunités de carrière et de la qualité des conditions de vie et de travail offertes par la Suède. D’autant plus qu’à partir des années 1740 les sculpteurs français ne manquent plus d’ouvrage à Paris. La proposition suédoise offre, en effet, au sculpteur « l’honneur de travailler à un Palazzo Réale, [sans compter] la bordure qu’il pourroit donner à son mérite en se faisant apostropher peintre du Roy et Professeur de l’Académie Royale de pein- 121 Sjuttonhundratal | 2015 ture et de sculpture de Suède ».27 Le surintendant Carl Fredrik Adelcrantz réussit ainsi à recruter L’Archevêque en lui offrant le privilège, très recherché à l’époque, de réaliser une statue équestre du Roi.28 Les sculpteurs obtiennent également des conditions matérielles beaucoup plus avantageuses. Leur logement individuel est pris en charge de même que le chauffage, le luminaire voire l’atelier de travail et le matériel.29 Bouchardon obtiendra, par exemple, la fourniture de « tous les outils, ferrement, matériaux et autres ustencils nécessaires à la profession », Masreliez la fourniture de « matières nécèssaires pour ses ouvrages d’où bois, plomb, bron- ze, cire, plastre, stuc, papier, carton, et génèralement toutes les autres matières, matèriaux, outils », alors que L’Archevêque profite de la fourniture d’un atelier, d’outils, de matières premières et même d’une exemption d’impôt.30 Anne-Sophie Michel | Une communauté d’artistes et d’artisans français à l’étranger Afin de réaliser l’ensemble du décor, les Suédois ont donc d’abord engagé des équipes d’ornemanistes capables de travailler dans une certaine unité artistique, avant de leur préférer des sculpteurs expérimentés. Comme en Russie, les Français se voient également confier la conduite des travaux de sculpture.33 D’après les contrats, les Français « conduiront le travail des autres ouvriers de leur profession soit français ayant fait le voyage avec eux soit suédois ou autres qui pourront leur être adjoint a Stockholm ».34 La conduite des travaux de sculpture apparaît comme une charge pour laquelle ils perçoivent une rémunération spécifique. Ain- si, ils forment et animent de véritables équipes de travail qui constituent autant d’ateliers de production. Ils se composent de maîtres français, de compagnons, d’ouvriers et de manœuvres suédois, tous placés sous la direction de la surin- tendance des Bâtiments. Ils communiquent notamment par l’intermédiaire d’un tisserand français, Dubois, installé à Stockholm depuis plusieurs années. Mais quel est exactement le rôle du surintendant dans l’élaboration du décor réalisé par les sculpteurs français ? A la différence de ce qui se passe en Russie ou au Danemark, le décor semble rester dans l’ensemble l’œuvre des architectes suédois secondés, pour sa réalisation, par les Français. Formés en France, ils sont capables de créer un décor rocaille français de très belle qualité. Cependant, comme pour les décors de boiseries parisiens, il est difficile d’affirmer qui de l’architecte ou de l’ornemaniste l’imagine et surtout le dessine.35 D’après les documents d’archives, il semble que les ornemanistes français aient travaillé tantôt d’après leurs propres dessins, tantôt d’après ceux de l’architecte.36 Selon Bruno Pons ou Göran Alm, les ornemanistes ont un mode de travail singulier.37 Il ne serait donc pas étonnant que la plupart réalisent les dessins des ornements, se distinguant ainsi des sculpteurs de figures pour qui la tutelle suédoise semble plus contraignante.38 Enfin, à côté de leur travail, les Français assument la formation des jeunes Suédois.39 Ce rôle assigné aux peintres comme aux sculpteurs résulte, au départ, d’un besoin pressant d’une main d’œuvre formée. A cette époque, il n’y a que des artisans, soumis à des statuts de corporations, dont quelques-uns se distinguent en travaillant au service du Roi. Les rôles assignés aux sculpteurs français Sur le chantier, le rôle des Français ne se limite pas à la décoration du château. Si la surintendance des Bâtiments recourt à leurs compétences artistiques, elle fait aussi appel à leurs expériences des grands chantiers parisiens et à leurs con- naissances théoriques. A leur arrivée, leur activité se concentre sur les décors de boiserie des appartements de l’aile Nord, le mobilier et les ornements des façades du château. Malgré leur destruction quasi-totale, il est certain que leur réalisation nécessita l’intervention de plusieurs équipes d’ornemanistes rompus à l’exercice. Peter Thornton considère d’ailleurs ces aménagements comme l’un des meilleurs exemples de l’art français hors de France.31 A partir des années 1740, date de l’ouverture de l’aile Sud, la nature des ouvra- ges change. Les Français ne se voient plus confier la réalisation de simples or- nements mais l’ensemble du décor d’où l’intervention, non plus de décorateurs, mais plutôt de bons sculpteurs. Le chantier de la salle des États (Rikssalen) et de la chapelle se présente ainsi comme l’alliance parfaite de la vision artistique de Nicodème Tessin, qui en donne les premiers plans, et du savoir-faire de ses succes- seurs qui réussissent habilement à le faire décorer. Jacques-Philippe Bouchardon y réalise une grande partie du décor sculpté en bois, stuc et pierre.32 Son talent et ses ouvrages lui valent d’être nommé Premier sculpteur du Roi de Suède et Di- recteur de l’Académie royale de Peinture et de Sculpture. La salle des États reçoit, quant à elle, un décor réalisé en partie par Charles-Guillaume Cousin, et achevé par Pierre-Hubert L’Archevêque vers 1754. Les intérieurs du château seront, par la suite, aménagés avec des pièces commandées pour la plupart directement dans les ateliers parisiens. 122 Anne-Sophie Michel | Une communauté d’artistes et d’artisans français à l’étranger Anne-Sophie Michel | Une communauté d’artistes et d’artisans français à l’étranger Anne-Sophie Michel | Une communauté d’artistes et d’artisans français à l’étranger Le peintre Guillaume Taraval et le sculpteur An- toine Belette sont les premiers à leur dispenser au sein même du château des cours libres et gratuits cinq jours par semaine afin de leur apprendre à modeler et dessi- ner. Les élèves commencent à travailler d’après les dessins des Français, les mou- lages d’Antiques rapportés par Tessin de Paris puis progressivement d’après des modèles vivants. Il faut cependant attendre 1735 pour que leur école de dessin devienne une véritable Académie royale, et les années 1750 pour que les profes- seurs reçoivent un traitement. Bien qu’insuffisant, il rappelle que l’enseignement fait désormais partie intégrante de leur mission. Officiellement reconnue, l’Aca- 123 Sjuttonhundratal | 2015 démie est soumise à l’autorité du surintendant des Bâtiments. Dès lors, le travail de construction du château et celui de l’Académie commencent à se distinguer. Grâce à Carl Gustaf Tessin et Carl Hårleman le budget s’étoffe peu à peu d’où la diversification de l’enseignement (gravure, anatomie, perspective), la création de prix et de bourses d’étude. En outre, les cours sont dispensés par des professeurs spécialisés à partir d’un corpus de dessin réuni par Tessin et Hårleman au cours de leurs voyages en France. La réforme des années 1770 vise, comme nous le verrons, à réaliser des économies en remplaçant les Français par leurs élèves puis, à terme, à doter la Suède d’une véritable élite artistique. Le recours aux artistes français n’étant plus nécessaire, il est rapidement condamné, si bien qu’en 1765, L’Arch- evêque et Masreliez sont les derniers encore actifs à la cour suédoise.40 démie est soumise à l’autorité du surintendant des Bâtiments. Dès lors, le travail de construction du château et celui de l’Académie commencent à se distinguer. Grâce à Carl Gustaf Tessin et Carl Hårleman le budget s’étoffe peu à peu d’où la diversification de l’enseignement (gravure, anatomie, perspective), la création de prix et de bourses d’étude. En outre, les cours sont dispensés par des professeurs spécialisés à partir d’un corpus de dessin réuni par Tessin et Hårleman au cours de leurs voyages en France. La réforme des années 1770 vise, comme nous le verrons, à réaliser des économies en remplaçant les Français par leurs élèves puis, à terme, à doter la Suède d’une véritable élite artistique. Le recours aux artistes français n’étant plus nécessaire, il est rapidement condamné, si bien qu’en 1765, L’Arch- evêque et Masreliez sont les derniers encore actifs à la cour suédoise.40 Anne-Sophie Michel | Une communauté d’artistes et d’artisans français à l’étranger La professionnalisation des activités artistiques apparaît encore plus nettement au XVIIIe siècle lors des recrutements à travers les avantages concédés, comme sur le chantier où les ateliers se structurent. La Suède suit donc le mouvement initié par la France où les activités de peintre et sculpteur se détachent progressivement du métier.43 En effet, à partir des années 1740, les Suédois ne cherchent plus à recruter des familles d’artisans susceptibles de s’établir en Suède mais plutôt à bénéficier rapidement et temporairement d’équipes de travail spécialisées et opé- rationnelles. Les Français sont alors minutieusement choisis et partent seuls pour y séjourner pendant le temps de leur contrat. Leurs femmes et leurs enfants res- tent désormais en France, comme le suggère la possibilité qui leur est laissée de faire toucher une partie de leur pension à la personne de leur choix. À l’exception des Masreliez, aucun d’ailleurs ne s’y établit vraiment définitivement. Par le biais de ce recrutement ciblé, la surintendance des Bâtiments dispose d’un savoir-faire spécifique qui répond précisément aux besoins du chantier. p p p De la même manière, la spécialisation des tâches était encore embryonnaire alors même que Tessin le jeune avait eu recours à des artisans spécialisés dans la décoration. D’après Linda Hinners cette spécialisation, bien qu’en voie d’af- firmation, était loin d’être la règle dans la pratique des ateliers familiaux.44 Il faut attendre le siècle suivant pour percevoir une affirmation de ce phénomène. Il se traduit par une véritable répartition des tâches au sein des différents ate- liers de sculpture du château et, inéluctablement, l’introduction de hiérarchies qui transparaissent surtout au niveau de la rémunération.45 L’idée de groupe, dominante jusque-là, s’efface peu à peu au profit de celle d’équipes spécialisées, structurées et organisées. Cette répartition est intéressante car elle témoigne, dans un souci d’efficacité, d’une volonté nouvelle de restreindre les domaines d’activité en distinguant notamment les sculpteurs de figure des sculpteurs d’ornement, même si la frontière n’est pas imperméable. Peut-être que cette répartition allait d’ailleurs bien au-delà et que certains sculpteurs étaient spé- cialisés dans un type de motif ou de mobilier. Il faut dire aussi que la réalisation du décor rocaille nécessite un savoir-faire artistique et technique très spécifique. Les spécificités de cette vague migratoire (1732–1765) La venue de cette seconde génération de Français a joué un rôle important dans la redéfinition du statut et de la place des artisans et des artistes au sein de la société suédoise. Amorcées un siècle plus tôt, la professionnalisation et la spécialisation des activités artistiques résultent d’une modernisation des structures imputables à la création de la surintendance des Bâtiments suédois et de l’Académie royale de Peinture et de Sculpture. Crée en 1697 à l’initiative de Nicodème Tessin le jeune, la surintendance des Bâtiments (Överintendentsämbetet) s’inspire du modèle français. Pour Lars Ljungström Son office n’était en fait qu’un cabinet d’architecte élargie ; où l’on assumait à l’occasion d’autres tâches artistiques tandis que le surintendant français, qui avait rang de ministre, commandait une multitude d’architectes, de dessinateurs, d’artisans et de techniciens. Il n’existait pas en Suède d’institution telle que les ateliers royaux français. C’est seulement pour le projet du nouveau château que le rôle du surintendant en vint à inclure en partie la coordination des artistes qualifiés qui y travaillaient.41 De ce fait, on assiste à un glissement des ateliers de travail traditionnels et fami- liaux vers des ateliers organisés, spécialisés et hiérarchisés. Cette évolution était déjà perceptible chez les premiers Français, et à Linda Hinners de noter à leur égard « une évolution du rôle de l’artiste et de la créativité artistique dans les débuts de l’ère moderne tels qu’ils ont été discutés par les historiens de l’art Baxandall, Alpers et Wittkower ».42 124 Anne-Sophie Michel | Une communauté d’artistes et d’artisans français à l’étranger Sjuttonhundratal | 2015 Sjuttonhundratal | 2015 Avec la création d’une véritable Académie royale de Peinture et de Sculpture, la rupture semble véritablement consommée. En effet, depuis 1735 l’Académie se structure progressivement jusqu’en 1773, date à laquelle elle reçoit ses premiers règlements officiels. Protégée par le Roi et subventionnée par l’Etat, elle est dé- sormais en mesure de s’installer dans de nouveaux locaux et de recruter plusieurs professeurs suédois. Comme en France, cette institution concurrente de la corpo- ration va peu à peu établir une sélection pour créer une élite artistique, et redéfinir les critères de cette compétence dans un sens plus intellectuel que manuel. Ainsi, elle assure la promotion sociale des arts et légitime le nouveau statut d’artiste. Cette idée se retrouve parfaitement dans la nature de l’enseignement dispensé après le départ des Français. Très théorique, il s’intellectualise et se spécialise permettant aux Suédois de former autant des peintres et des sculpteurs que des architectes et des graveurs. Directeur de l’Académie de 1768 à 1778, Pierre-Hu- bert L’Archevêque parachève sa transformation sur le modèle parisien et forme ainsi les premiers grands maîtres de la sculpture suédoise à l’image de l’œuvre de Nicolas François Gillet en Russie.46 A la lumière de ce développement, le château royal de Stockholm apparaît comme le fruit des relations artistiques franco-suédoises, et la France comme une source d’inspiration autant qu’une pépinière de sculpteurs dans laquelle la surintendance des Bâtiments suédois puise pour répondre aux besoins spécifiques du chantier. Il semble ainsi que ce soit surtout des raisons pragmatiques et éco- nomiques qui aient conduit les Suédois à se tourner vers elle. Au-delà du modèle artistique, ils y ont également puisé les principes d’une gestion efficace et mo- derne d’un chantier. Les rouages administratifs indispensables à la conduite d’une véritable politique artistique sont mis en place dès lors que la Suède se dote d’un bureau de la surintendance des Bâtiments et d’une Académie royale. Ces évolu- tions se répercutent bientôt sur le statut et le rôle des artistes dans la mesure où le mouvement de professionnalisation et de spécialisation des activités les éloigne définitivement de l’univers artisanal. En recrutant des équipes de travail compé- tentes, formées et autonomes, la Suède est alors en mesure de réaliser un décor à la française. Elle se distingue de ses voisins polonais ou allemands qui, à la même époque, ont plutôt fait le choix d’importer les décors. Anne-Sophie Michel | Une communauté d’artistes et d’artisans français à l’étranger Les charges et fonctions administratives telles que Premier sculpteur du Roi, Conducteur des travaux, Professeur de sculpture viennent enfin reconnaître le rôle de chacun dans le cadre du chantier du château royal, et introduire une hiérarchie tout à fait nouvelle dans l’organisation du travail. La maîtrise perd alors peu à peu de son sens au profit de l’expérience et de la capacité d’inven- tion. Ainsi, la professionnalisation et la spécialisation des activités artistiques, induites par la présence des sculpteurs français, les éloignent définitivement au cours du siècle de l’univers artisanal de la génération précédente. 125 Notes 1. Cf. Stéphane Castellucio (dir.), Le commerce du luxe à Paris aux XVIIe siècle et XVIIIe siècle, échanges Nationaux et Internationaux (Bern, 2009). ( ) 2. A partir du XVIIe siècle, la France à l’apogée de sa puissance politique et culturelle do- mine l’Europe. De son côté la Suède, de par sa fragile situation géographique et politique, est à la recherche à la fois d’un puissant allié et d’un modèle politique pour s’affirmer sur la scène internationale. Dans ce rapport, souvent déséquilibré mais toutefois complémen- taire entre ces deux nations, la France apparaît comme un modèle en termes de goût, d’art de vivre et d’orientation artistique. Pour autant, ce rayonnement culturel ne résulte pas d’une emprise politique française qui aurait conduit, par son impérialisme, à une francisa- tion de l’élite suédoise. En effet, avec la création d’un Etat puissant, le besoin de paraître allait bientôt se faire ressentir chez les nobles suédois réunis autour du parti politique des Chapeaux. Les échanges commerciaux doublés d’échanges artistiques sont alors encouragés par une demande sans cesse croissante, appelée à resserrer les liens entre les deux pays. Cette circulation des idées comme des objets résulte de réseaux amicaux et commerciaux qui alimentent un véritable marché du luxe à destination de Stockholm. Sur ce sujet voir Marianne et Jean-François Battail, Une amitié millénaire : les relations entre la France et la Suède à travers les âges (Paris, 1993) ; Influences : relations culturelles entre la France et la Suède, actes pu- bliés par Gunnar von Proschwitz (Göteborg, 1988). 3. En 1771, le château royal est habitable mais les travaux d’aménagement se poursui- vent. La taxe prélevée pour la construction du château (Slottsbyggnadshjälpen) ne sera d’ailleurs abolie qu’en 1809. 4. Le soleil et l’étoile du Nord : La France et la Suède au XVIIIe siècle, association française d’action artistique, ministère des affaires étrangères (Paris, 1994). 5. Linda Hinners, De fransöske hantwerkarna på Stockholms Slott : yrkesroller, organisation, arbetspro- cesser, Stockholms Universitet (Stockholm, 2012) ; Linda Hinners & Martin Olin, « Les appartements royaux du château de Stockholm », Appartement princier – architecture – décor – cérémonial (à paraître) ; Linda Hinners, Linnéa Rollenhagen-Tilly & Anne-Sophie Mi- chel, « Paris à Stockholm, des échanges franco-suédois de la fin du XVIIe siècle au milieu du XVIIIe siècle », Bulletin de la Société d’Histoire de Paris et de l’Île-de-France, 139, 2012, pp. Sjuttonhundratal | 2015 Loin de témoigner d’un phénomène d’imitation servile, cette recherche confirme l’idée d’une adaptation circonstanciée et éphémère du modèle artistique français dans une perspective de transfert culturel. Affirmant ses propres compétences, la Suède s’émancipera d’ailleurs rapidement, comme en témoigne la création du style national gustavien par la nouvelle génération d’artistes formés par les Français au sein de la nouvelle Académie royale de Peinture et de Sculpture. 126 Anne-Sophie Michel | Une communauté d’artistes et d’artisans français à l’étranger Anne-Sophie Michel | Une communauté d’artistes et d’artisans français à l’étranger Notes 58–78 ; Pour une étude complète des artistes français présents sur le chantier du château royal cf. : Martin Olsson (ed.), Stockholm slotts historia, vol. 3 (Stockholm, 1940). 6. Cf. Pierre Lespinasse, Les artistes français en Scandinavie, coll. Société de l’histoire de l’Art français, (Paris, 1913) ; Louis Dussieux, Les artistes français à l’étranger (Paris, 1946) ; « Les études de transfert visent à étudier les interactions entre cultures et sociétés – ou frac- tions et groupes à l’intérieur d’une société – dans leur dynamique historique, à rendre compte des conditions qui ont marqué leur déclenchement et leur déroulement, à analyser les phénomènes d’émission, de diffusion, de réception et de réinterprétation qui les cons- tituent, enfin à décortiquer les mécanismes symboliques à travers lesquels se recomposent 127 Sjuttonhundratal | 2015 les groupes sociaux et les structures qui les sous-tendent », Sylvie Mesure & Patrick Savi- daan, Dictionnaire des Sciences humaines (Paris, 2006). les groupes sociaux et les structures qui les sous-tendent », Sylvie Mesure & Patrick Savi- daan, Dictionnaire des Sciences humaines (Paris, 2006). 7. Cf. Jérôme de La Gorce, « Le voyage des objets d’art de Paris à Stockholm à la fin du règne de Louis XIV », Le commerce du luxe à Paris aux XVIIe et XVIIIe siècles, pp. 321–334 ; Linda Hinners, « Nicodème Tessin le jeune et le marché de luxe parisien à la fin du règne de Louis XIV », Le commerce du luxe à Paris aux XVIIe et XVIIIe siècles, pp. 231–248 ; ou en- core : Marianne Roland-Michel, « Les achats du Comte de Tessin », Revue de l’Art, 1987, n°77, p. 28. DOI: http://dx.doi.org/10.3406/rvart.1987.347649 ; Gunnar Von Pros- chwitz, Tableaux de Paris et de la Cour de France 1739–1742, lettres inédites de Carl Gustaf, Comte de Tessin (Paris, 1983) ; Magnus Olausson, « Count Tessin, Mariette and the Crozat sale », Art Bulletin of Nationalmuseum Stockholm, vol. 19, 2012, pp. 145–156. 8. Cf. Linnéa Rollenhagen-Tilly, « Carl Johan Cronstedt’s stay in Paris (1732–1735) : Instruction, Contacts and Purchases », Art Bulletin of Nationalmuseum Stockholm, vol. 15, 2008, pp. 101–108 ; Ces dates correspondent au lancement des travaux de construction du château royal et à l’emménagement de la famille royale. Elles se caractérisent donc par une intense activité artistique autour du chantier du château royal ; Riksarkivet, Stoc- kholm, Ericsbergarkivet, Tessinsamlingen II, Correspondances. 13. RA, EA, TS, E5723, Lettre de Carl Gustaf Tessin à Carl Hårleman, Paris, le 7–18/11 1740. 12. Les Français recrutés entre ces dates sont: Jean Bourguignon, Charles-Guillaume Cousin, Jacques-Philippe Bouchardon, Pierre Saint Laurent, Pierre Leloup, Pierre et Si- mon Pontaléon, Jacques-Adrien Masreliez, Jean-Baptiste Maréchal, Pierre-Hubert L’Ar- chevêque. Notes E723, E724, E725, E726, E727, E728, E729, E730, E731, E732, E733, E734, E735, E736, E737, E738, E739, E740; Autografsamlingen, vol. 1–260. Papiers Carl Gustaf Tessin et Carl Hårleman ; Gunnar von Proschwitz, Tableaux de Paris et de la Cour de France 1739–1742, lettres inédites de Carl Gustaf, Comte de Tessin (Paris, 1983). 9. RA, EA, TS, E7537 : Lettre de Carl Johan Cronstedt à Carl Hårleman, Paris, le 14/07 1735. Il évoque la possibilité de recruter un sculpteur de Bruxelles : Johan Baptista Vanderhaegen ; Dans deux brouillons de lettres de Cronstedt à Hårleman, il est ques- tion d’un Flamand : Michiel Van der Voort (1667–1737) ou Michiel François van der Voort (1714–1777). RA, EA, TS, E5723, Lettre de Carl Gustaf Tessin à Carl Hårle- man, Venise, le 5–16/06 1736 : il évoque un sculpteur italien nommé Gaï. En revanche, ils réussirent très tôt à recruter deux peintres italiens : Alessandro Feretti et Domenico Francia. 10. RA, EA, TS, E5723, Lettre de Carl Hårleman à Carl Gustaf Tessin, Paris, le 22/02 1732. 11. RA, EA, TS, E5723, Lettre de Carl Hårleman à Carl Gustaf Tessin, Paris, le 15/05 1732. 12. Les Français recrutés entre ces dates sont: Jean Bourguignon, Charles-Guillaume Cousin, Jacques-Philippe Bouchardon, Pierre Saint Laurent, Pierre Leloup, Pierre et Si- mon Pontaléon, Jacques-Adrien Masreliez, Jean-Baptiste Maréchal, Pierre-Hubert L’Ar- chevêque. 128 Anne-Sophie Michel | Une communauté d’artistes et d’artisans français à l’étranger 14. Au total, ce corpus comprend onze contrats conservés dans les Archives Nationales, Paris, Minutier Central, ET/CVXIII/373, ET/XVIII/381, ET/XVIII/397 et ET/I/405 et deux autres contrats conservés dans les Archives du Château royal, Slottsarkivet, Slotts- byggnadsdeputationen, vol. II : 19 (artistes étrangers). 15. Cf. Antoine Schnapper, Le métier de peintre au grand siècle (Paris, 2004). 15. Cf. Antoine Schnapper, Le métier de peintre au grand siècle (Paris, 2004). 16. Cf. Lespinasse 1913. Le récit du voyage du premier groupe de Français parti au XVIIe siècle et revenu à cause du manque d’ouvrage et sans fortune, constitue un élément impor- tant. Il en est de même de celui des Français partie en Russie à qui on avait tout promis mais rien donné une fois sur place. 16. Cf. Lespinasse 1913. Le récit du voyage du premier groupe de Français parti au XVIIe siècle et revenu à cause du manque d’ouvrage et sans fortune, constitue un élément impor- tant. Notes Il en est de même de celui des Français partie en Russie à qui on avait tout promis mais rien donné une fois sur place. 17. RA, EA, TS, E5723, Lettre de Carl Hårleman à Carl Gustaf Tessin, Paris, le 15/05 1732. 18. RA, EA, TS, E5723, Lettre de Carl Hårleman à Carl Gustaf Tessin, Paris, 15/05 1732. 19. Cf. Jean-Pierre Poussou, Anne Mezin & Yves Perret-Gentil (dir.), L’influence française en Russie au XVIIIe siècle (Paris, 2004). Dans une lettre de Carl Hårleman à Carl Gustaf Tessin, Paris, en date du 15 mai 1732, Hårleman explique à Tessin : « J’ai mesme vu une lettre du Roy de Pologne par laquelle il offre huit milles francs à Oudry, jugés mon très cher Surintendant si l’on doit tant s’écrier si j’ai accordé 6000 livres à Taraval ». 20. Le logement de Rännarebanan, à Hötorget, est utilisé pour la fonte des sculptures en bronze et le logement des sculpteurs. Pourtant, les premiers ouvriers présents sur le chantier semblent résider au château. La mise à disposition d’un logement apparaît donc bien comme un avantage concédé aux Français. RA, EA, TS, E5723, Lettre Carl Hårleman à Carl Gustaf Tessin, Paris, 15/05 1732. « Le logement de Rännarebanan a été de tout temps celui des sculpteurs. La maison comme vous le savez est encore actuellement au château et ils en tiendront beaucoup dans celle la et elle est fort commode pour le jour et aux sculpteurs et aux peintres ». 20. Le logement de Rännarebanan, à Hötorget, est utilisé pour la fonte des sculptures en bronze et le logement des sculpteurs. Pourtant, les premiers ouvriers présents sur le chantier semblent résider au château. La mise à disposition d’un logement apparaît donc bien comme un avantage concédé aux Français. RA, EA, TS, E5723, Lettre Carl Hårleman à Carl Gustaf Tessin, Paris, 15/05 1732. « Le logement de Rännarebanan a été de tout temps celui des sculpteurs. La maison comme vous le savez est encore actuellement au château et ils en tiendront beaucoup dans celle la et elle est fort commode pour le jour et aux sculpteurs et aux peintres ». 21. RA, EA, TS, E5723, Lettre Carl Hårleman à Carl Gustaf Tessin, Paris, 30/05 1732. 22. Frédéric Tiberghien, Versailles : le chantier de Louis XIV (1662–1715) (Paris, 2002), p. 161. 21. RA, EA, TS, E5723, Lettre Carl Hårleman à Carl Gustaf Tessin, Paris, 30/05 1732. 22. Notes Frédéric Tiberghien, Versailles : le chantier de Louis XIV (1662–1715) (Paris, 2002), p. 161. 23. Cf. Barbro Lindqvist, Rokoko, opera och bibliskt skuggspel : katolska kulturarbetare i 1700-talets Stockholm, http://www.signum.se/archive/read.php?id=3662. 23. Cf. Barbro Lindqvist, Rokoko, opera och bibliskt skuggspel : katolska kulturarbetare i 1700-talets Stockholm, http://www.signum.se/archive/read.php?id=3662. 24. Cf. Tiberghien 2002, passim. 24. Cf. Tiberghien 2002, passim. 25. AN, MC, Placards et affiches, 6C6–6C13. 25. AN, MC, Placards et affiches, 6C6–6C13. 26. Slottsarkivet, Slottsbyggnadsdeputationen, vol. II : 19 : artistes étrangers. 27. RA, EA, TS, E5740, Lettre de Carl Gustaf Tessin à Carl Hårleman, Venise, le 18– 29/08 1736. Et SA, SBD, vol. II : 19 : artistes étrangers. Il est possible de lire dans une lettre non datée « J’eus l’honneur de vous présenter un mémoire pour l’augmentation de ma pension, eu égard de la hauteur du change, de la cherté des vivres, et de tous les autres besoins de la vie […] Loin de voir l’accomplissement de mes souhaits, le mal n’a fait qu’empirer depuis et me met absolument dans la nécessité ou de prendre de nouveau en- 129 Sjuttonhundratal | 2015 gagement avec vous Messieurs, en ce cas que mes service vous soyent agréables, ou, sinon, de me retirer, dans ma patrie, où mes parents et mes amis m’appellent depuis longtemps et où la paix et les ouvrages considérables, que le Roi et les princes font faire, m’ouvrent une carrière assez vaste pour exceller mon talent avec honneur et profit ». 28. Cette statue sera d’ailleurs la première statue équestre du Roi en Suède. Cf. Ragnar Josephson, L’Archevêques ungdom : hans studier i Rom och hans första verksamhet i Frankrike (Stoc- kholm, 1974); Johan Cederlund, Skulptören Pierre Hubert L’Archevêque 1721–1778 (Stoc- kholm, 2003). 29. SA, SBD, vol. II : 19, Quittances de Jean Bourguignon. 29. SA, SBD, vol. II : 19, Quittances de Jean Bourguignon. 30. Contrat de Jacques-Philippe Bouchardon cité par Andreas Lindblom, Jacques-Philippe Bouchardon och de franska bildhuggarna vid Stockholms slott under rokokotiden (Uppsala, 1924), p. 180 ; SA, SBD, vol. II : 19 : Contrat Adrien Masreliez. 31. Le soleil et l’étoile du Nord, pp. 104–105. 32. Lindblom 1924. 32. Lindblom 1924. 33. Cf. Marie-Liesse Pierre-Dulau, Trois artistes lorrains à Saint-Pétersbourg au XVIIIe siècle, « L’influence française en Russie au XVIIIe siècle », pp. 131–157. 34. AN, MC, LXVIII-373, 15/05 1732 ; SA, SB, vol. concédés aux artistes étrangers, l’emploi de jeunes Suédois revenus de leur voyage d’étude étant préférable. concédés aux artistes étrangers, l’emploi de jeunes Suédois revenus de leur voyage d’étude étant préférable. p 41. Lars Ljungström, « Surintendant et intendant de cour », Le soleil et l’étoile du Nord, p. 78. 42. Hinners 2012, p. 298. 43. Cf. Jean Chatelus, La Condition du peintre à Paris au 18e siècle (Lille, 1988); Nathalie Hei- nich, Du peintre à l’artiste, artisans et Académiciens à l’âge classique (Paris, 1993). 44. Cf. Hinners 2012; Heinich 1993; Schnapper 2004. 45. Les rémunérations des sculpteurs : A. Belette : 3400 livres, M. Lelièvre : 3400 livres, M. Gardy : 2000 livres, J. Bourguignon : 3400 livres, C-G. Cousin : 2500 livres, J-P. Bou- chardon : 5000 livres, A. Masreliez : 4500 livres, P-H. L’Archevêque 6000 livres. 46. Poussou 2004, pp. 131–157. 45. Les rémunérations des sculpteurs : A. Belette : 3400 livres, M. Lelièvre : 3400 livres, M. Gardy : 2000 livres, J. Bourguignon : 3400 livres, C-G. Cousin : 2500 livres, J-P. Bou- chardon : 5000 livres, A. Masreliez : 4500 livres, P-H. L’Archevêque 6000 livres. 46. Poussou 2004, pp. 131–157. Notes II : 19, 19/04 1735 ; AN, MC, XVIII-581, 23/01 1734 ; SA, SBD, vol. II : 19, 16/04 1748. XVIII-581, 23/01 1734 ; SA, SBD, vol. II : 19, 16/04 1748. XVIII-581, 23/01 1734 ; SA, SBD, vol. II : 19, 16/04 1748. 35. Cf. Bruno Pons, De Paris à Versailles, 1699–1736, les sculpteurs ornemanistes parisiens et l’art décoratif des bâtiments du roi (Paris, 1986) ; Bertrand Jestaz, Jules-Hardouin Mansart (Barcelone, 2008). 36. On a retrouvé au Garde-meuble un moulage en plâtre de détail de la chambre du prince royal. Selon les comptes ces cartouches furent exécutés d’après un dessin d’Adrien Masreliez de 1748. 37. Göran Alm, « L’atelier de sculpture d’ornement du château de Stockholm », Le soleil et l’étoile du Nord, pp. 104–105 ; Göran Alm, Franskt blev svenskt: den franska konstnärsfamiljen Masreliez i Sverige under 1700-talet (Stockholm, 1991). 38. Cf. Anne-Sophie Michel, Une communauté d’artistes et d’artisans Français à l’étranger : Le cas des sculpteurs français au château royal de Stockholm au XVIIIe siècle, Mémoire Master 2, sous la direction d’Alain Mérot (Paris, 2012). 39. Pour une histoire de l’Académie : cf. Ludvig Looström, Den svenska konstakademien under första århundradet af hennes tillvaro 1735–1835 : ett bidnag till den svenska konstens historia (Stoc- kholm, 1887). 40. Selon M. Lespinasse, un patriotisme mal inspiré imputait à crime l’appel fait par Tessin à des étrangers. Un clan se forma contre leurs partisans. En témoignent plusieurs faits comme le refus de la municipalité de Stockholm de concéder aux Français un local destiné à les loger plus convenablement ou la cabale dont fut victime L’Archevêque, ac- cusé de négligence et d’incapacité. Il sera d’ailleurs le dernier Français à avoir occupé une place prépondérante au sein de l’Académie. A partir de 1762 la Commission écrit au Roi qu’il est désormais inutile de grever les fonds de la construction de lourds appointements 130 Anne-Sophie Michel | Une communauté d’artistes et d’artisans français à l’étranger concédés aux artistes étrangers, l’emploi de jeunes Suédois revenus de leur voyage d’étude étant préférable. 41. Lars Ljungström, « Surintendant et intendant de cour », Le soleil et l’étoile du Nord, p. 78. 42. Hinners 2012, p. 298. 43. Cf. Jean Chatelus, La Condition du peintre à Paris au 18e siècle (Lille, 1988); Nathalie Hei- nich, Du peintre à l’artiste, artisans et Académiciens à l’âge classique (Paris, 1993). 44. Cf. Hinners 2012; Heinich 1993; Schnapper 2004. 45. Notes Les rémunérations des sculpteurs : A. Belette : 3400 livres, M. Lelièvre : 3400 livres, M. Gardy : 2000 livres, J. Bourguignon : 3400 livres, C-G. Cousin : 2500 livres, J-P. Bou- chardon : 5000 livres, A. Masreliez : 4500 livres, P-H. L’Archevêque 6000 livres. 46. Poussou 2004, pp. 131–157. concédés aux artistes étrangers, l’emploi de jeunes Suédois revenus de leur voyage d’étude étant préférable. Summary: A Community of French Artists and Craftsmen Abroad: A Case Study of the Sculptors of the Royal Palace of Stockholm in the Eighteenth Century Following the approach of Linda Hinners’s research, this article comprises a study of French sculptors who worked on the construction of Stockholm’s royal palace in the eighteenth century. Indeed, between 1732 and 1765, the superintendent of royal buildings had recruited, through the action of social networks, thirty French sculptors. To encourage them to leave France, the superintendent offered them very attractive conditions of life and work, and the prospects of a career. Once there, these sculptors created the royal palace decoration from the sketches of the Swedish architects. Beyond their artistic ability, the Swedes utilized their great experience of construction work and technical know-how. Soon, they took over the management of the sculpture works and training of young Swedish craftsmen present on the site. With the recruitment of French experts, the Swedes therefore had skilled and knowledgeable work teams, which created autonomous production workshops. These latter also underwent a modernization process induced by the creation of the Superintendence of royal buildings and the Swedish Royal Acad- emy. Thus, the French appear to have been the actors of a modern artistic policy that allowed Sweden to utilize the French aesthetic model. Keywords: cultural transfer; French sculptors; Royal Palace of Stockholm; Swedish artistic policy. 131
https://openalex.org/W2924273089
https://ora.ox.ac.uk/objects/uuid:1433057e-1858-4d32-a080-a4fd67d3081b/files/m7560dad7468d0a75921d2c19cb1f569e
English
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The Clinical Features, Pathogenesis and Methotrexate Therapy of Chronic Chikungunya Arthritis
Viruses
2,019
cc-by
8,045
Received: 24 February 2019; Accepted: 19 March 2019; Published: 22 March 2019 Abstract: Chikungunya fever (CHIKF) is an emerging viral infection that has spread widely, along with its Aedes vectors, throughout the tropics and beyond, causing explosive epidemics of acute illness and persistent disabling arthritis. The rheumatic symptoms associated with chikungunya virus (CHIKV) infection include polyarthralgia, polyarthritis, morning stiffness, joint edema, and erythema. Chronic CHIK arthritis (CCA) often causes severe pain and associated disability. The pathogenesis of CCA is not well understood. Proposed hypotheses include the persistence of a low level of replicating virus in the joints, the persistence of viral RNA in the synovium, and the induction of autoimmunity. In this review, we describe the main hypotheses of CCA pathogenesis, some of which support methotrexate (MTX) treatment which has been shown to be effective in preliminary studies in CCA. Keywords: chikungunya virus; chronic chikungunya arthritis; pathogenesis; methotrexate J. Kennedy Amaral 1, Peter C. Taylor 2, Mauro Martins Teixeira 3, Thomas E. “Tem” Morrison 4 and Robert T. Schoen 5,* J. Kennedy Amaral 1, Peter C. Taylor 2, Mauro Martins Teixeira 3, Thomas E. “Tem” Morrison 4 and Robert T. Schoen 5,* . Kennedy Amaral 1, Peter C. Taylor 2, Mauro Martins Teixeira 3, Thomas E. “Tem” Morrison 4 and Robert T. Schoen 5,* 1 Department of Infectious Diseases and Tropical Medicine, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais 31270-901, Brazil; jkennedy-@hotmail.com 2 Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Windmill Road, Oxford, OX3 7LDR, UK; peter.taylor@kennedy.ox.ac.uk 1 Department of Infectious Diseases and Tropical Medicine, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais 31270-901, Brazil; jkennedy-@hotmail.com 2 Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Windmill Road, Oxford, OX3 7LDR, UK; peter.taylor@kennedy.ox.ac.uk Belo Horizonte, Minas Gerais 31270 901, Brazil; jkennedy @hotmail.com 2 Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Windmill Road, Oxford, OX3 7LDR, UK; peter.taylor@kennedy.ox.ac.uk p y y 3 Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais 31270-901, Brazil; mmtex.ufmg@gmail.com 4 Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045, USA; Thomas.morrison@udenver.edu 5 Section of Rheumatology, Allery and Immunology, Yale University School of Medicine, New Haven, CT 06510, USA * Correspondence: robert.schoen@yale.edu * Correspondence: robert.schoen@yale.edu   viruses viruses viruses viruses viruses 1. Introduction Chikungunya virus (CHIKV) is a small (60–70 nm, 12 Kb), single-stranded positive-sense RNA virus in the Alphavirus genus of the Togaviridae family [1,2]. Like many other arboviral diseases, chikungunya fever (CHIKF) is transmitted by mosquito vectors, primarily Aedes aegypti and Aedes albopictus [3]. CHIKF is an infectious febrile illness often accompanied by acute and chronic arthritis. The name “chikungunya” in the Makonde dialect refers to the stooped posture assumed by many patients who experience disabling joint pain [4]. CHIKV was isolated in 1952 during an outbreak in the Makonde Plateau, Tanzania (Africa), but CHIKF may be much older [5]. A disease similar to CHIKF was reported in Zanzibar in 1823, and since then, sporadic outbreaks have primarily been reported in Africa and parts of Asia. In the 21st century, CHIKF became a global epidemic. The disease occurred in Kenya in 2004 (500,000 cases), and in 2005, spread across countries in the Indian Ocean, including Reunion Island (266,000 cases) and India (1.4 million cases) [3,6,7]. In 2013, CHIKF was reported in the Caribbean island of Saint Martin and spread to 45 countries in the Americas (with more than 3 million cases) [6]. In Brazil alone, almost 500,000 cases of CHIKF have been reported since 2014 [8]. www.mdpi.com/journal/viruses Viruses 2019, 11, 289; doi:10.3390/v11030289 Viruses 2019, 11, 289 2 of 12 CHIKV infection typically occurs in two phases. In the first phase, 2 to 6 days after the mosquito bite, infected patients develop a high fever, arthralgia and arthritis, headache, maculopapular rash, and intense fatigue, often accompanied by anorexia, nausea, vomiting, and diarrhea [9]. In most reports, almost all of the infected patients became symptomatic, but high rates of asymptomatic infection have also been recorded in Thailand (47.1%) and Kenya (45.1%) with east/central/south African viral lineages [10]. Acute CHIKF lasts approximately 10 days [4]. Neurological complications, including encephalitis, optic neuritis, facial paralysis, sensorineural deafness, and Guillain–Barré syndrome, can occur in a variable proportion of patients [11]. Less frequently, CHIKF causes myocarditis, cardiac arrhythmia, severe sepsis, septic shock, and renal failure [3]. After the acute phase, the disease resolves in some patients, but 25%–40% develop chronic arthritis that includes musculoskeletal pain, arthralgia, or frank arthritis, lasting weeks or months after the acute attack. These symptoms can be relapsing or persistent and are unrelated to previous rheumatic disease, which is absent in the majority of patients [12,13]. 1. Introduction CHIKF has become a global epidemic, causing acute febrile illness, chronic painful arthritis, and severe economic harm to the affected communities [4,7]. Unfortunately, at present, the only available treatment for acute infection is supportive—hydration, and use of opioid analgesics, paracetamol/acetaminophen and non-steroidal anti-inflammatory drugs (NSAIDs). Aspirin has been avoided due to the risk of bleeding, especially in patients who were co-infected with dengue or at risk of Reye’s syndrome. Similarly, corticosteroids have been avoided due to the concern for immunosuppression and potential exacerbation of the viral infection [4]. Despite the gaps in understanding of the mechanism of viral replication and pathogenesis of both acute and chronic disease, various antiviral therapeutics have been investigated [14,15]. These efforts include preclinical studies of the traditional antiviral compounds, synthesis of designer molecules, in silico high-throughput screening for existing products with efficacy against CHIKV, nucleic acid compounds, therapeutic monoclonal antibodies, and drugs that target host cell proteins [15]. Several compounds are in early clinical trials, including ribavirin, 6-azauridine, glycyrrhizin, and interferon. There has also been interest in vaccine development, including virus-like particle and measles-vectored vaccines that have reached phase 2 clinical trials [16]. However, at the present time, no antiviral therapies or licensed vaccines are available to lessen the impact of this epidemic [15,17]. As its pathogenesis is uncertain, there is no existing consensus as to how chronic chikungunya arthritis (CCA) should be treated. In addition to symptomatic treatment with NSAIDs, a variety of inflammation suppressing therapies, including corticosteroids, chloroquine (CHQ), hydroxychloroquine (HCQ), sulfasalazine (SSZ), methotrexate (MTX), and immune-modulating biologic agents including anti-TNF agents, B-lymphocyte depletion (rituximab), and interleukin-6 receptor inhibition (tocilizumab), have been used [13,18,19]. The treatment strategy has been empirical and no high-quality, controlled, randomized trials have assessed these interventions in CCA [19]. In this report, we discuss the clinical features and pathogenesis of CCA, including evidence for a persistent viral infection versus a post-infectious inflammatory disease. We considered MTX as a therapeutic option for CCA, based on the current understanding of its pathogenesis and evaluated the MTX studies that have been conducted in CCA [20]. Due to the severity and chronicity of CHIK arthritis, it is important to develop effective treatment [21]. We are optimistic about the use of MTX in CCA, based on favorable, albeit limited, clinical data [19,20,22]. 2. Chronic Chikungunya Arthritis The transition from CHIKF to CCA is variable. In many patients, arthrialgias/arthritis began at the onset of disease and is unremitting. In others, there is transient improvement after CHIKF followed by persisitent arthritis [4,12,23]. In several studies, 25% to 62% of patients had arthritic symptoms 18 months after the onset of CHIKF [23,24]. Even at the 36-month follow-up, the prevalence of arthritis has been reported to be as high as 60% [12,13,25]. Among 152 Colombian patients evaluated at 26 weeks after disease onset, persistent arthritis was found in 54%, morning stiffness in 49%, joint Viruses 2019, 11, 289 3 of 12 edema in 41%, and both polyarthralgia and morning stiffness in 38% [26]. In a Reunion Island study (2005–2006) of 88 patients, chronic arthritis occurred in 93%, 57%, and 47% at 3, 15, and 24 months, respectively [24]. In another Colombian cohort (2014–2015), persistent arthritis was less frequent, with 12% affected at the 18-month follow-up [23]. Viruses 2018, 10, x 3 of 12 Reunion Island study (2005–2006) of 88 patients, chronic arthritis occurred in 93%, 57%, and 47% at Rheumatic symptoms associated with CHIKV infection include polyarthralgia, polyarthritis, morning stiffness, joint edema, and joint redness. CHIK arthritis often causes severe pain and associated disability [21,27]. The most commonly affected joints are hands, knees, wrists, ankles, and shoulders [22,24,26,28]. For example, Borgherini et al. [24] reported the frequency of joints affected as hands (57%), knees (57%), wrists (50%), ankles (46%), and shoulders (45%). Mathew et al. [29] described the involvement of knees (83%), ankles (62%), and elbows (59%). The pattern of symmetric polyarthralgia/polyarthritis of large and small joints, especially in the hands, knees, shoulders, wrists, and ankles, has been frequently described [26,28–31]. 3, 15, and 24 months, respectively [24]. In another Colombian cohort (2014–2015), persistent arthritis was less frequent, with 12% affected at the 18-month follow-up [23]. Rheumatic symptoms associated with CHIKV infection include polyarthralgia, polyarthritis, morning stiffness, joint edema, and joint redness. CHIK arthritis often causes severe pain and associated disability [21,27]. The most commonly affected joints are hands, knees, wrists, ankles, and shoulders [22,24,26,28]. For example, Borgherini et al. [24] reported the frequency of joints affected as hands (57%), knees (57%), wrists (50%), ankles (46%), and shoulders (45%). Mathew et al. [29] described the involvement of knees (83%), ankles (62%), and elbows (59%). 2. Chronic Chikungunya Arthritis The pattern of symmetric polyarthralgia/polyarthritis of large and small joints especially in the hands knees shoulders wrists A number of reports characterized CCA as “post-chikungunya chronic inflammatory rheumatism” (CIR-CHIK) [12,18,26,29,30,32]. Javelle et al. [13] described 159 patients who were symptomatic for at least 2 years, 112 of whom developed CIR-CHIK classified as mimicking four clinical patterns—rheumatoid arthritis (RA), seronegative spondyloarthritis (SpA), fibromyalgia (FM), or undifferentiated polyarthritis (UP), defined as the presence of inflammatory arthritis affecting more than four joints of greater than 6 weeks duration in the absence of an alternative diagnosis. Among these 112 patients, 33 fulfilled classification criteria for spondyloarthritis, 40 for RA, and 21 for undifferentiated polyarthritis [13,31]. In a group of 437 patients with CHIKF (India, 2011), 57% developed post-viral polyarthralgia, 22% inflammatory polyarthritis, and 19.5% tenosynovitis during a 15-month period [29]. Several of these patients’ diseases were reported to mimic RA and psoriatic arthritis (PsA) [13,30,33]. polyarthralgia/polyarthritis of large and small joints, especially in the hands, knees, shoulders, wrists, and ankles, has been frequently described [26,28–31]. A number of reports characterized CCA as “post-chikungunya chronic inflammatory rheumatism” (CIR-CHIK) [12,18,26,29,30,32]. Javelle et al. [13] described 159 patients who were symptomatic for at least 2 years, 112 of whom developed CIR-CHIK classified as mimicking four clinical patterns—rheumatoid arthritis (RA), seronegative spondyloarthritis (SpA), fibromyalgia (FM), or undifferentiated polyarthritis (UP), defined as the presence of inflammatory arthritis affecting more than four joints of greater than 6 weeks duration in the absence of an alternative diagnosis. Among these 112 patients, 33 fulfilled classification criteria for spondyloarthritis, 40 for RA, and 21 for undifferentiated polyarthritis [13,31]. In a group of 437 patients with CHIKF (India, 2011), 57% developed post-viral polyarthralgia, 22% inflammatory polyarthritis, and 19.5% tenosynovitis during a 15-month period [29]. Several of these patients’ diseases were reported to Similarly, Schilte et al. [25] studied 180 patients with CHIKV arthritis of 36 months duration in whom the hands, wrists, ankles, and knees were most affected. Around 60%–80% of the patients had intermittent arthritis, while arthritis was unremitting in 20%–40%. Among 173 patients with CHIKV evaluated at 27.5 months, Essackjee et al. [33] reported that 78.6% had persistent musculoskeletal symptoms and 5% met the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria for RA. mimic RA and psoriatic arthritis (PsA) [13,30,33]. Similarly, Schilte et al. 2. Chronic Chikungunya Arthritis [25] studied 180 patients with CHIKV arthritis of 36 months duration in whom the hands, wrists, ankles, and knees were most affected. Around 60%–80% of the patients had intermittent arthritis, while arthritis was unremitting in 20%–40%. Among 173 patients with CHIKV evaluated at 27.5 months, Essackjee et al.[33] reported that 78.6% had persistent musculoskeletal symptoms and 5% met the American College of Rheumatology/European League Against Rh ti (ACR/EULAR) it i f RA Other reports have also focused on CCA as an “RA mimic”. Among the 203 patients with CHIKF who developed joint pain, 36% (34/94) met the ACR/EULAR criteria for RA [34]. In other reports, RA mimics have been even more frequent. In 39 Colombian patients with CHIKF, 90% developed arthritis that met the RA ACR/EULAR criteria [35]. In a small cohort of 10 American relief workers who developed CHIKF in Haiti, 8 of 10 fulfilled the ACR/EULAR criteria for seronegative RA, presenting with morning stiffness and polyarthritis, especially in the hands, wrists, feet, and ankles [36]. A typical example of symmetrical polyarthritis of the hands—reminiscent of RA—in one of our patients is shown in Figure 1. Another parallel to RA has been the finding of subchondral bone erosions, joint effusions, and joint thickening in patients with CCA [34,37]. Rheumatism (ACR/EULAR) criteria for RA. Other reports have also focused on CCA as an “RA mimic”. Among the 203 patients with CHIKF who developed joint pain, 36% (34/94) met the ACR/EULAR criteria for RA [34]. In other reports, RA mimics have been even more frequent. In 39 Colombian patients with CHIKF, 90% developed arthritis that met the RA ACR/EULAR criteria [35]. In a small cohort of 10 American relief workers who developed CHIKF in Haiti, 8 of 10 fulfilled the ACR/EULAR criteria for seronegative RA, presenting with morning stiffness and polyarthritis, especially in the hands, wrists, feet, and ankles [36]. A typical example of symmetrical polyarthritis of the hands—reminiscent of RA—in one of our patients is shown in Figure 1. Another parallel to RA has been the finding of subchondral bone erosions, joint effusions, and joint thickening in patients with CCA [34,37]. Figure 1. Woman, 82 years old, 2 years after CHIKV infection. Intense arthritis of metacarpophalangeal joints and wrist. Figure 1. Woman, 82 years old, 2 years after CHIKV infection. Intense arthritis of metacarpophalangeal joints and wrist. Figure 1. Woman, 82 years old, 2 years after CHIKV infection. 2. Chronic Chikungunya Arthritis Intense arthritis of metacarpophalangeal joints and wrist. Figure 1. Woman, 82 years old, 2 years after CHIKV infection. Intense arthritis of metacarpophalangeal joints and wrist. 4 of 12 Viruses 2019, 11, 289 Among reports of CCA patients whose disease mimics RA, the rates of rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody (CCP) positivity have varied widely. In an Indian study, 13 of 95 patients and 4 of 67 patients were respectively positive for RF and anti-CCP antibody [38]. In other reports, RF positivity has varied between 25% and 43%, and anti-CCP antibody positivity has been less frequent [39]. We observed a patient who developed RA symptoms 3 months after CHIKF who was positive for both RF and anti-CCP antibody. In addition, this patient developed neutropenia and splenomegaly within 1 year after the onset of her arthritis, establishing the diagnosis of Felty’s syndrome [40]. On the other hand, in a Colombian cohort of 109 CCA patients, 98% had no detectable RF or anti-CCP antibodies [12]. In the Haitian report, none of the 8 RA mimic patients had RF or anti-CCP antibody positivity [36], and of the 22 prospectively evaluated Reunion Island patients with long-term arthralgia, none were positive for anti-CCP antibody [13,31]. There may be two different groups of chronic arthritis patients being reported—CCA patients who are negative for RF and anti-CCP antibody, and another group that are more frequently positive for RF and anti-CCP antibody and actually suffering from two diseases, CHIKF followed by new onset of RA. Recently in our clinic, we evaluated a cohort of 50 Brazilian patients seen with CCA that we defined as arthritis/arthralgia, persisting for more than 3 months after the onset of CHIKF [41]. Our patients, 90% of whom were self-referred, experienced chronic pain and disability prior to evaluation in our clinic. The mean time between the onset of CHIKF and the first visit to us was 14.2 months, similar to the delays of 8 months to 2 years in treatment of CCA reported elsewhere [41]. Thirty of our patients (60%) had arthralgia while 20 patients (40%) also had arthritis, with clinically evident synovitis (Figure 1). Arthralgia was most common in the hands (56%), ankles (48%), and knees (44%). Arthralgia was polyarticular (>4 joints) (76%) or oligoarticular (2–4 joints) (24%). Of the patients with arthritis, all 20 had hand involvement. 2. Chronic Chikungunya Arthritis Other joints with arthritis included wrists in 16 (32%), ankles in 12 (24%), and ankles and knees in 9 (18%) patients. Overall, both large and small joints of the upper and lower extremities were affected. Morning stiffness, low back pain, and neck pain were reported by 3 (6%), 8 (16%), and 3 (6%) patients, respectively. The ACR criteria for RA were met by 11 (22%) of the patients, and another 7 (14%) met the criteria for FM [41]. We evaluated whether pre-existing rheumatic disease in our patients impacted the subsequent clinical expression of CCA and found that pre-existing rheumatic diseases could aggravate the pain in CCA patients. Similarly, among 159 cases of CHIK-CIR analyzed by Javelle et al. [12], 50 (31%) had previous “rheumatic and musculoskeletal disorders”, including 6 with “chronic inflammatory rheumatism” [12]. Sissoko [32] observed osteoarthritis in 38 of 147 (26%) patients who were followed for 15 months. In a small study, Zeana [42] reported preexisting rheumatic disease, including osteoarthritis, carpal tunnel syndrome, retrocalcaneal bursitis, and lateral epicondylitis. Essackjee [33] found that among 173 patients followed for more than 2 years after CHIKV infection, 27 (15.6%) had some pre-existing musculoskeletal disease. In our cohort, the rate of pre-existing rheumatic disease was significant (22 of 50; 44%), but there was no relationship between these conditions and the clinical expression of CHIK arthritis or the response to treatment [41]. Suggested risk factors for progression to long-term disease include female sex, age greater than 45, diabetes mellitus, hypertension, dyslipidemia, and previous rheumatic disease [43]. In addition, one study found that more severe initial CHIKV infection predicted a greater likelihood of long-term arthritis [44]. 3. Replication Cycle of Chikungunya Virus The transmission cycle of CHIKV requires the infection of female mosquitoes through a virus-containing blood meal and, following an appropriate extrinsic incubation period, transmission to another vertebrate host during subsequent feeding [1,4]. The ability of CHIKV to bind human cells and to replicate in cell cultures was documented, and the general features are similar to other alphaviruses [45–47]. CHIKV is internalized into human target cells mainly as a result of Viruses 2019, 11, 289 5 of 12 receptor-mediated endocytosis involving clathrin-dependent mechanisms, delivering the virus to endosomes from which the viral capsid is released into the cytosol [48–50]. The latter process is triggered by the low pH environment in the endosome that induces conformational changes in the viral E1 and E2 glycoproteins. These conformational changes result in exposure of the E1 fusion loop, which inserts into the host membrane and promotes fusion of the viral envelope and endosomal membrane. The synthesis of viral RNA occurs in the replication complexes found in the bulb-shaped invaginations of the plasma membrane, termed spherules, and includes the production of genomic positive-sense viral RNA, as well as a subgenomic positive-sense viral RNA, that encodes the structural polyprotein (capsid, E3, E2, 6K/TF, E1) from a negative-sense template. Following release of the capsid protein via autoproteolysis, the E1 and E2 glycoproteins are trafficked into the endoplasmic reticulum, transported through the Golgi, processed in the trans Golgi network, and finally transported to the plasma membrane where virus assembly and egress occur [49,51]. CHIKV is highly cytopathic in human cell cultures, and the infected cells rapidly undergo apoptosis [52]. Alphavirus replication strongly affects the fundamental processes of cellular physiology, with inhibition of cell transcription and translation, and redirection of cellular resources for the synthesis of viral proteins and viral genomes [52–54]. Studies have shown that CHIKV infection has been associated with extensive cell death and the release of high levels of infectious virus [46,54]. In human cell cultures, CHIKV elicits an autophagic response that promotes viral replication [55–57]. A study showed that the ability of CHIKV to induce apoptosis depends on the ability of the virus to replicate [55]. There are clues that the completion of the apoptotic process is an important element for the efficient propagation of the virus [58]. 4. Pathogenesis of Chronic Chikungunya Arthritis There are intriguing similarities in the immunological phenotypes of peripheral blood mononuclear cells of patients with RA and CCA (CCA), but the pathobiology of CCA is not well understood. Proposed hypotheses include the persistence of a low level of replicating virus in the joints, the persistence of viral RNA in synovium, and the induction of autoimmunity [63]. 3. Replication Cycle of Chikungunya Virus Inhibition of the apoptotic process by pan-caspase inhibitors (a family of intracellular cysteine proteases critical to several cellular functions, including apoptosis and inflammation) limits the number of CHIKV-infected cells [55,59]. A study showed that blood monocytes were the main targets of CHIKV during acute phase infection and that macrophages and B lymphocytes were also infected with the virus, contrary to previous reports [60]. CHIKV infection rapidly results in the induction of type I interferons (IFNs) and the production of proinflammatory cytokines as part of the innate cellular immune response [61,62]. 4.1. Cytokine/Chemokine Responses In some studies, CCA is associated with high levels of circulating IL-6, GM-CSF, IFN-α, and IL-17 [64,65]. In a study of the involvement of inflammatory cytokines and chemokines, plasma levels of IL-6 and GM-CSF were significantly higher in patients with persistent arthralgia compared with those who had recovered [66]. IL-6 is involved in the joint inflammation associated with RA and increases the production of cartilage-destroying enzymes. In addition to IL-6, CCA patients have other systemic markers of inflammation, such as the IFN-induced chemokines MIG/CXCL-9 and IP-10/CXCL-10, as well as proinflammatory cytokines including IL-1β, IL-1RA, CCL-2/MCP-1, CCL-3/MIP-1a, CCL-4/MIP-1b, and IL-12 [64]. MCP-1/CCL-2 is a major chemoattractant for monocytes and macrophages, and is strongly expressed during acute infection in humans and animal models [1,67]. In addition, the worsening severity of CHIKF has been associated with increased plasma levels of IL-1β and IL-6 and a reduced level in RANTES. As in RA, high levels of IL-1β may also mediate the development of abrupt and persistent arthralgia [68]. In a recent study, levels of all cytokines that were elevated in patients with CCA (IL1 -RA, IL-6, IL-8, MIP-1a, MIP-1b, and MCP-1) returned to Viruses 2019, 11, 289 6 of 12 control levels following disease resolution, indicating that elevations of these cytokines were specific for patients who develop chronic arthritis [65]. Viruses 2018, 10, x 6 of 12 lymphocytes, acting as a growth factor for natural killer (NK) cells, increasing their cytotoxic action A study of the cytokine profile of patients in the acute and chronic phases of CHIKF identified dramatically elevated levels of IL-12 in chronic patients [64]. IL-12 is essential for the synthesis of Th1 lymphocytes, acting as a growth factor for natural killer (NK) cells, increasing their cytotoxic action [69]. In the synovial tissue analysis of patients with CCA, there was evidence of active monocyte/macrophage trafficking and several abnormal histological findings in the synovial membrane, including synovial lining hyperplasia, vascular proliferation, and infiltration of macrophages [64]. y p y g g g y [69]. In the synovial tissue analysis of patients with CCA, there was evidence of active monocyte/macrophage trafficking and several abnormal histological findings in the synovial membrane, including synovial lining hyperplasia, vascular proliferation, and infiltration of macrophages [64]. Most parenchymal cells express interleukin-17 receptors. 4.1. Cytokine/Chemokine Responses Signaling through these receptors induces target cells to produce proinflammatory factors, such as IL-1, IL-6, IL-8, TNF, and matrix metalloproteinases capable of destroying the extracellular matrix and causing bone resorption Most parenchymal cells express interleukin-17 receptors. Signaling through these receptors induces target cells to produce proinflammatory factors, such as IL-1, IL-6, IL-8, TNF, and matrix metalloproteinases capable of destroying the extracellular matrix and causing bone resorption [2,62,70]. This helps in understanding cases of bone erosion and joint damage in patients with CCA (Figure 2). p p y g g p [2,62,70]. This helps in understanding cases of bone erosion and joint damage in patients with CCA (Figure 2). Figure 2. Cytokines that participate in the pathogenic process of acute and chronic phases of CHIKV infection and likely mechanism of action of MTX therapy. Figure 2. Cytokines that participate in the pathogenic process of acute and chronic phases of CHIKV infection and likely mechanism of action of MTX therapy. Figure 2. Cytokines that participate in the pathogenic process of acute and chronic phases of CHIKV infection and likely mechanism of action of MTX therapy. Figure 2. Cytokines that participate in the pathogenic process of acute and chronic phases of CHIKV infection and likely mechanism of action of MTX therapy. 4 2 Cellular Response 4.2. Cellular Response p An inefficient antiviral response due to disturbed immune cell function (NK, T cell, B cell, etc.) may be a possible reason for the persistence of the virus and/or chronic arthralgia [71]. For example, in one study, the expression of NKG2A and CD94 inhibitory receptors on natural killer (NK)/natural killer T cell (NKT) cells from chronic CHIK patients was elevated [71]. Another study that examined human synovial biopsies with CCA showed a high fraction of activated CD69 + CD4 + T cells, suggesting that these cells may contribute to chronic disease [64]. Indeed, a study reported the reduced frequency of NK-like T cells, lower expression of perforin + NK, and higher expression of TNF-α + NK-like T and IFN-γ + NK-like T cells as markers of chronic arthritic diseases [72] An inefficient antiviral response due to disturbed immune cell function (NK, T cell, B cell, etc.) may be a possible reason for the persistence of the virus and/or chronic arthralgia [71]. For example, in one study, the expression of NKG2A and CD94 inhibitory receptors on natural killer (NK)/natural killer T cell (NKT) cells from chronic CHIK patients was elevated [71]. Another study that examined human synovial biopsies with CCA showed a high fraction of activated CD69 + CD4 + T cells, suggesting that these cells may contribute to chronic disease [64]. Indeed, a study reported the reduced frequency of NK-like T cells, lower expression of perforin + NK, and higher expression of TNF-α + NK-like T and IFN-γ + NK-like T cells as markers of chronic arthritic diseases [72]. 5. Methotrexate Therapy of Chronic Chikungunya Arthritis To explain the anti-inflammatory properties of MTX, several mechanisms of action have been suggested, including the inhibition of purine and pyrimidine synthesis, suppression of transmethylation reactions with accumulation of polyamines, reduction of antigen-dependent T-cell proliferation, and promotion of adenosine release with adenosine-mediated suppression of inflammation [77,78]. In RA, the anti-inflammatory effects of MTX seem to be related to an extracellular increase in adenosine and its interaction with specific cell surface receptors. This is followed by the inhibition of purine and pyrimidine synthesis, mediated by the production of interleukin 8 (IL8)/CXCL8 by peripheral blood mononuclear cells (PBMC), the secretion of IL6 by human monocytes, and the inhibition of other proinflammatory cytokines, including IL-1, IL-2, IL-8, IL-12, and TNF-α [78,79]. In addition, MTX reduces the production of IL-4, IL-6, IL-13, TNF-α, interferon gamma (IFNγ), and granulocyte-macrophage colony-stimulating factor (GM-CSF), due to the de novo synthesis of purines and pyrimidines [80]. Studies in animal models have shown that a low dose of MTX promotes intracellular accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an intermediate in purine synthesis, and that the accumulation of AICAR is associated with increased adenosine release in inflammatory exudates; adenosine mediates the anti-inflammatory effects of MTX in animal models of both acute inflammation and adjuvant arthritis [77,81]. As CCA is similar to RA [26,30,36] and has a similar pathogenesis [2,62–64], the improvement of patients with CCA using MTX [20,41] could be due to the increase in adenosine, in addition to the decrease in levels of inflammatory interleukins. A recent study revealed that MTX treatment is probably safe and does not affect the antiviral and inflammatory responses of primary human synovial fibroblasts (HSF). MTX alone did not increase the capacity of CHIKV to infect and replicate in HSF [82]. This fact is important because the immunomodulatory activity of MTX in the context of viral persistence has been of concern. We conducted a systematic review to evaluate the efficacy and safety of MTX in CCA [20]. Among 131 possibly relevant available studies, six met our evaluation criteria. Of these, four were retrospective studies, one was an uncontrolled prospective study, and one was an unblinded randomized clinical trial comparing MTX monotherapy to MTX combined with sulfasalazine and hydroxychloroquine. Based on our review of published studies of MTX in CCA, we believe that (a) MTX treatment has been safe and (b) the evidence warrants high-quality randomized clinical trials of MTX in CCA [20]. 4 3 Autoimmunity 4.3. Autoimmunity 4.3. Autoimmunity Experiments using non-human primates and mice demonstrated that the RNA and CHIKV antigen remained detectable in musculoskeletal and other tissues for months after infection. In mice, the long-term persistence of viral RNA was associated with chronic inflammatory pathology and immune activation [73]. In the mouse model, chronic arthritis may be related to persistent, replicating, transcriptionally active CHIKV RNA [74]. In contrast, in an analysis of 33 patients 22 months after acute infection, no viral RNA or proteins were identified in the synovial fluid, Experiments using non-human primates and mice demonstrated that the RNA and CHIKV antigen remained detectable in musculoskeletal and other tissues for months after infection. In mice, the long-term persistence of viral RNA was associated with chronic inflammatory pathology and immune activation [73]. In the mouse model, chronic arthritis may be related to persistent, replicating, transcriptionally active CHIKV RNA [74]. In contrast, in an analysis of 33 patients 22 months after acute infection, no viral RNA or proteins were identified in the synovial fluid, suggesting that viral Viruses 2019, 11, 289 7 of 12 persistence may not be necessary for persistent arthritis [75]. The authors suggested that autoantigens or autoreactive lymphocytes could be present in the synovium or muscle tissue, contributing to the CCA. Immunohistology on muscle biopsies from two CHIKV-infected patients with a myositic syndrome showed that viral antigens were found exclusively inside skeletal muscle progenitor cells; viral RNA has also been detected in synovial and muscle tissue biopsies collected from patients with CCA [51,63,76]. 5. Methotrexate Therapy of Chronic Chikungunya Arthritis As noted, we recently evaluated the effectiveness of MTX in pain reduction using a visual analog scale (VAS) (0–10) with higher values indicating more severe pain [41]. We treated 48 Brazilian CCA patients who were seen 14.2 (SD, 4.2) months after the onset of disease. Sixty percent of the patients had arthralgia. Forty percent also had arthritis. The patients received MTX 7.5 mg/week with folic acid, with dose escalations for refractory symptoms at 4 weeks. The final mean MTX dose was 9.2 (SD, 3.2) mg/week. The mean reductions in pain at 4 and 8 weeks, compared to baseline, were 4.3 (3.0) (p < 0.0001) and 4.5 (2.6) (p < 0.0001), respectively [41] Viruses 2019, 11, 289 8 of 12 6. Conclusions The reported improvement of CCA patients treated with MTX may be a pathway for understanding the uncertain pathogenesis of this emerging form of arthritis. The similarities in the clinical and pathogenic characteristics of CCA and RA make MTX an essential drug to evaluate in CCA. A better understanding of the treatment of CCA should provide further insights, not only in this disease, but in other forms of inflammatory arthritis, including RA. Author Contributions: All authors have made substantial contributions to the conception of the work and have drafted the work or substantively revised it; A.N.D. has approved the submitted version; they agree to be personally accountable for their contributions and for questions related to the accuracy or integrity of any part of the work. Conceptualization, J.K.A., P.C.T., M.M.T., T.E.T.M. and R.T.S.; Writing—Original Draft Preparation, J.K.A., P.C.T., M.M.T., T.E.T.M. and R.T.S.; Writing—Review & Editing, J.K.A., P.C.T., M.M.T., T.E.T.M. and R.T.S. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. 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Physicians India 2011, 59, 83–86. 23. Chang, A.Y.; Encinales, L.; Porras, A.; Pacheco, N.; Reid, S.P.; Martins, K.A.; Simon, G.L. Frequency of Chronic Joint Pain Following Chikungunya Virus Infection. Arthritis Rheumatol. 2018. [CrossRef] 24. Borgherini, G.; Poubeau, P.; Jossaume, A.; Gouix, A.; Cotte, L.; Michault, A.; Paganin, F. Persistent Arthralgia Associated with Chikungunya Virus: A Study of 88 Adult Patients on Reunion Island. Clin. Infect. Dis. 2008, 47, 469–475. [CrossRef] [PubMed] 25. Schilte, C.; Staikovsky, F.; Couderc, T.; Madec, Y.; Carpentier, F.; Kassab, S.; Michault, A. Chikungunya Virus-associated Long-term Arthralgia: A 36-month Prospective Longitudinal Study. PLoS Negl. Trop Dis. 2013, 7, e2137. [CrossRef] 26. Rodriguez-Morales, A.J.; Gil-Restrepo, A.F.; Ramírez-Jaramillo, V.; Montoya-Arias, C.P.; Acevedo-Mendoza, W.F.; Bedoya-Arias, J.E.; Lagos-Grisales, G.J. Post-chikungunya chronic inflammatory rheumatism: Results from a retrospective follow-up study of 283 adult and child cases in La Virginia, Risaralda, Colombia. F1000Research 2016, 5, 360. [CrossRef] 27. References De Andrade, D.C.; Jean, S.; Clavelou, P.; Dallel, R.; Bouhassira, D. Chronic pain associated with the Chikungunya Fever: Long lasting burden of an acute illness. BMC Infect Dis. 2010, 10. [CrossRef] 28. Kularatne, S.A.; Weerasinghe, S.C.; Gihan, C.; Wickramasinghe, S.; Dharmarathne, S.; Abeyrathna, A.; Jayalath, T. Epidemiology, Clinical Manifestations, and Long-Term Outcomes of a Major Outbreak of Chikungunya in a Hamlet in Sri Lanka, in 2007: A Longitudinal Cohort Study. J. Trop Med. 2012, 2012, 1–6. [CrossRef] 29. Mathew, A.J.; Goyal, V.; George, E.; Thekkemuriyil, D.V.; Jayakumar, B.; Chopra, A. Rheumatic-musculoskeletal pain and disorders in a naïve group of individuals 15 months following a Chikungunya viral epidemic in south India: A population based observational study. Int. J. Clin. Pract. 2011, 65, 1306–1312. [CrossRef] 30. Bouquillard, É.; Combe, B. A report of 21 cases of rheumatoid arthritis following Chikungunya fever. A mean follow-up of two years. Joint. Bone Spine. 2009, 76, 654–657. [CrossRef] 31. Javelle, E.; Ribera, A.; Degasne, I.; Marimoutou, C.; Simon, F. Clinical spectrum of post-chikungunya rheumatic musculoskeletal disorders and use of disease-modifying antirheumatic drugs to treat the chronic inflammatory entities: 6-year experience from Reunion Island. BMC Infect Dis. 2014, 14 (Suppl. 2), O20. [CrossRef] 32. Sissoko, D.; Malvy, D.; Ezzedine, K.; Renault, P.; Moscetti, F.; Ledrans, M.; Pierre, V. Post epidemic chikungunya disease on Reunion Island: Course of rheumatic manifestations and associated factors over a 15-month period. PLoS Negl. Trop Dis. 2009, 3, e389. [CrossRef] 10 of 12 Viruses 2019, 11, 289 10 of 12 33. Essackjee, K.; Goorah, S.; Ramchurn, S.K.; Cheeneebash, J.; Walker-Bone, K. Prevalence of and risk factors for chronic arthralgia and rheumatoid-like polyarthritis more than 2 years after infection with chikungunya virus. Postgrad. Med. J. 2013, 89, 440–447. [CrossRef] 34. Manimunda, S.P.; Vijayachari, P.; Uppoor, R.; Sugunan, A.P.; Singh, S.S.; Rai, S.K.; Guruprasad, D.R. Clinical progression of chikungunya fever during acute and chronic arthritic stages and the changes in joint morphology as revealed by imaging. Trans. R Soc. Trop Med. Hyg. 2010, 104, 392–399. [CrossRef] Clinical progression of chikungunya fever during acute and chronic arthritic stages and the changes in joint morphology as revealed by imaging. Trans. R Soc. Trop Med. Hyg. 2010, 104, 392–399. [CrossRef] 35. Rodriguez-Morales, A.J.; Villamil-Gomez, W.; Merlano-Espinosa, M.; Simone-Kleber, L. Post-chikungunya chronic arthralgia: A first retrospective follow-up study of 39 cases in Colombia. Clin. Rheumatol. 2015, 35, 831–832. [CrossRef] 36. References Miner, J.J.; Aw Yeang, H.X.; Fox, J.M.; Taffner, S.; Malkova, O.N.; Oh, S.T.; Kim, A.H.J.; Diamond, M.S.; Lenschow, D.J.; Yokoyama, W.M. Brief report: Chikungunya viral arthritis in the united states: A mimic of seronegative rheumatoid arthritis. Arthritis Rheumatol. 2015, 67, 1214–1220. [CrossRef] 37. Chaaithanya, I.K.; Muruganandam, N.; Raghuraj, U.; Sugunan, A.P.; Rajesh, R.; Anwesh, M.; Vijayachari, P. Chronic inflammatory arthritis with persisting bony erosions in patients following chikungunya infection. Indian J. Med. Res. 2014, 140, 142–145, Retrieved March 10, 2017. 38. Chopra, A.; Anuradha, V.; Lagoo-Joshi, V.; Kunjir, V.; Salvi, S.; Saluja, M. Chikungunya virus aches and pains: An emerging challenge. Arthritis Rheum. 2008, 58, 2921–2922. [CrossRef] 39. Horcada, M.L.; Díaz-Calderón, C.; Garrido, L. Chikungunya Fever. Rheumatic Manifestations of an Emerging Disease in Europe. Reumatol. Clin. (English Edition). 2015, 11, 161–164. [CrossRef] 40. Amaral, J.K.; Schoen, R.T. A Case Report of Chikungunya Fever, Rheumatoid Arthritis, and Felty’s Syndrome. Rheumatol. Ther. 2018. [CrossRef] 41. Amaral, J.K.; Bingham, C.O.; Schoen, R.T. Successful Methotrexate Treatment of Chronic Chikungunya Arthritis. J. Clin. Rheumatol. 2018, 1. [CrossRef] 42. Zeana, C.; Kelly, P.; Heredia, W.; Cifuentes, A.; Franchin, G.; Purswani, M.; Hagmann, S.H. Post-chikungunya rheumatic disorders in travelers after return from the Caribbean. Travel Med. Infect. Dis. 2016, 14, 21–25. [CrossRef] [PubMed] 43. Gérardin, P.; Fianu, A.; Michault, A.; Mussard, C.; Boussaïd, K.; Rollot, O.; Grivard, P.; Kassab, S.; Bouquillard, E.; Borgherini, G.; et al. Predictors of chikungunya rheumatism: A prognostic survey ancillary to the TELECHIK cohort study. Arthritis Res. Ther. 2013, 15, R9. [CrossRef] [PubMed] 44. Yaseen, H.M.; Simon, F.; Deparis, X.; Marimoutou, C. Identification of initial severity determinants to predict arthritis after chikungunya infection in a cohort of French gendarmes. BMC Musculoskelet Disord. 2014, 15. [CrossRef] 45. Cunningham, A.L.; Fraser, J. Ross river virus infection of human synovial cells in vitro. Aust. J. Exp. Biol. Med. Sci. 1985, 63, 197–204. [CrossRef] 46. Linn, M.L.; Aaskov, J.G.; Suhrbier, A. Antibody-dependent enhancement and persistence in macrophages of an arbovirus associated with arthritis. J. Gen. Virol. 1996, 77, 407–411. [CrossRef] [PubMed] 47. Lee, R.C.; Hapuarachchi, H.C.; Chen, K.C.; Hussain, K.M.; Chen, H.; Low, S.L.; Chu, J.J. Mosquito Cellular Factors and Functions in Mediating the Infectious entry of Chikungunya Virus. PLoS Negl. Trop Dis. 2013, 7, e2050. [CrossRef] 48. Bernard, E.; Solignat, M.; Gay, B.; Chazal, N.; Higgs, S.; Devaux, C.; Briant, L. Endocytosis of Chikungunya Virus into Mammalian Cells: Role of Clathrin and Early Endosomal Compartments. References PLoS ONE 2010, 5, e11479. [CrossRef] 49. Wichit, S.; Hamel, R.; Bernard, E.; Talignani, L.; Diop, F.; Ferraris, P.; Missé, D. Imipramine Inhibits Chikungunya Virus Replication in Human Skin Fibroblasts through Interference with Intracellular Cholesterol Trafficking. Sci. Rep. 2017, 7. [CrossRef] 0. Kielian, M. Membrane fusion And the Alphavirus Life Cycle. Adv. Virus. Res. 1995, 113–151. [CrossRef 51. Ozden, S.; Huerre, M.; Riviere, J.; Coffey, L.L.; Afonso, P.V.; Mouly, V.; Ceccaldi, P. Human Muscle Satellite Cells as Targets of Chikungunya Virus Infection. PLoS ONE. 2007, 2, e527. [CrossRef] 52. Sourisseau, M.; Schwartz, O. Characterization of reemerging chikungunya virus. Plos Pathog. 2007, 3, 0804–0817. [CrossRef] 53. Ashok, A.; Atwood, W.J. Contrasting Roles of Endosomal pH and the Cytoskeleton in Infection of Human Glial Cells by JC Virus and Simian Virus 40. J. Virol. 2003, 77, 1347–1356. [CrossRef] 11 of 12 Viruses 2019, 11, 289 54. Krejbich-Trotot, P.; Denizot, M.; Hoarau, J.; Jaffar-Bandjee, M.; Das, T.; Gasque, P. Chikungunya virus mobilizes the apoptotic machinery to invade host cell defenses. FASEB J. 2011, 25, 314–325. [CrossRef] 55. Judith, D.; Mostowy, S.; Bourai, M.; Gangneux, N.; Lelek, M.; Lucas-Hourani, M.; Lecuit, M. Species-specific impact of the autophagy machinery on Chikungunya virus infection. EMBO Rep. 2013, 14, 534–544. [CrossRef] 56. Krejbich-Trotot, P.; Gay, B.; Li-Pat-Yuen, G.; Hoarau, J.; Jaffar-Bandjee, M.; Briant, L.; Denizot, M. Chikungunya triggers an autophagic process which promotes viral replication. Virol. J. 2011, 8, 432. [CrossRef] 57. Tang, B.L. The cell biology of Chikungunya virus infection. Cell Microbiol. 2012, 14, 1354–1363. [CrossRef] 58. Callus, B.A.; Vaux, D.L. Caspase inhibitors: Viral, cellular and chemical. Cell Death Differ. 2006, 14, 73–78. [CrossRef] 57. Tang, B.L. The cell biology of Chikungunya virus infection. Cell Microbiol. 2012, 14, 1354–1363. [Cro 58. Callus, B.A.; Vaux, D.L. Caspase inhibitors: Viral, cellular and chemical. Cell Death Differ. 2006, 14, 73–78. [CrossRef] 59. Schilte, C.; Couderc, T.; Chretien, F.; Sourisseau, M.; Gangneux, N.; Guivel-Benhassine, F.; Kraxner, A.; Tschopp, J.; Higgs, S.; Michault, A.; et al. Type I IFN controls chikungunya virus via its action on nonhematopoietic cells. J. Exp. Med. 2010, 207, 429–442. [CrossRef] 60. Her, Z.; Malleret, B.; Chan, M.; Ong, E.K.; Wong, S.C.; Kwek, D.J.; Ng, L.F. Active Infection of Human Blood Monocytes by Chikungunya Virus Triggers an Innate Immune Response. J. Immunol. 2010, 184, 5903–5913. [CrossRef] 61. Chirathaworn, C.L.; Poovorawan, Y.; Lertmaharit, S.; Wuttirattanakowit, N. Cytokine levels in patients with chikungunya virus infection. Asian Pac. J. References Trop Med. 2013, 6, 631–634. [CrossRef] 62. Miner, J.J.; Lenschow, D.J. Editorial: Lessons Learned From Chikungunya in the Americas. Arthritis Rheumatol. 2018, 70, 477–479. [CrossRef] 63. Hoarau, J.J.; Jaffar Bandjee, M.C.; Krejbich Trotot, P.; Das, T.; Li-Pat-Yuen, G.; Dassa, B.; Guichard, E.; Ribera, A.; Henni, T.; Tallet, F.; et al. Persistent chronic inflammation and infection by chikungunya arthritogenic alphavirus in spite of a robust host immune response. J. Immunol. 2010, 184, 5914–5927. [CrossRef] 64. Chaaitanya, I.K.; Muruganandam, N.; Sundaram, S.G.; Kawalekar, O.; Sugunan, A.P.; Manimunda, S.P.; Ghosal, S.R.; Muthumani, K.; Vijayachari, P. Role of pro-inflammatory cytokines and chemokines in chronic arthropathy in CHIKV infection. Viral. Immunol. 2011, 24, 265–271. [CrossRef] 65. Chow, A.; Her, Z.; Ong, E.K.; Chen, J.; Dimatatac, F.; Kwek, D.J.; Ng, L.F. Persistent Arthralgia Induced by Chikungunya Virus Infection is Associated with Interleukin-6 and Granulocyte Macrophage Colony-Stimulating Factor. J. Infect Dis. 2011, 203, 149–157. [CrossRef] 66. Dupuis-Maguiraga, L.; Noret, M.; Brun, S.; Le Grand, R.; Gras, G.; Roques, P. Chikungunya Disease: Infection-Associated Markers from the Acute to the Chronic Phase of Arbovirus-Induced Arthralgia. PLoS Negl. Trop Dis. 2012, 6, e1446. [CrossRef] 67. Labadie, K.; Larcher, T.; Joubert, C.; Mannioui, A.; Delache, B.; Brochard, P.; Roques, P. Chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages. J. Clin. Invest. 2010, 120, 894–906. [CrossRef] 68. Ng, L.F.; Chow, A.; Sun, Y.; Kwek, D.J.; Lim, P.; Dimatatac, F.; Leo, Y. IL-1β, IL-6, and RANTES as Biomarkers of Chikungunya Severity. PLoS ONE 2009, 4, e4261. [CrossRef] 69. Spadaro, A.; Scrivo, R.; Rinaldi, T.; Riccieri, V.; Sili Scavalli, A.; Taccari, E.; Valesini, G. The role of Interleukin-12 in immune-mediated rheumatic diseases. Reumatismo 2011, 54. [CrossRef] 70. Miossec, P.; Korn, T.; Kuchroo, V.K. Interleukin-17 and Type 17 Helper, T. Cells. N. Engl. J. Med. 2009, 361, 888–898. [CrossRef] 71. Thanapati, S.; Ganu, M.A.; Tripathy, A.S. Differential inhibitory and activating NK cell receptor levels and NK/NKT-like cell functionality in chronic and recovered stages of chikungunya. PLoS ONE 2017, 12, e0188342. [CrossRef] 72. Thanapati, S.; Ganu, M.; Giri, P.; Kulkarni, S.; Sharma, M.; Babar, P.; Tripathy, A.S. Impaired NK cell functionality and increased TNF-α production as biomarkers of chronic chikungunya arthritis and rheumatoid arthritis. Hum. Immunol. 2017, 78, 370–374. [CrossRef] 73. McCarthy, M.K.; Morrison, T.E. Chronic chikungunya virus musculoskeletal disease: What are the underlying mechanisms? Future Microbiol. 2016, 11, 331–334. [CrossRef] 74. References Poo, Y.S.; Rudd, P.A.; Gardner, J.; Wilson, J.A.; Larcher, T.; Suhrbier, A. Multiple immune factors are involved in controlling acute and chronic chikungunya virus infection. PLoS Negl. Trop Dis. 2014, 8, e3354. [CrossRef] Viruses 2019, 11, 289 12 of 12 75. Chang, A.Y.; Martins, K.A.; Encinales, L.; Reid, S.P.; Acuña, M.; Encinales, C.; Firestein, G.S. Chikungunya Arthritis Mechanisms in the Americas. Arthritis Rheumatol. 2018, 70, 585–593. [CrossRef] 76. McCarthy, M.K.; Morrison, T.E. Persistent RNA virus infections: Do PAMPS drive chronic disease? Curr. Opin. Virol. 2017, 23, 8–15. [CrossRef] 77. Tian, H.; Cronstein, B. Understanding the mechanisms of action of methotrexate: Implications for the treatment of rheumatoid arthritis. Bull NYU Hosp. Jt. Dis. 2007, 65, 168–173. 78. Swierkot, J.; Szechinski, J. Methotrexate in rheumatoid arthritis. Pharmacol. Rep. 2006, 58, 473–492. 79. Cutolo, M. Anti-inflammatory mechanisms of methotrexate in rheumatoid arthritis. Ann. Rheum. Dis. 2001, 60, 729–735. [CrossRef] 80. Wessels, J.A.; Huizinga, T.W.; Guchelaar, H. Recent insights in the pharmacological actions of methotrexate in the treatment of rheumatoid arthritis. Rheumatology (Oxford). 2007, 47, 249–255. [CrossRef] 81. Montesinos, M.C.; Yap, J.S.; Desai, A.; Posadas, I.; McCrary, C.T.; Cronstein, B.N. Reversal of the antiinflammatory effects of methotrexate by the nonselective adenosine receptor antagonists theophylline and caffeine: Evidence that the antiinflammatory effects of methotrexate are mediated via multiple adenosine receptors in rat adjuvant arthritis. Arthritis Rheum. 2000, 43, 656–663. [PubMed] 82. Bedoui, Y.; Giry, C.; Jaffar-Bandjee, M.; Selambarom, J.; Guiraud, P.; Gasque, P. Immunomodulatory drug methotrexate used to treat patients with chronic inflammatory rheumatisms post-chikungunya does not impair the synovial antiviral and bone repair responses. PLoS Negl. Trop Dis. 2018, 12, e0006634. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2766812273
https://figshare.com/articles/journal_contribution/QbD-driven_development_and_evaluation_of_nanostructured_lipid_carriers_NLCs_of_Olmesartan_medoxomil_employing_multivariate_statistical_techniques/5579695/1/files/9702496.pdf
English
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QbD-driven development and evaluation of nanostructured lipid carriers (NLCs) of Olmesartan medoxomil employing multivariate statistical techniques
Drug development and industrial pharmacy
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Supplementary Data Tables Supplementary Data Tables Table S1: Comparison of predicted and experimental values of the CQAs of NLCs Composition in coded levels X1 (ratio) : X2 (%) Response variable Predicted value Experimental value % Prediction Error Optimized-NLCs (0.30 : 0.16) Particle Size 229.4 232.9 1.50 Zeta Potential -20.6 -21.7 5.07 Entrapment Eff. 74.71 76.94 2.90 Drug Rel. (Q4h) 83.4 82.19 -1.47 VAL 1 (0.32 : 0.08) Particle Size 225.5 229.78 1.86 Zeta Potential -21.7 -19.67 -10.32 Entrapment Eff. 72.6 73.58 1.33 Drug Rel. (Q4h) 81.5 82.21 0.86 VAL 2 (0.38 : 0.34) Particle Size 224.7 221.58 -1.41 Zeta Potential -20.5 -21.58 5.00 Entrapment Eff. 77.5 76.68 -1.07 Drug Rel. (Q4h) 82.7 82.57 -0.16 Overall percentage error (Mean ± S.D.) = 0.54 ± 1.56 VAL 1 and VAL 2 are the validation formulations Table S1: Comparison of predicted and experimental values of the CQAs of NLCs Supplementary Data Figures Supplementary Data Figures Figure S1: Perturbation charts for the CQA, viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h pp y g Figure S1: Perturbation charts for the CQA, viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S2: Normal plot of residuals for the CQAs viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S1: Perturbation charts for the CQA, viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S2: Normal plot of residuals for the CQAs viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S2: Normal plot of residuals for the CQAs viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S3: Predicted versus Run plots for CQAs viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S4: Residual versus Run plots for the CQAs viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S3: Predicted versus Run plots for CQAs viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S3: Predicted versus Run plots for CQAs viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S4: Residual versus Run plots for the CQAs viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S4: Residual versus Run plots for the CQAs viz. Supplementary Data Tables (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S5: Leverage plots for the CQAs viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S6: Cook’s distance plots for the CQAs viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S5: Leverage plots for the CQAs viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S5: Leverage plots for the CQAs viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S5: Leverage plots for the CQAs viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S6: Cook’s distance plots for the CQAs viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S7: Box-Cox plots for the CQAs viz. (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S7: Box-Cox plots for the CQAs viz. Supplementary Data Tables (A) particle size, (B) zeta potential, (C) entrapment efficiency, (D) Q4h Figure S8: Linear and residual plots between the observed and predicted values of CQAs, (A-B) particle size, (C-D) zeta potential, (E-F) entrapment efficiency, (G-H) Q4h y = 1.005x R = 0.999 85 190 295 85 190 295 Predicted Observed -5.0 -2.5 0.0 2.5 5.0 85 190 295 %Residuals Observed y = 1.012x R = 0.993 18 29 40 18 29 40 Predicted Observed -5.0 -2.5 0.0 2.5 5.0 18 29 40 %Residuals Observed y = 1.001x R = 0.980 72 85 98 72 85 98 Predicted Observed -2.5 0.0 2.5 72 85 98 %Residuals Observed y = 1.098x R = 0.963 72 79 86 72 79 86 Predicted Observed -2.5 0.0 2.5 72 79 86 %Residuals Observed (A) (B) (C) (D) (E) (F) (G) (H) -5.0 -2.5 0.0 2.5 5.0 85 190 295 %Residuals Observed (B) y = 1.005x R = 0.999 85 190 295 85 190 295 Predicted Observed (A) (B) (B) -5.0 -2.5 0.0 2.5 5.0 18 29 40 %Residuals Observed (D) y = 1.012x R = 0.993 18 29 40 18 29 40 Predicted Observed (C) (D) (D) -2.5 0.0 2.5 72 85 98 %Residuals Observed (F) y = 1.001x R = 0.980 72 85 98 72 85 98 Predicted Observed (E) (F) (E) (F) -2.5 0.0 2.5 72 79 86 %Residuals Observed (H) y = 1.098x R = 0.963 72 79 86 72 79 86 Predicted Observed (G) (H) (H) (G) Figure S8: Linear and residual plots between the observed and predicted values of CQAs, (A-B) particle size, (C-D) zeta potential, (E-F) entrapment efficiency, (G-H) Q4h
W4385504176.txt
https://journals.wlb-stuttgart.de/ojs/index.php/sh/article/download/7153/7040
de
"Schreiben für das Reich Gottes"
Schwäbische Heimat
2,023
cc-by
4,860
«Schreiben für das Reich Gottes» Werner Raupp Christian Gottlob Barth, ein schaffiger Württemberger Im Jahre 1945 erschien in Stuttgart die 483. und letzte Auflage eines Büchleins, das erstmals 1832 in Calw im Schwarzwald unter dem Titel Zweymal zwey und fünfzig biblische Geschichten für Schule und Familien das Licht der Welt erblickt hatte. Bis zum Ende des Zweiten Weltkrieges hatte das 216seitige Werk Dut- zende von Übersetzungen erlebt und war ein internationaler Bestseller unter den Schulbüchern geworden. Als ursprünglicher Verfasser gilt Christian Gottlob Barth, der rührige Pietistenkopf und Gründer des Calwer Verlags, dessen Geburtstag sich in 1 diesem Jahr zum 200. Male jährt. Jahrzehnte Vier Stadt Calw und er in der den nahe und Stammheim Möttlingen als gewirkt: hat in Hermann-Hesse- gelegenen Dörfern Weise vielfältiger in Pfarrer, Erweckungsprediger, Dichter, Schriftsteller und Verleger wie auch als Förderer der Inneren und äußeren Mission und als Naturforscher. Vor allem durch die biblischen Geschichten hat seinen großen Erfolg er erzielt und den Calwer Verlag, der mittlerweile in Stuttgart beheimatet ist, weltweit bekannt gemacht. Geboren 1799 in Stuttgart, die Studium der Theologie in Tübingen (1747-1811), dem Ur-Urgroßvater Hermann Hesses, vom Pietistenführer geleitet wurde. Von In der damals zwanzigtausend Einwohner zählen- den Landeshauptstadt begann auch am 31. Juli 1799 humanistische Johann Christian 1810 an besuchte er Gundert dann das Gymnasium, die damals Stuttgarter beste Schule des Landes. Vom Bildungseifer ergriffen, Barths Lebenslauf. Seine Vorfahren väterlicherseits galt er als ein von Ehrgeiz zerfressener Schüler (so sein gehörten einem alteingesessenen Stuttgarter Bürger- Mitschüler, der Jurist und Politiker Robert von Mohl). geschlecht an und waren Strumpfstricker tätig. Der als eine Vater, begabte Natur, war Gipser und introvertierte Mutter, Weingärtner eine und künstlerisch Maler. Die ernste, geborene Engelmann, Über die Schule hinaus entfaltete Barth eine rege Betriebsamkeit. So verfaßte er bereits mit zehn Jahren eine kleine Sammlung Biblischer Geschichten zur Aufmunterung für die Seele, kommt aus Kirchheim am Neckar. Ebenso wie die von Großeltern waren auch die Eltern religiös veranlagt Wochenmarkt für einen und hatte gehörten pietistischen die Württembergische Zu seinem Friedrich Verwandtschaftskreis Böhringer (1791-1867), Verwaltungsrat men Kreisen an, die 1812 Bibelgesellschaft gründeten. zählt ein in einer dem Groschen Auflage Stuttgarter feilbot. Damit gleichsam sein literarisches Erstlingswerk vorgelegt: ein nicht zu überhörendes Präludium der zwanzig Jahre später erscheinenden Zweymal zwey langjähriger und fünfzig biblischen Geschichten. 1817 ließ er sodann dieser Gesellschaft, der 1817 zusam- mit einem Vetter Barths in Stuttgart eine Apo- «Boehringer Ingelheim KG Chemische Fabrik» (gegr. 1885) wurde. Den ersten Schulunterricht erhielt der geistig fünf weitere Schriften pietistischen Inhalts folgen, in denen sich seine dem romantisch-idealistischen Lebensgefühl erwachsende religiöse Einbildungskraft äußert. Die Johann rege und vielfältig begabte Barth in der deutschen Schule, Schwäbische Heimat 99/3 er er Christian theke gründete, die die Keimzelle der weltbekannten zwanzig die Exemplaren auf Eine Heinrich Scene aus wichtigste war Jung-Stilling: ein Nachruf auf Stillings Siegesfeier. der Geisterwelt, ein in Hexametern abgefaßtes Epos. 341 Die malerische Universitätsstadt Tübingen bot verwundert es kaum, zeugnis der Theologie bewohnte er das berühmte Evangeli- des Mystizismus gelandet. vom in seinem wenn es Abgangs- Herbst 1821 heißt, er wäre auf Irrwegen Barth von 1817 bis 1821 eine neue Bleibe. Als Student sche Stift, wo drei Jahrzehnte zuvor das schwäbische Dreigestirn Hegel, Hölderlin und Schelling studiert «So viele wie möglich zu retten» hatte. Zu seinen Studiengenossen zählte unter ande- als erweckter Pfarrer in Möttlingen und rem - Unterhaugstett der Dichter Wilhelm Hauff, mit dem er kurzzeitig das Zimmer bewohnte. Dem aufklärerischen Zeit- Für seine nach dem Vikariat 1824 beginnende Lauf- in jenen Jahren an der evan- bahn als Pfarrer von Möttlingen und für seine schrif- geist verpflichtet, war gelisch-theologischen Fakultät der von «biblischer tenmissionarischen Projekte war der Boden bereitet: Denkgläubigkeit» bestimmte «biblisch-verständige» Es war die Zeit der sogenannten Erweckungsbewe- Supranaturalismus der sogenannten Alteren Tübin- gung («Erweckungspietismus»), die nach den Befrei- Schule ger Dieser vorherrschend. konnte den mystisch-spiritualistisch gesinnten Barth zwar nicht entscheidend prägen, ihn aber dennoch beeinflussen. ungskriegen von 1813 bis 1815 als kontinentalprotestantische Bewegung einsetzt und den Zeitraum des Vormärz umfaßt. Wie ernst Barth anfangs sein Studium nahm, zeigt sein es «Morgenstudienplan» vom Januar 1818, worin heißt: Zuerst, wenn ich vom Bette komme, Uhr, lese ich zwei Kapitel im Brief an um drei die Römer im fran- In 18. wurzelte Württemberg Dessen Jahrhunderts. Traditionen erwuchsen schen sie im Pietismus des biblizistisch-spekulative weitgehend dem schwäbi«religiösen Überbegabt- Volkscharakter: der zösischen Testament, sodann zwei Psalmen hebräisch und heit» des sinnierenden schwäbischen Menschen, der holländisch, ferner 30-40 Verse aus der Illias [...], endlich für ein Kapitel aus Seneca, Cicero und aus meiner spanischen «prädisponiert» pietistische Frömmigkeitsform gleichsam sei (Ernst Kretschmar). Angeführt wurde der mit der Naturwissenschaft und erwarb sich schließ- Johann Albrecht lich den Ruf eines «gärenden Polyhistors». Christoph Oetinger (1702-1782), dessen theosophi- Grammatik. Damit mehr Je länger, je durch seine nicht die genug, beschäftigte er sich aber wurden seine Studien Aktivitäten für das «Reich vielfältigen der an Gründung des Tübinger (1819), sondern predigte als auch Missionsvereins Sonntag für Sonntag redegewandter Himmelstürmer und griff mit zwei Flugschriften in den heftigen Streit um die 1819 gegründete ein. Erfüllt Pietistenkolonie war er dabei von Korntal bei Leonberg mystisch-spiritualisti- den von «Schwabenvätern» und Friedrich Bengel (1687-1752) sche Gedanken nicht Barth, sondern auch nur dem Deutschen Idealismus Impulse verliehen. Gottes» beeinträchtigt, das das zentrale Motiv seines Wirkens werden sollte. So beteiligte er sich nicht nur Pietismus Dieser und die Romantik indes beeinflußten auch die Erweckungsbewegung, die sich in ihrer Gesamtheit als irrationaler Protest gegen die mit Aufklärung und Französischer Revolution einsetzende Säkulari- sierung des sozialen und religiösen Lebens ausweist. Damit einhergehend hielt sie Hüterin des Christentums servilen Dienerin der am Ancien regime als fest und Gott «von mutierte zur eingesetzten» schem, kirchenkritischem Sendungsbewußtsein, das restaurativen ein wurde sie eine Rechristianisierungsbewegung: eine geistunmittelbares Christentum beschwörte. So Monarchie. Je länger, desto mehr antimodernistische Bewegung, die im süddeutschen Raum hauptsächlich Kleinbürgertum die Bauernschaft erfaßte und und das biedermeierliches Gepräge annahm. Mit ängstlichem Blick sah man in Liberalismus Werner Raupp Christian Gottlob Barth Werner Raupp Christian Gottlob Barth STUDIEN ZU LEBEN UND — Ej Studien zu Leben und Werk Demokratie (Revolution am Werk, die Christi Wiederkunft Reich Gottes heraufführen sollten. QF 16. 1998. WERK und 294 SöitSH DM 98,-ÖS 715,-SFR 88,ISBN 3-7668-3579-3 calwer l Calwer Verlag ■ 70599 Stuttgart I und damit das Gespeist waren derartige Hoffnungen von neutestamentlich-apokalyptischer Mythologie. Somit galt es, mittels der missionarischen und diakonischen Tat noch zu retten, was * von 1848/49) antichristliche und endzeitliche Vorboten zu retten sei. Die eschatologische (endzeitliche) Krönung bildete die Christianisierung und die damit einhergehende Zivilisierung der sogenannten gottverlassenen heidniVölker: die schen Mission, die bekanntlich in Übersee als liierte Partnerin des Kolonialismus verheerende 342 Schwäbische Heimat 99/3 Für die Festschrift «Hundert Jahre Kinderrettungsanstalt Stammheim-Calw» hat O. Elsässer diese überblickende Dar- stellung geschaffen. kulturelle Schäden anrichtete. Die dort erzielten Erfolge dienten als Kompensation für das in der Hei- Erfolgreich als Missionspublizist, Kinderbuchautor und Volksschriftsteller mat verlorengehende Terrain. «Soviele wie möglich zu retten» - dies war auch Bedingt war dieser nicht Fehlschlag zuletzt das Ziel von Barths Wirken in den etwa 800 Einwoh- durch seine vielfältigen «Reich-Gottes-Projekte», die zählenden Dörfern Möttlingen und Unterhaug- Barths geschäftigen Geist neben der Gemeindearbeit ner stett, wo er vierzehn Werk und Jahre amtierte. Er ging eifrig ans vermochte die «erwecken»: durch die Gemeinde zunächst zu Einrichtung von Missions- in Atem hielten. So gründete er 1826 im Calwer Vor- ort Stammheim eine Dies «Kinder-Rettungsanstalt». allerdings erst der Auftakt. Zwei Jahre war und Jugendkreisen sowie durch die Förderung der später Armenpflege; zudem verzichtete er auf die Stolge- Reich-Gottes-Arbeit aufzunehmen, bühren bei Taufe und Hochzeit. und Reich-Gottes-Predig- Barth sodann seine planmäßige um zurückzudrängen und Aufklärung Im Mittelpunkt standen seine mit Feuereifer vor- getragenen Bekehrungs- begann die Gottes gottlose tausend- jähriges Königreich vorzubereiten. Dazu ging er an die Grenze des Menschenmöglichen und entfachte als ten, die weit über die Gemeinde hinaus zahlreiche Autor und Dichter wie auch als Publizist und Verle- Gottesdienstbesucher anzogen. Mitgerissen von sei- ger ein Schnellfeuer, das von Calw aus gleichsam die Gegenwart Gottes nem jahrzehnte- vermitteln- lang hell aufflammte und ihn zu einem Bahnbrecher dem Pathos, konnte mitunter die ganze Kirche mit ihm des kirchlichen Pressewesens, der Volksmission und weinen. Mit Vorliebe prangerte er dabei das Tanzen der Missionsbewegung machte. Möglich geworden und die Kirchweihfeste als Fleischeslust an. Aufsehen war dies durch den in jenen Jahren heraufziehenden erregte seine im Frühjahr 1828 bewirkte Heilung eines epileptischen Mädchens, die er als «Exorzis- mus» aufgefaßt hatte. gemeindeübergreifende Erweckung einstellte, die allerdings auch bald wieder abflaute, nachdem Barth, wie gepredigt selbst feststellte, er hatte. Das und sich am die Gemeinde idealisierten totVor- bild neutestamentlicher Gemeindebildung orientie- Den um Anfang wer ren. Dargeboten Schwäbische Heimat 99/3 werden begeisternder Briefe teilen, Pietisten ließ sich freilich nicht realisieren. nahezu 150% ansteigen ließ. machte das Calwer Missionsblatt (1828 Verlags werden sollte. Es erschien vierzehntägig romantische begeisterten Bücherpro- in einer Auflage von zunächst viertausend Exempla- meindlichen» von der die bis 1918), das zu einem der «Flaggschiffe» des Cal- rende, hochgesteckte Ziel: die Bildung einer «urge- Mustergemeinde Zeitungsfrühling, duktion im deutschsprachigen Raum zwischen 1820 und 1850 So kam es nicht von ungefähr, daß sich schon bald eine Bücher- zahllose von darin eine riesige Fülle Missionaren aus allen Erd- Erfolgsbilanzen und Bekehrungsgeschichten, ten zu Münchhausiaden anschwellen. vor allem die nicht sel- Getragen wer- 343 den sie die weitgehend von eschatologischen Motiven, den Kolonialismus als heilsgeschichtliche Führungen Gottes feiern und von abendländisch-kulturellem Superioritätsgefühl erfüllt sind. Demnach sind die Heiden wie Unmündige und Kinder, die einen Pfleger und Vormund haben müssen. Mit derartigen diskriminierenden Vorstellungen, die zu damaliger Zeit weit verbreitet waren, erlangte das Missionsblatt großen Erfolg. Mit einer Auflage schließlich 16 000 von Exemplaren (im Jahre 1847) wurde es weit über Württemberg hinaus neben dem Barmer Missionsblatt zum meistgelesenen Missions- periodikum im deutschsprachigen hatte Barth eine neue Raum. kirchlich-journalistische Lite- raturgattung mitgeschaffen. Über das hinaus sen Damit Missionswe- plante er schließlich, eine politisch-christ- liche Tageszeitung zu begründen, worüber er sich mit dem preußischen Kultusminister Friedrich Eichhorn besprach. Neben der publizistischen Tätigkeit trat Barth auch als Dichter auf den Plan und erwarb sich als Mitbegründer der Gattung des Missionsliedes Anerkennung er im in der evangelischen Welt. Noch heute ist Evangelischen Gesangbuch vertreten, unter anderem mit einigen Strophen des weithin bekannten Liedes Sonne denen er der Gerechtigkeit (EG 262/263), in die verschlafene Christenheit wachzurütteln sucht: Weck die tote Christenheit aus dem Bischofstraße 4 in Calw, das Domizil des Calwer Verlagsvereins seit 1854 und Wohnung von Christian Gottlob Barth. Schlaf der Sicherheit; mache Deinen Ruhm bekannt überall im - ganzen Land. Erbarm' Dich Herr. trauen und Moral; Bibel und Weg; Auch auf dem Gebiet der Kinder- und Jugendlite- Armut und Gebet weisen stets den Monarchie Demokrat sein) werden Jein Christ kann kein gottgegeben akzeptiert; als ratur hat Barth eine Marktlücke entdeckt und eine nicht selten erscheint Gott als ein deus ex Ära mitbegründet. der auf wundersame Weise eingreift. Immerhin tritt vom lang Bereits Armen Heinrich mehrfach von seine Erzählung erste 1828, die ein Jahrhundert aufgelegt und übersetzt wurde, machte ihn über Deutschland hinaus bekannt. Sie schildert die providentiell-heilsgeschichtliche «Lebens- Waisenknaben, in der eigene Kindheit widerspiegelt, die er führung» eines sich seine sehnsüchtig nacherlebt. Fortan trat Barth als Verfasser des Armen missionarischem zu Drang zu zu Sie verkünden gleichsam ein individu- Damit avancierte Barth emigrieren. stantischen Pendant schriftstellers gen erlebten. spürbarer ein dennoch weisen ten Teilen als «Fluchtliteratur» aus, die dem Leser die und Biographien folgen, und Möglichkeit eröffnen, in eine pietistische Überwelt Heinrich auf und ließ (v. a. bei Johann Friedrich Stein- Übersetzun- - sich derlei heilsgeschichtliche Erzählungen in wei- kopf in Stuttgart) Dutzende von heilsgeschichtlichen die zumeist auch Bekennermut sozialer Aktivität machina, zu des Christoph einem der Jugendbuchautoren zum katholischen von Schmid prote- Lieblings(1768-1854) erfolgreichsten Kinder- und des 19. Jahrhunderts. Erfolgreich war Barth auch als Publizist mit sei- elles Reich Gottes, in dem sich das pietistische See- nen lenleben mit der äußeren Geschichte verknüpft. schrift zur Förderung wahrer Bildung (1836-1951). Ver- Oft in eindrucksvolle Naturschilderungen einge- rahmt, sind sie Jugend-Blättern, einer achtzigseitigen Monats- suchten seine Kinderschriften vor allem das Gemüt vom Leitgedanken bestimmt: Alles anzusprechen, so beabsichtigten diese Blätter alles kommt von oben! Und so erscheint Barths pietistische menschliche Wissen vom Wort Gottes her zu verstehen Gesinnung stereotyp: Überall herrschen 344 Gottver- und damit eine christlich-restaurative Weitsicht zu Schwäbische Heimat 99/3 propagieren. Dazu dienten besonders geschichtliche und naturkundliche Beiträge, die spürbar vom aufder klärerischen Denken damaligen Zeit geprägt sind, das Barth ja vehement bekämpfte. Und so weist er sich gleichsam als biblisch-verständiger Physiko- zwanzig illu- strierte Traktate, die Barth aus dem Englischen über- verbreitete der Zunächst setzte; sechzehn weitere, ihm verfaßte Num- von In den ersten drei Jahren fanden sie in folgten. mern Verein mehr als einer Million Exemplaren dankbare Abneh- Theologe aus: als durchaus moderner Zeitgenosse mer. und als wahrer Aufklärer. lange nicht zufrieden. Nachdem er noch den finanz- - Der «Zeitgeist», den Barth bekämpfen ausgezogen zu war, sprach aus ihm selbst. Damit aber geschäftige Barth noch gewinnen konnte, Pietismus kräftigen Stuttgarter gründete Auch als Volksschriftsteller fand Barth, besonders der war 5. Juli 1833 den «Calwer Verlagsver- er am ein», mit dem er endlich sein großes Lebenswerk mit seinen historischen Darstellungen, große Beach- gefunden hatte. Dessen Ziel war es, zum Zwecke der tung. Zunächst verfaßte er die heilsgeschichtlich ori- christlichen entierte Christliche Kirchengeschichte (1835), die bis in zum und Jahrhundert hinein nahezu dreißig Auflagen vierzig Übersetzungen erlebte. Ihr folgte die unser Allgemeinen Weltgeschichte (1837), die von Adam und Eva bis Gegenwart eine skurrile biblizistische zur Geschichtsinterpretation darbietet. 1843 erschien Volksbildung Kinder- möglichst wohlfeilsten und Preis zu Schulschriften verbreiten, um damit eine Sündfluth christlicher Bücher auszulösen, um die unchristlichen Bücher hinwegzuschwemmen. Nach der Gründung übernahm der Verein das Calwer Missionsblatt und ließ weitere Bücher und Zeitschriften folgen: unter anderem eine Biblische sodann die beliebte Geschichte Württembergs, die in Geographie für Schulen und Familien (1836), das Calwer der Einleitung ABC-Buch (1838), das mit originellen Beispielen aus mit der stupenden Bemerkung auf- wartet: Es gibt zwei gelobte Länder in der ist das andere Palästina, zum ist Welt, das eine Württemberg. Sie wurde Klassiker und erlebte 1986 eine, von Hansmar- tin Decker-Hauff initiierte, weitere Neuauflage. Weite Verbreitung Christliche Gedichte, fanden schließlich die die Schönheiten die unbeschwerte Kindheit und das der unter den aus Missionsblatt für Kinder (1842-1918) und schließlich das weitverbreitete Handbuch der Bibeler- klärung (2 Bde, 1849/50). 1836 erhielt der Verlagsverein die Konzession der Rechte einer Buchhandlung. Natur, In jenem Jahr hat auch das Konsistorium einige Cal- kleinbürger- theologische Gelegenheitsschriften, er anderem Calwer Barths lich-idyllische Familienglück besingen. Zudem veröffentlichte der Bibel versehene Calwer Rechenbuch (1840), das mythologisch-christologi- wer Bücher für die Volksschulen in Württemberg empfohlen. Eines dieser Werke fünfzig waren die Zweymal zwey und biblischen Geschichten von 1832. Der alttesta- schen Traditionen schöpfenden Traktat Der Engel des mentliche Teil stammt Bundes (1845), womit er mit dem berühmten Philo- Ludwig Gottlob Hochstetter (1790-1863), sophen Friedrich Wilhelm Schelling die Klingen zu testamentliche von Barth. Die mit Holzschnitten ver- kreuzen suchte. zierten vom testamentlichen und Leiter des Calwer Verlags, der die Hermann- neu- der Schöpfung über die alt- Kriegs- Wundergeschichten Seinen bleibenden Erfolg erzielte Barth als Gründer der Geschichten, die sich eng an den Bibeltext anlehnen, reichen von Gründer und Leiter des Calwer Verlags Simmozheimer Pfarrer bis und hin neutestamentlichen zur Ausbreitung des Evangeliums durch die Apostel. Der Erfolg des Büchleins war grandios. Hesse-Stadt weit über Deutschland hinaus bekannt erschien bereits machte. Dabei verbanden sich seine dertste, 1945 schließlich die 483. und letzte Auflage. schöpferischen Ideen und sein Unternehmergeist mit der Fähigkeit, andere für seine Vorhaben zu gewinnen. Die Grün- Nicht die hundertste, genug damit, fand setzungen 1902 1854 die vierhun- das Buch durch seine Über- in mindestens 87 Sprachen weltweite Ver- dung im Jahr 1833 geht zurück auf den 1827 entstan- breitung, die hauptsächlich auf der englischen Ver- denen sogenannten «Blätter-Verein», sodann auf den sion Dr. Barths Bible Stories basierten. Dabei wurden «Calwer Traktat-Verein», den Barth 1829 initiierte. auch Sprachen kleinerer Volksgruppen bedacht wie Dieser ist ein Ableger der Londoner etwa das Fidschi, das Religious Tract Society (gegr. 1799), die den Verlag jahrzehntelang unterstützte. Getragen wurde er von mehreren Pfarrern aus der Umgebung und von wohlhabenden Calwer Bürgern: vor allem vom Strumpffabrikanten Inupik oder auch das Sorbi- Insgesamt kursierten wohl in über tausend Gesamtauflagen nahezu fünf Millionen Exemplare sche. und machten Barths Biblische Geschichten Weltbestseller: zu zu Jakob Ludwig Federhaff (1764-1833), der führenden und Gestalt des Schrift. Bis zum heutigen Tag zählt es auch Calwer Pietismus, der anfangs auch verlegte. Schwäbische Heimat 99/3 die Schriften Koran wohl einem der im 19. Jahrhundert nach Bibel mit am weitesten verbreiteten zu den erfolgreichsten Büchern evangelischer Provenienz. 345 Das «Calwer Missionsblatt» erschien von 1828 bis 1918 alle vierzehn Tage und war das «Flagg- schiff» des Calwer Verlags. Mitte des vorigen Jahrhunderts erreichte es eine Auflage von 16000 Exemplaren. Ein Der Calwer Reich-Gottes-Manager besonderer haus bildete die aus wuchs auch Barth Aufgaben Mit seinen endgültig den engen Grenzen des Möttlinger Kirchspiels über, um widmen sich ganz Kurz zuvor ihm war der von Ehrendoktor-Diplom verliehen worden, das er mittels monetärer Beigaben beantragt hatte. Verlagshaus (zunächst in der Bischofs- straße 60, seit 1854 in der Bischofstr. 4) als Autor und seine Verleger machte Calw sionarischen Zudem «Reich-Gottes-Geschäfte» fort und Mittelpunkt Bewegung, die im zum versorgte er die Reich-Gottes-Manager So wurde der er im das besonders Exponaten, die er von seinen Missi- onsfreunden aus der fernen Welt zugesandt bekam. - Das Reich Gottes sollte auch in der Natur sichtbar werden. Eine weitere Attraktion war die «gewaltige» Bibliothek, die in späteren Jahren auch lagshaus einige Jahre ihresgleichen sucht. sie Staatliche Museum für Naturkunde in Stuttgart, ton- 19. schwäbische ist bewundern. zu württembergischen und bayerischen Naturkundemuseen, Hermann Deutschland Basel in Kulturen einer schriftenmis- Jahrhundert in Heute Sammlung würdigte. der nenweise mit Mit herkulischer Arbeitsamkeit führte er sodann Verlags- angelegte «Naturalien- Baedekers Reiseführer als in der Welt einzig dastehende Greifswalder Universität das im Calwer sogar Museum können. zu die 1838 nach Calw Frühjahr im Barth sammlung» aus dem Bereich von Fauna und Flora, der überhandnehmenden Arbeit heraus und siedelte im Anziehungspunkt von Hesse in Staunen den jungen versetzte, der im Ver- seiner Kindheit verbracht hatte. und residierte Seine Arbeit am blätterüberladenen Schreibtisch in Calw gleichsam als ein pietistischer König. Als ein unterbrach Barth ständig durch Reisen, die ihn auch solcher wußte er nach England und Schottland führten, wo er für den nicht Libanon und genießen, sondern nur Wein und Walfischzunge auch vom zu Verlagsverein spartanisch zu Überdies fuhr er nach Österreich und Ungarn, wo er von durchaus Feigen Grönland leben, wenn er etwa jahrelang in seinem Arbeitszimmer in einer drei Uhr Hängematte nächtigte, frühmorgens sein Lebensmotto Tagewerk begann machte: hasse die Ruhe.» Und er «Odi und sich tranquillitatem so verwundert es um zum Ich - das zuvor überkonfessionellen wurde, den pflegte. In Barth diese einem Kaiser Tao Kuang. weltweit verstreuten, Freundeskreis mit jährlich über aufgesucht 1200 u. a. Briefen des warb. Unterstützung Verlags gründete. Im verkehrte im preußischen Königshaus, wo er 1843 Portofreiheit für die Calwer Zeitschriften erwirkte. In Württemberg und Baden indes reiste zum anderen und trug er von als herz- ergreifender Prediger in bilderreicher Sprache begeisternde Reden vor. Diese glichen zumeist einer anzie- henden Weltumseglung vom (so die Schwäbische Chronik 27. 8.1843) und riefen zur tatkräftigen Anteil- nahme am weltweiten Missionswerk auf. Auf diesen er Festen, die in den 1840er Jahren den Charakter eines den chinesischen Volksfestes annahmen, erreichte er wöchentlich bis «Reich-Gottes-Agentur» auch Staatsoberhäupter ein, 346 jetzt Verlagshaus zu einer «Wallfahrtsstätte», die je von Zweigstelle einem Missionsfest das Möttlinger Pfarrhaus wurde länger, je mehr kleine finanzielle Inland besuchte er auch politische Oberhäupter und nicht, wenn für die Ehe keine Zeit gefunden hat. Wie eine um band zu 8000 Zuhörer. Er galt sogar als Lieblingsprediger Schwäbische Heimat 99/3 des Landes, der in umlaufenden Gedichten gefeiert benkopf: als eine entschlossene, leicht knorrige, aber wurde: auch empfindsame, sinnierende Kein frommer Karren kommt in Fahrt, trat er vor den man einem Besuch in Calw bemerkt nicht gespannt den Barth. - Natur. Äußerlich wie der Schriftsteller Wolfgang Menzel nach - als stattlicher Mann Wo sonst ein muntres Posthorn klingt, auf, der Doctor Barth Hosianna singt. Mitte hielt zwischen einem feinen Weltmann und einem der ein Toupet trug und in seinem Aussehen die ernsten Geistlichen. So strahlte er auf manche seiner Auch der fromme Karren der Basler Mission kam nicht zuletzt erst dank Barth in volle Fahrt, der mit ihr «heimlicher den Jahren war. Zudem unterstützte sam eine Aura des Geheimnisvollen, die ihm den eines «Reich- Inspektor*» geworden Anschein eines «Oetinger redivivus», mehrere diakonische Gottes-Gnostikers», verlieh. Andere erblickten darin er Einrichtungen, besonders das Stammheimer Kinderheim, Zeitgenossen eine geistliche Erhabenheit aus, gleich- einen Anflug von narzistischer Koketterie. das bis zu seinem Tod von 400 Kindern durch- laufen wurde. Das Reich Gottes sollte auch im - Leiblichen Gestalt gewinnen. Schöpfung und Naturgeschichte als «organisch-heilsgeschichtliche» Veranstaltung Den schwersten Karren aber hatte er mit dem Ver- lagsverein zu ziehen, der bis 1860 über drei Millio- Und eben vom heilsgeschichtlich-eschatologischen nen Schriften und Traktate verbreitet hatte und eine Reich-Gottes-Verständnis weithin beachtete Institution der Erweckungsbewe- scher Provenienz gung geworden war. Mit diesem massenhaften Schrifttum war es Barth gelungen, ein antimodernistisch-christliches Volksbildungsprogramm sein Denken Sinne der war und sein württembergisch-pietisti- Barths christliche Existenz, Schaffen, getragen. Ganz im Erweckungsbewegung stellt dieses Den- durch- zuführen. Er hatte damit in Württemberg, aber auch im süddeutschen gesamten Schweiz einen der immensen Raum Beitrag biedermeierlichen von Jahrhundert geleistet. Weit der Ausbildung Wertvorstellungen Kultur geprägten kleinbürgerlich-pietistischen 19. in und zur über seine im Heimat hinaus hat er auch mitgeholfen, der heimischen Mis- sionsbewegung die Bahn erzielten die zahlreichen Schriften bei der zu brechen; überdies Übersetzungen der Calwer Missionierung nichtchristlicher Völker weltweit Erfolg. Und so steht Barth seit den 1850er Jahren als international bekannte Persönlichkeit auf dem Zenit seines Lebens. Vielfach wurde er für seine Verdienste als christlich-restaurativer Schriftsteller und Publizist von geehrt: europäischen Regierungen einem Dutzend von Ritterkreuzen und mit Medaillen, die augenfällig die Liaison zwischen Monarchie und Erweckungsbewegung bekunden. Auszeichnungen wurden ihm auch von wissenschaftlichen Gesell- schaften und Naturwissenschaftlern zuteil, die sogar Pflanzen und Tiere nach ihm benannten, wie das orthotrichum barthii, Außerdem lebt er ein in Labrador Moos in weiter, etwa Grönland. wo er sich durch den Kauf einer kleinen Insel ein kolonialistisches Denkmal setzte, die noch heute «Barth-Island» heißt. So tritt er als eine bewunderte, aber auch als eine schillernde Persönlichkeit auf. Er wurde nicht nur als «Reich-Gottes-Streiter» von kritischen gefeiert, Zeitgenossen als sondern auch «pietistischer Zyklop» beäugt. So erscheint er als originaler Schwa- Schwäbische Heimat 99/3 Barths «Biblische Geschichten» erschienen bis 1945 in 483 Auflagen und wurden in 87 Sprachen übersetzt. 347 ken eine auf das praktische Christentum abzielende Testaments und «Frömmigkeits-Theologie» dar, die ein mystisch-spi- gen ritualistisches Gepräge besaß und sich aus seiner tief waren wenn aus, geht von rassistischen Vorstellunschreibt: Die Indianer Amerikas er wie die Völker Kanaans reif zur Ausrottung, nach- Urzeiten das Evangelium ablehnten und es angelegten schwäbisch-religiösen Natur erschloß. dem sie Aus deren unversiegbaren Quellen konnte er lebens- mit dem abscheulichsten Götzendienst vertauscht hatten vor lang schöpfen (Ich fühle es seelenvoll) und eine geist- und wegen ihrer daraus erwachsenden hochgetriebe- leibliche Verwandlung in die göttliche Natur erleben: in nen Laster ohnehin einer inneren eine individuelle Reich-Gottes-Existenz. gen. Ähnlich Mit der Verwandlung ging eine «Erleuchtung des Glaubens» einher: ein an Oetinger erinnerndes theo- sophisches «Gesamtsystem», das in der «Offenbarungsorganismus» vorliege. Bibel als Vom naturalismus als rationaler Außenseite Supra- geschützt, kurios Barths Bild der wie Fäulniß entgegenginVergangenheit die sieht gegenwärtigen Welt aus, die auf dem letzten Loch pfeift. So kreierte er im Anschluß an die neutestamentliche Apokalypse einen len Fahrplan. Als königstreuer phantasievol- Deutscher hatte er vermochte Barth mittels dieses konsequenten Bibli- darin in der zismus deutschen Erzfeind Frankreich den Ausgangspunkt eindeutige sogar naturwissenschaftliche gottlosen Revolution von 1848 und im Erkenntnisse zu gewinnen, wie etwa die der uneinge- des Antichristen wiedererkannt, der in Kürze auftre- schränkten ten und Christi Wiederkunft einleiten würde. In Gültigkeit des geozentrischen Weltbil- die- apokalyptischen Drama hatte er sich selbst eine des. Er hatte somit die gesamte Weltwirklichkeit in das sem Prokrustesbett die wichtige Rolle zugedacht. Er verstand seine vielfäl- bildete schichte unmittelbar zu gestalten und das Kommen der Bibel gezwängt, zwischen Deckel eines antiken Buches. des Zielpunkt tige biblischen Lehrsystems das präexistente Reich Gottes, das sich als eine von Schöpfung bis der zur Vollendung der das zur konkreten des Reiches Gottes zu Beitrag, um Heilsge- beschleunigen. Ein schwäbisch-eschatologischer Pietist Veranstaltung («Heilsökonomie Gottes») offen- bare. Diese münde alsbald nach Christi Wiederkunft in als Welt- und Naturgeschichte währende organisch-heilsgeschichtliche Arbeit «Tausendjährige Reich» ein, um schließlich Vollendung zu gelangen: auf der «Neuen Erde», auf der sodann Gott und die versöhnte Menschheit in geistleiblicher Natur alles in allem seien. Das dem schwäbischen Seelenleben tief innewohnende Vernach langen sich hier zu geistig-leiblicher ebenso Wort, Fortschrittsglaube, «Ganzheit» meldet aber der aufklärerische der mit biblisch-mythologischer Apokalyptik friedlich Hand in Hand geht. Zugleich schwingt im das Hintergrund Geschichtsverständnis des organologische Deutschen mit. Und insofern läßt sich Barths Idealismus Heilsgeschichte gleichsam als ein biblizistisches Pendant zu Hegels Geschichtskonstruktion verstehen: Was dieser mit So hatte sich Barth als Calwer Reich-Gottes-Manager mit seinem Überwelt Denken und Wirken eine «geschaffen»: eine von pietistische Gigantomanie geprägte eschatologische Gesamt-Wirklichkeit. Verdunkelt sie durch eine inhumane heilsgeTheologie, erhellt durch seine soziale war schichtliche die Tatkraft, ihn als Gründer der Stammheimer Anstalt zu einem Wegbereiter der Inneren Mission in Württemberg mochte in er werden ließ. Diese der Ausübung Eschatologie ver- der schwäbischen Tugenden gleichsam zu «erleben»; im biblizistischen «Sinnieren» erwies er und im asketischen «Schaffen». Dabei Glaubwürdigkeit, weil Glaube und Tun ausnehmend kongruent waren. Mit aller Kraft hatte Barth versucht, das Rad der «Weltgeist» meint, ist bei Barth das «Reich Gottes». Zeit zurückzudrehen. Der von der Moderne herauf- Dank seiner biblizistischen Brille vermochte geführten Entzauberung der Welt hatte er allerdings die Durchsetzung schauen des Reiches und daraus ein Gottes zu er über- vollständiges Bild der Kir- mittels der neutestamentlichen Mythologie lediglich eine Wieder-Verzauberung entgegenzusetzen ver- chen- und Weltgeschichte zu gewinnen. Hinsichtlich mocht: eine Illusion namens «Reich Gottes». Damit dieser hatte er eine Flucht ins 18. Jahrhundert angetreten geht er von alttestamentlich-theokratischen Vorstellungen aus und sieht das Schicksal der Völker und in ihrem Glaubenskühnheit wurde det. Er lichem Glaubensgehorsam Gott gegenüber begrün- kann dabei nicht Kolonialismus die Conquista, bei der Tode kamen, als nur reden, von heilsgeschicht- sondern auch etwa vierzig Millionen Indianer zu notwendiges heilsgeschichtliches Gericht Gottes bezeichnen. Er orientiert sich dabei an den schauderhaften 348 Genozid-Erzählungen des Alten große Erfolge erzielt. Seine - leidenschaftliche allerdings nicht belohnt: Das so sehnlich erwartete und mit einer Legion von Calwer Schriften «herbeigeschriebene» Reich Gottes war nicht gekommen. Der Siegeszug der Säkulari- sierung ging unaufhaltsam weiter. Und so drehte sich auch die Weltenkugel weiter, als Barth, abgearbeitet und von mehreren Krankhei- Schwäbische Heimat 99/3 ten geplagt, am 12. November 1862 im Alter von 63 Jahren verstarb. Immerhin wurde hinaus Deutschland als weit er über internationale Gestalt der Erweckungsbewegung mit einer Legion von Nekrologen bedacht, Weltweit ein Begriff: unter anderem von einer Der Calwer Verlagsverein englischen Ausstellung Zeitung, die titelte: Dr. Barth was a Champion ofthegos- V pel in Germany. Sein Nachfolger als Verlagsleiter wurde dem Gepräge ein Verlag verlieh. Schwiegersohn, ? Christian Gottlob Barth O -•- kirchlich-wissenschaftliches Beerbt dem wurde er von seinem Missionsprediger Johannes Hesse (1847-1916), dem Vater des Dichters. 1920 zog der Verlagsverein nach Stuttgart um; seit 1952 trägt er Geburtstag des Gründers der Sprachwissenschaftler Hermann Gundert (1814-1893), der zum 200. Ww den Namen «Calwer Verlag». Auch Barths zweite Gründung, heute, 200 die Stammheimer Anstalt, führt bis Jahre nach seinem Geburtstag, ihre Arbeit fort, seit 1978 als Sprachheilzentrum. Ausstellung noch bis 24.10.1999 Geöffnet sonntags 14-17 Uhr Cuw ANMERKUNG: 1 Näheres zu Barth v.a. in: Werner Raupp, C. G.Barth. Studien zu Palais Vischer Leben und Werk. Stuttgart 1998 (Bibliogr.); ders., Art.: «Barth, C.G.», in: Biographisch-Bibliographisches Museum der Stadt Calw Kirchenlexikon, Bd. 16 (erseh. 1999); Bischofstraße 48 ■ 75365 Calw ders., Christian Gottlob Barth und die Anfänge des Calwer Ver- lags, in: Der Calwer Verlagsverein. Literatur aus Calw Telefon (07051) 92 6995 oder 167-260 für die Welt. Hrsg, von der Stadt Calw. Calw 1999 (Kleine Reihe 8. Palais Vischer, Museum der Stadt.) Vgl. auch C.-G.-Barth-Archiv (Privatarchiv Werner Lutz Reichardt Raupp, Dusslingen), das Quellenmaterial aus weltweit achtzig Archiven Ortsnamenbuch des Ostalbkreises umfaßt. Teil I: A - Teil II: M L 1999. 419 Seiten. Fester Fadenheftung DM 74,- ISBN 3-17-015351-X Gesamtwerk ich empfinde diesen Anblick in meinem Innersten: ♦ DM 136- Quellennachweis und unter Berücksichtigung der mundartlichen Form wird die sprachgeschichtliche Entwicklung jetzt von Millionen Zungen jedes Ortsnamens dargestellt. Ein Hallelujah stimmt: Geist, und schwöre hier, Dein Leben Und Deine Kräfte Gott zu weih'n: Lebensbilder aus Und Du, Allvater, der sie mir gegeben, Band wenn ich einst aus Baden-Württemberg 19: 1998. 400 Seiten. Leinen DM 54,ISBN 3-17-015060-X Erhalte meine Seele rein! Daß, ISBN 3-17-015353-6 erhobener Belege der historischen Ortsnamenformen mit Am Fuße seines Thrones klimmt, B lick' auf mein ♦ ISBN 3-17-015352-8 Ausgehend von einer Aufreihung hauptsächlich im Archiv Staub, der zu Gott emporgedrungen Zu Gott, dem Z Fester Einband/ Einband/Fadenheftung DM 74,- O Freund! Ich fühle es seelenvoll, - 1999. 402 Seiten, 1 Karte. diesem Thal der Zähren (Tränen) In diesem Band werden 18 Biographien vorgestellt: u.a. Zur Ruhe jenes Lebens geh', über Der Fruchtstaub vieler Carl Mez, den Erfinder Siegfried Junghans, den Pfarrer guter Aehren und Kommunisten Erwin Eckert, den Nordamerika-Pionier Um meinen Grabeshügel weh'! «Weihegesang» von Christian und Gottlob Barth, natur-mystisches Erlebnis Indianer-Diplomat Conrad Weiser. ent- standen im Oktober 1816 beim Betrachten der aufge- henden Sonne. Ein Markgraf Christoph von Baden, den Unternehmer und Kohlhammer W. Kohlhammer GmbH ■ 70549 Stuttgart Motto seines Lebens. Schwäbische Heimat 99 /3 349
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Analysis of the effects of importin α1 on the nuclear translocation of IL-1α in HeLa cells
Scientific reports
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Analysis of the effects of importin α1 on the nuclear translocation of IL‑1α in HeLa cells OPEN Akiko Yamada 1,2,8*, Kiyotaka Wake 3,4,8, Saya Imaoka 1,2, Mitsuru Motoyoshi 3,5, Takenori Yamamoto 6,7 & Masatake Asano 1,2 Interleukin-1α (IL-1α), a cytokine released by necrotic cells, causes sterile inflammation. On the other hand, IL-1α is present in the nucleus and also regulates the expression of many proteins. A protein substrate containing a classical nuclear localization signal (cNLS) typically forms a substrate/importin α/β complex, which is subsequently transported to the nucleus. To the best of our knowledge, no study has directly investigated whether IL-1α—which includes cNLS—is imported into the nucleus in an importin α/β-dependent manner. In this study, we noted that all detected importin α subtypes interacted with IL-1α. In HeLa cells, importin α1-mediated nuclear translocation of IL-1α occurred at steady state and was independent of importin β1. Importin α1 not only was engaged in IL-1α nuclear transport but also concurrently functioned as a molecule that regulated IL-1α protein level in the cell. Furthermore, we discussed the underlying mechanism of IL-1α nuclear translocation by importin α1 based on our findings. Abbreviations cNLS Classical nuclear localization signal HAX-1 HCLS1-associated protein X-1 IL-1α Interleukin 1 alpha IL-1R1 IL-1 receptor type 1 IMAC Immobilized metal affinity chromatography Importin α Importin subunit alpha Importin β Importin subunit beta SDS-PAGE Sodium dodecylsulfate-polyacrylamide gel electrophoresis Y2H Yeast two-hybrid Abbreviations cNLS Classical nuclear localization signal HAX-1 HCLS1-associated protein X-1 IL-1α Interleukin 1 alpha IL-1R1 IL-1 receptor type 1 IMAC Immobilized metal affinity chromatography Importin α Importin subunit alpha Importin β Importin subunit beta SDS-PAGE Sodium dodecylsulfate-polyacrylamide gel electrophoresis Y2H Yeast two-hybrid Interleukin-1α (IL-1α), a member of the IL-1 family that is extensively present in mesenchymal-derived tissues and epithelial ­cells1. The precursor of IL-1α has a molecular weight of approximately 33 kDa and is cleaved by proteases into a 17-kDa mature form and a 16-kDa N-terminal form called a ­propiece2,3. However, most IL-1α is present in its precursor form, the majority of which is in intracellular proteins or membrane ­forms4,5. The activity of the precursor form of IL-1α is inhibited intracellularly by IL-1 receptor type 2, a decoy ­receptor6,7. When cells undergo necrosis, the precursor form of IL-1α is immediately released extracellularly, acting as an “alarmin” and informing the loss of membrane integrity to nearby ­cells1. Extracellularly released IL-1α precur- sor binds to IL-1 receptor type 1 (IL-1R1) on neighboring cells, stimulating the production of cytokines such as IL-68, and promoting tissue ­inflammation4,5. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports/ Furthermore, IL-1α is known for its role as a dual-function cytokine; it has a nuclear localization signal (NLS) ­sequence9 and can be transported to the ­nucleus10. In the nucleus, IL-1α activates NF-κB and AP-1 in an IL-1R1-independent manner, promoting the production of inflammatory cytokines such as IL-6 and IL-811. In addition, IL-1α was reported to interact with histone acetyl transferase complexes in vitro12,13, with mRNA splicing-related proteins, promoting apoptosis of malignant tumor ­cells14, and that IL-1α expression lowers cell growth rates and migratory potential in vascular endothelial ­cells15,16. These findings suggested that IL-1α is involved in regulation of the expression of various proteins in the nucleus. Therefore, regulating the nuclear translocation of IL-1α is effective for the control of intranuclear IL-1α-related diseases.hi f The mechanisms of IL-1α nuclear localization are unknown. Several findings regarding IL-1α nuclear trans- port were previously reported. Cohen et al. discovered that acetylation of IL-1α at Lys82 promoted the nuclear localization of IL-1α during genotoxic stress in the murine macrophage cell line RAW 264.717. Yin et al. found that HAX-1 interacted with three domains of the N-terminus of IL-1α, including the NLS-containing domain, in HEK293 cells using an immunoprecipitation ­assay18. According to Kawaguchi et al., HAX-1 knockdown in systemic sclerosis fibroblasts reduced nuclear IL-1α ­levels19. However, the mechanism through which Lys82 of IL-1α and HAX-1 are involved in the nuclear localization of IL-1α requires further elucidation. On the con- trary, the most prevalent nuclear localization signal, namely the classical NLS (cNLS; KVLKKRRL)9, is found in IL-1α20,21. In the cytoplasm, cNLS-containing molecule and importin α, a cargo receptor, and importin β1, a carrier molecule, form a complex, passing through the nuclear–pore complex by utilizing the gradient of the small G proteins Ran-GDP and Ran-GTP22–24. Luheshi et al. reported that the nuclear translocation of IL-1α is Ran-dependent upon analyzing COS-7 cells coexpressing IL-1α with RanQ69L, a dominant-negative isoform of Ran lacking the ability to hydrolyze ­GTP25. Sahni et al. studied the influence of disease-associated mutations on the protein interactome using a comprehensive yeast two-hybrid (Y2H) screening assay, which revealed that importin α7 interacts with IL-1α26. The findings that IL-1α is transported to the nucleus via Ran-GDP/GTP gradient and that IL-1α interacts with the importin α family in yeast cells suggest that IL-1α is transported to the nucleus in an importin α dependent manner. Analysis of the effects of importin α1 on the nuclear translocation of IL‑1α in HeLa cells OPEN 1Department of Pathology, Nihon University School of Dentistry, 1‑8‑13, Kanda‑Surugadai, Chiyoda‑ku, Tokyo  101‑8310, Japan. 2Division of Immunology and Pathobiology, Dental Research Center, Nihon University School of Dentistry, 1‑8‑13, Kanda‑Surugadai, Chiyoda‑ku, Tokyo 101‑8310, Japan. 3Department of Orthodontics, Nihon University School of Dentistry, 1‑8‑13, Kanda‑Surugadai, Chiyoda‑ku, Tokyo 101‑8310, Japan. 4Division of Oral Structural and Functional Biology, Nihon University Graduate School of Dentistry, 1‑8‑13, Kanda‑Surugadai, Chiyoda‑ku, Tokyo  101‑8310, Japan. 5Division of Clinical Research, Dental Research Center, Nihon University School of Dentistry, 1‑8‑13, Kanda‑Surugadai, Chiyoda‑ku, Tokyo  101‑8310, Japan. 6Division of Molecular Target and Gene Therapy Products, National Institute of Health Sciences, 3‑25‑26, Tonomachi, Kawasaki‑ku, Kawasaki‑shi, Kanagawa 210‑9501, Japan. 7Institute for Genome Research, Tokushima University, Kuramotocho‑3, Tokushima  770‑8503, Japan. 8These authors contributed equally: Akiko Yamada and Kiyotaka Wake. *email: yamamoto.akiko@nihon-u.ac.jp | https://doi.org/10.1038/s41598-024-51521-w Scientific Reports | (2024) 14:1322 IL‑1α expression and cNLS‑dependent nuclear transport of IL‑1α in HeLa cells IL‑1α expression and cNLS‑dependent nuclear transport of IL‑1α in HeLa cells IL‑1α expression and cNLS‑dependent nuclear transport of IL‑1α in HeLa cells In this study, using HeLa cells expressing IL-1α, we examined the interactions between importin α family proteins and IL-1α. Since endogenous IL-1α may interfere with the interaction of expressed IL-1α and endogenous impor- tin α, it is desirable to use cells with low endogenous IL-1α for sensitive detection of interactions. We performed Western blotting using lysate from HeLa cells and an anti-human IL-1α antibody. Consequently, immunoreactive bands were not detected for the precursor (33 kDa) or mature (C-terminal fragment: 17 kDa) fragments (lane of the empty vector in leftmost panel, Fig. 1a). This result showed that HeLa cells contain little or no endogenous IL-1α, making them suitable for this analysis. Then, Western blotting was performed using cell lysate obtained from HeLa cells that were transfected with human IL-1α fused with a HiBiT-tag at the N-terminus and a His-tag at the C-terminus (Fig. 1b). When detected with anti-IL-1α, anti-His antibodies, and HiBiT-tag luminescence, a distinct band of approximately 33 kDa was observed in each result, which is similar to the molecular weight of the IL-1α precursor (Fig. 1a). In contrast to the precursor IL-1α, the 17 kDa form of IL-1α was detected with low or no signal intensity. These findings are consistent with previous studies reporting that IL-1α mainly exists intracellularly as a precursor. Therefore, the IL-1α precursor was targeted for further analysis in this study.l h Previously, immunofluorescence microscopy was utilized to explore the subcellular localization of GFP- fused IL-1α in HeLa cells; the findings demonstrated that GFP-fused IL-1α localizes to the nucleus in a cNLS- dependent ­manner25,29. The biochemical analysis in this study also revealed that IL-1α localization to the nucleus is cNLS-dependent. As shown in Fig. 1c, we prepared a construct of cNLS-deleted IL-1α fused with HiBiT, His tags (ΔNLS-IL-1α), and then transiently transfected it into HeLa cells. From the cell lysate, nuclear and cyto- plasmic fractions were taken and put through Western blotting with an anti-His antibody. As a result, the signal intensity of IL-1α band in the nuclear fraction was reduced by the deletion of cNLS, whereas that of IL-1α band in the cytoplasmic fraction was enhanced by the deletion of cNLS (Fig. 1d). This suggests that deleting cNLS reduces IL-1α nuclear translocation. www.nature.com/scientificreports/ However, in addition to whether IL-1α interacts with the importin α family in mammalian cells, the actual functional involvement of importin α in the nuclear translocation of IL-1α was not examined in the mammalian or yeast cells in aforementioned reports.h y p The importin α family is also called karyopherin α, and there are seven subtypes in humans, which are clas- sified into three subfamilies, α1, α2, and α3, based on the homology of the amino acid sequence (Table 1). There is approximately 50% homology between subfamilies, and within each subfamily, there is approximately 80% homology between subtypes except for importin α1 and importin α8. Each subtype is known to exhibit tissue- dependent expression (Table 1)27; furthermore these subtypes are expected to bind to intracellular substances and viruses in vivo in a substrate-specific ­manner28. In this study, we examined the interactions of all detectable importin α subtypes with IL-1α in a mammalian cell line (HeLa) and discussed the mechanism by which the importin α family participates in IL-1α nuclear translocation in mammalian cells. 2 Scientific Reports | (2024) 14:1322 | https://doi.org/10.1038/s41598-024-51521-w Table 1. The subtypes of human importin α family. N/A not applicable. Gene name Protein name Subfamily Accession no. Molecular weight (Da) Tissue specificity Function References on interaction with IL-1α mRNA (NCBI) Protein (UniProt) KPNA2 Importin subunit alpha-1 α2 NM_002266.4 P52292 57,862 Expressed ubiqui- tously Functions in nuclear protein import as an adapter protein for nuclear receptor importin β1 N/A KPNA4 Importin subunit alpha-3 α3 NM_002268.5 O00629 57,887 Highly expressed in testis, ovary, small intestine, heart, skeletal muscle, lung and pancreas, but barely detect- able in kidney, thymus, colon and peripheral blood leukocytes N/A KPNA3 Importin subunit alpha-4 α3 NM_002267.4 O00505 57,811 Ubiquitous Highest levels in heart and skeletal muscle N/A KPNA1 Importin subunit alpha-5 α1 NM_002264.4 P52294 60,222 Expressed ubiqui- tously N/A KPNA5 Importin subunit alpha-6 α1 NM_002269.3 O15131 60,666 Testis N/A KPNA6 Importin subunit alpha-7 α1 NM_012316.5 O60684 60,030 Widely expressed 26 KPNA7 Importin subunit alpha-8 α2 NM_001145715.3 A9QM74 56,938 unknown Functions in nuclear protein import N/A https://doi.org/10.1038/s41598-024-51521-w www.nature.com/scientificreports/ Analysis of interaction between IL‑1α and importin α family proteinsi Analysis of interaction between IL‑1α and importin α family proteins To confirm whether the nuclear transport of IL-1α is mediated by the importin protein complex, we first analyzed the interaction of IL-1α with each importin α family protein. Initially, we performed a coimmunoprecipita- tion assay with an IL-1α-specific antibody. The importin α subtypes and antibody heavy chains have similar molecular weights (57–61 kDa) (Table 1); therefore, in addition to detecting importin α, the heavy chains of the antibody used for precipitation were detected nonspecifically. This masked the detection of importin α (results not shown). In order to provide clearer results, cobalt-based immobilized metal ion affinity chromatography (IMAC) was conducted on HeLa cells transfected with His-tag-fused IL-1α to isolate the His-tag fused protein complex in the current study. y To verify whether His-tag fused protein was isolated, the fractions obtained during IMAC were subjected to Western blotting using anti-His antibody, and a clear single band was detected corresponding to the molecular weight of IL-1α in the elution fraction expected to contain the His-tag fused protein (Fig. 2a). In contrast, no band was detected in the flow-through fraction (Fig. 2a). GAPDH (which does not interact with IL-1α) was detected with a stronger signal intensity in the flow-through fraction, whereas extremely low signal intensity was detected in the elution fraction (Fig. 2a). Moreover, HAX-1, which has been shown to interact with IL-1α in HEK293 ­cells18, was found in the elution fraction with a high signal intensity (Fig. 2b). These findings suggest that this assay can evaluate the interactions of the importin protein with IL-1α. To investigate the interaction of IL-1α with each importin α subtype using this assay, importin α1, α3, α4, α5, α6, and α7 in the flow-through and elution fractions were analyzed via Western blotting using each specific antibody (Supplementary Table 1). The results showed that for all importin α subtypes, a stronger signal was detected in the elution fraction than in the flow-through fraction (Fig. 2b). The anti-importin α8 antibody, however, was unable to detect importin α8 in HeLa cell lysates (data not shown). Thus, it is revealed that importin α1, α3, α4, α5, α6, and α7 interact with IL-1α in HeLa cells. IL‑1α expression and cNLS‑dependent nuclear transport of IL‑1α in HeLa cells In general, molecules with a molecular weight of around ≤ 40 kDa may diffuse passively across the nuclear membrane ­pore30; accordingly, since the molecular weight of IL-1α is below this threshold, it is possible that the detection of IL-1α in the nuclear fraction, even in the absence of cNLS, is due to free diffusion of IL-1α into the nucleus. This biochemical approach could therefore be used to assess the cNLS-dependent nuclear localization of IL-1α. These findings supported the utility of this expression system for identifying the molecules involved in nuclear translocation. Effect of importin α1 on the intracellular behavior of IL‑1α h b h f Among the importin α subtypes, the overexpression of importin α1 in various cancers, including breast, lung, esophageal, squamous cell, colon, prostate, and cervical cancers, has been reported in several ­studies31–35. Impor- tin α1 expression is also higher in HeLa cells than in normal human cervical epithelial ­cells36; thus, importin α1 is considered to be functional in HeLa cells. Therefore, the present study focused on the effect of importin α1 on the nuclear translocation of IL-1α. Following that, we used different approaches to confirm the interaction between importin α1 and IL-1α, including coimmunoprecipitation of HeLa cells expressing His-tag-fused IL-1α and GST pulldown assay with GST-tag-fused importin α1 and His-tag-fused IL-1α; both approaches confirmed the interaction between importin α1 and IL-1α (Supplementary Figs. S2, S3). To examine the effect of importin α1 on the subcellular localization of IL-1α, we expressed His-tag fused IL-1α in HeLa cells transfected with siRNA targeting importin α1. The quantity of importin α1 protein was significantly reduced by siRNA transfection (Fig. 3a). Interestingly, in Western blotting using equal amounts of protein, IL-1α protein expression was also significantly decreased by importin α1 knockdown, whereas there was no difference in the expression of β-actin (Fig. 3a). To investigate the influence of importin α1 on IL-1α nuclear translocation, nuclear and cytoplasmic https://doi.org/10.1038/s41598-024-51521-w Scientific Reports | (2024) 14:1322 | www.nature.com/scientificreports/ i Figure 1. Construction of an expression system comprising HiBiT-tag- and His-tag fused IL-1α in HeLa cells and analysis of nuclear translocation of cNLS-deleted IL-1α. (a) At 24 h after transfection of HeLa cells with an empty vector or a vector incorporating N-terminal HiBiT-tag- and C-terminal His-tag fused IL-1α, the cells were collected and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting with anti-IL-1α, anti-His, and anti-GAPDH antibodies or detection with LgBiT and its substrate. (b) Schematic of the IL-1α gene fused with a HiBiT-tag at the N-terminus and a His-tag at the C-terminus. pcDNA incorporating N-terminal HiBiT-tag and C-terminal His-tag-fused IL-1α plasmid was transiently expressed in HeLa cells. (c) Schematic representation of precursor IL-1α with the deleted cNLS sequence (KVLKKRRL). pcDNA incorporating N-terminal HiBiT-tag- and C-terminal His-tag fused IL-1α plasmid or a plasmid with deleted cNLS sequence from the full-length IL-1α plasmid were transiently expressed in HeLa cells. (d) Nuclear and cytoplasmic fractions of the HeLa cells expressing full-length IL-1α and cNLS- deleted IL-1α were subjected to immunoblotting with an anti-His, anti-lamin B1 and anti-β-actin antibodies. Effect of importin α1 on the intracellular behavior of IL‑1α h b h f Arrowheads indicate bands corresponding to the expected molecular weight of each protein. We confirmed the reproducibility of the results (Supplementary Fig. S1). The full-length blots are shown in Supplementary Information, Fig. 2. Figure 2. Identification of importin α subtypes interacting with IL-1α. The isolation of His-tag-fused IL-1α via IMAC was conducted using cell lysates from HeLa cells expressing His-tag-fused IL-1α. (a) Immunoblotting was performed using anti-His and anti-GAPDH antibodies; FT represents the flow-through fraction, and Elution represents the imidazole elution fraction. (b) anti-HAX-1, anti-importin α1, anti-importin α3, anti-importin α4, anti-importin α5, anti-importin α6, and anti-importin α7 antibodies were used for immunoblotting. Arrowheads indicate bands corresponding to the expected molecular weight of each protein. We confirmed the reproducibility of the results (Supplementary Fig. S1). The full-length blots are shown in Supplementary Information, Fig. 2. Figure 2. Identification of importin α subtypes interacting with IL-1α. The isolation of His-tag-fused IL-1α via IMAC was conducted using cell lysates from HeLa cells expressing His-tag-fused IL-1α. (a) Immunoblotting was performed using anti-His and anti-GAPDH antibodies; FT represents the flow-through fraction, and Elution represents the imidazole elution fraction. (b) anti-HAX-1, anti-importin α1, anti-importin α3, anti-importin α4, anti-importin α5, anti-importin α6, and anti-importin α7 antibodies were used for immunoblotting. Arrowheads indicate bands corresponding to the expected molecular weight of each protein. We confirmed the reproducibility of the results (Supplementary Fig. S1). The full-length blots are shown in Supplementary Information, Fig. 2. Figure 2. Identification of importin α subtypes interacting with IL-1α. The isolation of His-tag-fused IL-1α via IMAC was conducted using cell lysates from HeLa cells expressing His-tag-fused IL-1α. (a) Immunoblotting was performed using anti-His and anti-GAPDH antibodies; FT represents the flow-through fraction, and Elution represents the imidazole elution fraction. (b) anti-HAX-1, anti-importin α1, anti-importin α3, anti-importin α4, anti-importin α5, anti-importin α6, and anti-importin α7 antibodies were used for immunoblotting. Arrowheads indicate bands corresponding to the expected molecular weight of each protein. We confirmed the reproducibility of the results (Supplementary Fig. S1). The full-length blots are shown in Supplementary Information, Fig. 2. protein in the nuclear fraction to that in the cytoplasmic fraction), was substantially reduced by knockdown of importin α1 (Fig. 3c). This indicated that importin α1 is related to the transport system of IL-1α in HeLa cells. Effect of importin α1 on the intracellular behavior of IL‑1α h b h f The full-length blots are shown in Supplementary Information, Fig. 1. Figure 1. Construction of an expression system comprising HiBiT-tag- and His-tag fused IL-1α in HeLa cells and analysis of nuclear translocation of cNLS-deleted IL-1α. (a) At 24 h after transfection of HeLa cells with an empty vector or a vector incorporating N-terminal HiBiT-tag- and C-terminal His-tag fused IL-1α, the cells were collected and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting with anti-IL-1α, anti-His, and anti-GAPDH antibodies or detection with LgBiT and its substrate. (b) Schematic of the IL-1α gene fused with a HiBiT-tag at the N-terminus and a His-tag at the C-terminus. pcDNA incorporating N-terminal HiBiT-tag and C-terminal His-tag-fused IL-1α plasmid was transiently expressed in HeLa cells. (c) Schematic representation of precursor IL-1α with the deleted cNLS sequence (KVLKKRRL). pcDNA incorporating N-terminal HiBiT-tag- and C-terminal His-tag fused IL-1α plasmid or a plasmid with deleted cNLS sequence from the full-length IL-1α plasmid were transiently expressed in HeLa cells. (d) Nuclear and cytoplasmic fractions of the HeLa cells expressing full-length IL-1α and cNLS- deleted IL-1α were subjected to immunoblotting with an anti-His, anti-lamin B1 and anti-β-actin antibodies. The full-length blots are shown in Supplementary Information, Fig. 1. fractions were extracted from the cell lysate, and IL-1α was identified by Western blotting (Fig. 3b). In this analysis, although the protein amounts of the importin α1 siRNA-treated and negative siRNA-treated samples used were equal, the IL-1α signal intensities in the nuclear and cytoplasmic fractions of HeLa cells treated with importin α1 siRNA were lower than those in the nuclear and cytoplasmic fractions of HeLa cells treated with negative siRNA. By contrast, the signal intensities of lamin B1 in the nuclear fraction and β-actin in the cyto- plasmic fraction were unchanged. This indicated that the knockdown of importin α1 can decrease the protein amount of IL-1α in HeLa cells. In addition, the nuclear/cytoplasmic ratio (i.e., the ratio of the amount of IL-1α https://doi.org/10.1038/s41598-024-51521-w Scientific Reports | (2024) 14:1322 | www.nature.com/scientificreports/ Figure 2. Identification of importin α subtypes interacting with IL-1α. The isolation of His-tag-fused IL-1α via IMAC was conducted using cell lysates from HeLa cells expressing His-tag-fused IL-1α. (a) Immunoblotting was performed using anti-His and anti-GAPDH antibodies; FT represents the flow-through fraction, and Elution represents the imidazole elution fraction. (b) anti-HAX-1, anti-importin α1, anti-importin α3, anti-importin α4, anti-importin α5, anti-importin α6, and anti-importin α7 antibodies were used for immunoblotting. Effect of importin α1 on the intracellular behavior of IL‑1α h b h f To see whether IL-1α nuclear translocation was specifically regulated by importin α1, we investigated the effect of importin α4, which has been also shown to interact with IL-1α (Fig. 2b). The results showed that the knockdown of importin α4 had little effect on the protein amount and the nuclear translocation of IL-1α (Supplementary Fig. S4a,d). To further confirm the redundancy among importin α subtypes, we analyzed the effects of other importin α subtypes (importin α3, importin α5, and importin α7) on the nuclear transport of IL-1α. However, importin α6 was excluded from this analysis because it is expressed specifically in the testes. The results indicated that in addition to importin α1, knockdown of the gene expression of other importin α subtypes reduced the amount of IL-1α protein transported to the nucleus (Supplementary Fig. S5). Analysis of the behavior of importin β1 in IL‑1α nuclear transport (b) The nuclear and cytoplasmic fractions of HeLa cells transfected with importin α1 siRNA or negative-control siRNA and His-tag-fused IL-1α were obtained, and each fraction was then subjected to immunoblotting using anti-importin α1, anti-His, anti-lamin B1, and anti- β-actin antibodies. (c) IL-1α protein levels were measured in the nuclear and cytoplasmic fractions. Results are expressed as the nuclear/cytoplasmic ratio. Data are presented as the mean ± SD. Significant difference (P < 0.05) based on an unpaired Student’s t-test is indicated by asterisk (n ≥ 3). The full-length blots are shown in Supplementary Information, Fig. 3. Figure 3. Analysis of the effect of importin α1 on IL-1α expression and nuclear translocation. (a) Cell lysates from HeLa cells transfected with 5 nM negative-control siRNA or 2.5 or 5 nM importin α1 siRNA and then transfected 24 h later with His-tag-fused IL-1α were subjected to SDS-PAGE and immunoblotting with anti- importin α1, anti-His, and anti-β-actin antibodies. (b) The nuclear and cytoplasmic fractions of HeLa cells transfected with importin α1 siRNA or negative-control siRNA and His-tag-fused IL-1α were obtained, and each fraction was then subjected to immunoblotting using anti-importin α1, anti-His, anti-lamin B1, and anti- β-actin antibodies. (c) IL-1α protein levels were measured in the nuclear and cytoplasmic fractions. Results are expressed as the nuclear/cytoplasmic ratio. Data are presented as the mean ± SD. Significant difference (P < 0.05) based on an unpaired Student’s t-test is indicated by asterisk (n ≥ 3). The full-length blots are shown in Supplementary Information, Fig. 3. not part of the IL-1α complex in HeLa cells. In addition, when importin β1 was knocked down using siRNA in HeLa cells expressing IL-1α, the nuclear translocation of IL-1α was not inhibited, reflecting non-interaction of importin β1 with IL-1α (Supplementary Fig. S4d). Furthermore, the nuclear translocation of IL-1α was analyzed in the presence of the importin β1 inhibitor importazole in HeLa cells expressing IL-1α. The nuclear transloca- tion of p65, a substrate for importin β1 transport, was suppressed in the presence of 50 µM importazole, whereas IL-1α nuclear transport was unaffected (Supplementary Fig. S4e). These results indicated that importin β1 is not involved in the importin α-mediated nuclear transport of IL-1α. Analysis of the behavior of importin β1 in IL‑1α nuclear transport To determine whether importin β1 was needed for nuclear transport of the IL-1α complex, we examined whether importin β1 was present in the complex. First, we examined the interaction between IL-1α and importin β1 by performing IMAC analysis using HeLa cells expressing His-tag fused IL-1α. The results showed that importin β1 was mainly detected in the flow-through fraction, which behaved similarly to GAPDH (Fig. 4a). Results suggested that importin β1 did not interact with the IL-1α complex. Importin β1 was further investigated using coimmunoprecipitation with an anti-importin β1 antibody. Consequently, importin β1 was detected in the pellet fraction, and so was importin α1. In addition, p65, which was transported to the nucleus by the importin α1/β1 complex, was coprecipitated with importin β1. However, IL-1α was detected in the supernatant fraction, exhibit- ing similar behavior as the negative control GAPDH (Fig. 4b). These findings demonstrated that importin β1 was https://doi.org/10.1038/s41598-024-51521-w Scientific Reports | (2024) 14:1322 www.nature.com/scientificreports/ Figure 3. Analysis of the effect of importin α1 on IL-1α expression and nuclear translocation. (a) Cell lysates from HeLa cells transfected with 5 nM negative-control siRNA or 2.5 or 5 nM importin α1 siRNA and then transfected 24 h later with His-tag-fused IL-1α were subjected to SDS-PAGE and immunoblotting with anti- importin α1, anti-His, and anti-β-actin antibodies. (b) The nuclear and cytoplasmic fractions of HeLa cells transfected with importin α1 siRNA or negative-control siRNA and His-tag-fused IL-1α were obtained, and each fraction was then subjected to immunoblotting using anti-importin α1, anti-His, anti-lamin B1, and anti- β-actin antibodies. (c) IL-1α protein levels were measured in the nuclear and cytoplasmic fractions. Results are expressed as the nuclear/cytoplasmic ratio. Data are presented as the mean ± SD. Significant difference (P < 0.05) based on an unpaired Student’s t-test is indicated by asterisk (n ≥ 3). The full-length blots are shown in Supplementary Information, Fig. 3. Figure 3. Analysis of the effect of importin α1 on IL-1α expression and nuclear translocation. (a) Cell lysates from HeLa cells transfected with 5 nM negative-control siRNA or 2.5 or 5 nM importin α1 siRNA and then transfected 24 h later with His-tag-fused IL-1α were subjected to SDS-PAGE and immunoblotting with anti- importin α1, anti-His, and anti-β-actin antibodies. Discussion To evaluate the substrate specificity of the importin α family, detailed analysis based on the absolute abundance of each importin α subtype is needed in the future. By contrast, Wan et al. detected multiple importin α subtypes in the same fraction in a proteomic analysis of isolated fractions obtained using various separation carriers, indicating that they interact with each ­another37. Furthermore, we also confirmed that the interaction between importin α1 and importin α3 using immunologic analysis (Supplementary Fig. S6). Thus, the elution of each importin α subtype together with IL-1α might reflect interactions between different importin α subtypes; namely, importin α1 and importin α3 might interact and function in a cooperative manner in vivo. Future studies on the interactions of importin α1 with subtypes other than importin α3 that considers the absolute abundance of each protein are needed. Furthermore, whether each importin α subtype binds directly or indirectly to IL-1α remains to be elucidated, and the contribution of the interactions between different importin α subtypes to the transport function of IL-1α requires additional study. I l i ti bi d t th NLS f d i ti β1 bi d t i ti f i l indicates that importin α subtypes are widely involved in the nuclear transport of IL-1α. However, the nuclear transport of IL-1α was unaffected by importin α4 knockdown (Supplementary Figs. S4d, S5b). These results suggest that importin α4 does not play a regulatory role in the nuclear transport of IL-1α. Although importin α4 might have a regulatory function in the nuclear transport of IL-1α, it is possible that its expression will be extremely low, in which case it will have a negligible effect on transport. To evaluate the substrate specificity of the importin α family, detailed analysis based on the absolute abundance of each importin α subtype is needed in the future. By contrast, Wan et al. detected multiple importin α subtypes in the same fraction in a proteomic analysis of isolated fractions obtained using various separation carriers, indicating that they interact with each ­another37. Furthermore, we also confirmed that the interaction between importin α1 and importin α3 using immunologic analysis (Supplementary Fig. S6). Thus, the elution of each importin α subtype together with IL-1α might reflect interactions between different importin α subtypes; namely, importin α1 and importin α3 might interact and function in a cooperative manner in vivo. Discussion Previously, Sahni et al. showed that IL-1α interacts with importin α7 using the Y2H screening ­assay26. However, the results were obtained from a comprehensive analysis opposed to an experiment focused solely on IL-1α, and it was possible that this interaction would not be replicated in mammalian cells. Therefore, it was necessary to verify the interaction between IL-1α and importin α subtypes in an experimental system using mammalian cells, which was not performed in the previous study. For the first time, we studied interactions between IL-1α and importin α subtypes in mammalian cells and discovered that IL-1α interacts with all detectable importin α subtypes, namely importin α1, α3, α4, α5, α6, and α7 (Fig. 2b). The results suggest that these subtypes are possibly involved in the nuclear translocation of IL-1α. However, the function of the importin α family in the nuclear translocation of IL-1α has not been investigated. Our analysis showed that reducing the expression of importin α1 suppressed IL-1α nuclear localization, demonstrating that among the importin α subtypes, at least importin α1 is involved in IL-1α nuclear translocation (Fig. 3b,c). d d h f l l d b h b d h g Redundancy in the importin α family was also examined because each importin α subtype interacted with IL-1α (Fig. 2b). We found that in addition to knockdown of importin α1, knockdown of most other importin α subtypes also reduced the amount of IL-1α protein transported to the nucleus (Supplementary Fig. S5b). This https://doi.org/10.1038/s41598-024-51521-w Scientific Reports | (2024) 14:1322 | www.nature.com/scientificreports/ Figure 4. Analysis of the behavior of importin β1 in IL-1α nuclear translocation. (a) After isolation of His- tag-fused protein via IMAC from HeLa cells expressing His-tag-fused IL-1α, the obtained fractions during IMAC were analyzed using Western blotting with anti-His, anti-importin β1, and anti-GAPDH antibodies. Input lysate of HeLa cells before IMAC, FT flow-through fraction, Elution imidazole elution fraction. (b) Coimmunoprecipitation was performed with an anti-importin β1 antibody using HeLa cells expressing His-tag- fused IL-1α, and the input, supernatant fraction, and pellet fraction were analyzed by Western blotting using anti-importin β1, anti-importin α1, anti-p65, anti-His, and anti-GAPDH antibodies. Input lysate of HeLa cells before immunoprecipitation, Sup supernatant fraction, Pellet pellet fraction. The full-length blots are shown in Supplementary Information, Fig. 4. Figure 4. Analysis of the behavior of importin β1 in IL-1α nuclear translocation. Discussion (a) After isolation of His- tag-fused protein via IMAC from HeLa cells expressing His-tag-fused IL-1α, the obtained fractions during IMAC were analyzed using Western blotting with anti-His, anti-importin β1, and anti-GAPDH antibodies. Input lysate of HeLa cells before IMAC, FT flow-through fraction, Elution imidazole elution fraction. (b) Coimmunoprecipitation was performed with an anti-importin β1 antibody using HeLa cells expressing His-tag- fused IL-1α, and the input, supernatant fraction, and pellet fraction were analyzed by Western blotting using anti-importin β1, anti-importin α1, anti-p65, anti-His, and anti-GAPDH antibodies. Input lysate of HeLa cells before immunoprecipitation, Sup supernatant fraction, Pellet pellet fraction. The full-length blots are shown in Supplementary Information, Fig. 4. Figure 4. Analysis of the behavior of importin β1 in IL-1α nuclear translocation. (a) After isolation of His- tag-fused protein via IMAC from HeLa cells expressing His-tag-fused IL-1α, the obtained fractions during IMAC were analyzed using Western blotting with anti-His, anti-importin β1, and anti-GAPDH antibodies. Input lysate of HeLa cells before IMAC, FT flow-through fraction, Elution imidazole elution fraction. (b) Coimmunoprecipitation was performed with an anti-importin β1 antibody using HeLa cells expressing His-tag- fused IL-1α, and the input, supernatant fraction, and pellet fraction were analyzed by Western blotting using anti-importin β1, anti-importin α1, anti-p65, anti-His, and anti-GAPDH antibodies. Input lysate of HeLa cells before immunoprecipitation, Sup supernatant fraction, Pellet pellet fraction. The full-length blots are shown in Supplementary Information, Fig. 4. indicates that importin α subtypes are widely involved in the nuclear transport of IL-1α. However, the nuclear transport of IL-1α was unaffected by importin α4 knockdown (Supplementary Figs. S4d, S5b). These results suggest that importin α4 does not play a regulatory role in the nuclear transport of IL-1α. Although importin α4 might have a regulatory function in the nuclear transport of IL-1α, it is possible that its expression will be extremely low, in which case it will have a negligible effect on transport. To evaluate the substrate specificity of the importin α family, detailed analysis based on the absolute abundance of each importin α subtype is needed in the future. By contrast, Wan et al. detected multiple importin α subtypes in the same fraction in a proteomic analysis of isolated fractions obtained using various separation carriers, indicating that they interact with each ­another37. Furthermore, we also confirmed that the interaction between importin α1 and importin α3 using immunologic analysis (Supplementary Fig. S6). Discussion Thus, the elution of each importin α subtype together with IL-1α might reflect interactions between different importin α subtypes; namely, importin α1 and importin α3 might interact and function in a cooperative manner in vivo. Future studies on the interactions of importin α1 with subtypes other than importin α3 that considers the absolute abundance of each protein are needed. Furthermore, whether each importin α subtype binds directly or indirectly to IL-1α remains to be elucidated, and the contribution of the interactions between different importin α subtypes to the transport function of IL-1α requires additional study. In general, importin α binds to the cNLS of a cargo, and importin β1 binds to importin α, forming a complex that is transported into the nucleus in an importin β1-dependent manner (Fig. 5, left)38–43. The cNLS is usually found in the coils, loops, or intrinsically disordered regions so that its binding importin α can be facilitated. The NLS sequence of IL-1α (amino acids 79–86) is located in intrinsically disordered regions according to the AlphaFold model; therefore, this sequence can interact with importin α as a cNLS. In the present study, we demonstrated the following: (1) The cNLS deletion in IL-1α prevents the nuclear translocation of IL-1α (Fig. 1d); (2) importin α1 interacts with IL-1α, forming a complex (Fig. 2b); and (3) importin β1 is not included in the complex, suggesting that importin β1 does not involve IL-1α nuclear translocation (Fig. 4, Fig. S4d,e). These suggest that IL-1α translocates into the nucleus depending on importin α1 but independently of importin β1. In Arabidopsis thaliana, importin α binds with cNLS and mediates nuclear import independently of importin β1, but the mechanism is ­unclear44. What mechanism underlies the importin β1-independent nuclear translocation of IL-1α in HeLa cells? Importin α has an cNLS-binding domain as well as an importin β-binding domain (IBB) that indicates that importin α subtypes are widely involved in the nuclear transport of IL-1α. However, the nuclear transport of IL-1α was unaffected by importin α4 knockdown (Supplementary Figs. S4d, S5b). These results suggest that importin α4 does not play a regulatory role in the nuclear transport of IL-1α. Although importin α4 might have a regulatory function in the nuclear transport of IL-1α, it is possible that its expression will be extremely low, in which case it will have a negligible effect on transport. Discussion Although HAX-1 is present in the ­nucleus19,48–51 and has been implicated in the nuclear transport of IL-1α by binding to the region containing the ­cNLS19,52, the specific transport mechanism is unclear, and further elucida- tion is required. It is possible that the unknown protein shown in Fig. 5, which presents a model of the regulation of the nuclear transport of IL-1α, plays a significant role in the nuclear transport of IL-1α mediated by importin α1. Conversely, Kotera et al. reported that importin α alone can transport ­Ca2+/calmodulin-dependent protein kinase type IV, which does not contain cNLS, to the ­nucleus53. The possibility of IL-1α nuclear translocation by importin α itself needs additional investigation.h This research indicated that importin α1 knockdown decreased the protein level of IL-1α in HeLa cells (Fig. 3). Recently, in addition to nuclear transport capacity, importin α1 is also noted for its various other ­functions54,55, such as the regulation of gene ­expression56, cell ­differentiation57,58, and spindle ­assembly59. In particular, the func- tions regarding protein polymerization and folding have been postulated: importin α/β inhibits the fibrillization of TDP-43, which is associated with amyotrophic lateral sclerosis and Alzheimer’s ­disease60,61, and in influenza A virus, importin α5 acts as a chaperone that inhibits the aggregation of ­nucleoprotein62. Hence, the interaction of importin α1 with IL-1α may also assist in the stabilization of IL-1α protein. The deletion of the cNLS in IL-1α lowered the quantity of IL-1α protein in the nucleus but had no impact on the overall quantity of IL-1α protein in the cell (Fig. 1d). Importin α1 therefore contributes to the regulation of IL-1α protein level by interacting with a domain other than the cNLS of IL-1α. This means that importin α1 interacts with IL-1α at multiple sites, similar to how HAX-1 interacts with IL-1α18,19. Ainscough et al., reported that IL-1α was polyubiquitinated and exposed to proteasomal degradation in murine dendritic ­cells63. Although polyubiquitinated sites of IL-1α have not been identified, the cNLS sequence containing multiple lysine residues may correspond to polyubiquitinated sites, i.e., by masking the cNLS of IL-1α, importin α1 might be protecting IL-1α from proteasomal degradation. Importin α4 interacts with IL-1α (Fig. 2b), but knockdown did not lower IL-1α expression (Supplementary Fig. S4d). There can be substrate specificity between importin α subtypes for the regulation of IL-1α protein level. Discussion Right side: the IL-1α binds to importin α1 and an unknown protein, and is subsequently transported into the nucleus independently of importin β1. binds to importin β1. The IBB domain also has cNLS-like properties, so both domains interact intramolecularly to prevent importin α from binding to importin β145–47. After the cNLS of a cargo binds to the cNLS-binding domain of importin α, the IBB domain of importin α will become free to bind importin β1. In our findings, cNLS deletion significantly inhibited IL-1α nuclear translocation, suggesting that importin α1 binds to the cNLS of IL-1α, resulting in nuclear translocation despite the absence of importin β1 in the transport complex. This indicates that importin β1 cannot bind to importin α1, which is already attached to IL-1α’s cNLS. One suggested mechanism is that an unknown protein in the IL-1α nuclear transport complex inhibits the interaction between importin α1 and importin β1 (Fig. 5, right). binds to importin β1. The IBB domain also has cNLS-like properties, so both domains interact intramolecularly to prevent importin α from binding to importin β145–47. After the cNLS of a cargo binds to the cNLS-binding domain of importin α, the IBB domain of importin α will become free to bind importin β1. In our findings, cNLS deletion significantly inhibited IL-1α nuclear translocation, suggesting that importin α1 binds to the cNLS of IL-1α, resulting in nuclear translocation despite the absence of importin β1 in the transport complex. This indicates that importin β1 cannot bind to importin α1, which is already attached to IL-1α’s cNLS. One suggested mechanism is that an unknown protein in the IL-1α nuclear transport complex inhibits the interaction between importin α1 and importin β1 (Fig. 5, right). p p β g g To further clarify whether HAX-1, which interacted with IL-1α (Fig. 2b), and its knockdown prevented IL-1α nuclear translocation (Supplementary Fig. S4d), is also involved in importin α1-mediated IL-1α translocation, the interaction between importin α1 and HAX-1 was examined by immunoprecipitation using anti-importin α1 and anti-HAX-1 antibodies. The results revealed no coprecipitation of importin α1 and HAX-1 (Supplementary Fig. S7). These results demonstrate that importin α1 and HAX-1 do not interact. Therefore, it was indicated that HAX-1 is involved in the nuclear translocation of IL-1α through a pathway independent of importin α1. Discussion Future studies on the interactions of importin α1 with subtypes other than importin α3 that considers the absolute abundance of each protein are needed. Furthermore, whether each importin α subtype binds directly or indirectly to IL-1α remains to be elucidated, and the contribution of the interactions between different importin α subtypes to the transport function of IL-1α requires additional study. f p yp p q y In general, importin α binds to the cNLS of a cargo, and importin β1 binds to importin α, forming a complex that is transported into the nucleus in an importin β1-dependent manner (Fig. 5, left)38–43. The cNLS is usually found in the coils, loops, or intrinsically disordered regions so that its binding importin α can be facilitated. The NLS sequence of IL-1α (amino acids 79–86) is located in intrinsically disordered regions according to the AlphaFold model; therefore, this sequence can interact with importin α as a cNLS. In the present study, we demonstrated the following: (1) The cNLS deletion in IL-1α prevents the nuclear translocation of IL-1α (Fig. 1d); (2) importin α1 interacts with IL-1α, forming a complex (Fig. 2b); and (3) importin β1 is not included in the complex, suggesting that importin β1 does not involve IL-1α nuclear translocation (Fig. 4, Fig. S4d,e). These suggest that IL-1α translocates into the nucleus depending on importin α1 but independently of importin β1. In Arabidopsis thaliana, importin α binds with cNLS and mediates nuclear import independently of importin β1, but the mechanism is ­unclear44. What mechanism underlies the importin β1-independent nuclear translocation of IL-1α in HeLa cells? Importin α has an cNLS-binding domain as well as an importin β-binding domain (IBB) that https://doi.org/10.1038/s41598-024-51521-w Scientific Reports | (2024) 14:1322 www.nature.com/scientificreports/ Figure 5. Components of complexes for nuclear translocation of IL-1α. Left side: general transport complex of substrate containing cNLS, which forms a complex with importin α, importin β1, and substrate transported to the nucleus in an importin β1-dependent manner. Right side: the IL-1α binds to importin α1 and an unknown protein, and is subsequently transported into the nucleus independently of importin β1. Figure 5. Components of complexes for nuclear translocation of IL-1α. Left side: general transport complex of substrate containing cNLS, which forms a complex with importin α, importin β1, and substrate transported to the nucleus in an importin β1-dependent manner. Cell culture and maintenance HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum, 50 μg/ mL streptomycin, and 50 U/mL penicillin (Sigma-Aldrich, St. Louis, MO) for 3 days in a humidified incubator (5% ­CO2, 37 °C). The cells were maintained by passage every 2–3 days. Construction of plasmid vectors Using the Quick-Change site-directed mutagenesis kit (Agilent, Böblingen, Germany), expression vector (HiBiT- IL-1α-His) containing the N-terminal HiBiT-tag (11 amino acids [VSGWRLFKKIS]) and the C-terminal His-tag was constructed, and pcDNA-IL-1α vector was used as a ­template64. The cNLS-deletion mutant of IL-1α (∆NLS) was constructed using the HiBiT-IL-1α-His vector as a template and the above-mentioned kit. Plasmids were sequenced to ensure that no undesired mutations were present. Transfection experiment p HeLa cells were seeded in a 6-well plate (5 × ­105 cells/well) 1 day before transfection with the expression vector. The expression vector was transfected according to the manufacturer’s instructions using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA). After incubation for 24 h, the transfected cells were collected and analyzed. For RNA interference, 2.5 and 5 nM siRNA targeting importin α1 (Silencer select ID s7922, Thermo Fisher Scientific), 5 nM siRNA targeting importin α4 (Silencer select ID s7923, Thermo Fisher Scientific), HAX-1 (Silencer select ID s20458, Thermo Fisher Scientific), importin β1 (Silencer select ID s7917, Thermo Fisher Sci- entific), and 5 nM universal negativecontrol siRNA (Silencer select ID 4390843, Thermo Fisher Scientific) were transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific) for 24 h before transfecting the cells with the expression vector, as per the manufacturer’s instructions. Materials and methods Reagents g Monoclonal mouse anti-IL-1α (sc-271618), anti-GAPDH (sc-47724), anti-importin α1 (sc-55538), anti-importin α4 (sc-514101), anti-importin α5 (sc-101292), anti-importin α7 (sc-390055) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Proteintech (Rosemont, IL, USA) provided polyclonal rabbit anti-β-actin (20536-1-AP) and anti-lamin B1 (12987-1-AP) antibodies, as well as monoclonal mouse anti-GST tag antibody (66001-2-Ig). Monoclonal mouse anti-His antibody (D291-3) was purchased from MBL (Tokyo, Japan). Monoclonal mouse anti-importin α3 (ab53751) and anti-importin β1 antibodies (ab2811) were purchased from Abcam (Cambridge, UK). Monoclonal mouse anti-importin α6 antibody (H00003841-M01) was purchased from Abnova (Taipei, Taiwan). Monoclonal rabbit anti-p65 antibody (8242) was purchased from Cell Signaling Technology (Danvers, MA, USA). For more information on anti-importin antibodies, see Supplementary Table 1. Verification of the specificity of the antibody against each importin α subtype is shown in Supplementary Fig. S8. Discussion However, if the protein expression of importin α4 is extremely low, its effect on protein expression of IL-1α might not be reflected in the experimental results. To elucidate the mechanism underlying reduction of IL-1α protein expression via suppression of importin α1 expression and to discuss the substrate specificity of importin α family, a comprehensive analysis of the changes in protein abundance associated with the suppression of importin α1 expression need to be performed. In addition, targeted proteomic analysis should be conducted to determine the abundance of importin α subtypes and other related analyses would be needed. https://doi.org/10.1038/s41598-024-51521-w Scientific Reports | (2024) 14:1322 www.nature.com/scientificreports/ To date, only HAX-1 has been identified as a molecule that directly regulates IL-1α nuclear translocation, and the mechanism of the importin α dependent nuclear translocation of IL-1α with cNLS has not been directly analyzed. This research showed that all detected importin α subtypes interact with IL-1α in HeLa cells. Among these subtypes, we discovered that at least importin α1-mediated nuclear translocation of IL-1α occurs and that the transport pathway is independent of importin β1. Moreover, importin α1 is involved in the regulation of the IL-1α protein level in HeLa cells. To clarify the mechanism by which the IL-1α-importin α1 complex is trans- located to the nucleus, detailed and careful analyses, including proteomic analysis, are required to identify the proteins that interact with the complex during the nuclear transport of IL-1α in a spatiotemporal manner. Our results imply that importin α1 may be valuable as a potential therapeutic target for all IL-1α-related diseases, whether intracellular and extracellular. Statistical analysis y Data are expressed as mean ± SD. Differences between two groups were assessed using an unpaired two-tailed Student’s t-test. Data were analyzed using the iBright imaging system. The results were considered significant at P < 0.05. www.nature.com/scientificreports/ used to collect the target protein complexes. Thereafter, the samples (the supernatant fraction obtained from the first wash, referred to as “Sup” and the fraction obtained from elution buffer, referred to as “Pellet”) were used for Western blotting. Alternatively, to confirm the interaction between importin α1 and IL-1α, HeLa cells transfected with empty vector or His-tag fused IL-1α gene were utilized for coimmunoprecipitation with anti-His antibody. The precipitates were collected using Protein G Sepharose™ 4 fast flow (GE Healthcare Bioscience, Piscataway, NJ) after incubating the cell lysate with anti-His antibody and were subjected to Western blotting analysis. t g y y j g y HeLa cells coexpressing His-tag-fused IL-1α and GST-fused importin α1 were utilized for GST pulldown assays. Glutathione SepharoseTM 4 Rapid Flow (GE Healthcare Bioscience) was utilized to capture GST-fused proteins. Western blotting using an anti-His antibody was used to detect IL-1α in GST-fusion protein complexes. Additional procedures included collecting His-tag fused proteins on Ni–NTA agarose beads (Qiagen, Hilden, Germany) and blotting the samples with an anti-GST antibody. Western blotting experiment g p HeLa cells transfected with the expression vector were washed twice with cold PBS before being lysed with cell lysis buffer (1% Triton X-100/10 mM Tris–HCl buffer [pH 8.0]). Cytoplasmic and nuclear extracts were obtained from the cell lysate using NE-PER™ nuclear and cytoplasmic extraction kit (Thermo Fisher Scientific). Protein concentrations were measured using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA), and then Western blotting was performed. Briefly, protein mixtures were resolved by SDS-PAGE under reducing conditions of 8% or 12% gels, which were then electrotransferred onto PVDF membranes. 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Oral Sci. 62, 340–343 (2020). 64. Sata, E. et al. A new enzyme-linked immunosorbent assay system against the N-terminal propiece of interleukin-1α. J. Oral Sci. 62, 340–343 (2020). Acknowledgementsh g The authors would like to thank Professor Imamoto, RIKEN Cellular Dynamics Laboratory for kindly providing anti-importin antibodies, and for helpful discussions and comments on this study. This work was supported by JSPS KAKENHI (Grant Numbers 18H02971 and 17K19759), and by Grant from Dental Research Center, School of Dentistry, Nihon University Japan. Author contributions A.Y. conceived and designed the experiments; A.Y., K.W., and S.I. performed the experiments; A.Y. and T.Y. analyzed the data; A.Y. wrote the paper; A.Y., K.W., S.I., M.M., T.Y. and M.A. reviewed and edited the paper; A.Y. supervised; A.Y. provided funding for the project; all authors approved the manuscript content and its final version. Competing interests h p g The authors declare no competing interests. Additional informationh Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​024-​51521-w. Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​024-​51521-w. Correspondence and requests for materials should be addressed to A.Y. Correspondence and requests for materials should be addressed to A.Y. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. © The Author(s) 2024 https://doi.org/10.1038/s41598-024-51521-w Scientific Reports | (2024) 14:1322 |
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Angiopoietins stimulate pancreatic islet development from stem cells
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OPEN Soujanya S. Karanth1, Shuofei Sun1, Huanjing Bi1, Kaiming Ye1,2 & Sha Jin1,2* In vitro differentiation of human induced pluripotent stem cells (iPSCs) into functional islets holds immense potential to create an unlimited source of islets for diabetes research and treatment. A continuous challenge in this field is to generate glucose-responsive mature islets. We herein report a previously undiscovered angiopoietin signal for in vitro islet development. We revealed, for the first time, that angiopoietins, including angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) permit the generation of islets from iPSCs with elevated glucose responsiveness, a hallmark of mature islets. Angiopoietin-stimulated islets exhibited glucose synchronized calcium ion influx in repetitive glucose challenges. Moreover, Ang2 augmented the expression of all islet hormones, including insulin, glucagon, somatostatin, and pancreatic polypeptide; and β cell transcription factors, including NKX6.1, MAFA, UCN3, and PDX1. Furthermore, we showed that the Ang2 stimulated islets were able to regulate insulin exocytosis through actin-filament polymerization and depolymerization upon glucose challenge, presumably through the CDC42-RAC1-gelsolin mediated insulin secretion signaling pathway. We also discovered the formation of endothelium within the islets under Ang2 stimulation. These results strongly suggest that angiopoietin acts as a signaling molecule to endorse in vitro islet development from iPSCs. Diabetes is a global epidemic posing significant challenges for human health and wellbeing. While islet trans- plantation is the most promising treatment and is seen as a ‘cure’ to ­diabetes1, the major hindrance is the scarcity of donor islets. Stem cells can be an unlimited source for generating islets. In particular, islets derived from human induced pluripotent cells (iPSCs) have an added advantage of the reduced risk of graft rejection, as these cells could be derived from the patient’s own ­cells2. These concepts have evolved with promising, positive and steady progress in the past decades. Earlier studies from our and other groups have showed the possibility of generating glucose-responsive insulin-secreting cells from iPSCs by recapitulating the in vivo developmental processes using a stepwise differentiation ­protocol3–13. More recently, efforts have been made for generating islet-like organoids, consisting of major islet hormone-secreting cell subsets, including α, β, δ, and pancreatic polypeptide (PP) from stem ­cells11–13.f p yp p Mounting evidence suggests that β cell alone may not offer a long-term solution to maintaining normogly- cemia in diabetic patients, as both type 1 (T1D) and 2 diabetes (T2D) are “bi-hormonal disorder” ­diseases14. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2021) 11:13558 OPEN α cell dysfunction is also a major cause of excessive hepatic glucose production and insulin resistance in both T1D and T2D ­patients15–17. In addition, the heterotypic interactions between α and β cells are pivotal to maintain- ing glucose homeostasis in the ­blood18–21. Somatostatin secreted from δ cells is crucial to balancing endocrine hormone ­production22. While PP cells are one of the most poorly understood cell types in islets, pancreatic polypeptide (PPY) secreted from PP cells is found to be correlated to somatostatin levels in response to eating and ­fasting23,24. These evidences strongly suggest the necessity of generating whole islets that encompass not only β, but also α, δ, and PP cells. This necessity becomes more profound when human islets are required for drug discovery and diabetes pathophysiological studies. y p p y g However, the generation of physiologically functional islets has had limited success, due in part to our poor understanding of tissue niches essential to islet assembly and development in vitro. Particularly, our knowledge of acquired microenvironments indispensable for multicellular islet differentiation and maturation is lacking. Hence, the directed iPSC islet development must be improved in order to realize robust production of islets from iPSCs for diabetes treatment, pathophysiological study, and drug discovery. y g y g y We have previously reported that decellularized pancreatic extracellular matrix (dpECM) contains instructive molecules, such as type V collagen, that endorse islet development from ­iPSCs11,13. Our proteomic analysis of dpECM revealed the presence of another signaling molecule, i.e. angiopoietin-2 (Ang2). Ang2 is an endothelial growth factor. It is also a modulator of endothelial ­permeability25. It has been reported that Ang2 contributes to 1Department of Biomedical Engineering, Binghamton University, State University of New York (SUNY), Binghamton, NY 13902, USA. 2Center of Biomanufacturing for Regenerative Medicine, Binghamton University, State University of New York (SUNY), Binghamton, NY 13902, USA. *email: sjin@binghamton.edu Scientific Reports | (2021) 11:13558 | https://doi.org/10.1038/s41598-021-92922-5 www.nature.com/scientificreports/ Figure 1. Outline of the stepwise differentiation procedure and key signature marker gene expressions during iPSC islet development. (a) A schematic diagram of a five-stage islet development protocol. (b,c, d) iPSC- derived cells were characterized for their pancreatic marker gene expressions at stage of definitive endoderm (b), pancreatic progenitor (c), and islet organoid (d). The expression levels were normalized to IMR90 cells. C: control; A: Ang2. Human pancreas RNA (hP) and human islet RNA (hI) were used as positive controls. OPEN Results were obtained from four independent differentiation experiments and shown as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001. Figure 1. Outline of the stepwise differentiation procedure and key signature marker gene expressions during iPSC islet development. (a) A schematic diagram of a five-stage islet development protocol. (b,c, d) iPSC- derived cells were characterized for their pancreatic marker gene expressions at stage of definitive endoderm (b), pancreatic progenitor (c), and islet organoid (d). The expression levels were normalized to IMR90 cells. C: control; A: Ang2. Human pancreas RNA (hP) and human islet RNA (hI) were used as positive controls. Results were obtained from four independent differentiation experiments and shown as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001. endothelial cell (EC) activation, the initiation of angiogenesis, and pancreatic ­vascularization25,26. In vivo, Ang2 is exclusively secreted from ECs and is stored in ECs’ Weibel–Palade bodies (small storage granules)27. The expres- sion of Ang2 is rapidly induced in stressed ECs to mitigate ­apoptosis28. Accumulating evidence shows an intimate interplay between the endothelium and endocrine cells of the pancreas, which influences the development, function, and survival of the ­pancreas29–33. ECs secrete cytokines, such as HGF, CTGF, and thrombospondin-1 to orchestrate organogenesis, although their complex and dynamic interactions are largely ­unknown34–36. While angiopoietins have been reported principally for their role in ­angiogenesis37,38 and non-vascular ­system39–41, to the best of our knowledge, the cellular actions of Ang2 on pancreatic islet development from stem cells have not been reported or explored. In this study, we investigated whether angiopoietin can act as a singling molecule to augment the matura- tion of islets during iPSC islet development. We discovered that the exposure of pancreatic progenitors to either angiopoietin-1 (Ang1) or Ang2 at late-stages of iPSC islet development led to the development of physiologically functional islets. These organoids were capable of regulating insulin exocytosis through dynamic polymerization and depolymerization of actin-filament (F-actin) upon glucose challenge. In addition, the angiopoietin signal elevated the glucose synchronized calcium ion ­(Ca2+) influx of iPSC-derived islet cells in repetitive glucose challenges. Likewise, it enhanced the expression of not only all islet hormones, including insulin, glucagon, somatostatin, and pancreatic polypeptide; but also β cell transcription factors, including NKX6.1, MAFA, UCN3, and PDX1. Mechanistic study indicated that Ang2 stimulated islets were able to regulate insulin exocytosis by activating the CDC42-RAC1-gelsolin-mediated insulin secretion pathway. OPEN Furthermore, Ang2 induced the for- mation of intra-islet endothelium. These results demonstrated, for the first time, a unique role that  angiopoietin plays in the iPSC islet development. Results Ang2 promotes in vitro islet development. A five-stage differentiation protocol developed in our pre- vious study was adopted with modification for this ­study11. As illustrated in Fig. 1a, Stage I generates definitive endoderm (DE) cells, whereas Stage II and Stage III produce pancreatic progenitors committed to endocrine lineage. Stage IV generates four major islet cell subsets, including α, β, δ, and PP cells. Stage V allows the forma- tion of islet cell clusters that mature into islets. Ang2 was added to the differentiation medium from Stage IV to Stage V. The timing of addition was chosen based on our pre-test. The islet cells were characterized by quantita- tive real-time PCR (qRT-PCR) at the early, intermediate, and end stages of the islet development (Fig. 1b–d). We observed substantially elevated expressions of DE marker genes SOX17 and FOXA2 (Fig. 1b), along with the loss of pluripotent gene marker OCT4 at Stage I. The expression levels of pancreatic progenitor marker genes PDX1, https://doi.org/10.1038/s41598-021-92922-5 Scientific Reports | (2021) 11:13558 | www.nature.com/scientificreports/ Figure 2. Representative organogenesis of iPSC-derived islet organoids. At the end of differentiation, the islets were immunofluorescently labeled for (a) C-peptide (CP, red) and glucagon (GCG, green), somatostatin (SST, green) and pancreatic polypeptide (PPY, red). (b) NKX6.1 (green) and CP (red), and MAFA (green) and CP (red). Cells were counterstained with DAPI (grey). Scale bars, 50 μm. Human islets (hIslet) served as a positive control. (c) Semi-quantitative analysis of cellularity of the islets. Image analysis was performed using ImageJ software (n = 7–16 images for each condition). Results are shown as mean ± SD. Different letters indicate significant differences between the groups and p-value represented as *p < 0.05; **p < 0.01; and ***p < 0.001 compared to the control group. Figure 2. Representative organogenesis of iPSC-derived islet organoids. At the end of differentiation, the islets were immunofluorescently labeled for (a) C-peptide (CP, red) and glucagon (GCG, green), somatostatin (SST, green) and pancreatic polypeptide (PPY, red). (b) NKX6.1 (green) and CP (red), and MAFA (green) and CP (red). Cells were counterstained with DAPI (grey). Scale bars, 50 μm. Human islets (hIslet) served as a positive control. (c) Semi-quantitative analysis of cellularity of the islets. Image analysis was performed using ImageJ software (n = 7–16 images for each condition). Results are shown as mean ± SD. Results Different letters indicate significant differences between the groups and p-value represented as *p < 0.05; **p < 0.01; and ***p < 0.001 compared to the control group. NKX6.1, and NGN3 were significantly high at Stage III (Fig. 1c). The expression levels of all islet hormone mark- ers, including insulin (INS), glucagon (GCG), somatostatin (SST), and pancreatic polypeptide (PPY) increased considerably at Stage V under Ang2 stimulation (Fig. 1d). In addition, the expressions of mature β cell tran- scription factors NKX6.1, MAFA, UCN3, and PDX1 elevated remarkably under the Ang2 stimulation (Fig. 1d), signifying that Ang2 may augment islet maturation during iPSC islet development.l g y g g y g g p Next, we examined the phenotype and tissue architecture of the islets by immunofluorescence staining of hormone-secreting islet cell subsets. As shown in Fig. 2a, we detected C-peptide (CP)+ β, GCG​+ α, ­SST+ δ, and ­PPY+ PP subsets in the islet organoids. Further analysis of C-peptide producing cells in the islets revealed the localization of mature β cell transcription factors, NKX6.1 and MAFA, inside cell nucleus (Fig. 2b). To deter- mine the cellularity of the islets, we analyzed the images (n = 7–16 at each experimental condition) using ImageJ software (Fig. 2c). We found that subsets of hormone-producing cells in the control and Ang2 groups were similar. Islets formed in the presence or absence of Ang2 all consisted of ­CP+/GCG​−, ­CP−/GCG​+, ­SST+/PPY− and ­SST−/PPY+. Remarkably, the percentages of ­CP+/NKX6.1+ and ­CP+/MAFA+ cells increased considerably in the Ang2 group. The islets in the Ang2 group contained 20.4 ± 5.7% C-peptide (CP)+/NKX6.1+ cells, 18.2 ± 6.9% ­CP+/MAFA+, whereas the control group encompassed only 8.8 ± 3.7% ­CP+/NKX6.1+ cells and 11.3 ± 2.4% cells ­CP+/MAFA+. Earlier studies by other groups have shown that cells co-expressing CP and NKX6.1 are mature β cells that possess a better glucose responsive ­ability42,43. These results revealed that β cells generated in the Ang2 group were more mature than those in the control group. Angiopoietins permit iPSC‑derived islet cells to release insulin in a glucose‑responsive man- ner. We next conducted a static parallel glucose-stimulated insulin secretion (p-GSIS) assay to ascertain the https://doi.org/10.1038/s41598-021-92922-5 Scientific Reports | (2021) 11:13558 | www.nature.com/scientificreports/ Figure 3. Angiopoietins promote the development of islets with elevated glucose-responsive insulin secretion capacity from differentiation of multiple pluripotent stem cell lines. Results (a) Parallel glucose-stimulated insulin secretion from islets generated in the absence or presence of Ang2 (n = 6). (b) Insulin stimulation index from consecutive three rounds of low (2 mM)–high (20 mM) glucose challenge measured from iPSC-derived islets by sequential glucose-stimulated insulin secretion analysis. Control (n = 12) and Ang2 (n = 14). (c) Stimulation index of insulin secretion from iPSC-derived islets in the presence of Ang2 at 0 (A0), 4 (A4), 20 (A20), 100 (A100) and 200 ng/ml (A200) (n = 4). Groups with different letters (a or b) represent significant differences. (d-f) Insulin stimulation index of islet organoids generated from iPSC line IMR90 (n = 4) (d), iPSC line DF4 (n = 6) (e), and hESC line H9 (n = 3) (f), respectively. (g) Insulin stimulation index of iPSC-derived islets generated in the presence of Ang1 (n = 4). All the results are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.00, and NS: not significant. Figure 3. Angiopoietins promote the development of islets with elevated glucose-responsive insulin secretion capacity from differentiation of multiple pluripotent stem cell lines. (a) Parallel glucose-stimulated insulin secretion from islets generated in the absence or presence of Ang2 (n = 6). (b) Insulin stimulation index from consecutive three rounds of low (2 mM)–high (20 mM) glucose challenge measured from iPSC-derived islets by sequential glucose-stimulated insulin secretion analysis. Control (n = 12) and Ang2 (n = 14). (c) Stimulation index of insulin secretion from iPSC-derived islets in the presence of Ang2 at 0 (A0), 4 (A4), 20 (A20), 100 (A100) and 200 ng/ml (A200) (n = 4). Groups with different letters (a or b) represent significant differences. (d-f) Insulin stimulation index of islet organoids generated from iPSC line IMR90 (n = 4) (d), iPSC line DF4 (n = 6) (e), and hESC line H9 (n = 3) (f), respectively. (g) Insulin stimulation index of iPSC-derived islets generated in the presence of Ang1 (n = 4). All the results are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.00, and NS: not significant. function of Ang2 stimulated islets (Fig. 3a). Islets generated in the absence of Ang2 served as a control. The control group exhibited a non-regulated insulin secretion. These cells released similar amounts of insulin when being challenged with either low (2 mM) or high glucose (20 mM). Results The stimulation index, which denotes the ratio of insulin secretion at high (20 mM) to low glucose (2 mM), was 1.0 ± 0.3 (Fig. 3b). Interestingly, the Ang2 group displayed an increased insulin release under a high glucose concentration and a decreased-basal level insulin release under a low glucose concentration. The insulin stimulation index was 2.5 ± 0.5-fold, equivalent to that observed from human islets 2.5 ± 0.3 (Fig. 3a–c). Furthermore, the stimulation index was examined by three rounds of serial challenges of 2 mM and 20 mM glucose. The control group failed to distinguish between a minor and a major increase in glucose, while the Ang2 group retained glucose responsiveness for all rounds of sequential low–high glucose challenges, as quantified by the stimulation index (Fig. 3a,b). It is worth noting that we did not use human donor islets to perform sequential GSIS (s-GSIS), due to the fact that other groups had shown that the stimulation index of human islets is maintained at 2.0 most of the time, although there is a high variability in an individual donor’s absolute magnitude of insulin ­secretion9. We did examine the stimulation index of human donor islets repeatedly. The results from these tests confirmed that the insulin stimulation index is maintained at 2.0 in all the tests we performed (data no shown).ff function of Ang2 stimulated islets (Fig. 3a). Islets generated in the absence of Ang2 served as a control. The control group exhibited a non-regulated insulin secretion. These cells released similar amounts of insulin when being challenged with either low (2 mM) or high glucose (20 mM). The stimulation index, which denotes the ratio of insulin secretion at high (20 mM) to low glucose (2 mM), was 1.0 ± 0.3 (Fig. 3b). Interestingly, the Ang2 group displayed an increased insulin release under a high glucose concentration and a decreased-basal level insulin release under a low glucose concentration. The insulin stimulation index was 2.5 ± 0.5-fold, equivalent to that observed from human islets 2.5 ± 0.3 (Fig. 3a–c). Furthermore, the stimulation index was examined by three rounds of serial challenges of 2 mM and 20 mM glucose. The control group failed to distinguish between a minor and a major increase in glucose, while the Ang2 group retained glucose responsiveness for all rounds of sequential low–high glucose challenges, as quantified by the stimulation index (Fig. 3a,b). www.nature.com/scientificreports/ formed from DF4 and H9 in the presence of 20 ng/ml of Ang2 was 1.8 ± 0.7 and 1.9 ± 0.4, respectively, which is comparable with that of IMR90-derived islets (Fig. 3d), suggesting that the effect of Ang2 on islet development is not cell line-specific. pi Ang1 is another major protein in the angiopoietin family. It shares 60% amino acid homology with ­Ang244. Therefore, we investigated whether Ang1 also plays a similar role in the islet development as Ang2. Remarkably, the islets developed in the presence of Ang1 (20 ng/ml) exhibited an insulin stimulation index of 2.4 ± 0.7, which is similar to that detected from Ang2 induced islets (Fig. 3g). Hence, similar to Ang2, Ang1 can also induce the development of glucose-responsive islets. These experimental results clearly demonstrated the augmentation of iPSC islet development by angiopoietin factors. p y g p It should be noted that there are no reliable markers to sort islets from non-islet cell ­clusters11. We found no significant difference in the total amount of insulin released from the control and Ang2 groups at high glucose level (Fig. 3a). Therefore, we did not focus on quantifying islet cells by flow cytometry; rather, we explicitly focused on assessing glucose-responsive insulin secretion from these islets, as described below. We sought to specifically examine the improved glucose responsiveness of the islets generated under the Ang2 stimulation. To this end, we extended our study to a rat β cell line RIN-5F to examine whether Ang2 signaling can augment these cells’ glucose sensitivity. RIN-5F line does not possess glucose sensitivity. It was reported that these cells secrete insulin in a non-glucose responsive ­fashion45. We cultured the cells in the presence of Ang2 from 0, 20, and 100 ng/ml for 3 days and then performed GSIS assay (Fig. S1). Ang2 stimulation failed to improve these cells’ glucose sensitivity, as they released the same amount of insulin regardless of glucose level change, which coincides with the previous ­report45. We speculated that Ang2 stimulation affects the maturation of β cells, but has no effect on the glucose-responsive insulin secretion capability of the terminally differentiated β cells. To further confirm these observations, we characterized glucose synchronous ­Ca2+ influx in Ang2 stimulated islets. Angiopoietins regulate dynamics of calcium ion influx in response to different glucose lev- els. The ­Ca2+ influx is intimately associated with glucose-stimulated insulin exocytosis in β ­cells46,47. www.nature.com/scientificreports/ An increase in blood glucose levels causes ­Ca2+ influx in β cells, which in turn leads to insulin exocytosis. To scruti- nize whether the glucose-responsive insulin secretion from the islets generated under the angiopoietin stimula- tion represents glucose level regulated ­Ca2+ influx, we performed semi-quantitative time series imaging analysis of Fluo-4 stained single β cells in the islets (Fig. 4a–c). Fluo-4 has been widely used as a ­Ca2+ ­indicator48. The representative ­Ca2+ influx in the control, Ang2, Ang1 groups, and human islets (hIslet) are shown in Fig. 4b. The ­Ca2+ influx in the control group remained constant with no rise upon high glucose challenge (Fig. 4d). In contrast, the ­Ca2+ influx in the Ang1 and Ang2 groups rose upon high glucose challenge. It dropped to a basal level at a low glucose level. This ­Ca2+ influx was similar to that observed in human islets. The area under the curve (a.u.c) for ­Ca2+ influx response at all low glucose levels was compared to the ­Ca2+ influx response curves detected upon high glucose challenges. The a.u.c of ­Ca2+ influx response under high glucose stimulations in the Ang1 and Ang2 groups was significantly greater than that at low glucose levels (Fig. 4d). After the three rounds of consecutive low–high glucose challenges, the islets were treated by KCl for islet depolarization. The human islets showed immediate response to KCl treatment in the form of a sharp increase in ­Ca2+ influx followed by a rapid drop. This might attribute to deterioration of these islets during donor isolation procedure, preservation, shipping, and repeated treatment in an imaging chamber. iPSC-derived islets in the Ang1 and Ang2 groups exhibited a certain degree of increase in ­Ca2+ influx after KCl challenge. The results suggested that these islets need to be matured further.lh We next carried out ­Ca2+ influx analysis of single cells inside the islets. The single cells that responded to cyclic low (2 mM) and high (20 mM) glucose challenges once (1), twice (2), and thrice (3) were distinguished as shown in Fig. 4e. In the control group, approximately 5% of cells responded all three times, 22% of cells responded twice, 60% of cells responded once, and 12% of cells did not respond to glucose level change anytime (Fig. 4f). In contrast, 42% of cells responded thrice, 25% of cells responded twice, and 33% of cells responded once in the Ang2 group. Results It is worth noting that we did not use human donor islets to perform sequential GSIS (s-GSIS), due to the fact that other groups had shown that the stimulation index of human islets is maintained at 2.0 most of the time, although there is a high variability in an individual donor’s absolute magnitude of insulin ­secretion9. We did examine the stimulation index of human donor islets repeatedly. The results from these tests confirmed that the insulin stimulation index is maintained at 2.0 in all the tests we performed (data no shown).ff p To determine whether the effect of Ang2 on islet development is dose-dependent, we differentiated iPSCs at varied concentrations of Ang2 from 0, 4, 20, 100, to 200 ng/ml. As shown in Fig. 3c, islet cells generated in the presence of Ang2 at 20 and 100 ng/ml showed a stimulation index of 2.5 ± 0.6 and 2.1 ± 0.3, respectively, which is equivalent to human donor islets 2.5 ± 0.3. The islets generated at 4 and 200 ng/ml of Ang2 showed no difference as compared to the control group. Hence, we confirmed that the effect of Ang2 on islet development is dose-dependent. To ensure rigor of the study, we determined the effect of Ang2 on islet development using another iPSC line DF4 and human embryonic stem cell (hESC) line H9. Different from IMR90 that was prepared from a female donor, DF4 was derived from a male donor. We differentiated both DF4 and H9 into islets under the exactly same conditions shown in Fig. 1a. As shown in Fig. 3e,f, the insulin stimulation index of the islets Scientific Reports | (2021) 11:13558 | https://doi.org/10.1038/s41598-021-92922-5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ In the Ang1 group, 18% of cells responded thrice to glucose level changes, 33% of cells responded twice, and 44% of cells responded once. As a positive control, 12% of human adult islet cells responded all three times, 73% of cells responded twice, and 14% of cells responded once. When the analysis was extended to multiple islet organoids in each group (Fig. 4g), the cells responding to every glucose challenge (Fig. 4g Res 3) were the highest in the Ang2 group with a percentage of 33.8 ± 6.6 cells. In the Ang2 group, the percentage of cells responding at least once (Res Atl 1) was 99.45 ± 1.0 and the percentage of cells responding at least twice was 73.58 ± 7.6. Collectively, the percentage of glucose-responsive cells generated under either Ang1 or Ang2 stimulation was significantly higher than the control group as indicated by all the responding frequencies of ­Ca2+ influx measured. In addition, the percentages of glucose-non-responsive cells (No Res) in the Ang1 and Ang2 groups were significantly lower than that in the control group. Ang2 facilitates F‑actin remodeling in response to glucose level changes. It has been reported that F-actin of β cells is involved in insulin granule mobilization and cell membrane fusion to facilitate insulin ­exocytosis49. We determined whether Ang2 facilitates F-actin rearrangement during insulin exocytosis upon glucose challenge. We observed that the F-actin polymerized into a thick barrier around the Ang2 stimulated β cells, restricting the insulin within the cells under low glucose condition (Fig. 5a). Under a high glucose condi- tion, the F-actin depolymerized into discontinuous thinner fragments, facilitating the insulin release from these cells (Fig. 5b). Conversely, no such F-actin rearrangements were observed in the control group. The F-actin remained depolymerized under both low and high glucose conditions. p y g g To quantitatively assess the F-actin remodeling, we performed a systematic characterization of the F-actin expression of individual β cells. We observed that the F-actin intensity at the cell edges of the control group Scientific Reports | (2021) 11:13558 | https://doi.org/10.1038/s41598-021-92922-5 www.nature.com/scientificreports/ Figure 4. Angiopoietins facilitate the development of islets with enhanced glucose-responsive cellular ­Ca2+ influx in insulin-secreting cells. (a) A regimen for imaging cellular ­Ca2+ influx. (b) Representative micrographs of the iPSC-derived islets stained with Fluo-4 upon low (2 mM) or high (20 mM) glucose challenge. Scale bars: 100 μm. www.nature.com/scientificreports/ (c) Imaging analysis of ­Ca2+ dynamics for a subset of cell populations within an islet organoid. (d) ­Ca2+ dynamics behavior depicted as average of the Fluo-4 fluorescence intensity obtained from each experimental group. These values were from a population of cells within the islets (n = 9) and donor islets (n = 4) over the entire course of sequential glucose stimulations. (e) Fluo-4 stained insulin-secreting cells responding to sequential low–high glucose stimulations all three, two, and one times were circled with magenta, blue, and yellow respectively. Red circles denote cells that did not respond to any glucose challenge. Scale bar: 100 μm. (f) Percentage of cells in the control, Ang2, and Ang1 groups, whose ­Ca2+ influx responded to sequential (low– high) glucose changes. Human islets (hIslet) served as a positive control. Red: nonresponding cells; yellow, blue, and magenta: cells’ ­Ca2+ influx responded to glucose change once, twice, and thrice. More than 400 cells were counted in each islet organoid. All of the experiments were performed in triplicate. (g) The cells’ ­Ca2+ influx changes responding to sequential low–high glucose level challenge. Three islets with single cell number n > 400 and human islets with cell number n > 250 were analyzed. No Res, Res Atl 1, Res Atl 2, and Res 3 denote to cells’ ­Ca2+ influx responded to glucose level changes zero time, at least once, at least twice, and all three times, respectively. *p < 0.05, **p < 0.01, and ***p < 0.001. Figure 4. Angiopoietins facilitate the development of islets with enhanced glucose-responsive cellular ­Ca2+ influx in insulin-secreting cells. (a) A regimen for imaging cellular ­Ca2+ influx. (b) Representative micrographs of the iPSC-derived islets stained with Fluo-4 upon low (2 mM) or high (20 mM) glucose challenge. Scale bars: 100 μm. (c) Imaging analysis of ­Ca2+ dynamics for a subset of cell populations within an islet organoid. (d) ­Ca2+ dynamics behavior depicted as average of the Fluo-4 fluorescence intensity obtained from each experimental group. These values were from a population of cells within the islets (n = 9) and donor islets (n = 4) over the entire course of sequential glucose stimulations. (e) Fluo-4 stained insulin-secreting cells responding to sequential low–high glucose stimulations all three, two, and one times were circled with magenta, blue, and yellow respectively. Red circles denote cells that did not respond to any glucose challenge. Scale bar: 100 μm. www.nature.com/scientificreports/ (f) Percentage of cells in the control, Ang2, and Ang1 groups, whose ­Ca2+ influx responded to sequential (low– high) glucose changes. Human islets (hIslet) served as a positive control. Red: nonresponding cells; yellow, blue, and magenta: cells’ ­Ca2+ influx responded to glucose change once, twice, and thrice. More than 400 cells were counted in each islet organoid. All of the experiments were performed in triplicate. (g) The cells’ ­Ca2+ influx changes responding to sequential low–high glucose level challenge. Three islets with single cell number n > 400 and human islets with cell number n > 250 were analyzed. No Res, Res Atl 1, Res Atl 2, and Res 3 denote to cells’ ­Ca2+ influx responded to glucose level changes zero time, at least once, at least twice, and all three times, respectively. *p < 0.05, **p < 0.01, and ***p < 0.001. remained around 200–750 arbitrary units (a.u) under both low and high glucose levels (Fig. 6). Remarkably, the F-actin intensity at the cell edges of the Ang2 group reached to 2500–3750 a.u. under a low glucose condition, but dropped to 500 a.u upon a high glucose challenge (Fig. 6b). The overall F-actin intensities under low and high glucose challenges in both control and Ang2 groups were compared by analyzing area under the F-actin intensities using approximately 15–30 β cells from each group (Fig. 6c). F-actin intensity under low glucose was significantly higher than that under high glucose in the Ang2 group. In contrast, the F-actin intensity of the con- trol group under low glucose was significantly lower as compared to that of the Ang2 group, suggesting defective glucose responsive F-actin rearrangement in islet cells generated in the absence of Ang2. Accordingly, Ang2 facili- tates the dynamic regulation of F-actin remodeling of insulin-producing cells for physiological insulin release. remained around 200–750 arbitrary units (a.u) under both low and high glucose levels (Fig. 6). Remarkably, the F-actin intensity at the cell edges of the Ang2 group reached to 2500–3750 a.u. under a low glucose condition, but dropped to 500 a.u upon a high glucose challenge (Fig. 6b). The overall F-actin intensities under low and high glucose challenges in both control and Ang2 groups were compared by analyzing area under the F-actin intensities using approximately 15–30 β cells from each group (Fig. 6c). F-actin intensity under low glucose was significantly higher than that under high glucose in the Ang2 group. www.nature.com/scientificreports/ In contrast, the F-actin intensity of the con- trol group under low glucose was significantly lower as compared to that of the Ang2 group, suggesting defective glucose responsive F-actin rearrangement in islet cells generated in the absence of Ang2. Accordingly, Ang2 facili- tates the dynamic regulation of F-actin remodeling of insulin-producing cells for physiological insulin release. https://doi.org/10.1038/s41598-021-92922-5 Scientific Reports | (2021) 11:13558 | www.nature.com/scientificreports/ Figure 5. Ang2 regulates glucose-responsive insulin secretion through dynamic F-actin remodeling. Representative micrographs of F-actin organization in insulin-secreting cells at (a) 2 mM and (b) 20 mM glucose challenge. White arrowheads indicate thick and highly polymerized F-actin. Yellow arrowheads indicate depolymerized F-actin. Scale bars: 50 µm. Figure 5. Ang2 regulates glucose-responsive insulin secretion through dynamic F-actin remodeling. Representative micrographs of F-actin organization in insulin-secreting cells at (a) 2 mM and (b) 20 mM glucose challenge. White arrowheads indicate thick and highly polymerized F-actin. Yellow arrowheads indicate depolymerized F-actin. Scale bars: 50 µm. Furthermore, we implemented radial profiling of F-actin intensity to validate the line scanning analysis using ImageJ software (Fig. 6d–f). The F-actin intensity was evaluated from 360 °C of encircling the insulin-producing cells (Fig. 6d). This yielded F-actin expression curves of mean intensity along the radius, i.e. from center to the edge of the β cells (Fig. 6e). The curves generated consisted of prominent peaks at the edge. At low glucose condi- tion, a higher F-actin intensity peak reflecting highly polymerized F-actin was observed towards the edge in the Ang2 group, as compared to that of the control group (Fig. 6e). The circularly analytical results are consistent with the line scanning analysis. Moreover, no difference was observed in the F-actin intensity between the control and Ang2 groups at high glucose condition (Fig. 6). g g p g g ( g ) It has been recognized that mature β cells utilize cell division control protein 42 (CDC42) and Ras-related C3 botulinum toxin substrate 1 (RAC1) to mobilize and dock insulin granules to the plasma ­membrane50,51. Gelsolin (GSN) has been reported to facilitate F-actin reorganization by mediating glucose-dependent actin depolymerization to release insulin from the ­cells52,53. Hence, we investigated whether Ang2 stimulated islet cells utilize these molecules to regulate glucose-responsive insulin release. www.nature.com/scientificreports/ We collected the islets at the end of the development and challenged them with low (2 mM) or high (20 mM) glucose, followed by assessing the expres- sion of CDC42 and RAC1 at high or low glucose. As shown in Fig. 6g, the ratio of CDC42 expressed at high to low glucose increased approximately 2.4-fold in the Ang2 group, as compared to that in the control group. There was no significant difference between the Ang2 and the human donor islet groups (Fig. 6g). RAC1 and GSN in the Ang2 group were also slightly increased as compared to that in the control group, and their sensitivity to glucose was similar to that of the human islets (Fig. 6g). These results suggested that Ang2 augmented the islets’ glucose-responsiveness by enhancing their insulin secretion pathway for insulin release. Ang2 induces endothelium formation during iPSC islet development. Ang2 is known to initi- ate angiogenesis and facilitate vascular ­permeability54,55. Pericytes interact with ECs in the islet endothelium ­environments56. To gain insights into the interplays between endothelium and islet organogenesis, we interro- gated whether Ang2 induces angiogenesis during iPSC islet development, leading to the production of mature islets. We examined the expression of an endothelial marker, vascular endothelial cadherin (VE-cadherin) and a pericyte marker NG2 in islets at Stage V by immunofluorescence microscopy. As expected, we detected a wide spread of VE-cadherin+ ECs and ­NG2+ pericytes within Ang2 stimulated islets (Fig. 7a,b), suggesting the forma- tion of intra-islet endothelium. Further analysis showed that the ratio of VE-cadherin area/DAPI area and the ratio of NG2 area/DAPI area were significantly higher in the Ang2 group (Fig. 7c,d). These results indicated that Ang2 induces initiation of new endothelial microenvironment within islet organoids. Discussion In this study, we report, for the first time, an inductive effect of angiopoietins on iPSC islet development and maturation. We demonstrated that Ang2 induced islet maturation and the formation of intra-islet endothelium. Mechanistic study revealed active F-actin remodeling aided by activated CDC42-RAC1-gelsolin pathway in addition to glucose level synchronous ­Ca2+ influx dynamics in Ang2 stimulated islets. By applying angiopoietin factors such as Ang1 or Ang2 at late-stages of islet development, we discovered that glucose sensitivity of the angiopoietin stimulated islets increased significantly. We showed that the islets generated without angiopoietin stimulation did not possess glucose-responsive insulin secretion capability. They secreted the same amount of insulin all the time even at a low glucose condition, indicating their immaturity and failure to release insulin in a physiological fashion. In contrast, the islets developed under angiopoietin stimulation were able to secrete insulin in a glucose-responsive manner. Through analysis of F-actin remodeling, a critical step of glucose- responsive insulin-secretion in mature islet cells, we found that insulin-secreting cells generated in the presence of angiopoietin signaling were able to repattern F-actin in response to glucose level changes to facilitate glucose- responsive insulin-release. As shown in Fig. 6a,d, insulin was constrained inside the cell cytoplasm by a thicker layer of F-actin surrounding the cell membrane of islets generated under Ang2 stimulation upon low glucose https://doi.org/10.1038/s41598-021-92922-5 Scientific Reports | (2021) 11:13558 | www.nature.com/scientificreports/ Figure 6. Characterization of F-actin patterning and key molecules involved in glucose-responsive insulin exocytosis. (a) Line scanning analysis of individual insulin secreting cells upon 2 mM or 20 mM glucose challenge (n = 15–30 cells at each condition). (b) F-actin intensity patterns across the individual insulin- secreting cell upon low or high glucose challenge. (c) Average F-actin intensity determined by line scanning of individual insulin-producing cells upon low or high glucose challenge (n = 15–30 cells at each condition). (d) Representative images of individual C-peptide producing cells analyzed in control and Ang2 groups upon 2 mM or 20 mM glucose challenge (n = 15–30 cells at each condition). (e) Representative curves of the integrated F-actin intensity from the center to edge of a β cell in the control and Ang2 groups. F-actin intensity in all 360 °C directions was characterized along the radius to obtain radial mean intensity. The x-axis represents distance from center to the edge of a β cell. Discussion These findings are highly significant, suggesting elevated physiological functionality of the islets generated under the angiopoietin stimulation. Furthermore, we observed more ­CP+/NKX6.1+ and ­CP+/MAFA+ cells formed in the Ang2 stimulated islets, and the insulin stimulation index in the Ang1 and Ang2 groups is comparable to that in human donor islets. To confirm angiopoietin signaling for islet development, we examined whether Ang2 changes the glucose sensitivity of terminally differentiated insulin-secreting cells such as RIN-5F cells. RIN-5F is a rat β cell line that does not possess glucose ­sensitivity45. It secretes insulin in a non-glucose-responsive fashion. Treating RIN-5F cells with Ang2 did not result in glucose-responsive insulin secretion in these cells (Fig. S1). The results sug- gested that Ang2 signaling does not regulate terminally differentiated cells’ glucose-responsive insulin-secretion. Interestingly, the analysis of mRNA expression level in the iPSC-derived organoids revealed that Ang2 signaling challenge. The F-actin layer was dispersed to enable the secretion of insulin upon high glucose challenge. This F-actin depolymerization is one of the characteristic physiological actions of mature β cells. These findings are highly significant, suggesting elevated physiological functionality of the islets generated under the angiopoietin stimulation. Furthermore, we observed more ­CP+/NKX6.1+ and ­CP+/MAFA+ cells formed in the Ang2 stimulated islets, and the insulin stimulation index in the Ang1 and Ang2 groups is comparable to that in human donor islets. To confirm angiopoietin signaling for islet development, we examined whether Ang2 changes the glucose sensitivity of terminally differentiated insulin-secreting cells such as RIN-5F cells. RIN-5F is a rat β cell line that does not possess glucose ­sensitivity45. It secretes insulin in a non-glucose-responsive fashion. Treating RIN-5F cells with Ang2 did not result in glucose-responsive insulin secretion in these cells (Fig. S1). The results sug- gested that Ang2 signaling does not regulate terminally differentiated cells’ glucose-responsive insulin-secretion. Interestingly, the analysis of mRNA expression level in the iPSC-derived organoids revealed that Ang2 signaling challenge. The F-actin layer was dispersed to enable the secretion of insulin upon high glucose challenge. This F-actin depolymerization is one of the characteristic physiological actions of mature β cells. These findings are highly significant, suggesting elevated physiological functionality of the islets generated under the angiopoietin stimulation. Discussion (f) Average F-actin intensity at low and high glucose conditions determined by radial profiling (n = 15–30 cells at each condition). (g) Relative gene expressions of key molecules involved in glucose-responsive insulin exocytosis. iPSC-derived islets generated in the presence or absence of Ang2 stimulation at Stage V were challenged with low (2 mM) or high (20 mM) glucose, followed by RNA extraction and qRT-PCR analysis The ratio of gene expression at high to low glucose was assessed Human Figure 6. Characterization of F-actin patterning and key molecules involved in glucose-responsive insulin Figure 6. Characterization of F-actin patterning and key molecules involved in glucose-responsive insulin Figure 6. Characterization of F-actin patterning and key molecules involved in glucose-responsive insulin exocytosis. (a) Line scanning analysis of individual insulin secreting cells upon 2 mM or 20 mM glucose challenge (n = 15–30 cells at each condition). (b) F-actin intensity patterns across the individual insulin- secreting cell upon low or high glucose challenge. (c) Average F-actin intensity determined by line scanning of individual insulin-producing cells upon low or high glucose challenge (n = 15–30 cells at each condition). (d) Representative images of individual C-peptide producing cells analyzed in control and Ang2 groups upon 2 mM or 20 mM glucose challenge (n = 15–30 cells at each condition). (e) Representative curves of the integrated F-actin intensity from the center to edge of a β cell in the control and Ang2 groups. F-actin intensity in all 360 °C directions was characterized along the radius to obtain radial mean intensity. The x-axis represents distance from center to the edge of a β cell. (f) Average F-actin intensity at low and high glucose conditions determined by radial profiling (n = 15–30 cells at each condition). (g) Relative gene expressions of key molecules involved in glucose-responsive insulin exocytosis. iPSC-derived islets generated in the presence or absence of Ang2 stimulation at Stage V were challenged with low (2 mM) or high (20 mM) glucose, followed by RNA extraction and qRT-PCR analysis. The ratio of gene expression at high to low glucose was assessed. Human donor islets (hIslet) served as controls. Results are shown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, NS: non-significant. challenge. The F-actin layer was dispersed to enable the secretion of insulin upon high glucose challenge. This F-actin depolymerization is one of the characteristic physiological actions of mature β cells. Discussion Furthermore, we observed more ­CP+/NKX6.1+ and ­CP+/MAFA+ cells formed in the Ang2 stimulated islets, and the insulin stimulation index in the Ang1 and Ang2 groups is comparable to that in human donor islets.i To confirm angiopoietin signaling for islet development, we examined whether Ang2 changes the glucose sensitivity of terminally differentiated insulin-secreting cells such as RIN-5F cells. RIN-5F is a rat β cell line that does not possess glucose ­sensitivity45. It secretes insulin in a non-glucose-responsive fashion. Treating RIN-5F cells with Ang2 did not result in glucose-responsive insulin secretion in these cells (Fig. S1). The results sug- gested that Ang2 signaling does not regulate terminally differentiated cells’ glucose-responsive insulin-secretion. Interestingly, the analysis of mRNA expression level in the iPSC-derived organoids revealed that Ang2 signaling Scientific Reports | (2021) 11:13558 | https://doi.org/10.1038/s41598-021-92922-5 www.nature.com/scientificreports/ Figure 7. Ang2 induces the generation of endothelial cells and pericytes in iPSC derived islet microenvironment. (a) Representative images of VE-cadherin and C-peptide immunofluorescence co-staining in islet-like organoids. (b) Representative images of NG2 and C-peptide immunofluorescence co-staining in islet-like organoids. (c,d) The ratio of VE-Cadherin+ endothelial area/DAPI area and ­NG2+ pericyte area/DAPI area in islet-like organoids. Scale bars: 50 µm. n = 19–35 islet organoids for each condition. Results are shown as mean ± SD. ****p < 0.0001. Figure 7. Ang2 induces the generation of endothelial cells and pericytes in iPSC derived islet microenvironment. (a) Representative images of VE-cadherin and C-peptide immunofluorescence co-staining in islet-like organoids. (b) Representative images of NG2 and C-peptide immunofluorescence co-staining in islet-like organoids. (c,d) The ratio of VE-Cadherin+ endothelial area/DAPI area and ­NG2+ pericyte area/DAPI area in islet-like organoids. Scale bars: 50 µm. n = 19–35 islet organoids for each condition. Results are shown as mean ± SD. ****p < 0.0001. significantly enhances not only insulin expression but also all the other major hormone gene expressions in these cells (Fig. 1d). Insulin secretion is controlled at various levels, including gene, metabolism, exocytotic, and cell–cell interaction ­levels57–59. The production of physiologically functional islets from iPSCs in vitro requires the improvement of insulin production and secretion at all levels. Our study demonstrated a critical role of angi- opoietin signaling in elevating insulin gene expression, insulin-secreting cells’ F-actin remodeling, and glucose synchronized ­Ca2+ influx of islet cells.h yl The biological function of iPSC-derived islet cells was determined by GSIS assay. We used approximately ~ 200 islets for the assay. Discussion In order to understand how insulin secretion is affected by Ang1 and Ang2, we determined glucose synchronized ­Ca2+ influx in insulin-secreting cells at the single cell level (Fig. 4e,f). We found that most cells in the control group failed to secrete insulin efficiently upon glucose challenge. Thus, overall glucose-respon- sive insulin-secretion from these islets was lower, as compared to those generated under either Ang1 or Ang2 stimulation. Interestingly, we observed the heterogeneity of the glucose synchronized ­Ca2+ influx in the islets (Fig. 4f,g). This is very similar to that observed in adult donor islets. Indeed, islet heterogeneity is not unusual in adult ­islets60. There are various sub-populations of β cells with heterogeneous gene and protein expression profiles and insulin secretion capacity in adult ­islets61. Our experimental results are promising. It has been shown that the transplantation of cells capable of glucose synchronized ­Ca2+ influx can reduce hyperglycemia in mice ­rapidly9.h p p g yl yp g y p y The cytoskeleton of a β cell has been reported to be involved in insulin granule mobilization, fusion with the cell membrane, and finally, in facilitation of insulin ­exocytosis49,62. We observed the dynamic polymerization and depolymerization of F-actin in Ang2 stimulated islets upon glucose challenge. This observation is consistent with previous ­reports63,64. In primary human islets, the F-actin of β cells at a basal glucose condition forms a ring beneath the cell surface to block the exocytosis of insulin ­granules63. Upon glucose stimulation, cells reorganize their filaments to mobilize the insulin granules out of the cells with help of proteins like ­gelsolin53,64. Previ- ous studies revealed that CDC42 acts as a modulator for glucose-responsive insulin secretion, which requires F-actin remodeling to mobilize ­granules50,65. Deletion of CDC42 gene in β cells decreased insulin expression and secretion in response to glucose stimulation in vitro and in vivo66. Moreover, RAC1 is in the downstream of CDC42 ­signaling67. The glucose-stimulated insulin secretion is decreased when RAC1 is inhibited and/or in β cell-specific RAC1 deficient ­mice51. These findings support our discovery on the unique role played by Ang2, demonstrating mechanistic insight of improved functionality of iPSC-derived islets under Ang2 stimulation for in vitro islet development as depicted in Fig. 8. p p g In addition, angiopoietins are known for their role in angiogenesis and ­vascularization37,38. For instance, Ang2 is involved in the maintenance of established vasculature, mainly in vascular remodeling and ­maturation68. Discussion A microvasculature in islets is a route for hormone secretion and profoundly essential for glucose and oxygen supply to execute the functions of islets. An endothelium helps induce pancreatic endocrine ­development32. In our study, we found the formation of vascular cellular components such as VE-Cadherin+ cells and ­NG2+ cells within the islets under the Ang2 signal. This endothelium sprouting by Ang2 might facilitate in vitro functional islet development. Taken together, the results indicated that Ang2 supports the initiation of endothelium for- mation along with islet differentiation which positively improves the functionality of the iPSC-derived islets.hf f The effect of angiopoietins has been established to be dose and context-dependent whose molecular mecha- nism is poorly ­understood37. Recent reports suggested their diverse roles in tissue homeostasis. In the context of tissue generation, Hu et al. stated Ang2’s role in liver generation, where it controls the proliferation of hepatic cell populations in vivo41. Park et al. have reported the role of Ang1 in regulating insulin secretion by stabilizing the microenvironment of islets in vivo70. Kitazawa et al. showed that angiopoietin-like protein-2 (ANGPTL2) Scientific Reports | (2021) 11:13558 | https://doi.org/10.1038/s41598-021-92922-5 www.nature.com/scientificreports/ Figure 8. Illustration of Ang2 effect on iPSC islet differentiation and iPSC-derived islet microenvironment. (a) Ang2 supports islet differentiation with the generation of β, α, δ, PP-cells and additionally, promotes the generation of endothelial and pericyte cells. (b-c) iPSC-derived islets under Ang2 cue showed ­Ca2+ influx and regulated activity of CDC42-RAC1 pathway synchronous with glucose level change, leading to F-actin remodeling aided by gelsolin for physiologically functional insulin exocytosis. Created with BioRender.com. Figure 8. Illustration of Ang2 effect on iPSC islet differentiation and iPSC-derived islet microenvironment. (a) Ang2 supports islet differentiation with the generation of β, α, δ, PP-cells and additionally, promotes the generation of endothelial and pericyte cells. (b-c) iPSC-derived islets under Ang2 cue showed ­Ca2+ influx and regulated activity of CDC42-RAC1 pathway synchronous with glucose level change, leading to F-actin remodeling aided by gelsolin for physiologically functional insulin exocytosis. Created with BioRender.com. improves insulin sensitivity in diabetic ­mice71. In this study, we reported a previously unexplored role of angi- opoietin factors played in generating mature islets from iPSCs. Our study provides new knowledge on controlling in vitro organoid development and maturation.f g p It is worth noting that the molecular mechanisms underlying the Ang2 effect, particularly Ang2-mediated signaling transduction pathway is unknown. Discussion More detailed characterizations are required to elucidate the mecha- nism of angiopoietin signaling. The studies would involve mapping Ang2 induced signaling transduction path- ways using RNA-sequencing and loss- and gain-of-function assays. Unlocking of the mechanisms will help build cues critical to the production of physiologically functional islets from iPSCs for diabetes research and treatment. www.nature.com/scientificreports/ Images were captured using a Zeiss LSM 880 with an airyscan microscope. For estimating the number of different cell types, images (n = 7–16) were quantified by ImageJ (version 1.52p, https://​imagej.​nih.​gov/​ij/​notes.​html; or version 1.53i, https://​imagej.​nih.​gov/​ij/​notes.​ html) using the Biovoxxel plugin v1.0 (https://​www.​biovo​xxel.​de/​devel​opment/). The images were processed to remove background and noise. DAPI stained nuclei were distinguished using the watershed function. To avoid over-segmentation, the watershed function was used only when required, especially when images contained a high number of merged cells. Cell number was estimated by counting cells larger than 250-pixel units. Variations in cell size were monitored with each image and cell size threshold was modified accordingly to avoid over- or under-estimation of cell numbers. Glucose stimulated insulin secretion analysis. Islets were washed with DPBS and equilibrated in Krebs–Ringer buffer (KRB, Boston BioProducts) containing 1 mM glucose for 4 h. Aggregates were rinsed again with KRB containing 1 mM glucose. For p-GSIS, organoids were incubated in KRB containing 2 mM glucose and 20 mM glucose at 37 °C separately for 30 min. For multiple s-GSIS, organoids were seeded on Matrigel (1:50) coated 48-well plates and cultured in Stage V differentiation media at 37 °C overnight prior to 4 h pre-incubation step. Attached organoids were challenged serially in KRB containing (2–20 mM)–(2–20 mM)–(2–20 mM) glu- cose for 20 min for each stimulation condition at 37 °C. Between the three rounds of consecutive 20–2 mM challenge, the aggregates were rinsed twice by incubating in KRB containing 1 mM glucose for 5 min with each wash. The supernatants were collected from the respective glucose treatments. The number of islets under each condition was counted under an Olympus phase contrast microscope. About 100–200 µm sized aggregates were counted as clusters. Insulin was measured using an insulin ELISA kit (Mercodia) according to the manufac- turer’s instructions. Insulin secretion can be normalized to single cells based on accounting of cell numbers or a measurement of DNA ­content9–11. We compared insulin secretion from the islets normalized based on organoid number or DNA content. Two normalizations gave us similar results. Thus, we presented the data based on islet ­number74. DNA of the islets was extracted using DNeasy Blood and Tissue Kit from QIAGEN following manu- facturer’s instructions. Human donor islets purchased from Prodo Laboratories, Inc. (Aliso Viejo, CA) were used as positive controls. www.nature.com/scientificreports/ from Stage I to Stage IV. At the end of Stage IV, cells were scrapped after Dispase treatment and transferred to a 24-well ultralow attachment plate (Corning Life Science) initiating Stage V. At Stage V, the differentiation media was prepared with CMRL supplement containing 2% bovine serum albumin (BSA, Sigma-Aldrich), 1 µM T3, 10 µM A5iII, 100 nM SiXX, and 10 mM Nic. The differentiation media at all stages were exchanged every two days. For suspension culture, half of the media was changed every two days. Angiopoietin proteins, Ang2 (Biole- gend) and Ang1 (Peprotech) were applied to differentiation media at 20 ng/ml or indicated concentration from Stage IV until the end of Stage V. TaqMan quantitative real‑time PCR. qRT-PCR was performed as described ­previously71,72. Briefly, RNA was extracted from cells using the RNeasy mini kit (Qiagen). Primer and probe sets targeting genes of interest were used as shown in Table S1. CFX Connect system (Bio-Rad) was used for qRT-PCR. Ct values for the target genes were normalized to internal housekeeping gene Cyclophilin ­A71,73. ΔΔCt method was employed to analyze the gene expression relative to undifferentiated cells. RNA extracted from human islets purchased from Prodo Laboratories, Inc. were used as a positive control. Immunostaining and confocal imaging microscopy. Islets were rinsed with Dulbecco phosphate buffered saline (DPBS) (HyClone) and fixed with 4% paraformaldehyde at room temperature for 1 to 2 h. The fixed aggregates were washed with phosphate buffered saline (PBS), and 30% sucrose (w/v) was added and stored at 4 °C overnight. Optimal Cutting Temperature compound (OCT) (ThermoFisher Scientific) was added to the samples and stored overnight at 4 °C. This was followed by snap freezing in liquid nitrogen. Thin sections, 14 µm, of the frozen samples were prepared on TruBond TM (Electron Microscopy Sciences) adhesive slides by cryosectioning. The OCT compound was removed by PBS wash. Foxp3/Transcription Factor Fixation/Permea- bilization kit (ThermoFisher Scientific) was used for permeabilization, blocking and antibody staining according to the manufacturer’s instruction. Primary antibodies were applied overnight at 4 °C. Excess antibodies were washed off three times with PBS at room temperature. Secondary antibodies were then applied for 1 h at room temperature. Excess antibodies were washed off three times with PBS at room temperature. The samples were stained for nucleus with Vectashield Mounting Medium containing DAPI (ThermoFisher Scientific). Antibodies used in immunofluorescence staining were shown in Table S2. Materials and methods Cell culture. iPSC lines IMR90, DF4, and hESC line H9 were obtained from WiCell Research Institute. Cells were cultured on 1:100 diluted Matrigel (Corning Life Science) coated dishes and maintained in the mTeSR1 medium (StemCell Technologies), which was replenished with fresh media every day as described ­elsewere71,72. The cells were passaged every 3 to 4 days into a 1:3 split ratio using Dispase (StemCell Technologies). To initi- ate differentiation, the cells were seeded as single cells at one to two million cells per well of 6-well plate after Accutase (StemCell Technologies) treatment. Rat β cells RIN-5F (ATCC CRL-2058) were maintained in RPMI 1640 supplemented with 10% fetal bovine serum. Growth medium was exchanged every 3 days and cells were passaged at 80% confluence. Stepwise differentiation. Differentiation procedures were developed and modified based on our previ- ous ­study11, which was initiated using optimized serum-free differentiation media with combinations of growth factors and signaling molecules (Fig. 1a). Stage I differentiation was initiated 24 h post single cell seeding. The DE cells were achieved by applying RPMI 1640 (Corning Life Science) supplemented with 50 ng/ml activin A (AA, PeproTech) and 1 mM sodium butyrate (SB, Sigma-Aldrich) for 24 h. From day 2 to day 5, the concentra- tion of SB was reduced to 0.5 mM as described ­previously73. At Stage II, the media formulation consisted of RPMI 1640, 1 µM retinoic acid (RA, Sigma-Aldrich), 50 ng/ml keratinocyte growth factor (KGF, PeproTech), 300 nM (-)-indolactam V (ILV, AdipoGen), 100 ng/ml noggin (Nog, PeproTech), and 125 µM ascorbic acid (VitC, Sigma-Aldrich). At Stage III, cells were cultured in DMEM high glucose (Gibco) containing 1 µM RA, 300 nM ILV, 200 nM LDN 193189 (LDN, Sigma-Aldrich), 1 µM 3,3′,5-triiodo-L-thyronine Sodium Salt (T3, Sigma-Aldrich), 10 µM ALK5 inhibitor II (A5iII, Enzo Life Sciences), 100 nM γ-secretase inhibitor XX (SiXX, Millipore), and 10 µg/ml heparin (HP, Sigma-Aldrich). At Stage IV, the differentiation media was prepared with RPMI 1640 containing 1 µM T3, 10 µM A5iII, 10 µg/ml HP, 1 mM N-acetyl cysteine (NCys, Sigma-Aldrich), 0.5 µM R428 (Selleck Chem), 10 µM Trolox (Tro, Enzo Life Sciences), 100 nM SiXX, and 10 mM nicotinamide (Nic, Sigma-Aldrich); and glucose (Gibco) was supplemented up to 20 mM. Serum-free B27 (Gibco) was added https://doi.org/10.1038/s41598-021-92922-5 Scientific Reports | (2021) 11:13558 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ was recorded for 5 min for each of the incubation conditions. During the 5-min time series, a snapshot of the aggregate was captured every 17 s. For the cell-cluster level analysis, a 40 × objective was used, and for single- cell analysis, 63 ×/40 × objectives were used. Image analysis was performed with ImageJ and MATLAB (Version R2018a). A total of 126 images per aggregate, which includes 18 images per glucose treatment, were analyzed to calculate average fluorescence intensity. Individual cells of the islet organoids were also selected as region of interest (ROI) for single cell analysis. When average a.u.c at high glucose was higher than the average a.u.c at low glucose in an individual cell, the cell was considered to be responding positively. Human donor islets were used as positive controls. F‑actin staining and image analysis. The islets generated were subjected to glucose challenges and the aggregates were fixed and stained following the immunostaining procedure mentioned above. The images were captured with Zeiss LSM 880. The β cells were identified by C-peptide staining and F-actin was visualized by Phalloidin Alexa 488 staining. Image analysis was carried out by line scan and radial profiling. To account for heterogeneity of F-actin within the β cell boundary, radial integrated intensity was assessed using ImageJ soft- ware which accounts for F-actin expression in circular of a β cell. The F-actin intensity along the center to edge represents intensities integrated around concentric circles within the ROI encompassing β cells. Statistical analysis. All experimental data were presented as mean ± standard deviation (SD) of at least three times of independent experiments. 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Scientific Reports | (2021) 11:13558 | https://doi.org/10.1038/s41598-021-92922-5 Competing interests h Competing interests  The authors declare no competing interests. Fundingh g This work was partially supported by National Science Foundation CBET1531944, CBET1928855, EEC1757846, and National Institute of Health 1R15EB027391-01. Competing interests  The authors declare no competing interests. Additional informationh Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​021-​92922-5. Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​021-​92922-5. Correspondence and requests for materials should be addressed to S.J. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. 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Research article Research article Earth Syst. Dynam., 13, 1215–1232, 2022 https://doi.org/10.5194/esd-13-1215-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Evaluating uncertainty in aerosol forcing of tropical precipitation shifts Amy H. Peace1,4, Ben B. B. Booth2, Leighton A. Regayre1, Ken S. Carslaw1, David M. H. Sexton2, Céline J. W. Bonfils3, and John W. Rostron2 1Institute for Climate and Atmospheric Science, University of Leeds, Leeds, UK 2Met Office Hadley Centre, Exeter, UK 3Lawrence Livermore National Laboratory, Livermore, CA, USA 4College of Engineering, Maths and Physical Science, University of Exeter, Exeter, UK Correspondence: Amy H. Peace (a.h.peace@exeter.ac.uk) Received: 14 March 2022 – Discussion started: 23 March 2022 Revised: 29 July 2022 – Accepted: 5 August 2022 – Published: 26 August 2022 Amy H. Peace1,4, Ben B. B. Booth2, Leighton A. Regayre1, Ken S. Carslaw1, David M. H. Sexton2, Céline J. W. Bonfils3, and John W. Rostron2 1Institute for Climate and Atmospheric Science, University of Leeds, Leeds, UK 2Met Office Hadley Centre, Exeter, UK 3Lawrence Livermore National Laboratory, Livermore, CA, USA 4College of Engineering, Maths and Physical Science, University of Exeter, Exeter, UK Received: 14 March 2022 – Discussion started: 23 March 2022 Revised: 29 July 2022 – Accepted: 5 August 2022 – Published: 26 August 2022 Abstract. An observed southward shift in tropical rainfall over land between 1950 and 1985, followed by a weaker recovery post-1985, has been attributed to anthropogenic aerosol radiative forcing and cooling of the Northern Hemisphere relative to the Southern Hemisphere. We might therefore expect models that have a strong historic hemispheric contrast in aerosol forcing to simulate a further northward tropical rainfall shift in the near-term future when anthropogenic aerosol emission reductions will predominantly warm the Northern Hemisphere. We investigate this paradigm using a perturbed parameter ensemble (PPE) of transient coupled ocean–atmosphere climate simulations that span a range of aerosol radiative forcing comparable to multi-model studies. In the 20th century, in our single-model ensemble, we find no relationship between the magnitude of pre-industrial to 1975 inter-hemispheric anthropogenic aerosol radiative forcing and tropical precipitation shifts. Instead, tropical precipitation shifts are associated with major volcanic eruptions and are strongly affected by internal variability. However, we do find a relationship between the magnitude of pre-industrial to 2005 inter- hemispheric anthropogenic aerosol radiative forcing and future tropical precipitation shifts over 2006 to 2060 un- der scenario RCP8.5. Our results suggest that projections of tropical precipitation shifts will be improved by reducing aerosol radiative forcing uncertainty, but predictive gains may be offset by temporary shifts in tropical precipitation caused by future major volcanic eruptions. Published by Copernicus Publications on behalf of the European Geosciences Union. 1 Introduction Multi-model studies show the strength of the hemispheric difference in aerosol radiative forcing correlates with the magnitude of tropical precipitation shifts in the 20th century. Coupled Model Inter-comparison Project 5 (CMIP5) models with relatively detailed representations of aerosol–cloud in- teractions simulate a further southward migration of tropical precipitation over 1950 to 1985 (Allen et al., 2015) and bet- ter reproduce decadal drivers of Indian rainfall (Choudhury et al., 2021). However, multi-model ensembles (MMEs) rep- resent a collection of models which vary not only in how they represent physical processes, but also in the complexity and range of processes that they represent at all. As such, it is hard to interpret differences across multi-model ensembles and link them back to processes. Whether or not aerosol re- ductions will be a main driver of tropical precipitation shifts in the future remains unclear, yet it is likely the large uncer- tainty in aerosol forcing will cause uncertainty in projected tropical precipitation shifts (Byrne et al., 2018). The influence of aerosol radiative forcing on tropical pre- cipitation shifts has not previously been investigated using perturbed parameter ensembles (PPEs). PPEs explore model uncertainties by perturbing influential uncertain model pa- rameters within their plausible ranges. An advantage of us- ing PPEs is that differences in climate response across the ensemble can often be linked back to known differences in the perturbed parameters – which can yield new insights into what causes spread in climate model response. Here, we make use of a PPE of the coupled ocean–atmosphere model HadGEM3-GC3.05 that was developed for UK Na- tional Climate Projections (UKCP18). In the PPE, 47 param- eters are perturbed across a range of model schemes. The per- turbed parameters resulted in an emergence of a wide range of aerosol forcings across the PPE, but a relatively smaller range of climate sensitivities, as shown in Fig. 1. In prin- ciple, the PPE may therefore be a useful tool to assess the relationship between anthropogenic aerosol radiative forcing Anthropogenic aerosol emissions are projected to decline in the future in response to increasingly stringent air qual- ity and climate change mitigation policies (Rao et al., 2017; van Vuuren et al., 2011b). Reductions in anthropogenic aerosol emissions will lead to a warming of climate rela- tive to present day that will primarily affect the Northern Hemisphere, and could lead to a northward shift in tropical precipitation (Allen, 2015; Rotstayn et al., 2015). 1 Introduction model simulations (Allen et al., 2015; Hwang et al., 2013; Williams et al., 2001; Rotstayn and Lohmann, 2002; Rot- stayn et al., 2000; Chang et al., 2011; Evans et al., 2020; Chemke and Dagan, 2018; Bonfils et al., 2020). Most no- tably, anthropogenic aerosol emissions that increased rapidly across Europe and North America up to the 1980s (Lamar- que et al., 2010) have been linked to an observed southward shift in tropical precipitation, which was associated with widespread drying of the Sahel between the 1950s and 1980s (Biasutti and Giannini, 2006; Allen et al., 2015; Kang et al., 2021; Herman et al., 2020; Ackerley et al., 2011; Booth et al., 2012; Dong et al., 2014; Hirasawa et al., 2020). Over recent decades there has been a partial northward return of The interaction of atmospheric aerosols with clouds and radi- ation is the largest cause of uncertainty in the radiative forc- ing of the Earth system over the industrial period (e.g. Bel- louin et al., 2020; Myhre et al., 2013). Atmospheric aerosols have short residence times of days to weeks; therefore the strongest radiative effects of aerosols occur relatively close to emission sources. The increase in anthropogenic aerosol emissions over the industrial era has therefore caused a neg- ative radiative forcing mainly in the Northern Hemisphere. This hemispheric nature of anthropogenic aerosol radiative forcing has been linked to observed shifts in tropical precip- itation and understood using idealised and transient climate Published by Copernicus Publications on behalf of the European Geosciences Union. A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts 1216 tropical precipitation alongside a modest recovery of rainfall over the Sahel (Giannini and Kaplan, 2019) and India, but increased drought in the northeast Brazilian region (Utida et al., 2019). Natural aerosols from major volcanic eruptions can also cause a negative radiative forcing primarily in one hemisphere, dependent on the latitude of the eruption (Hay- wood et al., 2013). additive effect on any northward tropical precipitation shift, rather than acting in opposing directions as seen over the 20th century, making attribution to a particular forcing agent even harder to disentangle (Friedman et al., 2013). Through- out the 21st century, the climate response to warming also adds a layer of complexity to the attribution of tropical pre- cipitation changes. 1 Introduction Climate feedbacks, such as sea-ice feed- backs or changes in Atlantic Meridional Overturning Circu- lation (AMOC) strength, can modulate the position of the ITCZ, leading to a tug-of-war between different forcing and feedback components (McFarlane and Frierson, 2017; Ma- malakis et al., 2021). Changes in large-scale circulation as- sociated with warming (e.g. wet-gets-wetter paradigm) can also affect the tropical precipitation distribution and could mask an identifiable signal for latitudinal tropical precipita- tion shifts (Friedman et al., 2013). Latitudinal shifts of tropical precipitation are intertwined with perturbations to the Hadley circulation and movement of the Intertropical Convergence Zone (ITCZ). The theo- retical energetic framework links the position of the ITCZ to the inter-hemispheric energy balance. As such, a pertur- bation to the inter-hemispheric energy balance, particularly in the extra-tropics, can trigger a shift in the position of the ITCZ and associated tropical precipitation (Kang et al., 2008, 2009, 2018). From an atmospheric perspective, the Hadley cell would adjust to transport more energy north- wards in response to the anomalous inter-hemispheric energy balance imposed by a cooling of the Northern Hemisphere, for example, by anthropogenic aerosol forcing (Hwang et al., 2013). From the perspective of a dynamical ocean, wind- driven shallow overturning cells also act to transport energy in the same direction as the atmosphere (Green and Mar- shall, 2017; Kang, 2020). Hence, in a framework where a dynamical ocean is taken into account, the atmospheric re- sponse of the ITCZ to an inter-hemispheric energy imbalance is partially dampened. In addition to aerosol radiative forc- ing, other forcing agents such as changes in high-latitude ice cover and ocean circulation can alter the inter-hemispheric energy balance (Chiang and Bitz, 2005; Chiang and Fried- man, 2012; Broccoli et al., 2006). Migrations in tropical pre- cipitation over the 20th century have also been linked to vari- ability in the difference in sea surface temperature between the Northern Hemisphere and Southern Hemisphere (Chiang and Friedman, 2012; Thompson et al., 2010). Because there are multiple drivers, there remains debate over whether the shifts in tropical precipitation observed over the 20th cen- tury can be attributed to anthropogenic aerosols, other forced responses, natural climate variability, or a combination of these. Large uncertainty in anthropogenic aerosol radiative forc- ing limits our understanding of historical changes in climate and the drivers of tropical precipitation and ITCZ migrations. 1 Introduction However, identifying the drivers of future tropical precipitation shifts is complex for a number of reasons. Warming of Northern Hemisphere land masses caused by greenhouse gases could too lead to a northward shift in tropical precipitation (Frier- son and Hwang, 2012; Friedman et al., 2013). If so, the rel- ative warming from anthropogenic aerosol reductions and warming from increasing greenhouse gases could have an Earth Syst. Dynam., 13, 1215–1232, 2022 https://doi.org/10.5194/esd-13-1215-2022 A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts 1217 Figure 1. Range of aerosol ERF (a) and equilibrium climate sensitivity (b) across the PPE and AR6. The PPE error bars show the 90 % range across 15 PPE members compared with the AR6 90 % “very likely” ranges from Forster et al. (2021). We use 13 PPE members in this study, excluding two shown in this figure due to model drifts. A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts 1217 A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts 1217 Figure 1. Range of aerosol ERF (a) and equilibrium climate sensitivity (b) across the PPE and AR6. The PPE error bars show the 90 % range across 15 PPE members compared with the AR6 90 % “very likely” ranges from Forster et al. (2021). We use 13 PPE members in this study, excluding two shown in this figure due to model drifts. uncertainty and tropical precipitation shifts over the 20th and 21st centuries. to explore the relationship between aerosol radiative forcing uncertainty and tropical precipitation shifts. The base model used in this work is version 3.05 of the UK Hadley Centre Unified Model (HadGEM3- GC3.05), which is a global coupled ocean–atmosphere model. HadGEM3-GC3.05 includes many of the main im- provements that were made to GC3.0 to create GC3.1 that was submitted to CMIP6 (Williams et al., 2018; Walters et al., 2019). The atmospheric component is HadGEM3- GA7.05 (Williams et al., 2018). The atmospheric and land components are configured at “N216” resolution (approx- imately 60 km horizontal spacing of grid boxes at mid- latitudes), with 85 vertical atmospheric levels. 1 Introduction HadGEM3- GC3.05 incorporates the modal version of the GLObal Ver- sion of Aerosol Processes (GLOMAP-mode), which sim- ulates new particle formation, gas-to-gas particle transfer, aerosol coagulation, cloud processing of aerosol, and aerosol deposition of sulfate, sea salt, dust, black carbon, and par- ticulate organic species (Mann et al., 2010). The ocean and sea-ice model components are NEMO and CICE respectively (Hewitt et al., 2011). The ocean component is eddy permit- ting with a resolution of 1/4◦and finer. Ocean components of global climate models (GCMs) that are higher resolution and resolve eddies (such as this model version) can affect the mean state of the ocean, climate variability, and climate re- sponse, in comparison to lower-resolution ocean components (Hewitt et al., 2020). 2.1 Perturbed parameter ensemble of HadGEM3-GC3.05 This study utilises a perturbed parameter ensemble (PPE) of climate model simulations that was designed to inform UK Climate Projections 2018 (UKCP18). One of the key aims of UKCP18 was to provide a flexible dataset with a small sample size to support a wide range of impact assess- ments (Sexton et al., 2021). The design of the PPE was there- fore focused on providing a small number of transient cou- pled ocean-atmosphere simulations that sample plausible and diverse climate changes for a given emissions scenario (Mur- phy et al., 2018; Yamazaki et al., 2021). The resulting PPE spans a large fraction of the AR6 very likely range of aerosol effective radiative forcing (ERF), but a smaller range of cli- mate sensitivity (Fig. 1). Similar to multi-model ensembles, the PPE therefore provides an opportunity to investigate the role of aerosol forcing uncertainty in tropical precipitation shifts in an “ensemble of opportunity”, but with the advan- tage that differences in climate responses can be linked back to underlying parameters/processes. Below we summarise the design of this PPE and how we utilise 13 PPE members Earth Syst. Dynam., 13, 1215–1232, 2022 https://doi.org/10.5194/esd-13-1215-2022 A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts 1218 A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts Figure 2. Schematic showing the stages in the design process used in UKCP18 to provide a small subset of model variants that sample a diverse climate response and are plausible when evaluated against historical climate. In this study, we use 13 PPE members of the aerosol ERF and transient coupled ocean–atmosphere experiments which are highlighted in the purple boxes. Figure 2. Schematic showing the stages in the design process used in UKCP18 to provide a small subset of model variants that sample a diverse climate response and are plausible when evaluated against historical climate. In this study, we use 13 PPE members of the aerosol ERF and transient coupled ocean–atmosphere experiments which are highlighted in the purple boxes. ing diversity in climate responses. Therefore, multi-stage fil- tering of an initial large sample of model variants was under- taken for UKCP18 to identify a plausible and diverse subset to be used in the transient simulations. We briefly summarise the stages of the filtering process below aided by Fig. 2.2 Quantifying shifts in tropical precipitation We use the 13 transient coupled ocean–atmosphere simu- lations described above to quantify shifts in tropical pre- cipitation over the 20th and 21st centuries. We define the latitudinal position of the ITCZ and tropical precipitation, 8ITCZ (degrees), as the median of annual mean precipita- tion (mm d−1) over area-weighted regional means between 20◦S and 20◦N. Several studies have used this definition to quantify changes in the position of the ITCZ and tropical pre- cipitation (Frierson and Hwang, 2012; Donohoe et al., 2013; Atwood et al., 2020; Evans et al., 2020; Moreno-Chamarro et al., 2020; Green et al., 2017; Green and Marshall, 2017). We calculate the linear trend of the 5-year rolling mean value of 8ITCZ over multi-decadal periods in the 20th and 21st cen- turies over three regions – global, Atlantic, and Pacific, where the Atlantic is defined as 75◦W to 30◦E and the Pacific as 150◦E to 75◦W. 2.1 Perturbed parameter ensemble of HadGEM3-GC3.05 Diversity was assessed using a combination of metrics from these ide- alised experiments, and transient coupled ocean–atmosphere simulations were created for the 25 most diverse, plausible model variants. The filtering process to identify the 25 model variants is described in further detail in Sexton et al. (2021). Lastly, the transient PPE simulations were filtered based on their performance over 1900 to 2005, as described in Ya- mazaki et al. (2021). This multi-stage process left 15 (out of an initial 2800) remaining model variants that sample known model uncertainties and hence provide, for a given emissions scenario, a range of climate responses. The aerosol ERF experiment that was used in the diversity filtering stage shows that these 15 model variants span a large range of aerosol ERF, as shown in Fig. 1. However, additional ide- alised 4 × CO2 coupled ocean-atmosphere simulations that were created following the completion of UKCP18 to diag- nose equilibrium climate sensitivity (ECS) for the PPE (Sex- ton et al., 2020) show that the 15 model variants sample a comparatively smaller range of climate sensitivity. We use 13 of the 15 remaining ensemble members in this study, ex- cluding a further two members on the basis that these mem- bers show steady weakening of the AMOC (Sexton et al., 2020). idea of how internal variability alone can generate uncer- tainty in climate responses for a similar model version. The HadGEM-GC3.1-MM ensemble is the same resolution as the PPE, whereas the HadGEM-GC3.1-LL ensemble is lower resolution (“N96”; approximately 135 km horizontal spacing of grid boxes at mid-latitudes). 2.1 Perturbed parameter ensemble of HadGEM3-GC3.05 Three idealised atmosphere-only experiments that are simi- lar to those used in CMIP5 (Taylor et al., 2012) were used to assess the diversity in climate response across the model variants: aerosol ERF between 1860 and 2005, ERF due to a quadrupling of CO2, and sea surface temperature (SST) patterns prescribed for a global warming of 4 ◦C. Diversity was assessed using a combination of metrics from these ide- alised experiments, and transient coupled ocean–atmosphere simulations were created for the 25 most diverse, plausible model variants. The filtering process to identify the 25 model variants is described in further detail in Sexton et al. (2021). Lastly, the transient PPE simulations were filtered based on their performance over 1900 to 2005, as described in Ya- mazaki et al. (2021). This multi-stage process left 15 (out of an initial 2800) remaining model variants that sample known model uncertainties and hence provide, for a given emissions scenario, a range of climate responses. The aerosol ERF experiment that was used in the diversity filtering stage shows that these 15 model variants span a large range of aerosol ERF, as shown in Fig. 1. However, additional ide- alised 4 × CO2 coupled ocean-atmosphere simulations that were created following the completion of UKCP18 to diag- nose equilibrium climate sensitivity (ECS) for the PPE (Sex- ton et al., 2020) show that the 15 model variants sample a comparatively smaller range of climate sensitivity. We use 13 of the 15 remaining ensemble members in this study, ex- cluding a further two members on the basis that these mem- bers show steady weakening of the AMOC (Sexton et al., 2020). to 2009). A remaining 557 model variants were then assessed for performance using 5-year (2005 to 2009) atmosphere- only simulations. This filtering process left 39 remaining model variants considered plausible. These 39 model vari- ants were reduced to a 25-member PPE of diverse mem- bers that would sample a broad range of climate responses. Three idealised atmosphere-only experiments that are simi- lar to those used in CMIP5 (Taylor et al., 2012) were used to assess the diversity in climate response across the model variants: aerosol ERF between 1860 and 2005, ERF due to a quadrupling of CO2, and sea surface temperature (SST) patterns prescribed for a global warming of 4 ◦C. 2.1 Perturbed parameter ensemble of HadGEM3-GC3.05 2 and direct the reader to the literature (Sexton et al., 2021; Ya- mazaki et al., 2021) where the process is described in full for further information. Experts selected 47 model parameters from the physical atmosphere, aerosol, and land surface model components of HadGEM3-GC3.05 to perturb across their uncertain ranges in order to sample the parametric model uncertainty in cli- mate projections. The 47 model parameters chosen to be per- turbed are from the model schemes representing convection, boundary layer, gravity wave drag, cloud radiative and mi- crophysical properties, and aerosol and land surface proper- ties. A description of perturbed parameters is given in Ta- ble S1 in the Supplement, and the selection process for these model schemes and parameters is described in further detail in Sexton et al. (2021). Initially, 2800 model parameter combinations were cre- ated that sample the uncertainty across the 47 perturbed parameters. Latin hypercube sampling was used to select the initial wave of parameter combinations. The Latin hy- percube sampling approach maximises the minimum dis- tance between points to ensure good coverage of the multi- dimensional “parameter space” created by simultaneously perturbing 47 model parameters. After the model variants were created, the filtering process first involved assessing the performance of model variants against observed climate variables using cheaper atmosphere-only experiments. The performance of the initial 2800 model variants was assessed using 5 d weather hind-casts (start dates covering 2008 Each model simulation in a PPE framework consists of a unique combination of perturbed model parameter values. We refer to each unique combination of parameter values as a “model variant”. Current computational constraints meant that a total of 25 transient coupled ocean–atmosphere sim- ulations could be produced for UKCP18. These 25 model variants were required to sample the uncertainty across the 47 perturbed parameters, whilst remaining plausible when evaluated against observed climate variables and represent- Earth Syst. Dynam., 13, 1215–1232, 2022 https://doi.org/10.5194/esd-13-1215-2022 1219 A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts to 2009). A remaining 557 model variants were then assessed for performance using 5-year (2005 to 2009) atmosphere- only simulations. This filtering process left 39 remaining model variants considered plausible. These 39 model vari- ants were reduced to a 25-member PPE of diverse mem- bers that would sample a broad range of climate responses. 2.3 Inter-hemispheric temperature and radiative forcing To study the drivers of the ITCZ shifts across the PPE, we calculate the Spearman rank correlation coefficient of the decadal trend in 8ITCZ with variables related to the inter- hemispheric energy balance. These variables include the trend in the inter-hemispheric difference in surface air tem- perature and implied total radiative forcing, plus the inter- hemispheric difference in historical anthropogenic aerosol ERF. We also evaluate the role of cloud and non-cloud short- wave radiative responses, as well as the role of aerosol optical depth (AOD). Historical emissions were prescribed in the ensemble members up to 2005, then forking into the Representa- tive Concentration Pathway (RCP) scenarios RCP8.5 and RCP2.6 to 2100. The RCPs provide a range of greenhouse gas (GHG) concentrations and emission pathways that span a range of total radiative forcing at 2100. RCP8.5 is a high- emissions scenario, with GHG emissions assumed to rise substantially out to 2100 (Riahi et al., 2011). In contrast, RCP2.6 assumes aggressive measures to substantially reduce future GHG emissions (van Vuuren et al., 2011a). The RCP scenarios assume successful implementation of air quality legislation, but RCP2.6 has approximately double the reduc- tion of air pollutant emissions by 2030 compared to RCP8.5 (Riahi et al., 2011). The inter-hemispheric difference is calculated as the difference between area-weighted Northern Hemisphere (0 to 60◦N) and Southern Hemisphere (0 to 60◦S) means and referred to as “inter-hemispheric” herein. The inter-hemispheric variables are calculated over both land and ocean, unless specified. Linear trends of the inter- hemispheric variables are calculated from a 5-year rolling mean. The implied total radiative forcing for the transient PPE was estimated at each grid box using the formula derived from Gregory and Forster (2008): In addition to model uncertainty that can be sampled us- ing PPEs or MMEs, uncertainty in climate projections can arise from scenario uncertainty and internal variability. https://doi.org/10.5194/esd-13-1215-2022 Earth Syst. Dynam., 13, 1215–1232, 2022 3.1 Tropical precipitation shifts over the 20th century We begin by examining the latitudinal shift of tropical pre- cipitation (8ITCZ) over the 20th century in our PPE. Fig- ure 3 shows the 5-year rolling mean evolution of 8ITCZ over the historical period in the global, Atlantic, and Pacific re- gional means. The PPE mean 8ITCZ migrates southwards over the 1940 to 1985 period (around 0.01◦latitude per year globally). This southward migration of tropical precipitation agrees with multi-model studies (CMIP3, CMIP5) and ob- servations of tropical precipitation over land that show trop- ical precipitation shifted southward in the second half of the 20th century (Hwang et al., 2013; Allen et al., 2015; Chung and Soden, 2017; Bonfils et al., 2020). The idealised simulations that were used to assess the di- versity of PPE members (Sect. 2.1) provide estimates of an- thropogenic aerosol ERF at 2005 relative to 1860 for each PPE member in the transient coupled ocean–atmosphere sim- ulations that we use to analyse tropical precipitation shifts. To better align with the historical time period analysed in this study, we completed additional simulations to provide estimates of anthropogenic aerosol ERF at 1975 relative to 1860 for our 13 PPE members. ERF was quantified as the change in radiative fluxes caused by changes in an- thropogenic aerosol emissions between 1860 and the indus- trial time period (1975 or 2005), with SSTs, sea-ice extent, and greenhouse gas concentrations held constant at 2005 to 2009 values (rather than pre-industrial values as in CMIP studies; Taylor et al., 2012). , ; , ) There are brief shifts in 8ITCZ in the years following major volcanic eruptions in the 20th century. The 8ITCZ time series without the 5-year smoothing applied is shown in Fig. S3 to more precisely illustrate this effect. There is a northward shift in 8ITCZ following the Southern Hemisphere eruption of Mt Agung in 1963 and a southward shift following the North- ern Hemisphere eruption of El Chichón in 1982. Hence, our ensemble mean time series of 8ITCZ agrees with the litera- ture (Haywood et al., 2013; Iles et al., 2013; Bonfils et al., 2020; Colose et al., 2016) showing the position of the ITCZ and tropical precipitation responds to volcanic eruptions and shifts away from the hemisphere with the maximum strato- spheric aerosol loadings. 3 Results and discussion For PPE members, shortwave cloud and non-cloud radia- tive responses were estimated using the approximate partial radiative perturbation (APRP) method (Taylor et al., 2007). The APRP method uses a single-layer radiative transfer model to decompose climate model output into three com- ponents: the change in shortwave radiation due to cloud, the change in shortwave radiation due to non-cloud atmospheric scattering and absorption, and the change in shortwave radia- tion due to surface albedo. Under this method, changes in the cloud component are solely due to changes in cloud proper- ties, whereas changes in the non-cloud component are due to changes in aerosols, ozone, and water vapour (Zelinka et al., 2014). 2.3 Inter-hemispheric temperature and radiative forcing We use the small four-member initial condition ensembles of HadGEM3-GC3.1-LL and HadGEM-GC3.1-MM that were submitted to CMIP6 (Andrews et al., 2020) to provide an (1) 1FIm = 1FTOA −λ1T, (1) where 1FIm is the implied radiative forcing of interest, FTOA is the change in annual mean net top-of-atmosphere flux relative to a reference period, 1T is the change in global annual mean surface air temperature relative to a where 1FIm is the implied radiative forcing of interest, FTOA is the change in annual mean net top-of-atmosphere flux relative to a reference period, 1T is the change in global annual mean surface air temperature relative to a Earth Syst. Dynam., 13, 1215–1232, 2022 https://doi.org/10.5194/esd-13-1215-2022 Earth Syst. Dynam., 13, 1215–1232, 2022 A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts 1220 tury and the inter-hemispheric 1860 to 2005 aerosol ERF when analysing the 21st century. Aerosol ERF values are shown in Figs. S1 and S2 in the Supplement. We do not have simulations from which to quantify the aerosol ERF in the near-term future. Hence, our analyses rely on the assumption that ensemble members with strong or weak near-present- day aerosol radiative forcing will also have a strong or weak response to changes in aerosol emissions over the near-term future time periods as anthropogenic aerosol emissions de- cline. reference period, and λ is the climate feedback parameter. In this convention, positive feedback components are rep- resented by a positive contribution to λ. In the PPE case, the value for λ was estimated using the approach in Gre- gory and Forster (2008), where for an abrupt 4 × CO2 ex- periment, λ is the regression slope between radiative forc- ing and global temperature change, taking account of model drifts in the control runs (Sexton et al., 2020). In the case of the small HadGEM3-GC3.1 initial condition ensembles, we estimate 1FIm using a 1850 to 1870 reference period and a feedback parameter value of −0.86 W m−2 K−1 (following Andrews et al., 2019). A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts 1221 Figure 3. Time series of 5-year rolling mean 8ITCZ anomaly rel- ative to the 1950 to 2000 reference period for (a) global, (b) At- lantic, and (c) Pacific regional means. The ensemble mean time se- ries is shown by the darker line, and the individual ensemble mem- bers are shown by the lighter lines. Major volcanic eruptions are marked with grey vertical lines with maximum aerosol loading in the NH – El Chichón (1982, Mexico, 17.21◦N) and Pinatubo (1991, Philippines, 15.08◦N) – and in the SH (Mt Agung, 1963, Indonesia, 8.20◦S). ternal variability from each ensemble member as is common in multi-model studies. For example, in Allen et al. (2015) models that had aerosol radiative forcing experiments also had 3 to 10 ensemble members in the transient all-forcing runs that were averaged to obtain the tropical precipitation shift for that model. We use the initial condition ensemble for HadGEM3-GC3.1 (a similar model version submitted to CMIP6; Andrews et al., 2020; Murphy et al., 2018) to esti- mate the spread in the 8ITCZ trend due to internal variability. p y In the 1950 to 1985 global mean, the initial condition en- semble spread covers close to half (54 %) of the spread in our PPE, which suggests that a large fraction of our PPE spread is caused by natural variability, but there is still a consider- able influence from perturbed parameters. However, region- ally, the initial condition ensemble covers close to or more than the entire spread in 1950 to 1985 tropical precipitation shifts in our PPE (121 % in the Atlantic, 83 % in the Pacific). In previous studies internal variability alone has been shown not to generate migrations in tropical precipitation consis- tent with observations over the 20th century (Chang et al., 2011; Allen et al., 2015). Conversely radiative forcing caused by anthropogenic aerosol, which predominantly cooled the Northern Hemisphere, peaking in the 1980s, has been impli- cated as a main driver of the migration of tropical precipi- tation southward up to the 1980s, followed by a northward recovery to the end of the 20th century. We note here that models incorporating both aerosol direct and indirect effects tend to better reproduce the historical changes in tempera- ture (Booth et al., 2012; Chung and Soden, 2017) and ITCZ location (Friedman et al., 2013; Allen et al., 2015; Bonfils et al., 2020). 3.1 Tropical precipitation shifts over the 20th century After 1985 (the period after which pollution controls are enforced in Europe and North Amer- ica), there is a northward migration of 8ITCZ to the end of the 20th century. In the time series, individual members dis- play greater inter-annual variability in the Pacific than in the Atlantic region. This could be due to different sequences of internal variability and/or different spatial–temporal evolu- tion of the forced signal in the Atlantic and Pacific regions (e.g. Diao et al., 2021). Aerosol and physical atmosphere parameters are both im- portant sources of uncertainty in aerosol ERF (Regayre et al., 2018). In our PPE, a total of eight aerosol emission and process parameters are perturbed in combination with mul- tiple physical atmosphere parameters that, amongst other re- sponses, affect aerosol forcing. Our 13 ensemble members span a range of global mean 1860 to 2005 aerosol ERF of −2.0 to −0.9 W m−2, which is larger and more negative than the spread in 1850 to 2014 aerosol radiative forcing across 17 CMIP6 models (−1.37 to −0.63 W m−2) (Smith et al., 2020) and similar to the estimated 1750 to 2014 aerosol ERF range from AR6 (−2.0 to −0.6 W m−2; medium confidence) (Forster et al., 2021). Figure 4a shows the 8ITCZ trend over 1950 to 1985 in in- dividual ensemble members and as a density function across the PPE and initial condition ensemble. The PPE mean shows a small southward shift in the 8ITCZ throughout this time pe- riod in each region. However, across the PPE, there are both southward and northward shifts (−0.03 to 0.01◦latitude per year globally). The spread in tropical precipitation shifts over We use the inter-hemispheric 1860 to 1975 aerosol ERF when analysing tropical precipitation shifts in the 20th cen- Earth Syst. Dynam., 13, 1215–1232, 2022 https://doi.org/10.5194/esd-13-1215-2022 A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts 3.1.1 Potential factors obscuring a relationship between tropical precipitation shifts and anthropogenic aerosol ERF in our PPE Figure 5c shows that for the initial condition ensembles of HadGEM3-GC3.1, where model realisations have the same pre-industrial to present-day aerosol forcing, a large spread in tropical precipitation shifts is possible in transient climate simulations due to internal variability. In the Atlantic region where we expect increases in aerosol emissions from 1950 to 1985 over Europe and North America to have an influence on precipitation, the spread in tropical precipitation shifts in the initial condition ensemble covers a larger range than the PPE. Hence, Fig. 5c suggests the relationship between pre- industrial to present-day aerosol ERF and tropical precipita- tion shifts is obscured by internal variability in our PPE. In addition, the HadGEM3-GC3.1 initial condition ensembles cover nearly all (88 % in the global mean) of the spread in the trend in the inter-hemispheric difference in temperature in our PPE, which may be the reason why there is also only a weak relationship between the trend in the inter-hemispheric difference in temperature during the 20th century and 1860 to 1975 anthropogenic aerosol ERF (Fig. S8). If we had an ini- tial condition ensemble for each PPE member or a larger sample size, the expected relationships may have emerged more strongly. The effect of internal variability is therefore likely one of the main reasons why there is not a relation- ship between inter-hemispheric aerosol forcing and tropical precipitation shifts over the 20th century in our ensemble. Figure 4. Trend in 5-year rolling mean 8ITCZ for (a) 1950 to 1985 and (b, c) 2006 to 2060 for global (left), Atlantic (middle) and Pa- cific (right) regional means. The violin plots in light purple (in b) and red (in c) are equivalent. The black lines within the violin plots show individual ensemble members (13 in the PPE, 8 in the initial condition ensemble), and the white dashed lines show the ensemble mean. The strength of relationships between the 8ITCZ trend and inter-hemispheric variables is also sensitive to the time pe- riod chosen, as shown in Table 1. The 1950 to 1985 time period, which has the strongest relationship between tropical precipitation shifts and both inter-hemispheric temperature and implied total forcing trends, encapsulates two major vol- canic eruptions. There is a weaker correlation in the longer time series or the time series excluding El Chichón. A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts Figure 4. Trend in 5-year rolling mean 8ITCZ for (a) 1950 to 1985 and (b, c) 2006 to 2060 for global (left), Atlantic (middle) and Pa- cific (right) regional means. The violin plots in light purple (in b) and red (in c) are equivalent. The black lines within the violin plots show individual ensemble members (13 in the PPE, 8 in the initial condition ensemble), and the white dashed lines show the ensemble mean. Northern Hemisphere. In CMIP5, models that had a stronger inter-hemispheric aerosol radiative forcing simulated further southward shifts in tropical precipitation over 1950 to 1985, with a correlation coefficient of r ≥0.71 across 13 mod- els (Allen et al., 2015). Despite these relationships, we do not see a strong relationship between the strength of inter- hemispheric aerosol ERF estimated from the atmosphere- only runs and the degree of southward shift in tropical pre- cipitation over 1950 to 1985 in our PPE. In the paragraphs below we evaluate several hypotheses for this weaker-than- expected relationship. A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts Our single model PPE spans a range in aerosol forcing and tropical precipitation shifts comparable to multi- model studies. Therefore, we investigate the relationship be- tween the uncertainty in the inter-hemispheric difference in aerosol forcing and tropical precipitation shifts in our PPE framework. Figure 5 shows the relationships between the 8ITCZ trend over 1950 to 1985 and the trend in inter-hemispheric (over 60◦S to 60◦N) surface air temperature (Fig. 5a), implied total radiative forcing (Fig. 5b), and 1860 to 1975 anthro- pogenic aerosol ERF (Fig. 5c). Figure S5 shows the corre- sponding plot but with inter-hemispheric variables calculated only over the ocean. A time series of inter-hemispheric tem- perature and AOD is shown in Figs. S6 and S7. There is a strong statistical relationship (r = 0.91 for global mean, r = 0.66 for regional means) between the trend in inter- hemispheric surface air temperature and the 8ITCZ trend over 1950 to 1985 (with an intercept near 0). As expected, the statistical relationship between the 8ITCZ trend and the trend in inter-hemispheric implied total forcing is also strong (r ≥0.63). An energetics framework explains how the ITCZ and corresponding latitudinal position of tropical precipita- tion shifts in response to changes in the inter-hemispheric distribution of energy (Kang et al., 2008, 2009, 2018). The perturbed cross-equatorial Hadley circulation rebalances the Figure 3. Time series of 5-year rolling mean 8ITCZ anomaly rel- ative to the 1950 to 2000 reference period for (a) global, (b) At- lantic, and (c) Pacific regional means. The ensemble mean time se- ries is shown by the darker line, and the individual ensemble mem- bers are shown by the lighter lines. Major volcanic eruptions are marked with grey vertical lines with maximum aerosol loading in the NH – El Chichón (1982, Mexico, 17.21◦N) and Pinatubo (1991, Philippines, 15.08◦N) – and in the SH (Mt Agung, 1963, Indonesia, 8.20◦S). 1950 to 1985 across our single-model ensemble is compara- ble to that over the same period from CMIP5 (see Sect. S1 in the Supplement and Fig. S4), which also displayed both south and northward shifts in tropical precipitation (Allen et al., 2015). We do not have an initial condition ensemble for each PPE member, so we cannot remove the effects of in- Earth Syst. Dynam., 13, 1215–1232, 2022 https://doi.org/10.5194/esd-13-1215-2022 A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts A. H. A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts 1223 A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts 1223 Figure 5. Scatter plot of the 1950 to 1985 trend in 5-year rolling mean 8ITCZ against the 1950 to 1985 trend in inter-hemispheric (over 60◦S to 60◦N) surface air temperature (a), implied total radiative forcing (b), and anthropogenic aerosol ERF (c) for global (left), Atlantic (middle), and Pacific (right) regional means. Anthropogenic aerosol ERF is calculated over 1860 to 1975 for the PPE and 1850 to 2014 for the initial condition ensemble. The Spearman rank correlation coefficient between variables is shown at the top left of each plot. Trend lines are shown in plots where r > 0.5. Figure 5. Scatter plot of the 1950 to 1985 trend in 5-year rolling mean 8ITCZ against the 1950 to 1985 trend in inter-hemispheric (over 60◦S to 60◦N) surface air temperature (a), implied total radiative forcing (b), and anthropogenic aerosol ERF (c) for global (left), Atlantic (middle), and Pacific (right) regional means. Anthropogenic aerosol ERF is calculated over 1860 to 1975 for the PPE and 1850 to 2014 for the initial condition ensemble. The Spearman rank correlation coefficient between variables is shown at the top left of each plot. Trend lines are shown in plots where r > 0.5. cesses and evolution of other climate forcers, and/or inter- nal variability (Voigt et al., 2017). Firstly, to investigate fur- ther we examined the relationship between 8ITCZ trend and time-evolving variables related to aerosol radiative effects, as shown in Table 2. Over 1950 to 1985, there is no clear relationship between the trend in 8ITCZ and the trend in the inter-hemispheric difference of either outgoing shortwave ra- diation at the top of the atmosphere (TOA) (Fig. S9) or to- tal AOD (Fig. S10). There is some suggestion of a relation- ship between tropical precipitation shifts and the trend in the inter-hemispheric difference of both shortwave non-cloud ra- likely to be related to the choice of analysis period as there is little evidence of stronger correlations with anthropogenic aerosol ERF estimates on other 20th century timescales. The anthropogenic aerosol ERF is quantified as the ra- diative change between two periods (1860 to 1975) using atmosphere-only simulations with SSTs and other climate forcers prescribed for 2005 to 2009. 3.1.1 Potential factors obscuring a relationship between tropical precipitation shifts and anthropogenic aerosol ERF in our PPE These results may indicate that volcanic rather than anthropogenic aerosol changes drive much of the coherent changes in inter- hemispheric temperature trends, implied total forcing trend, and tropical precipitation shifts from 1950 to 1985. However, the lack of a strong relationship between tropical precipita- tion shifts and historical anthropogenic aerosol ERF is un- energy asymmetry by transporting energy towards the cooler (energy deficient) hemisphere, and consequently moisture towards the warmer hemisphere. As such, Fig. 5a and b show that ensemble members that have greater cooling in the Northern Hemisphere and a larger difference in inter- hemispheric implied total radiative forcing over 1950 to 1985 simulate stronger southward shifts in tropical precipitation. This behaviour is in line with the energetics theory of a southward migration of the ITCZ due to an energy deficient Earth Syst. Dynam., 13, 1215–1232, 2022 https://doi.org/10.5194/esd-13-1215-2022 https://doi.org/10.5194/esd-13-1215-2022 A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts 1224 Table 1. Table of Spearman’s rank correlation coefficients for the trend in global mean 5-year rolling mean 8ITCZ and global inter- hemispheric (60◦S to 60◦N) variables shown in Fig. 5 with values for alternate time periods. Table 1. Table of Spearman’s rank correlation coefficients for the trend in global mean 5-year rolling mean 8ITCZ and global inter- hemispheric (60◦S to 60◦N) variables shown in Fig. 5 with values for alternate time periods. Time period Correlation with Correlation with Correlation with the trend in the trend in inter-hemispheric inter-hemispheric inter-hemispheric 1860 to 1975 surface air implied total anthropogenic temperature forcing aerosol ERF (◦C yr−1) (W m−2 yr−1) (W m−2) 1950 to 1985 (shown) r = 0.91 r = 0.64 r = −0.12 1950 to 1980 r = 0.56 r = 0.65 r = −0.04 1940 to 1985 r = 0.29 r = 0.48 r = −0.12 1940 to 1980 r = −0.01 r = 0.57 r = −0.07 1940 to 1975 r = 0.19 r = 0.77 r = −0.30 Table 2. Table of Spearman’s rank correlation coefficients for the 1950 to 1985 trend in global mean 5-year rolling mean 8IT additional global inter-hemispheric (60◦S to 60◦N) variables. Variable Correlation with 8ITCZ trend (latitude per year) Trend in inter-hemispheric total AOD (yr−1) r = −0.23 Trend in inter-hemispheric dust AOD (yr−1) r = −0.54 Trend in inter-hemispheric shortwave non-cloud radiative effect (W m−2 yr−1) r = 0.54 Trend in inter-hemispheric shortwave cloud radiative effect (W m−2 yr−1) r = −0.34 Trend in inter-hemispheric top of atmosphere outgoing shortwave flux (W m−2 yr−1) r = −0.34 Table 2. Table of Spearman’s rank correlation coefficients for the 1950 to 1985 trend in global mean 5-year rolling mean 8ITCZ and additional global inter-hemispheric (60◦S to 60◦N) variables. diative effect (Fig. S11) and dust AOD (Fig. S10), although these variables can also be affected by internal variability. These results with the time-evolving variables do not clarify how representative pre-industrial to 1975 aerosol ERF is of transient aerosol radiative effects in the coupled simulations. Secondly, studies have shown that the slow precipitation re- sponse to aerosol forcing is a more effective driver of ITCZ shifts than the fast precipitation response (Voigt et al., 2017; Zhang et al., 2021). A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts As such, the anthro- pogenic aerosol ERF might not be representative of the aerosol-driven climate response in transient climate simu- lations, due to the differences in time period, mediation of aerosol radiative effects by the coupling of ocean pro- https://doi.org/10.5194/esd-13-1215-2022 Earth Syst. Dynam., 13, 1215–1232, 2022 A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts The AMOC is projected to weaken under warming (Schleussner et al., 2014; Collins et al., 2019), and as AMOC weakening is likely to reduce a northward ITCZ shift (McFarlane and Frier- son, 2017), the effect would be more dominant in RCP8.5 than RCP2.6 due to the greater warming from increasing GHG emissions combined with warming from anthropogenic aerosol emission reductions. In addition, the strength of the AMOC over the 20th century has been linked to the strength of aerosol forcing, and thus aerosol forcing may also affect future projections (Menary et al., 2020; Hassan et al., 2021). Hence, the further northward migration of tropical precipita- tion up to 2060 in RCP2.6 in our ensemble is likely due to a combination of greater anthropogenic aerosol emission re- ductions in RCP2.6 compared to RCP8.5 and a greater dom- inance of processes in RCP8.5 that pull tropical precipita- tion southwards. For example, the slight southward migra- tion of 8ITCZ in the Pacific could be due to weakening of the Walker circulation, causing, in the eastern tropical Pacific Ocean, a regional southward shift of the ITCZ (Mamalakis et al., 2021). We note here that in both emission scenarios, the spread in 8ITCZ trend across the near-term future ensembles is smaller than over the historical period, due to the trend being calculated over a longer time period. A shorter future time period induces a larger spread in the 8ITCZ trend across the PPE (Fig. S13), which is comparable to the spread in the historical period. Figure 6 shows the relationship between our inter- hemispheric variables and the 8ITCZ trend over 2006 to 2060 in RCP2.6 and RCP8.5. Figure S14 shows a correspond- Overall, our analysis of 20th century latitudinal shifts in tropical precipitation shows that any relationship between these shifts and the hemispheric contrast in 1860 to 1975 an- thropogenic aerosol ERF is difficult to detect when account- ing for the effect of parametric model uncertainty and inter- nal variability. The latitudinal shift of tropical precipitation over 1950 to 1985 is, however, associated with the trend in inter-hemispheric surface temperature and implied total ra- diative forcing. It is also clear that major volcanic eruptions in the 20th century induced relatively short-lived shifts in tropical precipitation and contribute to a time period depen- dence of the strength of these relationships. A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts internal variability (superimposed on a forced signal) over the same period using the initial condition ensemble of HadGEM3-GC3.1 under emission scenario SSP5-8.5. The total radiative forcing levels at 2100 are designed to be the same in RCP8.5 and SSP5-8.5, but there are differences in the emissions scenarios of individual forcing agents (Gid- den et al., 2019), which could affect the evolution of tropi- cal precipitation shifts. Under RCP8.5, the PPE mean shows only a small change in 8ITCZ, with both north- and south- ward migrations in 8ITCZ across the ensemble. Hence, we find no robust evidence of a tropical precipitation shift un- der RCP8.5 by the mid-21st century, which is in agreement with the conclusions based on end-of-century ITCZ shifts in Byrne et al. (2018). The spread in 8ITCZ trend due to in- ternal variability under SSP5-8.5 in the initial condition en- semble is smaller than under RCP8.5 in our PPE globally and in the Pacific, but comparable in the Atlantic region. Fig- ure 4c contrasts the 8ITCZ trend over 2006 to 2060 between the RCP8.5 and RCP2.6 scenarios. By mid-century, tropical precipitation shifts further northward in RCP2.6, compared to RCP8.5. This northward migration in tropical precipita- tion for RCP2.6 is in line with Allen (2015), prior to the then southward shift between the middle and end of the 21st cen- tury. The largest difference in tropical precipitation shift be- tween emission scenarios is in the Atlantic, which may be re- lated to scenario dependence of AMOC strength. The AMOC is projected to weaken under warming (Schleussner et al., 2014; Collins et al., 2019), and as AMOC weakening is likely to reduce a northward ITCZ shift (McFarlane and Frier- son, 2017), the effect would be more dominant in RCP8.5 than RCP2.6 due to the greater warming from increasing GHG emissions combined with warming from anthropogenic aerosol emission reductions. In addition, the strength of the AMOC over the 20th century has been linked to the strength of aerosol forcing, and thus aerosol forcing may also affect future projections (Menary et al., 2020; Hassan et al., 2021). Hence, the further northward migration of tropical precipita- tion up to 2060 in RCP2.6 in our ensemble is likely due to a combination of greater anthropogenic aerosol emission re- ductions in RCP2.6 compared to RCP8.5 and a greater dom- inance of processes in RCP8.5 that pull tropical precipita- tion southwards. A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts For example, the slight southward migra- tion of 8ITCZ in the Pacific could be due to weakening of the Walker circulation, causing, in the eastern tropical Pacific Ocean, a regional southward shift of the ITCZ (Mamalakis et al., 2021). We note here that in both emission scenarios, the spread in 8ITCZ trend across the near-term future ensembles is smaller than over the historical period, due to the trend being calculated over a longer time period. A shorter future time period induces a larger spread in the 8ITCZ trend across the PPE (Fig. S13), which is comparable to the spread in the historical period. lantic and Pacific regions the uncertainty in tropical precip- itation shifts due to internal variability may be larger than parametric model uncertainty, so to more robustly quantify the effects of parameters we would need initial condition en- sembles for each parameter combinations of the 47 uncertain model parameters. internal variability (superimposed on a forced signal) over the same period using the initial condition ensemble of HadGEM3-GC3.1 under emission scenario SSP5-8.5. The total radiative forcing levels at 2100 are designed to be the same in RCP8.5 and SSP5-8.5, but there are differences in the emissions scenarios of individual forcing agents (Gid- den et al., 2019), which could affect the evolution of tropi- cal precipitation shifts. Under RCP8.5, the PPE mean shows only a small change in 8ITCZ, with both north- and south- ward migrations in 8ITCZ across the ensemble. Hence, we find no robust evidence of a tropical precipitation shift un- der RCP8.5 by the mid-21st century, which is in agreement with the conclusions based on end-of-century ITCZ shifts in Byrne et al. (2018). The spread in 8ITCZ trend due to in- ternal variability under SSP5-8.5 in the initial condition en- semble is smaller than under RCP8.5 in our PPE globally and in the Pacific, but comparable in the Atlantic region. Fig- ure 4c contrasts the 8ITCZ trend over 2006 to 2060 between the RCP8.5 and RCP2.6 scenarios. By mid-century, tropical precipitation shifts further northward in RCP2.6, compared to RCP8.5. This northward migration in tropical precipita- tion for RCP2.6 is in line with Allen (2015), prior to the then southward shift between the middle and end of the 21st cen- tury. The largest difference in tropical precipitation shift be- tween emission scenarios is in the Atlantic, which may be re- lated to scenario dependence of AMOC strength. A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts By definition aerosol ERF does not quan- tity the climate response to aerosol-mediated surface temper- ature changes, and hence alongside the time-evolving vari- ables related to aerosol radiative effects may not be a useful proxy for aerosol-driven slow precipitation changes (which occur through surface temperature change). However, such a line of thinking does not explain the difference between our results and those from a multi-model ensemble (Allen et al., 2015). ships may be indicative of important atmospheric param- eter effects on tropical precipitation shifts. For example, Kang et al. (2008, 2009) showed how tuning a convective parameter related to entrainment can alter cloud radiative properties and cause a range in magnitude of ITCZ shifts for a given inter-hemispheric thermal forcing. In our PPE, the parameter that controls shallow convective core radia- tive effects (cca_sh_knob) and the parameter that controls the sensitivity of mid-level convection to relative humidity and entrainment (ent_fac_md) have a relationship with the 8ITCZ trend over some regions. Hence, both these param- eters could modulate the sensitivity of ITCZ shifts through altering cloud radiative feedbacks. In the global and At- lantic means, the scaling of natural dimethyl sulfide emis- sions flux (ps_natl_dms_emiss), which could alter the hemi- spheric contrast of aerosol forcing, has a relationship with the 8ITCZ trend. Parameters from the land surface (related to soil moisture thresholds, psm; and altering the temperature dependence of photosynthesis, tupp_io) and the cloud mi- crophysics (aspect ratio of ice particles, ar) scheme also have a relationship with 8ITCZ trend over some regions. These re- sults are at best indications of possible parameter effects. Our correlations are calculated using only 13 ensemble members that conflate the uncertainty in 47 model parameters. So, fur- ther simulations would be needed to clarify parametric ef- fects on tropical precipitation shifts. In addition, in the At- Our 13 PPE members include the combined effects of per- turbations to eight parameters in the aerosol model, along- side many parameters in the physical atmosphere model. So in our PPE, any relationship between anthropogenic aerosol radiative forcing and tropical precipitation might be masked by the effect of perturbations to physical atmosphere param- eters. The strongest correlations between the 8ITCZ trend over 1950 to 1985 and individual perturbed parameters in our PPE are shown in Fig. S12. Some of these relation- https://doi.org/10.5194/esd-13-1215-2022 Earth Syst. Dynam., 13, 1215–1232, 2022 A. H. https://doi.org/10.5194/esd-13-1215-2022 A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts 1225 A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts 1226 Figure 6. Scatter plot of trend in 5-year rolling mean 8ITCZ in 2006 to 2060 against the trend in inter-hemispheric (60◦S–60◦N) surface air temperature (a) and 1860 to 2005 anthropogenic aerosol ERF (b, c) for global (left), Atlantic (middle), and Pacific regional means (right). The Spearman rank correlation coefficient is shown at the top left of each plot. Trend lines are shown when r > 0.5. Figure 6. Scatter plot of trend in 5-year rolling mean 8ITCZ in 2006 to 2060 against the trend in inter-hemispheric (60◦S–60◦N) surface air temperature (a) and 1860 to 2005 anthropogenic aerosol ERF (b, c) for global (left), Atlantic (middle), and Pacific regional means (right). The Spearman rank correlation coefficient is shown at the top left of each plot. Trend lines are shown when r > 0.5. ing figure with inter-hemispheric variables calculated only over the ocean. The top panel of Fig. 6 shows that there is a relationship between the 8ITCZ trend and the trend in inter-hemispheric surface temperature in each of the RCP ensembles (consistent with what we find for the historical period). For each of the RCP scenario ensembles the rela- tionship between the global 8ITCZ trend and the trend in inter-hemispheric surface temperature is stronger than we found in the longer historical trends (Table 1) but weaker than those beginning in 1950, which were most affected by volcanic eruptions (Fig. 5). Contrary to the historical period, we identify a relationship between the 8ITCZ trend over 2006 to 2060 and inter-hemispheric 1860 to 2005 aerosol ERF in the Pacific under RCP8.5 (r = −0.69). Emission reduc- tions in Asia will dominate future global reductions in an- thropogenic aerosol emissions (Lund et al., 2019) and align with our results showing that there is a strong relationship be- tween the magnitude of inter-hemispheric aerosol ERF and tropical precipitation shifts, particularly in the Pacific re- gion. However, the lower latitude of emission reductions over Asia, in comparison to Europe or North America, may af- fect the sensitivity of the ITCZ shift. There is no relationship in the Atlantic, and consequently the global mean response (r = −0.47) is weaker than the Pacific. Figure S8 shows there is also a suggestion of a relationship between the inter- hemispheric temperature trend and 1860 to 2005 aerosol ERF in the Pacific, which is slightly stronger over the ocean (not shown). 3.2 Tropical precipitation shifts up to mid-21st century Here, we examine the relationship between the 8ITCZ trend and our inter-hemispheric variables over 2006 to 2060 under emission scenarios RCP8.5 and RCP2.6. The reductions in anthropogenic aerosol emissions and consequential warming of the Northern Hemisphere in the 21st century have been projected to cause a northward shift in the position of ITCZ and tropical precipitation (Hwang et al., 2013; Allen, 2015). Although the warming caused by increasing CO2 emissions is more homogeneous, it can also lead to a migration in the position of the ITCZ and tropical precipitation. For exam- ple, climate responses to GHG-induced warming such as ice– albedo feedbacks, the land-dominated Northern Hemisphere warming, cloud, and ocean heat content changes may lead to a northward shift in the ITCZ, whereas responses such as AMOC weakening and enhanced longwave cooling may lead to a southward shift (McFarlane and Frierson, 2017). These drivers of tropical precipitation shifts in the 21st century will also act on different timescales. Results from multi-model studies have mixed conclusions on how zonal mean tropical precipitation will migrate in the future. Figure 3 shows the 5-year rolling mean evolution of 8ITCZ up to 2060 across our PPE. For scenario RCP8.5, the ensem- ble mean 8ITCZ remains steady globally and in the Atlantic up to the mid-21st century, with a slight southward migra- tion in the Pacific. For RCP2.6, there is a northward migra- tion of 8ITCZ up to mid-century globally and in the Atlantic, followed by a southward migration from 2050 to 2060. Yet in the Pacific, the northward migration ends around 2035 and is followed by a strong, but brief, southward migration. The 8ITCZ exhibits greater variability in scenario RCP2.6, which is most pronounced in the Pacific. Figure 4b and c show the 8ITCZ trend over 2006 to 2060 across individual members in our PPE and as a density func- tion. Figure 4b also shows an estimate of the impact of Figure 6 shows the relationship between our inter- hemispheric variables and the 8ITCZ trend over 2006 to 2060 in RCP2.6 and RCP8.5. Figure S14 shows a correspond- Earth Syst. Dynam., 13, 1215–1232, 2022 https://doi.org/10.5194/esd-13-1215-2022 A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts These results show that in our PPE, ensemble members that have a stronger difference in inter-hemispheric aerosol ERF over the industrial period, and more warming in the Northern Hemisphere in the near-term future under scenario RCP8.5, simulate further northward migrations in Earth Syst. Dynam., 13, 1215–1232, 2022 https://doi.org/10.5194/esd-13-1215-2022 A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts 1227 strongest relationship between tropical precipitation shifts and the hemispheric contrast in temperature and implied total radiative forcing over the same period (i.e. ensemble mem- bers with more cooling in the Northern Hemisphere simu- lated a further southward shift of the ITCZ). Both the long- term trends and the rapid response to volcanic eruptions are in line with the theoretical energetic framework and mod- elling studies that have shown the zonal mean position of the ITCZ and corresponding tropical precipitation migrate in response to an anomalous inter-hemispheric energy balance (Kang et al., 2018). tropical precipitation, particularly in the Pacific region. It is surprising, however, that there is no clear relationship be- tween the 8ITCZ trend and inter-hemispheric aerosol ERF in RCP2.6, where we expected faster aerosol emission reduc- tions to yield a clearer tropical precipitation response. Pos- sible causes of a stronger relationship between tropical pre- cipitation shifts and aerosol radiative forcing under RCP8.5 could be due to feedbacks between warming and aerosol ra- diative forcing. For example, aerosol residence times and as- sociated net radiative effects may increase in a warmer cli- mate (Bellouin et al., 2011; Takemura, 2020), which may amplify the effect of anthropogenic aerosol forcing on ITCZ position in RCP8.5 compared to RCP2.6. Despite a contemporaneous relationship between tropical precipitation shifts and the trend in the inter-hemispheric dif- ference in temperature and implied total forcing, we find no statistically significant relationship between the strength of inter-hemispheric 1860 to 1975 anthropogenic aerosol ERF and the magnitude of tropical precipitation shifts in the PPE over the 20th century, which contradicts results from CMIP5 (Allen et al., 2015). We propose multiple hypotheses for this different result. Overall, our results suggest that being unable to isolate forced changes from those due to internal variabil- ity (due to an absence of initial condition ensembles of our PPE members) and accounting for single-model uncertainty obscure the role of anthropogenic aerosol forced responses in our ensemble over the 20th century. A. H. Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts Our analysis of 21st century tropical precipitation shifts suggests that the uncertainty in the inter-hemispheric differ- ence in aerosol ERF contributes to the spread of projected tropical precipitation shifts across our ensemble in the near- term future under RCP8.5. This is especially the case in the Pacific regional mean, as near-term future aerosol reduction will be driven by reductions from Asia. Our analysis of the historical period showed that the position of tropical precip- itation can be strongly modulated by major volcanic erup- tions that lead to inter-hemispheric differences in tempera- ture. Hence, any predictive skill for future shifts in tropical precipitation will also be limited by the effect of any future major volcanic eruptions that induce differences in hemi- spheric energy balance. Drivers of future tropical precipitation shifts are harder to disentangle, as both forced responses and climate feedbacks due to warming will have a bearing on the direction and mag- nitude of ITCZ shifts over the 21st century (McFarlane and Frierson, 2017). In the near-term future (up to 2060) globally our ensemble mean shows a negligible migration in tropical precipitation in RCP8.5 and a further northward migration in tropical precipitation in RCP2.6. The further northward migration in RCP2.6 compared to RCP8.5 is likely due to a combination of a faster reduction of anthropogenic aerosol emissions, in combination with warming-induced feedbacks (such as AMOC weakening) having a greater modulation of the regional ITCZ position in RCP8.5. We do find en- semble members that have a stronger positive trend in inter- hemispheric temperature and forcing (i.e. due to more warm- ing in the Northern Hemisphere) simulate further northward migrations in tropical precipitation. Earth Syst. Dynam., 13, 1215–1232, 2022 https://doi.org/10.5194/esd-13-1215-2022 4 Conclusion Yet, it is surprising that this logic does not follow through to there being a stronger relationship between trop- ical precipitation shifts and aerosol forcing in RCP2.6, sug- gesting climate feedbacks due to warming can influence the sensitivity of the climate response to aerosol forcing (Take- mura, 2020; Nazarenko et al., 2017). Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/esd-13-1215-2022-supplement. Author contributions. AHP, BBBB, LAR, and KSC designed the idea for this study. The analysis of data and figure prepara- tion were completed by AHP. BBBB, DMHS, and JWR extracted data and performed additional aerosol forcing simulations for the HadGEM3-GC3.05 PPE. All co-authors provided discussion on the interpretation of results. AHP wrote the manuscript with advice from all co-authors. Overall, our study suggests the persistent uncertainty in aerosol ERF plays a role in how accurately we can project zonal mean tropical precipitation shifts in the near-term fu- ture under RCP8.5. However, any predictive skill for future tropical precipitation shifts will also be limited by the ef- fect of future major volcanic eruptions that can temporarily shift tropical precipitation. Our study presents open ques- tions on the role of anthropogenic aerosol radiative forcing in modulating tropical precipitation shifts over the histor- ical and future periods in climate models, which we can- not definitively answer here because our experiment is de- signed to sample single-model uncertainty and thus has a relatively small sample size and neglects the effects of in- ternal variability. Additional experiments that clarify the role of aerosols in near-term future tropical precipitation shifts are also needed. In a broader analysis involving multi-model and other single-model ensembles, we could further develop our understanding of the relationship between feedbacks due to warming, future aerosol forcing, and tropical precipitation shifts up to the mid-21st century across multiple emission scenarios. Hence, we suggest future work investigating the role between aerosol forcing and tropical precipitation shifts in the CMIP6 multi-model ensemble and other single-model ensembles that span a range of aerosol radiative forcing val- ues. Competing interests. The contact author has declared that none of the authors has any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Financial support. Amy H. 4 Conclusion The inter-hemispheric nature of anthropogenic aerosol radia- tive forcing associated with evolving anthropogenic aerosol emissions has been linked to driving tropical precipitation shifts during the latter half of the 20th century and over the 21st century (Allen, 2015; Allen et al., 2015; Hwang et al., 2013; Chang et al., 2011; Rotstayn et al., 2015; Chemke and Dagan, 2018). In the CMIP5 multi-model ensemble there is a strong correlation between the strength of pre-industrial to present-day inter-hemispheric aerosol forcing and the latitu- dinal shift in tropical precipitation over 1950 to 1985 (Allen et al., 2015). We have used a perturbed parameter ensemble of the HadGEM3-GC3.05 climate model that spans a range of aerosol forcing comparable to current-generation climate models to further investigate the relationship between anthro- pogenic aerosol forcing and tropical precipitation shifts. In contrast to the historical time period, we find a rela- tionship between the strength of inter-hemispheric 1860 to 2005 anthropogenic aerosol ERF (which we use as a proxy of present-day aerosol influence) and future tropical precip- itation shifts under RCP8.5. This relationship is strongest in the Pacific where Asian anthropogenic aerosols have a strong historical and future influence. On the premise that present- day anthropogenic aerosol influence is informative about fu- ture anthropogenic aerosol influence, this indicates ensem- ble members with a large hemispheric difference in historical aerosol radiative forcing will have a further northward trop- ical precipitation shift in response to future aerosol reduc- tions. Faster anthropogenic aerosol emission reductions are In the 20th century as anthropogenic aerosol emissions in- creased, our PPE mean shows a long-term southward mi- gration in the latitudinal position of tropical precipitation globally and in the Atlantic and Pacific up to around 1985 (e.g. 0.01◦latitude per year globally over 1940 to 1985). Over the 20th century there are also brief shifts in tropi- cal precipitation in response to major volcanic eruptions. Of the time periods we analysed, the 1950 to 1985 time period which encapsulates two major volcanic eruptions had the Earth Syst. 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Diao, C., Xu, Y., and Xie, S.-P.: Anthropogenic aerosol ef- fects on tropospheric circulation and sea surface temper- ature (1980–2020): separating the role of zonally asym- metric forcings, Atmos. Chem. Phys., 21, 18499–18518, https://doi.org/10.5194/acp-21-18499-2021, 2021. Biasutti, M. 4 Conclusion Peace et al.: Evaluating uncertainty in aerosol forcing of tropical precipitation shifts 1229 4 Conclusion Peace was funded by a doctoral training grant from the Natural Environment Research Coun- cil (NERC) with a CASE studentship with the Met Office Hadley Centre under grant no. NE/P010547/1. Leighton A. Regayre and Ken S. Carslaw received funding from the NERC under grant nos. NE/J024252/1 (GASSP) and NE/P013406/1 (A-CURE). Leighton A. Regayre and Ken S. Carslaw received funding from the European Union’s Horizon 2020 research and innovation pro- gramme under grant agreement no. 821205 (the FORCeS project). Ben B. B. Booth and David M. H. Sexton were supported by the Met Office Hadley Centre Climate Programme funded by BEIS. John W. Rostron was supported by the UK-China Re- search & Innovation Partnership Fund through the Met Office Cli- mate Science for Service Partnership (CSSP) China as part of the Newton Fund. The work contribution of Céline J. W. Bon- fils was performed under the auspices of the US Department of Energy (DOE) by Lawrence Livermore National Laboratory un- der contract no. DE-AC52–07NA27344. Céline J. W. Bonfils was also supported by the DOE Regional and Global Model Analy- sis Program under the PCMDI SFA. The JASMIN facility (https: //jasmin.ac.uk/, last access: 18 August 2022) via the Centre for En- vironmental Data Analysis was used for data processing, which is funded by the NERC and the UK Space Agency and delivered by the Science and Technology Facilities Council. Code and data availability. The gridded global precipi- tation and temperature data for the historical and RCP8.5 HadGEM3-GC3.05 PPE simulations used in this study are available in the CEDA Archive (https://catalogue.ceda.ac.uk/ uuid/97bc0c622a24489aa105f5b8a8efa3f0; Met Office Hadley Centre, 2018). Gridded global data for HadGEM3-GC3.1 initial condition ensembles that were submitted to CMIP6 are available for download (https://esgf-index1.ceda.ac.uk/projects/esgf-ceda/; ESGF-CEDA, 2022). The historical simulations are available under CMIP DECK and future SSP5-8.5 under ScenarioMIP. The model source IDs are HadGEM3-GC31-LL and HadGEM3- GC31-MM (Ridley et al., 2019a). The four members of each historical ensemble are identified by the following variant labels: r1i1p1f3 to r4i1p1f3. Simplified data and code required to re- produce the main figures in this article is provided on Zenodo (https://doi.org/10.5281/zenodo.6979591; Peace et al., 2022). 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Research and Discussion on the Relationships between Noise-Induced Hearing Loss and ATP2B2 Gene Polymorphism
International journal of genomics
2,019
cc-by
6,339
Hindawi International Journal of Genomics Volume 2019, Article ID 5048943, 8 pages https://doi.org/10.1155/2019/5048943 Hindawi International Journal of Genomics Volume 2019, Article ID 5048943, 8 pages https://doi.org/10.1155/2019/5048943 Suhao Zhang ,1 Enmin Ding ,2 Haoyang Yin ,3 Hengdong Zhang ,2 and Baoli Zhu 1,2,3 1School of Public Health, Nantong University, Nantong, Jiangsu Province, China 2Institute of Occupational Disease Prevention, Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, Jiangsu Province, China 3Center for Global Health, China International Cooperation Center for Environment and Human Health, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu Province, China Correspondence should be addressed to Baoli Zhu; zhubljscdc@126.com Received 30 August 2019; Accepted 23 October 2019; Published 1 December 2019 Academic Editor: Byung-Hoon Jeong Academic Editor: Byung-Hoon Jeong Copyright © 2019 Suhao Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright © 2019 Suhao Zhang et al. This is an open access article distributed under the Creative Commons A which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is Long-term and continuous noise exposure can result in noise-induced hearing loss (NIHL), which is a worldwide problem resulting from the interaction of environmental and genetic factors. The ATP2B2 gene polymorphism can destroy cochlear hair cells and increase the risk of NIHL. A case-control study of 760 Chinese textile workers was conducted to investigate the relationship between ATP2B2 polymorphisms and NIHL susceptibility. Venous blood was collected and questionnaires were conducted by professional physicians. A case group and a control group which were typed by individuals’ pure-tone audiometry test results were set. Three polymorphism sites of ATP2B2 were genotyped by using the PCR technique. Analysis results revealed that the C allele of rs3209637 (95%CI = 1:08–2.58, odds ratio ðORÞ = 1:67, P = 0:027) was a dangerous factor and could add to risks of NIHL in the Chinese employees. The data of stratified analysis revealed that individuals who are exposed to noise > 95 dB with the rs3209637 C genotype have a higher susceptibility to NIHL (OR = 1:34, 95%CI = 1:07–1.68). Multifactor dimensionality reduction analysis revealed that the interaction between rs14154 and rs3209637 is linked to increased NIHL risk, and for the interaction among rs14154, smoking and drinking had the same function (OR = 1:54 and 1.77, 95%CI = 1:15–2.07, 1.33–2.37, and P = 0:0037 and P < 0:0001, respectively). Our results suggest that genetic polymorphism rs3209637 C within ATP2B2 is a risk factor for NIHL among Chinese employees and rs3209637 C could be a potential biomarker for NIHL patients. 1. Background From previous studies, it can be concluded that NIHL is a multifactor disease influenced by external environmental fac- tors and internal genetic factors [2]. Both physical factors, such as noise, chemicals, and heat, and personal behaviors could change the susceptibility of NIHL, such as smoking, drinking, and medical factors [3–8]. Eliminating interference from external environmental factors, individuals always showed different degrees of hearing loss under the same level of noise exposure, indicating that genetic susceptibility is a significant catalyst in the development of NIHL [9, 10]. Sliwinska-Kowalska and Pawelczyk [1] found that gene- knockout mice have expressed more susceptibility to noise than their wild-type littermates and proved that genetic Noise is a common occupational hazard in modern society which can cause permanent and irreversible damage to the human hearing system. NIHL is a primary occupational disorder worldwide and the second most common type of sensorineural hearing impairment with the second highest incidence [1]. According to reports, there are 10 million people suffering from NIHL in the USA and the group of NIHL patients in China has expanded 77.8% in three years (2010-2012). With the increasing number of NIHL patients, NIHL has caused serious harm to workers’ health and socio- economic conditions and becomes an important aspect of occupational prevention and control. International Journal of Genomics 2 personal noise dosimeter. The Quest NoisePro DL multi- functional individual noise dosimeter (Quest, USA) was used to measure the individual noise exposure dose during work hours. Then, an 8-hour equivalent continuous sound level (A) was calculated. polymorphisms contribute to occurrence of NIHL. Many genetic experiments had demonstrated that single nucleotide polymorphisms (SNPs) in the DFNA5, FOXO3, heat shock protein 70, and EYA4 genes are genetic risk factors for NIHL in humans and can promote or reduce the occurrence of NIHL with the participation of external factors [10–12]. 2.4. Defining Normal and Hearing Loss Subjects. Participants were detected for audiometry and divided into the hearing loss group and the normal hearing group according to diag- nostic criteria for occupational noise-induced deafness in China (GBZ 49-2007). In this research, workers who contin- ued to be exposed to at least 85 dB noise during an 8-hour working day were defined as suffering from occupational noise exposure. Participants with bilateral threshold devia- tions of more than 25 dB at high and low frequencies were classified as the hearing loss groups. 1. Background Oppositely, participants with bilateral threshold deviations of less than 25 dB at high and low frequencies were classified as the normal hearing groups. We determined patient population and then matched the control groups based on sex, age, and degrees of noise exposure. 380 NIHL cases and 380 controls from the individ- uals met our requirements and were selected finally. NIHL with the participation of external factors [10 12]. ATPase, calcium-transporting, plasma membrane 2 (ATP2B2) belongs to the ATP2B gene family and lies on human chromosome 3p25.3 and encodes Ca2+ pump PMCA2 whichisscatteredaroundtheplasmamembraneandfunctions to pump Ca2+ out of the cell with ATP. High-level expression of ATP2B2 in cochlear outer hair cells maintains the homeo- stasis of intracellular calcium [13], and when individuals were exposedtonoise,theexpressionlevelofATP2B2wouldchange and lead to the changes of calcium concentration in hair cells and extracellular calcium concentration in the inner ear [14]. ATP2B2 can be an early warning gene for NIHL, and low expression of ATP2B2 would lead to neurodevelopmental defects of auditory systems and result in hearing loss [15, 16]. Although many animal studies were performed to reveal the associations between ATP2B2 and the auditory system, the associations within the population were rarely explored [17, 18]. Documenting previous experiments, we speculated that the polymorphisms in ATP2B2 could be one of the risk factors for NIHL. A case-control study was designed and conformed to analyze the potential link between ATP2B2 SNPs (rs1719571, rs14154, and rs3209637) and genetic sus- ceptibility to NIHL, and the mechanism by which ATP2B2 leads to NIHL is discussed in this article. p p ATPase, calcium-transporting, plasma membrane 2 (ATP2B2) belongs to the ATP2B gene family and lies on human chromosome 3p25.3 and encodes Ca2+ pump PMCA2 whichisscatteredaroundtheplasmamembraneandfunctions to pump Ca2+ out of the cell with ATP. High-level expression of ATP2B2 in cochlear outer hair cells maintains the homeo- stasis of intracellular calcium [13], and when individuals were exposedtonoise,theexpressionlevelofATP2B2wouldchange and lead to the changes of calcium concentration in hair cells and extracellular calcium concentration in the inner ear [14]. ATP2B2 can be an early warning gene for NIHL, and low expression of ATP2B2 would lead to neurodevelopmental defects of auditory systems and result in hearing loss [15, 16]. Although many animal studies were performed to reveal the associations between ATP2B2 and the auditory system, the associations within the population were rarely explored [17, 18]. 2. Materials and Methods 2.6. SNP Selection and Genotyping. ATP2B2 was selected as the target SNPs based on the 1000 Genomes Project and previous literature results. The standards for identifying SNPs included (a) minor allele frequency (MAF) of the Chinese Han population ðCHBÞ > 0:10 and (b) linkage disequilibrium r2 value >0.8. Haploview 4.2 software was adopted to measure the LD patterns of SNPs with r -squared value and distinguished three SNPs (rs1719571, rs14154, and rs3209637) in ATP2B2. Combining with a review of literature from PubMed and Web of Science, we found that the rs1719571, rs14154, and rs3209637 of the ATP2B2 gene were studied frequently and thus selected for genotyping. 2.1. Research Objectives. This study had achieved authoriza- tion from the Research Ethics Committee of Jiangsu Provin- cial Center for Disease Prevention and Control (JSCDC), and all the studies were conducted in accordance with correlative standards and rules. Each participant signed the informed consent before the studies. The industrial employees in a Chinese textile manufactory were recruited and underwent medical health examinations every year as executed by the JSCDC. Occupational health test projects principally consisted of physiological and biochemical examinations, routine physical examination, and pure-tone audiometry (PTA). In health tests, personal drug history, smoking status, drinking status, and commonly used medicines were investi- gated by questionnaire. The participants in the following situations were removed: people had diseases which could influence hearing thresholds, such as otitis media, diabetes, and nephropathy, and participants had taken or were taking ototoxic drugs. Afterwards, 760 individuals confirmed to our requirements and participated in the studies. Using predesigned commercial genotyping assays, poly- morphic genotypes were selected with ABI company TaqMan SNP genotyping assays. Samples were genotyped by an ABI 7900 HT real-time PCR system. Then, the outcomes were analyzed by the ABI 7900 system sequence detection software. 2.7. Statistical Analysis. SPSS 23.0 software (IBM, USA) was adopted to analyze the experimental data. The goodness-of- fit χ2 tests were used for the Hardy-Weinberg equilibrium rule of the SNPs in ATP2B2 genes in the control group. Classification variables are described in percentages, and continuous variables are described in the mean ± SD. Con- ditional logistic regression models were adjusted for age, sex, smoking, and drinking and used to calculate the odds ratios (ORs) and 95% confidence intervals (95% CI) for genotypes [12]. The interaction between gene and dangerous 2.2. PTA and NIHL Evaluation. 1. Background Documenting previous experiments, we speculated that the polymorphisms in ATP2B2 could be one of the risk factors for NIHL. A case-control study was designed and conformed to analyze the potential link between ATP2B2 SNPs (rs1719571, rs14154, and rs3209637) and genetic sus- ceptibility to NIHL, and the mechanism by which ATP2B2 leads to NIHL is discussed in this article. 2.5. DNA Extraction. Three milliliters of peripheral blood were collected in EDTA-containing anticoagulant tubes for DNA extraction and genotyping. DNA was extracted from participants’ blood samples by the QIAcube HT and QIAamp 96DNA QIAcube HT Kit under guidance of the manufacturers’ instructions. The abstracted DNA was stored up at -20°C for future use. 2. Materials and Methods Variables Case group (n = 380) Control group (n = 380) P n % n % Age (years) Mean ± SD 39:82 ± 6:59 40:09 ± 6:32 0.567a Sex Male 358 94.2 355 93.4 0.652b Female 22 5.8 25 6.6 Tobacco use Now 223 58.7 210 55.3 0.501b Ever 9 2.4 13 3.4 Never 148 38.9 157 41.3 Alcohol consumption Now 169 44.5 173 45.5 0.836b Ever 8 2.1 10 2.6 Never 203 53.4 197 51.8 Work time with noise (years) Mean ± SD 17:35 ± 7:60 16:94 ± 7:16 0.452a Expose level with noise (dB) Mean ± SD 89:52 ± 7:26 90:09 ± 6:98 0.269a High-frequency hearing threshold (dB) Mean ± SD 36:74 ± 10:23 13:66 ± 4:20 <0.001c ≤25 0 0.0 380 100.0 >25 380 100.0 0 0.0 aStudents’ t-test. bTwo-sided χ2 test. cFisher’s exact test. Table 2: General information of selected SNPs and Hardy-Weinberg test. SNP Alleles Chromosome Functional consequence MAF P for HWEb Control Databasea rs1719571 A/G 3:10327496 3′UTR 0.357 0.360 0.903 rs14154 C/G 3:10326429 3′UTR 0.378 0.383 0.751 rs3209637 C/T 3:10327264 3′UTR 0.463 0.465 0.959 aData from NCBI dbSNP. bP value of Hardy-Weinberg test. 3 International Journal of Genomics Table 1: Demographic characteristics of study subjects. Table 1: Demographic characteristics of study subjects. Variables Case group (n = 380) Control group (n = 380) P n % n % Age (years) Mean ± SD 39:82 ± 6:59 40:09 ± 6:32 0.567a Sex Male 358 94.2 355 93.4 0.652b Female 22 5.8 25 6.6 Tobacco use Now 223 58.7 210 55.3 0.501b Ever 9 2.4 13 3.4 Never 148 38.9 157 41.3 Alcohol consumption Now 169 44.5 173 45.5 0.836b Ever 8 2.1 10 2.6 Never 203 53.4 197 51.8 Work time with noise (years) Mean ± SD 17:35 ± 7:60 16:94 ± 7:16 0.452a Expose level with noise (dB) Mean ± SD 89:52 ± 7:26 90:09 ± 6:98 0.269a High-frequency hearing threshold (dB) Mean ± SD 36:74 ± 10:23 13:66 ± 4:20 <0.001c ≤25 0 0.0 380 100.0 >25 380 100.0 0 0.0 aStudents’ t-test. bTwo-sided χ2 test. cFisher’s exact test. Table 2: General information of selected SNPs and Hardy-Weinberg test. Table 2: General information of selected SNPs and Hardy-Weinberg test. 2. Materials and Methods SNP Alleles Chromosome Functional consequence MAF P for HWEb Control Databasea rs1719571 A/G 3:10327496 3′UTR 0.357 0.360 0.903 rs14154 C/G 3:10326429 3′UTR 0.378 0.383 0.751 rs3209637 C/T 3:10327264 3′UTR 0.463 0.465 0.959 aData from NCBI dbSNP. bP value of Hardy-Weinberg test. exposed to low-frequency noise (13:66 ± 4:20; P < 0:001). The results of three chosen SNPs and Hardy-Weinberg tests are shown in Table 2, and χ2 tests indicated every chosen SNP was in Hardy-Weinberg equilibrium (P > 0:05). factors was analyzed by multifactor dimensionality reduction (MDR). We adopted P < 0:05 as the standard for measuring statistical significance. 2. Materials and Methods Each participant avoided noise exposure for at least 12 hours and was tested for the PTA experiment in a soundproof room by using an audiom- eter (Madsen, Taastrup, Denmark). 2.3. Individual Noise Exposure Measurement. The random sampling method is adopted to select workers to wear a International Journal of Genomics International Journal of Genomics 3 3 factors was analyzed by multifactor dimensionality reduction (MDR). We adopted P < 0:05 as the standard for measuring statistical significance. 3. Results 3.1. Demographic Characteristics of Study Individuals and Hardy-Weinberg Tests of ATP2B2 Genotype. The common features, such as age, sex, smoking status, drinking status, working time with noise, noise level, and high-frequency hearing thresholds of the NIHL groups and controls, are shown in Table 1. Comparing the general characteristics between the NIHL groups and control groups, no signifi- cant differences were found between different populations (P > 0:05) except the hearing threshold. The case groups exposed to high-frequency noise have a higher average hear- ing threshold (36:74 ± 10:23) than the control groups exposed to low-frequency noise (13:66 ± 4:20; P < 0:001). The results of three chosen SNPs and Hardy-Weinberg tests are shown in Table 2, and χ2 tests indicated every chosen SNP was in Hardy-Weinberg equilibrium (P > 0:05). 3.2. Association between ATP2B2 SNPs and Risks of NIHL. The relationship between ATP2B2 and the NIHL risk was analyzed by multivariate analysis. Three ATP2B2 SNPs were chosen to genotype in the 760 noise-exposed employees (380 NIHL patients and 380 controls). The genotype and allele distributions of rs1719571, rs14154, and rs3209637 under recessive, dominant, codominant, and allelic models are shown in Table 3. Table 3 showed significant variances of genotype frequencies of rs3209637 existed obviously between the cases and controls under the codominant model (P = 0:027). People with a higher proportion of the ATP2B2 C allele are more susceptible to NIHL with reference to the Table 1: Demographic characteristics of study subjects. 3. Results 3.2. Association between ATP2B2 SNPs and Risks of NIHL. The relationship between ATP2B2 and the NIHL risk was analyzed by multivariate analysis. Three ATP2B2 SNPs were chosen to genotype in the 760 noise-exposed employees (380 NIHL patients and 380 controls). The genotype and allele distributions of rs1719571, rs14154, and rs3209637 under recessive, dominant, codominant, and allelic models are shown in Table 3. Table 3 showed significant variances of genotype frequencies of rs3209637 existed obviously between the cases and controls under the codominant model (P = 0:027). People with a higher proportion of the ATP2B2 C allele are more susceptible to NIHL with reference to the 3.1. Demographic Characteristics of Study Individuals and Hardy-Weinberg Tests of ATP2B2 Genotype. The common features, such as age, sex, smoking status, drinking status, working time with noise, noise level, and high-frequency hearing thresholds of the NIHL groups and controls, are shown in Table 1. Comparing the general characteristics between the NIHL groups and control groups, no signifi- cant differences were found between different populations (P > 0:05) except the hearing threshold. The case groups exposed to high-frequency noise have a higher average hear- ing threshold (36:74 ± 10:23) than the control groups International Journal of Genomics International Journal of Genomics 4 Table 3: Distribution of three polymorphisms and the association with NIHL. Table 3: Distribution of three polymorphisms and the association with NIHL. 3. Results Genetic models Genotypes Case group Control group Pa Adjusted OR (95% CI)b n = 380 % n = 380 % rs1719571 Codominant AA 141 37.1 161 42.4 0.332 1.00 (ref.) AG 183 48.2 167 43.9 1.25 (0.92-1.70) GG 56 14.7 52 13.7 1.26 (0.81-1.96) Dominant AA 141 37.1 161 42.4 0.138 1.00 (ref.) GG+AG 239 62.9 219 57.6 1.25 (0.93-1.67) Recessive AG+AA 324 85.3 328 86.3 0.678 1.00 (ref.) GG 56 14.7 52 13.7 1.11 (0.74-1.68) Alleles A 465 61.2 489 64.3 0.203 1.00 (ref.) G 295 38.8 271 35.7 1.15 (0.94-1.42) rs14154 Codominant CC 119 31.3 144 37.9 0.129 1.00 (ref.) CG 211 55.5 185 48.7 1.39 (1.02-1.90) GG 50 13.2 51 13.4 1.21 (0.76-1.92) Dominant CC 119 31.3 144 37.9 0.057 1.00 (ref.) CG+GG 261 68.7 236 62.1 1.35 (1.00-1.83) Recessive CG+CC 330 86.8 329 86.6 0.915 1.00 (ref.) GG 50 13.2 51 13.4 0.99 (0.65-1.51) Alleles C 449 59.1 473 62.2 0.208 1.00 (ref.) G 311 40.9 287 37.8 1.15 (0.94-1.42) rs3209637 Codominant TT 83 21.8 92 24.2 0.027 1.00 (ref.) CT 203 53.4 224 58.9 1.01 (0.71-1.44) CC 94 24.7 64 16.8 1.67 (1.08-2.58) Dominant TT 83 21.8 92 24.2 0.438 1.00 (ref.) CC+CT 297 78.2 288 75.8 1.15 (0.82-1.62) Recessive CT+TT 286 75.3 316 83.2 0.007 1.00 (ref.) CC 94 24.7 64 16.8 1.66 (1.16-2.37) Alleles T 369 48.6 408 53.7 0.045 1.00 (ref.) C 391 51.4 352 46.3 1.24 (1.01-1.52) aTwo-sided χ2 test. bAdjusted for age, sex, tobacco use, and alcohol consumption in the logistic regression model. ATP2B2 T allele (P = 0:033). Moreover, the rs3209637 CC was significantly related to enhancive NIHL risks under the recessive model (P = 0:007). Logistic regression analysis adjusted for sex, age, smoking, and drinking revealed that the rs3209637 CC genotype increased NIHL hazard (OR = 1:66, 95%CI = 1:16–2.37). Under the allelic model, the rs3209637 C (95%CI = 1:01–1.52, OR = 1:24) allele revealed an enhancive risk for NIHL (P = 0:045). 3.4. Interactions among SNPs and Dangerous Factors. The consequences of the multifactor dimensionality analysis of interaction among three SNPs and other factors are expressed in Table 5. The analysis revealed that for the inter- actions between rs14154 and rs3209637 and among rs14154, smoking and drinking were associated with increased NIHL risk (OR = 1:54 and 1.77, P = 0:0037 and P < 0:0001, cor- respondingly). Diagrams of the best-fit model are shown in Figure 1. 3. Results 3.3. Interactions between rs1719571, rs14154, and rs3209637 Polymorphisms and Cumulative Noise Exposure. The analysis results of interactions between the SNPs and cumulative noise exposure were analyzed under an allelic model and are exhibited in Table 4. 95 dB was the cumulative average noise exposure and chosen as the standard. There was an obviously significant variance between the case and control groups with rs3209637 C when individuals’ cumulative noise exposure was >95 dB (95%CI = 1:07–1.68, OR = 1:34, and P = 0:015). 4. Discussion SNP, the mutation of single nucleotides, is an ordinary phe- nomenon in the human genome and there are 15 million SNPs in all humans [19]. Although the SNPs distribute in humans widely, SNPs do not distribute homogeneously and most SNPs are in noncoding areas of the genome. Currently, there are many methods to detect the SNPs, such as allele- specific PCR. Except for the allele-specific PCR, scientists International Journal of Genomics 5 Table 4: Stratified analysis of SNPs in the allelic model. SNPs Group Alleles Cumulative noise exposure (dBA) ≤95 >95 rs1719571 Case group G 67 228 A 109 356 Control group G 43 228 A 91 398 Pa 0.276 0.373 Adjusted OR (95% CI)b 1.32 (0.82-2.13) 1.12 (0.89-1.42) rs14154 Case group G 70 241 C 106 343 Control group G 47 240 C 87 386 Pa 0.398 0.298 Adjusted OR (95% CI)b 1.24 (0.77-1.98) 1.14 (0.91-1.44) rs3209637 Case group C 88 303 T 88 281 Control group C 71 281 T 63 345 Pa 0.602 0.015 Adjusted OR (95% CI)b 0.90 (0.57-1.42) 1.34 (1.07-1.68) aTwo-sided χ2 test. bAdjusted for age, sex, tobacco use, and alcohol consumption in logistic regression model. aTwo-sided χ2 test. bAdjusted for age, sex, tobacco use, and alcohol consumption in logistic regression model Table 5: Analysis of the interaction of the 3 selected SNPs by MDR. Best model Training balanced accuracy Testing balanced accuracy Cross-validation consistency P OR (95% CI) rs3209637 0.5408 0.5105 7/10 0.0073 1.62 (1.14-2.32) rs14154∗rs3209637 0.5541 0.5158 6/10 0.0037 1.54 (1.15-2.07) rs14154∗smoke∗drink 0.5756 0.4711 5/10 <0.0001 1.77 (1.33-2.37) Table 5: Analysis of the interaction of the 3 selected SNPs by MDR. phisms could increase the NIHL susceptibility in the Chinese employees. The stratified analysis of SNPs in the alle- lic model showed NIHL risk of individuals increased when subjects’ cumulative noise exposure is >95 dB. This result showed the addition effect between cumulative noise expo- sure and rs3209637 C genotype of ATP2B2. Finally, the inter- action between rs14154 and rs3209637 increased the risk of NIHL. The rs3209637 also interacted with smoking and drinking and led to the enhanced hazard of NIHL. Our study is an association research demonstrating that the ATP2B2 genes would enhance the NIHL risk among the Chinese population. also adopt numerous ways to perform researches, for instance, cleaved amplified polymorphic sequence (CAPS), single-strand conformational polymorphism (SSCP), and denaturing gradient gel electrophoresis (DGGE) [14]. 4. Discussion Eliminating the effects of noise, alcohol could cause hearing loss and the influence was partic- ularly significant at 1000 Hz [31]. An experiment in Hong Kong found the association between drinking and NIHL and confirmed the conclusion that drinking would increase the risk of NIHL [32]. Our results were consistent with the previous experimental results and confirmed drinking and smoking were both risk factors. Our studies expanded the sample size and discussed the interaction between risk factors and genotypes. The experimental results show that there were additive effects between individual factors and SNPs, which is consistent with the conclusions of previous experiments. Ca2+ to the endolymph without the natural activator calmod- ulin [24], and mutant PMCA2 expressed in CHO cells led to calcium extrusion impairment [16]. ATP2B2 was induced by exogenous Zn2+ to change its expression and caused cell death by disrupting calcium homeostasis [25]. Disruption of calcium homeostasis was an important incentive to many diseases, like breast cancers, autism, deafness, and ASD [13, 16, 24, 26, 27]. Ca2+ to the endolymph without the natural activator calmod- ulin [24], and mutant PMCA2 expressed in CHO cells led to calcium extrusion impairment [16]. ATP2B2 was induced by exogenous Zn2+ to change its expression and caused cell death by disrupting calcium homeostasis [25]. Disruption of calcium homeostasis was an important incentive to many diseases, like breast cancers, autism, deafness, and ASD [13, 16, 24, 26, 27]. [ ] The scientists considered that ATP2B2 could be the key role during the development of NIHL and found that the absence of ATP2B2 could decrease PMCA2 concentration, affect the calcium homeostasis in hair cells, and finally cause defects of auditory systems [15, 16]. ATP2b has four sub- types: PMCA1, PMCA2, PMCA3, and PMCA4. These four subtypes are highly similar and consist of 75-80% amino acids and are distributed in different areas of the human body. PMCA1 and PMCA4 are widely distributed in the whole body, and PMCA2 mainly exists in the sensory cells of the inner ear. Many animal studies were conducted to research the association between PMCA2 and NIHL. PMCA2 can regulate the intracellular Ca2+ concentration in hair cells of static fibers. High concentration of Ca2+ in hair cells could produce toxicity and damage hair cells. As a result, the physiological functions of static fibers are damaged, the damaged static fibers would influence the human auditory system. 4. Discussion High risk cells are expressed by the black shadow if the ratio of the number of cases to the number of controls exceeds the preset value T, as low risk cells by light shadow if not more than the threshold, and empty cells with no shadow which means no cases and controls. The multifactor cells labeled as “high risk” or “low risk” are then used to assess the classification and predication accuracy, thus identifying the best model in the subsequent steps (drink 1: now; drink 2: ever; drink 3: never; smoke 1: now; smoke 2: ever; smoke 3: never). 2 Drink rs14154 0.0 0.0 0.0 1.0 1.0 1.0 2.0 2.0 2.0 2.0 2.0 5.0 GG CG CC 1 0.0 1.0 4.0 3.0 2.0 4.0 36.0 43.0 76.0 51.0 22.0 12.0 13.0 14.0 17.0 27.0 8.0 9.0 GG CG CC 1 2 3 Smoke rs14154 rs14154 GG 7.0 13.0 45.0 47.0 34.0 35.0 1.0 1.0 11.0 16.0 51.0 63.0 42.0 29.0 0.0 0.0 2.0 3.0 CG CC 3 Figure 1: Best-fit model gained by the analysis of MDR. The implications of the bars and background color in each multifactor cell are as follows. The left bars represent the sum of scores in the case and the right represents the control. High risk cells are expressed by the black shadow if the ratio of the number of cases to the number of controls exceeds the preset value T, as low risk cells by light shadow if not more than the threshold, and empty cells with no shadow which means no cases and controls. The multifactor cells labeled as “high risk” or “low risk” are then used to assess the classification and predication accuracy, thus identifying the best model in the subsequent steps (drink 1: now; drink 2: ever; drink 3: never; smoke 1: now; smoke 2: ever; smoke 3: never). In the current study, smoking and drinking were both risk factors of NIHL. The interaction among smoking, drinking, and rs3209637 had an association with NIHL. Mohammad et al. [29] had suggested smoking is a contributing factor to hearing loss. In Iran, Mofateh et al. [30] also found smoking had an additive role in hearing loss interacting with massive noise exposure in high frequency. The same situation hap- pened in Sarawak and 20 packs of cigarettes per year were found as the risk factor [29]. 4. Discussion According to the literature review, although there are many animal experiments researching the surface relation- ship between ATP2B2 and NIHL and studies on the Chinese population were rare, human mutation in ATP2B2 which described a young deaf subject presented a variant in the ATP2B2 gene associated with a variant in the Cadherin 23 gene [20]. In this study, we adopted TaqMan genotyping to analyze the three selected ATP2B2 SNPs (rs1719571, rs14154, and rs3209637) in 380 NIHL cases and 380 controls and found statistically significant associations between the three SNPs and NIHL hazard. Demographic characteristic calculations for the population ensured homogeneity between the two groups. The significant difference between the case group and the control group showed that the noise would change the high-frequency hearing threshold (P < 0:001). We also found that the rs3209637 C genotype of ATP2B2 (OR = 1:24, 95%CI = 1:01–1.52, P = 0:045) was significantly relative with a higher NIHL risk in which the result is a pow- erful evidence for our hypothesis that ATP2B2 polymor- p p Till now, the mechanism of NIHL is still unclear, but numerous scientists widely accept that the DNA impairment in cochlear hair cells seriously damages the growth of NIHL, especially when individuals are exposed to massive noise [21]. Many scientists also put forward that the apoptosis and necrosis of inner ear cells which were caused by the oxi- dative stress or metabolic products and structural damage which directly affected the cochlear structure may be the most possible etiopathogenesis of NIHL [21–23]. Some knockout mouse research demonstrated that the ATP2B2 gene encodes PMCA2 [17]. PMCA2 can pump International Journal of Genomics 6 1 2 Drink rs14154 rs14154 GG 7.0 13.0 45.0 47.0 34.0 35.0 0.0 0.0 0.0 0.0 1.0 1.0 1.0 1.0 11.0 16.0 51.0 63.0 42.0 29.0 0.0 0.0 1.0 1.0 4.0 3.0 2.0 4.0 36.0 43.0 76.0 51.0 22.0 12.0 13.0 14.0 17.0 27.0 8.0 9.0 2.0 2.0 2.0 2.0 2.0 3.0 2.0 5.0 CG CC GG CG CC GG CG CC 1 2 3 Smoke rs14154 3 Figure 1: Best-fit model gained by the analysis of MDR. The implications of the bars and background color in each multifactor cell are as follows. The left bars represent the sum of scores in the case and the right represents the control. 4. Discussion PMCA2 acts as a beneficial gene by pumping excessive Ca2+ out of hair cells to maintain the homeostasis of Ca2+ concentration in the auditory sys- tem. The genotype rs3209637 is located in 3′-UTR within ATP2B2 and could change the conserved miR-137 binding site at the end of the human ATP2B2 distal 3′UTR. mir- 137 has an important role in the human auditory system, which may demonstrate the association between rs3209637 polymorphism and NIHL [28]. p p There are still many shortcomings in our experiments. First of all, our study’s sample size may be larger than sizes in previous studies. But the effectiveness of statistical tests in our study was inadequate to discuss the small biological effects of individual SNPs. Thus, the consequent targets are performing more massive sample size researches, cohort studies, and functional experiments in the future. We could research the function of the ATP2B2 polymorphisms on NIHL hazard through these experiments. Secondly, the can- didates in our study were mostly recruited from Chinese employees and had regional restrictions. The researches had racial limitations, and experimental results were more applicable to Chinese employees and cannot be extended to other populations. 7 International Journal of Genomics International Journal of Genomics Conflicts of Interest No potential conflict of interest relevant to this article was reported. [11] X. Zhang, Y. Liu, L. Zhang et al., “Associations of genetic variations in EYA4, GRHL2 and DFNA5 with noise-induced hearing loss in Chinese population: a case-control study,” Environmental Health, vol. 14, no. 1, p. 77, 2015. Data Availability [9] E. A. Da Costa, J. C. Castro, and M. E. G. Macedo, “Iris pigmentation and susceptibility to noise-induced hearing loss,” International Journal of Audiology, vol. 47, no. 3, pp. 115–118, 2008. The data used to support the findings of this study are avail- able from the corresponding author upon request. [10] N. C. Chang, C. K. Ho, H. Y. Lin, M. L. Yu, C. Y. Chien, and K. Y. Ho, “Association of polymorphisms of heat shock pro- tein 70 with susceptibility to noise-induced hearing loss in the Taiwanese population,” Audiology and Neurotology, vol. 16, no. 3, pp. 168–174, 2011. Authors’ Contributions Suhao Zhang performed the experiments and wrote the paper. Enmin Ding, Haoyang Yin, and Hengdong Zhang collected the specimens and statistically analyzed the data. Baoli Zhu designed the research and wrote the paper. All authors read and approved the final manuscript. Suhao Zhang, Enmin Ding, and Haoyang Yin contributed equally to this work. [12] H. Guo, E. Ding, Y. Bai et al., “Association of genetic variations in FOXO3 gene with susceptibility to noise induced hearing loss in a Chinese population,” PLoS One, vol. 12, no. 12, article e0189186, 2017. [13] W. Yang, J. Liu, F. Zheng et al., “The evidence for association of ATP2B2 polymorphisms with autism in Chinese Han population,” PLoS One, vol. 8, no. 4, article e61021, 2013. [14] E. Ding, J. Liu, H. Shen et al., “Notch polymorphisms associ- ated with sensitivity of noise induced hearing loss among Chinese textile factory workers,” BMC Medical Genetics, vol. 19, no. 1, p. 168, 2018. 5. Conclusion [6] D. Henderson, M. Subramaniam, and F. A. Boettcher, “Indi- vidual susceptibility to noise-induced hearing loss: an old topic revisited,” Ear and Hearing, vol. 14, no. 3, pp. 152–168, 1993. In general, the rs3209637 C genotype of ATP2B2 may lead to a greatly increased incidence of NIHL. The analysis also demonstrates that ATP2B2 SNPs (rs1719571, rs14154, and rs3209637) have a great effect in NIHL and these SNPs could be researched and developed to play a great role in the prevention of NIHL by functioning as a biomarker. [7] H. Virokannas and H. Anttonen, “Dose-response relationship between smoking and impairment of hearing acuity in workers exposed to noise,” Scandinavian Audiology, vol. 24, no. 4, pp. 211–216, 2009. [8] N. C. Chang, M. L. Yu, K. Y. Ho, and C. K. 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GABAAR-mediated tonic inhibition differentially modulates intrinsic excitability of VIP- and SST- expressing interneurons in layers 2/3 of the somatosensory cortex
Frontiers in cellular neuroscience
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OPEN ACCESS OPEN ACCESS EDITED BY Dirk Feldmeyer, Helmholtz Association of German Research Centres (HZ), Germany REVIEWED BY Ludovic Tricoire, Sorbonne Universités, France Christian Wozny, Medical School Hamburg, Germany *CORRESPONDENCE Joanna Urban-Ciecko j.ciecko@nencki.edu.pl RECEIVED 31 July 2023 ACCEPTED 25 September 2023 PUBLISHED 12 October 2023 CITATION Bogaj K, Kaplon R and Urban-Ciecko J (2023) GABAAR-mediated tonic inhibition differentially modulates intrinsic excitability of VIP- and SST- expressing interneurons in layers 2/3 of the somatosensory cortex. Front. Cell. Neurosci. 17:1270219. doi: 10.3389/fncel.2023.1270219 EDITED BY Dirk Feldmeyer, Helmholtz Association of German Research Centres (HZ), Germany Extrasynaptic GABAA receptors (GABAARs) mediating tonic inhibition are thought to play an important role in the regulation of neuronal excitability. However, little is known about a cell type-specific tonic inhibition in molecularly distinctive types of GABAergic interneurons in the mammalian neocortex. Here, we used whole-cell patch-clamp techniques in brain slices prepared from transgenic mice expressing red fluorescent protein (TdTomato) in vasoactive intestinal polypeptide- or somatostatin- positive interneurons (VIP-INs and SST-INs, respectively) to investigate tonic and phasic GABAAR-mediated inhibition as well as effects of GABAA inhibition on intrinsic excitability of these interneurons in layers 2/3 (L2/3) of the somatosensory (barrel) cortex. We found that tonic inhibition was stronger in VIP-INs compared to SST-INs. Contrary to the literature data, tonic inhibition in SST-INs was comparable to pyramidal (Pyr) neurons. Next, tonic inhibition in both interneuron types was dependent on the activity of delta subunit-containing GABAARs. Finally, the GABAAR activity decreased intrinsic excitability of VIP-INs but not SST-INs. Altogether, our data indicate that GABAAR-mediated inhibition modulates neocortical interneurons in a type-specific manner. In contrast to L2/3 VIP-INs, intrinsic excitability of L2/3 SST-INs is immune to the GABAAR-mediated inhibition. CITATION Bogaj K, Kaplon R and Urban-Ciecko J (2023) GABAAR-mediated tonic inhibition differentially modulates intrinsic excitability of VIP- and SST- expressing interneurons in layers 2/3 of the somatosensory cortex. Front. Cell. Neurosci. 17:1270219. doi: 10.3389/fncel.2023.1270219 Front. Cell. Neurosci. 17:1270219. doi: 10.3389/fncel.2023.1270219 COPYRIGHT © 2023 Bogaj, Kaplon and Urban-Ciecko. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. KEYWORDS TYPE  Original Research PUBLISHED  12 October 2023 DOI  10.3389/fncel.2023.1270219 TYPE  Original Research PUBLISHED  12 October 2023 DOI  10.3389/fncel.2023.1270219 TYPE  Original Research PUBLISHED  12 October 2023 DOI  10.3389/fncel.2023.1270219 barrel cortex, interneurons, tonic inhibition, excitability, GABA, somatostatin interneurons, VIP interneurons frontiersin.org Introduction GABA (gamma-aminobutyric acid) is the main inhibitory neurotransmitter in the cerebral cortex and plays an essential role in the regulation of the neuronal activity through two types of receptors (GABAARs and GABABRs). Both classes of receptors evoke two forms of inhibition: phasic and tonic (Lee and Maguire, 2014). Phasic inhibition is mediated by GABAARs and GABABRs located within the postsynaptic and perisynaptic membrane, whereas tonic inhibition is mediated by the extrasynaptic receptors (Glykys and Mody, 2007). It has been shown that tonic inhibition might be activated by ambient GABA that diffuses throughout the extracellular space and binds to the extrasynaptic GABAA and GABABRs (Farrant and Nusser, 2005). GABAARs mediating tonic inhibition are composed of unique subunit types (Belelli et al., 2009). Most extrasynaptic GABAARs contain alpha 5, alpha 4 or delta subunits (Belelli et al., 2009). GABAARs with these subunits display a high affinity for Frontiers in Cellular Neuroscience 01 frontiersin.org Bogaj et al. 10.3389/fncel.2023.1270219 10.3389/fncel.2023.1270219 10.3389/fncel.2023.1270219 GABA and a low efficacy (Saxena and Macdonald, 1994). Extrasynaptic GABAARs also are weakly desensitizing (Saxena and Macdonald, 1994). These features allow detection of micromolar concentrations of ambient neurotransmitter and enable high potential for allosteric modulation. Cell-type specific expression of specific GABAAR subunits mediating tonic inhibition in particular neuronal types are still under investigation. GABA and a low efficacy (Saxena and Macdonald, 1994). Extrasynaptic GABAARs also are weakly desensitizing (Saxena and Macdonald, 1994). These features allow detection of micromolar concentrations of ambient neurotransmitter and enable high potential for allosteric modulation. Cell-type specific expression of specific GABAAR subunits mediating tonic inhibition in particular neuronal types are still under investigation. Here, we used whole-cell patch-clamp techniques in brain slices prepared from transgenic mice expressing red fluorescent protein (TdTomato) in VIP- or SST-INs and investigated tonic and phasic GABAAR-mediated inhibition in these interneurons in layers 2/3 of the somatosensory (barrel) cortex. Then we analyzed the effect of GABAAR-mediated inhibition on their intrinsic excitability. We found that L2/3 VIP- and SST-INs of the barrel cortex displayed tonic GABAAR inhibition at different levels – higher in VIP- than in SST-INs. In both interneuron types, tonic inhibition was mediated by delta subunit-containing GABAARs. GABAARs modulated intrinsic excitability in an interneuron type-specific manner. The GABAAR activity decreased intrinsic excitability of VIP-INs but had no effect on the excitability of SST-INs. Frontiers in Cellular Neuroscience Animals GABAergic interneurons form a broad spectrum of subtypes based on the morphology, electrophysiological properties, the expression of characteristic molecular markers and specific functions in the network (Rudy et al., 2011). Two types of the GABAergic interneurons (VIP- and SST-INs) are involved in a disinhibitory loop in the neocortex (Pfeffer et al., 2013; Jiang et al., 2015, 2023). Namely, L2/3 VIP-INs inhibit L2/3 SST-INs and in turn release Pyr neurons from SST-IN-mediated inhibition. Such a disinhibitory effect has been found to be essential in learning process (Lee et al., 2013). However, VIP-INs also are reciprocally inhibited by SST-INs (Jiang et al., 2015). Thus, L2/3 VIP- and SST-INs play specific roles in the neocortex. Both interneuron types possess specific electrophysiological properties and various firing phenotypes (Prönneke et al., 2015, 2019; Liguz-Lecznar et al., 2016; Jiang et al., 2023) and can be further divided into many subgroups depending on the molecular, electrophysiological, morphological or functional features (Rudy et al., 2011; Liguz-Lecznar et al., 2016; Urban-Ciecko and Barth, 2016; Gouwens et al., 2020; Hostetler et al., 2023; Jiang et al., 2023; Munguba et al., 2023). The following strains of mice were used: (1) Sst-IRES-Cre (Jackson Labs stock #013044); (2) Vip-IRES-Cre (Jackson Labs stock #010908); (3) Ai14 mice (Jackson Labs stock #007908). Experiments were performed in offspring of Sst-IRES-Cre or Vip-IRES-Cre crossed to Ai14 (floxed-Tdt) reporter mice; Sst-Cre::Ai14 mice and VIP-Cre::Ai14 mice, respectively. All transgenes were used as heterozygotes and both sexes were used. Mice were housed under controlled light cycles (12-h light–dark cycles) with an ad libitum access to food and water. Ethical approval All procedures were conducted in accordance with the Act on the Protection of Animals Used for Scientific or Educational Purposes in Poland (Act of 15 January 2015, changed 17 November 2021; directive 2010/63/EU) and were approved by Polish Ministry of Environment (Dec. No 47/2019). The study was reported in agreement with ARRIVE guidelines. Introduction Altogether, our study reveals that molecularly distinct interneurons with the very similar firing phenotypes are differentially modulated by tonic GABAAR-mediated inhibition. GABAAR-mediated inhibition can differentially modulate neuronal excitability (Bryson et al., 2020). However, the role of tonic inhibition in specific neuronal types is still not fully understood. The effect of GABA on neuronal excitability is determined by the reversal potential for Cl−, which is the major ion mediating the GABAAR current. In general, in adult neurons, the reversal potential for Cl− is set close to the resting membrane potential. However, the resting membrane potential and the reversal potential for Cl− vary among cell types, therefore, the effect of GABA can be either hyperpolarizing or depolarizing (Hausselt et al., 2007; Khirug et al., 2008). Previous works have shown that tonic inhibition decreases the excitability of Pyr cell by increasing rheobase (Mitchell and Silver, 2003; Pavlov et al., 2009; Silver, 2010). The effect of tonic inhibition on interneuron excitability varies depending on the interneuron type (Song et al., 2011; Pavlov et al., 2014; Bryson et al., 2020). Depolarizing effect of GABA has been found in fast spiking (FS) CA3 stratum lucidum interneurons (Pavlov et al., 2014) and in other interneurons in the cerebellum (Chavas and Marty, 2003), amygdala (Woodruff et al., 2006) and striatum (Bracci and Panzeri, 2006). Studies using biophysically detailed neuron models have predicted that effects of tonic inhibition on interneuron excitability is determined by the variation in the electrophysiological properties of specific interneurons (Pavlov et al., 2014). Diverse firing properties of L2/3 VIP- and SST-INs Membrane parameters were measured every 10 s using a 100 ms-pulse of −10 mV and − 10 pA in voltage- and current-mode, respectively. To study different types of GABAergic interneurons, we used transgenic mice with fluorescently labeled interneurons. Before the analysis of tonic current, we checked firing phenotypes of fluorescently labeled interneurons because we were interested whether L2/3 VIP- and SST-INs might be differentiated according to their firing patterns in the high [Cl−] internal solution used for the analysis of tonic inhibition. We found that both L2/3 VIP- and SST-INs exhibit a variety of firing patterns (Figure 1 and Supplementary Figure 1) in this condition. According to Petilla terminology (Petilla Interneuron Nomenclature Group, 2008), both types of interneurons displayed accommodating firing (AC), low-threshold spiking (LTS) firing patterns with rebound spiking after hyperpolarizing current step (Figures 1A,B and Supplementary Figures 1A,B) or irregular spiking (IR). A part of SST-INs showed fast spiking (FS) patterns (Supplementary Figure 1B). In contrast to the recordings in the low [Cl−] internal solution, there were no L2/3 VIP-INs with the burst firing in the high [Cl−] internal solution (Supplementary Figure 1A). These results indicate that different recording conditions might influence the activity of particular channels and thus the overall properties of the firing phenotypes. Finally, putative Pyr neurons responded with a regular spiking phenotype without rebound spikes (Figure 1C). Tonic currents were recorded in voltage-clamp mode at the holding potential of −70 mV. The value of the tonic current was calculated as the baseline shift after the application of drugs and normalized to the whole-cell membrane capacitance (the current density). The baseline (holding) current was measured every 10 s and the baseline shift was calculated as a change in the holding current after 10 min with the appropriate drug. The current noise was measured as the standard deviation (SD) of the holding current (Bright and Smart, 2013). Spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in voltage-clamp at 0 mV in the cesium gluconate-based internal solution. Intrinsic excitability was assessed using square pulses of 500 ms of an amplitude increasing up to maximal firing frequency (steps of 10 pA). To control for potential effects of GABAAR agents on the resting membrane potential, the membrane potential of interneurons was maintained at −70 mV across different pharmacological conditions. Brain slice preparation At the age of P18–P30, where P0 indicates the day of birth, mice were anaesthetized with isoflurane and killed by decapitation using the procedure in accordance with the Polish Animal Protection Act (Act of 15 January 2015, changed 17 November 2021; directive 2010/63/EU). Previous studies have shown no tonic inhibition in L2/3 SST-INs in the mouse frontoparietal cortex (Vardya et al., 2008) and a weak tonic inhibition in L2/3 SST-INs in the barrel cortex (Donato et al., 2023). However, in these studies, transgenic “GIN” mice or X98 mouse line that express enhanced green florescent protein in a subset of SST-INs were used (Oliva et al., 2000). Other studies, using Sst-Cre mouse line have revealed a weak tonic inhibition in hippocampal SST-INs (Huang et  al., 2023; Wyroślak et  al., 2023). To our best knowledge, tonic inhibition has not yet been studied in VIP-INs. Brain slices (350 μm thick) were cut in an “across-row” procedure in which the anterior end of the brain was cut along a 45° plane toward the midline (Finnerty et al., 1999). Slices were cut at 2–4°C, then recovered and maintained at 32°C in artificial cerebrospinal fluid (ACSF) composed of (in mM): 113 NaCl, 2.5 KCl, 1.3 MgSO4, 2.5 CaCl2, 1 NaH2PO4, 26.2 NaHCO3, 11 glucose equilibrated with 95/5% O2/CO2. Recordings were performed at 32°C in ACSF of the same composition as above. 02 frontiersin.org Bogaj et al. 10.3389/fncel.2023.1270219 Whole-cell recording Somata of L2/3 neurons in the somatosensory (barrel) cortex were targeted for whole-cell recordings. Neurons were classified as Pyr neurons according to Pyr-like soma shape, the presence of an apical dendrite, as well as based on regular spiking pattern in response to 500 ms suprathreshold intracellular current injection. SST-INs and VIP-INs were identified using fluorescent reporter gene expression in Sst-Cre::Ai14 and Vip-Cre::Ai14 mice, respectively. For analysis of tonic GABAAR inhibition, a high [Cl−] internal solution was used (in mM): 140 KCl, 1 MgCl2, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, pH 7.25–7.35, 290 mOsm (Urban-Ciecko et al., 2010; Urban-Ciecko and Mozrzymas, 2011). For study of intrinsic excitability, a low [Cl−] internal solution was composed of (in mM): 125 potassium gluconate, 2 KCl, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, and 0.3 Na-GTP, at pH 7.25–7.35 and 290 mOsm (Urban-Ciecko et al., 2015, 2018). For recordings of sIPSCs, the internal solution contained (in mM): 130 cesium gluconate, 10 HEPES, 0.5 EGTA, 8 NaCl, 10 tetraethylammonium chloride, 5 QX-314, 4 Mg-ATP, and 0.3 Na-GTP, at pH 7.25–7.35, 290 mOsm (Kanigowski et al., 2023). Patch electrodes were made from borosilicate glass and had 2–4 MOhm or 4–6 MOhm when filled with the high [Cl−] internal solution and the low [Cl−] solution, respectively. The GABAAR blocker picrotoxin (PTX, 100 μM) and a delta subunit-containing GABAAR superagonist (4,5,6,7- tetrahydroisoxazolo[5,4-c]pyridin-3-ol, THIP, 20 μM) were bath applied for at least 10 min to assess drug effects. All the pharmacological agents were purchased from Tocris. Frontiers in Cellular Neuroscience Data analysis Population data are presented as mean ± SD. One cell in a slice and 1–3 cells were analyzed in an individual mouse. The normality distribution was tested with the Shapiro–Wilk test and equal variance was analyzed with Brown-Forsythe test. For comparison of multiple groups, a one way ANOVA (parametric) was used, followed by a Tukey post hoc test, or a Kruskal Wallis test (nonparametric) was used, followed by a Dunn’s test. For comparison of two variables in multiple groups a two way ANOVA was used with Holm-Sidak’s test. To compare an effect of a drug within a cell, a two-tailed paired t-test or Wilcoxon test were used depending on the normality distribution. Statistical significance was defined as *p < 0.05, **p ≤ 0.01, and ***p ≤ 0.001. Electrophysiological data were acquired by Multiclamp  700B (Molecular Devices) and digitized with Digidata 1550B (Molecular Devices). The data were filtered at 3 kHz, digitized at 20 kHz and collected by pClamp (Molecular Devices). Series and input resistances were analyzed online, recordings were discarded when access or series resistances were unstable more than 20%. frontiersin.org Diverse firing properties of L2/3 VIP- and SST-INs For intrinsic excitability, a curve of the relation between the frequency of action potentials and the intensity of the injected current (F-I curve) was plotted for every neuron in control ACSF and after the drug application. The effect of the drug on excitability was analyzed as the difference in the rheobase, the maximal firing frequency and the difference of the spiking frequency at the same current steps in the comparison to control ACSF. The rheobase means a step of the injected current at the minimal intensity that evoked at least one spike. Analysis of electrophysiological properties such as the resting membrane potential (Figure 1D), the input resistance (Figure 1E) and the membrane capacitance (Figure 1F) revealed significant differences between VIP- and SST-INs and Pyr cells. In agreement with the literature data (Gentet et al., 2010, 2012; Urban-Ciecko et  al., 2015; Dobrzanski et  al., 2022; Kanigowski et  al., 2023) 03 frontiersin.org 10.3389/fncel.2023.1270219 Bogaj et al. FIGURE 1 Firing phenotypes of L2/3 VIP- and SST-INs are very similar in a high [Cl−]-based internal solution. (A) Example traces of VIP-IN firing responses after the somatic current injection of a 500  ms-long pulse at the intensity of; 20 pA and −200 pA (left traces); 100 pA (middle trace) and 200 pA (right trace). Current steps shown below firing traces. (B) The same as in (A) but for SST-IN. (C) The same as in (A) but for a pyramidal (Pyr) neuron. (D) The comparison of mean (±SD) resting membrane potentials in 3 types of neurons. (E) The same as in (D) but for the input resistance. (F) The same as in (D) but for the membrane capacitance. *p  <  0.05, **p ≤  0.01, and ***p ≤ 0.001. FIGURE 1 Firing phenotypes of L2/3 VIP- and SST-INs are very similar in a high [Cl−]-based internal solution. (A) Example traces of VIP-IN firing responses after the somatic current injection of a 500  ms-long pulse at the intensity of; 20 pA and −200 pA (left traces); 100 pA (middle trace) and 200 pA (right trace). Current steps shown below firing traces. (B) The same as in (A) but for SST-IN. (C) The same as in (A) but for a pyramidal (Pyr) neuron. (D) The comparison of mean (±SD) resting membrane potentials in 3 types of neurons. (E) The same as in (D) but for the input resistance. (F) The same as in (D) but for the membrane capacitance. Diverse firing properties of L2/3 VIP- and SST-INs The input resistance was the highest in VIP-INs (268.98 ± 62.60 MOhm, n = 17 cells in 17 mice) in comparison to SST-INs (167.33 ± 38.39 MOhm, n = 21 cells in 21 mice, p < 0.001, one way ANOVA with Tukey test, Figure  1E) and Pyr (107.15 ± 60.09 MOhm, n = 13 cells in 7 mice, p < 0.001), also SST-INs had higher input resistance than Pyr cells (p = 0.007). In contrast, the whole- cell membrane capacitance was the lowest in VIP-INs (14.32 ± 2.70 pF, n = 38 cells in 19 mice) compared to SST-INs (33.40 ± 9.14 pF, n = 49 cells in 21 mice) and Pyr (48.61 ± 18.06 pF, n = 23 cells in 15 mice, p < 0.001, Kruskal-Wallis with Dunn’s tests, Figure 1F), whereas SST and Pyr did not differ in the membrane capacitance (p = 0.07). Diverse firing properties of L2/3 VIP- and SST-INs *p  <  0.05, **p ≤  0.01, and ***p ≤ 0.001. Summarizing, analysis of GABAergic interneurons according to their firing patterns would not discriminate between molecularly distinct interneurons, even though they have statistically different basic electrophysiological properties of the membrane. It is worthwhile to mention that an LTS firing phenotype had been assumed to be characteristic for SST-INs in previous studies before the era of transgenic tools, for rev (Liguz-Lecznar et al., 2016). Here, we show that this assumption is not accurate. interneurons exhibited more depolarized resting membrane potential than Pyr cells. SST-INs had the most depolarized resting membrane potentials (−52.00 ± 5.84 mV, n = 21 cells in 21 mice) compared to VIP-INs (−57.57 ± 6.61 mV, n = 21 cells in 19 mice) and Pyr neurons (−68.60 ± 5.17 mV, n = 10 cells in 8 mice, p = 0.012 for VIP-INs versus SST-INs, p < 0.001 for VIP-INs and SST-INs versus Pyr, one way ANOVA with Tukey test, Figure 1D). The input resistance was the highest in VIP-INs (268.98 ± 62.60 MOhm, n = 17 cells in 17 mice) in comparison to SST-INs (167.33 ± 38.39 MOhm, n = 21 cells in 21 mice, p < 0.001, one way ANOVA with Tukey test, Figure  1E) and Pyr (107.15 ± 60.09 MOhm, n = 13 cells in 7 mice, p < 0.001), also SST-INs had higher input resistance than Pyr cells (p = 0.007). In contrast, the whole- cell membrane capacitance was the lowest in VIP-INs (14.32 ± 2.70 pF, n = 38 cells in 19 mice) compared to SST-INs (33.40 ± 9.14 pF, n = 49 cells in 21 mice) and Pyr (48.61 ± 18.06 pF, n = 23 cells in 15 mice, p < 0.001, Kruskal-Wallis with Dunn’s tests, Figure 1F), whereas SST and Pyr did not differ in the membrane capacitance (p = 0.07). interneurons exhibited more depolarized resting membrane potential than Pyr cells. SST-INs had the most depolarized resting membrane potentials (−52.00 ± 5.84 mV, n = 21 cells in 21 mice) compared to VIP-INs (−57.57 ± 6.61 mV, n = 21 cells in 19 mice) and Pyr neurons (−68.60 ± 5.17 mV, n = 10 cells in 8 mice, p = 0.012 for VIP-INs versus SST-INs, p < 0.001 for VIP-INs and SST-INs versus Pyr, one way ANOVA with Tukey test, Figure 1D). Frontiers in Cellular Neuroscience frontiersin.org L2/3 VIP-INs exhibit stronger tonic GABAAR-mediated inhibition than SST-INs Because tonic GABAAR inhibition has previously been studied in Pyr neurons (Drasbek and Jensen, 2006; Vardya et al., 2008; Urban- Ciecko et al., 2010), we used these cells for the comparison to assess tonic inhibition in distinct interneuron types (Figure 2). After bath application of the GABAAR blocker (PTX), we observed a shift in baseline current and a reduction in the current noise in both 04 Frontiers in Cellular Neuroscience frontiersin.org 10.3389/fncel.2023.1270219 Bogaj et al. FIGURE 2 Tonic GABAAR-mediated inhibition is stronger in L2/3 VIP-INs than in SST-INs. (A) Example traces of current recordings in control (CTRL) ACSF and after picrotoxin (PTX) application in VIP-IN (left), the baseline holding current measured every 10 s and plotted against time was used for the estimation of the tonic current (right). (B) The same as in (A) but for SST-IN. (C) The same as in (A) but for a pyramidal (Pyr) neuron. (D) The comparison of mean (±SD) tonic current density in the presence of the GABAAR blocker (PTX) in VIP-, SST-INs and Pyr neurons. (E) With-in cell comparison and mean (±SD) input resistance of VIP-INs in CTRL and after PTX. (F) The same as in (E) but for SST-INs. (G) The same as in (E) but for Pyr neurons. *p < 0.05, **p ≤  0.01, and ***p ≤  0.001. FIGURE 2 Tonic GABAAR-mediated inhibition is stronger in L2/3 VIP-INs than in SST-INs. (A) Example traces of current recordings in control (CTRL) ACSF and after picrotoxin (PTX) application in VIP-IN (left), the baseline holding current measured every 10 s and plotted against time was used for the estimation of the tonic current (right). (B) The same as in (A) but for SST-IN. (C) The same as in (A) but for a pyramidal (Pyr) neuron. (D) The comparison of mean (±SD) tonic current density in the presence of the GABAAR blocker (PTX) in VIP-, SST-INs and Pyr neurons. (E) With-in cell comparison and mean (±SD) input resistance of VIP-INs in CTRL and after PTX. (F) The same as in (E) but for SST-INs. (G) The same as in (E) but for Pyr neurons. *p < 0.05, **p ≤  0.01, and ***p ≤  0.001. 1.17 ± 1.00 pA/pF, n = 11 cells in 6 females, p < 0.001), whereas the current density in SST-INs was similar to Pyr cells (p = 0.556, Figure  2D). Frontiers in Cellular Neuroscience frontiersin.org L2/3 VIP-INs exhibit stronger tonic GABAAR-mediated inhibition than SST-INs Because the strength of tonic inhibition might be  sex-dependent (Urban-Ciecko and Mozrzymas, 2011), we compared the values of the current density between males and females, and we found no differences in tonic inhibition in these 3 types of neurons in regards to the sex of animals (p = 0.817, two way interneuron types (Figures 2A,B and Supplementary Figures 2A,B) and in Pyr (Figure 2C and Supplementary Figure 2C). The current density was larger in VIP-INs (2.56 ± 1.69 pA/pF, n = 9 cells in 4 males and 2.17 ± 1.38 pA/pF, n = 8 cells in 5 females, Figure 2D) than in SST-INs (0.68 ± 0.84 pA/pF, n = 10 cells in 6 males and 0.43 ± 0.26 pA/ pF, n = 11 cells in 6 females, p < 0.001, two way ANOVA with Holm Sidak’s test) and Pyr (0.34 ± 0.38 pA/pF, n = 7 cells in 5 males and 05 frontiersin.org Bogaj et al. 10.3389/fncel.2023.1270219 10.3389/fncel.2023.1270219 17.46 ± 2.94 pA, n = 13 cells in 9 females, Figure 4B) compared to SST-INs (26.69 ± 7.01 pA, n = 14 cells in 12 males and 22.91 ± 7.24 pA, n = 17 cells in 13 females) and Pyr (32.72 ± 4.02 pA, n = 6 cells in 6 males and 29.54 ± 5.71 pA, n = 7 cells in 6 females, p < 0.001, two way ANOVA with Holm Sidak’s test). Also, sIPSC amplitude was smaller in SST-INs than in Pyr cells (p < 0.001, two way ANOVA with Holm Sidak’s test). In case of sIPSC frequency (Figure 4C), there was no difference between VIP-INs (4.40 ± 1.84 Hz, n = 21 cells in 10 males and 4.17 ± 2.90 Hz, n = 13 cells in 9 females) and SST-INs (3.88 ± 3.72 Hz, n = 14 cells in 12 males and 2.70 ± 2.79 Hz, n = 17 cells in 13 females, p = 0.51, two way ANOVA with Holm Sidak’s test). However, Pyr cells exhibited the highest sIPSC frequency (14.91 ± 9.20 Hz, n = 6 cells in 6 males and 15.13 ± 5.43 Hz, n = 7 cells in 6 females, p < 0.001, two way ANOVA with Holm Sidak’s test). L2/3 VIP-INs exhibit stronger tonic GABAAR-mediated inhibition than SST-INs Finally, there were no differences between males and females regarding the amplitude and the frequency of sIPSCs (p = 0.092 for the amplitude and p = 0.525 for the frequency, two way ANOVA with Holm Sidak’s test). ANOVA with Holm Sidak’s test, Figure  2D). Furthermore, we observed that the application of PTX increased the input resistance in each neuronal type (for VIP-INs from 268.98 ± 62.60 MOhm in CTRL to 301.05 ± 72.13 MOhm in PTX, n = 21 cells in 19 mice, p < 0.001, Wilcoxon test, Figure 2E; for SST-INs from 167.33 ± 38.39 MOhm in CTRL to 193.05 ± 54.28 MOhm in PTX, n = 17 cells in 17 mice, p = 0.004, two-tailed paired t-test, Figure 2F; for Pyr cells from 107.15 ± 60.09 MOhm in CTRL to 126.77 ± 82.36 MOhm in PTX, n = 13 cells in 7 mice, p < 0.001, Wilcoxon test, Figure 2G). Because there were no differences in tonic inhibition concerning the sex of mice (Figure 2D), we pooled data from males and females for the analysis of the input resistance (Figures 2E–G). ANOVA with Holm Sidak’s test, Figure  2D). Furthermore, we observed that the application of PTX increased the input resistance in each neuronal type (for VIP-INs from 268.98 ± 62.60 MOhm in CTRL to 301.05 ± 72.13 MOhm in PTX, n = 21 cells in 19 mice, p < 0.001, Wilcoxon test, Figure 2E; for SST-INs from 167.33 ± 38.39 MOhm in CTRL to 193.05 ± 54.28 MOhm in PTX, n = 17 cells in 17 mice, p = 0.004, two-tailed paired t-test, Figure 2F; for Pyr cells from 107.15 ± 60.09 MOhm in CTRL to 126.77 ± 82.36 MOhm in PTX, n = 13 cells in 7 mice, p < 0.001, Wilcoxon test, Figure 2G). Because there were no differences in tonic inhibition concerning the sex of mice (Figure 2D), we pooled data from males and females for the analysis of the input resistance (Figures 2E–G). Altogether, our results indicate that GABAARs influence membrane properties of both interneuron types and Pyr cells in a way that would promote a decrease of neuronal excitability. Intrinsic excitability of L2/3 VIP-INs is controlled by GABAARs Tonic inhibition is thought to regulate neuronal excitability, however, the effect might be cell type-specific (Bryson et al., 2020). To study the effect of GABAergic inhibition on intrinsic excitability of L2/3 VIP-INs, we used a canonical internal solution, the K-gluconate- based internal solution with a low [Cl−] concentration. In this condition, VIP-INs displayed a variety of the firing patterns (Supplementary Figure  1A), such as burst spiking (Figure  5A), irregular spiking or continuous adapting according to the terminology published in Jiang et al. (2023). To investigate the impact of GABAergic inhibition upon VIP-IN excitability, we created a curve of the relation between the frequency of action potentials (AP) and the intensity of the injected current (F-I curve) for every neuron in control ACSF and after the application of PTX (Figure 5B). Because there were no differences in phasic and tonic GABAAR-mediated inhibition in respect to the sex of animals (Figures 2–4), we pooled data from males and females for the analysis of neuronal excitability. We observed that PTX shifted the F-I curve to higher AP frequency in lower current intensities (n = 25 cells in 13 mice, p < 0.05, two-tailed paired t-test, Figures  5A,B). Also, PTX application increased the input resistance from 269.79 ± 68.76 MOhm to 296.19 ± 85.86 MOhm (n = 17 cells in 13 mice, p = 0.049, two-tailed paired t-test, Figure 5C), depolarized the resting membrane potential from −54.94 ± 6.23 mV to −50.48 ± 8.84 mV (n = 17 cells in 13 mice, p < 0.001, Wilcoxon test, Figure 5D) and decreased the rheobase from 41.20 ± 14.81 pA to 29.20 ± 15.79 pA (n = 25 cells in 13 mice, p < 0.001, Wilcoxon test, Figure  5E) in VIP-INs. However, the maximal Our data indicate that L2/3 VIP- and SST-INs possess different levels of tonic inhibition mediated by delta subunit- containing GABAARs. Tonic inhibition in L2/3 VIP- and SST-INs is mediated by delta subunit-containing GABAARs Summarizing, the analysis showed larger sIPSC amplitude in SST-INs than in VIP-INs but no difference in the frequency of sIPSCs. Taking into account the assumption that the event amplitude is shaped by the receptor number and the receptor subunit composition whereas the frequency of events depends on the number of synaptic inputs (Glasgow et al., 2019), our results suggest that these interneuron types might have different number of synaptic GABAARs or subunit compositions of these receptors but comparable numbers of inhibitory synaptic inputs. To check whether tonic inhibition in L2/3 VIP- and SST-INs is mediated by delta subunit-containing GABAARs, we bath applied THIP which is a selective GABAAR agonist with a preference for delta subunit-containing GABAARs (Hansen et al., 2004). THIP induced a baseline shift and an increase in the current noise in both VIP- (Figure 3A and Supplementary Figure 3A) and SST-INs (Figure 3B and Supplementary Figure 3B). The effect was reversible after wash out (Figures 3A,B). The current density evoked by THIP was larger in VIP-INs (6.80 ± 2.48 pA/pF, n = 11 cells in 5 males and 8.26 ± 4.15 pA/ pF, n = 10 cells in 5 females, Figure 3C) than in SST-INs (3.16 ± 2.36 pA/pF, n = 11 cells in 5 males and 1.94 ± 0.67 pA/pF, n = 7 cells in 6 females, p < 0.001, two way ANOVA with Holm Sidak’s test) and there was no difference between males and females (p = 0.831, two way ANOVA with Holm Sidak’s test). Moreover, THIP reduced the input resistance in both types of the interneurons (for VIP-INs from 244.48 ± 83.89 MOhm in CTRL to 204.43 ± 81.66 MOhm in THIP, n = 21 cells in 10 mice, p = 0.003, Wilcoxon test, Figure 3D; for SST-INs from 168.72 ± 69.29 MOhm in CTRL to 126.92 ± 64.75 MOhm in THIP, n = 25 cells in 21 mice, p < 0.001, Wilcoxon test, Figure 3E). Because there were no differences in tonic inhibition in regards to the sex of animals (Figure 3C), we pooled data from males and females for the analysis of the input resistance (Figures 3D,E). Frontiers in Cellular Neuroscience frontiersin.org L2/3 VIP-INs have weaker phasic GABAAR-mediated inhibition than SST-INs Similarly to VIP-INs, we  observed a variety of SST-IN firing phenotypes, which were regular, low-threshold spiking with rebound spikes, accommodating without rebound spikes (Figure 6A and Supplementary Figure 1B), occasionally fast-spiking (Petilla Interneuron Nomenclature Group, 2008; Liguz-Lecznar et al., 2016; Urban-Ciecko and Barth, 2016). Here, the same as for VIP-INs, we  pooled data from males and females for the analysis of excitability, because there were no differences in phasic and tonic inhibition of SST-INs in regards to the sex of animals (Figures 2–4). Surprisingly, for SST-INs we did not observe any differences in F-I curve between CTRL and after bath application of PTX (n = 7 cells, p > 0.05, two-tailed paired t-test, Figures 6A,B). Also, there were no changes in the input resistance (243.32 ± 53.37 MOhm in CTRL and 242.93 ± 37.06 MOhm in PTX, excitability of these interneurons (Figure 6). Similarly to VIP-INs, we  observed a variety of SST-IN firing phenotypes, which were regular, low-threshold spiking with rebound spikes, accommodating without rebound spikes (Figure 6A and Supplementary Figure 1B), occasionally fast-spiking (Petilla Interneuron Nomenclature Group, 2008; Liguz-Lecznar et al., 2016; Urban-Ciecko and Barth, 2016). Taking into account that the rheobase might be the best predictor of the changes in the intrinsic excitability (Bryson et al., 2020), our data show that GABAergic (presumably mostly tonic) inhibition decreases the excitability of L2/3 VIP-INs. L2/3 VIP-INs have weaker phasic GABAAR-mediated inhibition than SST-INs To compare whether differences in tonic inhibition of L2/3 SST- and VIP-INs are also accompanied by differences in synaptic (phasic) GABAergic inhibition, we recorded sIPSCs in these interneurons. For better comparison, in some cases recordings were performed in an interneuron and a neighboring Pyr cell within the same slice (Figure 4). We observed that the mean amplitude of sIPSCs was smaller in VIP-INs (16.99 ± 3.20 pA, n = 21 cells in 10 males and 06 frontiersin.org 10.3389/fncel.2023.1270219 Bogaj et al. FIGURE 3 Tonic inhibition in L2/3 VIP- and SST-INs depends on delta subunit-containing GABAARs. (A) Example traces of current recording in control (CTRL) ACSF, after the application of a selective GABAAR agonist with a preference for delta subunit-containing GABAARs (4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridin-3-ol, THIP) and wash-out condition (left), the baseline holding current measured every 10 s and plotted against time was used for the estimation of the tonic current in VIP-IN (right). (B) The same as in (A) but for SST-IN. (C) The comparison of mean (±SD) tonic current density in the presence of THIP in VIP- and SST-INs. (D) With-in cell comparison and mean (±SD) input resistance of VIP-INs in CTRL and after THIP. (E) The same as in (D) but for SST-INs. *p  <  0.05, **p  ≤  0.01, and ***p ≤  0.001. FIGURE 3 Tonic inhibition in L2/3 VIP- and SST-INs depends on delta subunit-containing GABAARs. (A) Example traces of current recording in control (CTRL) ACSF, after the application of a selective GABAAR agonist with a preference for delta subunit-containing GABAARs (4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridin-3-ol, THIP) and wash-out condition (left), the baseline holding current measured every 10 s and plotted against time was used for the estimation of the tonic current in VIP-IN (right). (B) The same as in (A) but for SST-IN. (C) The comparison of mean (±SD) tonic current density in the presence of THIP in VIP- and SST-INs. (D) With-in cell comparison and mean (±SD) input resistance of VIP-INs in CTRL and after THIP. (E) The same as in (D) but for SST-INs. *p  <  0.05, **p  ≤  0.01, and ***p ≤  0.001. frequency of APs was slightly reduced after PTX (from 62.00 ± 16.56 Hz in CTLR to 59.50 ± 20.20 Hz in PTX, n = 25 cells in 13, p = 0.038, Wilcoxon test, Figure 5F). excitability of these interneurons (Figure 6). Intrinsic excitability of SST-INs is immune to the activity of GABAARs Because L2/3 SST-INs had a low intensity of tonic inhibition (Figures 2, 3) we wondered if PTX application affected the intrinsic 07 Frontiers in Cellular Neuroscience frontiersin.org Bogaj et al. 10.3389/fncel.2023.1270219 FIGURE 4 Synaptic (phasic) inhibition is weaker in L2/3 VIP-INs than in SST-INs. (A) Example traces of sIPSC recordings in VIP-, SST-IN and Pyr. (B) The mean (±SD) amplitude of sIPSCs recorded in these neurons. (C) The same as in (B) but for the mean (±SD) frequency. *p < 0.05, **p ≤ 0.01, and ***p ≤ 0.001. FIGURE 4 Synaptic (phasic) inhibition is weaker in L2/3 VIP-INs than in SST-INs. (A) Example traces of sIPSC recordings in VIP-, SST-IN and Pyr. (B) The mean (±SD) amplitude of sIPSCs recorded in these neurons. (C) The same as in (B) but for the mean (±SD) frequency. *p < 0.05, **p ≤ 0.01, and ***p ≤ 0.001. Next, pharmacological activation of delta subunit-containing GABAARs by THIP showed that both interneuron types displayed tonic inhibition which was mediated by delta subunit and the level of delta subunit-induced tonic inhibition was around twice higher in L2/3 VIP-INs than in SST-INs. Thus, the differences in the values of tonic inhibition between VIP- and SST-INs might mainly come from the differential expression of the delta-subunit containing GABAARs in these interneurons. It has been found that hippocampal SST-INs show a very low expression of delta subunits and this is accompanied by a relatively low intensity of tonic current mediated by the delta- subunit containing GABAARs (Huang et al., 2023). Delta subunit- containing GABAARs have been found to be regulated by estrogens (Shen et al., 2005) and neurosteroids (Reddy and Kulkarni, 1999; Maguire et al., 2005; Wiltgen et al., 2005). Indeed, tonic inhibition mediated by these receptors was larger in L4 regular spiking (presumably Pyr neurons) and FS interneurons in the barrel cortex in females than in males (Urban-Ciecko and Mozrzymas, 2011). Nonetheless, in the present study there was no difference in the level of tonic inhibition between females and males. We hypothesize that the effect of the sex on tonic inhibition might be age-dependent because here mice were younger (P18-30) than in the prior work (P35-49) (Urban-Ciecko and Mozrzymas, 2011). Tonic inhibition can be mediated by GABAARs with other subunits. Intrinsic excitability of SST-INs is immune to the activity of GABAARs It has been found that alpha 5 subunit-containing GABAARs mediate tonic inhibition in L2/3 Pyr neurons but not in L2/3 PV-INs nor L2/3 SST-INs in the barrel cortex of X98 transgenic mouse line (Donato et al., 2023). Alpha 5-containing GABAARs have been revealed to be  extrasynaptic, mainly mediating tonic inhibition (Caraiscos et al., 2004). However, alpha 5-containing GABAARs mediate also synaptic inhibition coming from X98 SST-INs to L2/3 Pyr cells but not to L1 interneurons of the barrel cortex (Donato et al., 2023). What is more, synaptic inhibition originating from VIP-INs to SST-INs is mediated by the n = 7 cells in 7 mice, p = 0.578, Wilcoxon test, Figure 6C), the resting membrane potential (−49.77 ± 5.72 mV in CTRL and − 51.84 ± 7.71 mV in PTX, n = 7 cells in 7 mice, p = 0.479, two-tailed paired t-test, Figure 6D), rheobase (91.43 ± 21.16 pA in CTRL and 88.57 ± 18.64 pA in PTX, n = 7 cells in 7 mice, p = 0.457, two-tailed paired t-test, Figure 6E) and the maximal frequency in SST-INs (58.57 ± 20.32 Hz in CTRL and 48.86 ± 16.04 Hz in PTX, n = 7 cells in 7 mice, p = 0.136, two-tailed paired t-test, Figure 6F). n = 7 cells in 7 mice, p = 0.578, Wilcoxon test, Figure 6C), the resting membrane potential (−49.77 ± 5.72 mV in CTRL and − 51.84 ± 7.71 mV in PTX, n = 7 cells in 7 mice, p = 0.479, two-tailed paired t-test, Figure 6D), rheobase (91.43 ± 21.16 pA in CTRL and 88.57 ± 18.64 pA in PTX, n = 7 cells in 7 mice, p = 0.457, two-tailed paired t-test, Figure 6E) and the maximal frequency in SST-INs (58.57 ± 20.32 Hz in CTRL and 48.86 ± 16.04 Hz in PTX, n = 7 cells in 7 mice, p = 0.136, two-tailed paired t-test, Figure 6F). These data indicate that despite the presence of a low intensity of GABAAR-mediated tonic inhibition in L2/3 SST-INs, ambient GABA does not modulate the intrinsic excitability of these interneurons. frontiersin.org Donato et al., 2023). Discussion (C) With-in cell comparison and mean (±SD) input resistance under baseline condition (CTRL) and in the presence of PTX. (D) The same as in (C) but for membrane potential. (E) The same as in (C) but for rheobase. (F) The same as in (C) but for maximal frequency. *p < 0.05, **p ≤ 0.01, and ***p ≤ 0.001. receptors with this subunit in the hippocampus (Magnin et al., 2019) but not in the barrel cortex (Donato et al., 2023) indicating not only synapse- but brain area-specificity as well. Altogether, our results indicate that GABAARs regulate interneuron excitability in a cell type-specific manner. L2/3 VIP-INs show relatively strong tonic inhibition and it reduces VIP-IN excitability, whereas L2/3 SST-INs display very weak tonic inhibition, which is unable to modulate the intrinsic excitability of SST-INs. We also analyzed synaptic inhibition of both interneuron types and compared it to Pyr cells, because previous studies have reported that SST-INs in “GIN” mice possess minimal synaptic inhibition (Vardya et al., 2008). Here, we found that in fact sIPSCs had lower amplitude in VIP-INs than in SST-INs. In contrast, sIPSC frequency was comparable in both interneuron types. Also, sIPSCs were larger and more frequent in Pyr cells in comparison to VIP- and SST-INs, suggesting that synaptic inhibition is stronger in excitatory neurons than in these both interneuron types. Literature data have shown that the effect of tonic inhibition on interneuron excitability is unclear and varies depending on the interneuron type (Pavlov et al., 2009, 2014; Song et al., 2011; Bryson et al., 2020). Surprisingly, depolarizing effect of GABA has been found in fast spiking (FS) CA3 stratum lucidum interneurons (Pavlov et al., 2014) and in other interneurons in the cerebellum (Chavas and Marty, 2003), amygdala (Woodruff et al., 2006) and striatum (Bracci and Panzeri, 2006). The combination of dynamic clamp experiments with neural network simulations has shown that the strength of tonic inhibition in the interneurons might control interneuron firing pattern and synchronization in the CA3 hippocampal network (Pavlov et al., 2014). Thus, an influence of tonic inhibition on the firing output of the interneurons is an essential factor for rhythm generation in the brain. Finally, we observed that both the blockade and the activation of GABAARs had effects on the input resistance and the resting membrane potential suggesting that tonic inhibition might have essential effects on neuronal excitability. Frontiers in Cellular Neuroscience Discussion GABAARs regulate both fast synaptic (phasic) transmission as well as tonic (persistent) extrasynaptic inhibition (Farrant and Nusser, 2005; Glykys and Mody, 2007; Belelli et al., 2009). The tonic activation of GABAARs can regulate intrinsic excitability in a neuron-specific manner (Pavlov et  al., 2009, 2014; Song et  al., 2011). Here we investigated whether L2/3 SST-INs and VIP-INs might be regulated by tonic GABAAR inhibition. We  compared tonic inhibition in interneurons to neighboring Pyr neurons, since tonic inhibition in Pyr cells has been studied before (Drasbek and Jensen, 2006; Vardya et al., 2008; Urban-Ciecko et  al., 2010). Pharmacological blockade of GABAARs by PTX revealed tonic inhibition in both VIP- and SST-INs, however, the level of this inhibition was different in these cells. Namely, VIP-INs showed approximately two-fold stronger tonic inhibition than SST-INs. Surprisingly, tonic inhibition in SST-INs was comparable to Pyr cells in contrast to previous study, where a weak or no tonic inhibition in SST-INs has been found (Vardya et al., 2008; Donato et al., 2023). Frontiers in Cellular Neuroscience 08 frontiersin.org Bogaj et al. 10.3389/fncel.2023.1270219 FIGURE 5 GABAARs decrease intrinsic excitability of L2/3 VIP-INs. (A) Example traces of VIP-IN firing responses after the somatic current injection of a 500 ms- long pulse at 3 different intensities (20, 60, 120 pA) in CTRL ACSF (black traces) and after PTX application (red traces). (B) Summary plot of the mean firing frequency (±SD) in response to current injections (from 0 to 120 pA) from VIP-INs recorded in control ACSF and after PTX. (C) With-in cell comparison and mean (±SD) input resistance under baseline condition (CTRL) and in the presence of PTX. (D) The same as in (C) but for membrane potential. (E) The same as in (C) but for rheobase. (F) The same as in (C) but for maximal frequency. *p < 0.05, **p ≤ 0.01, and ***p ≤ 0.001. RE 5 FIGURE 5 GABAARs decrease intrinsic excitability of L2/3 VIP-INs. (A) Example traces of VIP-IN firing responses after the somatic current injection of a 500 ms- long pulse at 3 different intensities (20, 60, 120 pA) in CTRL ACSF (black traces) and after PTX application (red traces). (B) Summary plot of the mean firing frequency (±SD) in response to current injections (from 0 to 120 pA) from VIP-INs recorded in control ACSF and after PTX. frontiersin.org Discussion However, pharmacological blockade of GABAARs by PTX decreased the rheobase only in VIP-INs and had no effect on SST-INs, indicating that tonic GABAARs decrease intrinsic excitability of VIP-INs but not SST-INs. 09 frontiersin.org 10.3389/fncel.2023.1270219 Bogaj et al. FIGURE 6 GABAARs has no impact on intrinsic excitability of SST-INs. (A) Example traces of SST-IN firing responses after the somatic current injection of a 500  ms-long pulse at 3 different intensities (80, 120, 200 pA) in CTRL ACSF (black traces) and after PTX application (red traces). (B) Summary plot of the firing frequency (±SD) in response to current injections (from 0 to 250 pA) from SST-INs recorded in control ACSF and after PTX. (C) With-in cell comparison and mean (±SD) input resistance under baseline condition (CTRL) and in the presence of PTX. (D) The same as in (C) but for membrane potential. (E) The same as in (C) but for rheobase. (F) The same as in (C) but for maximal frequency. *p < 0.05, **p ≤ 0.01, and ***p ≤ 0.001. FIGURE 6 GABAARs has no impact on intrinsic excitability of SST-INs. (A) Example traces of SST-IN firing responses after the somatic current injection of a 500  ms-long pulse at 3 different intensities (80, 120, 200 pA) in CTRL ACSF (black traces) and after PTX application (red traces). (B) Summary plot of the firing frequency (±SD) in response to current injections (from 0 to 250 pA) from SST-INs recorded in control ACSF and after PTX. (C) With-in cell comparison and mean (±SD) input resistance under baseline condition (CTRL) and in the presence of PTX. (D) The same as in (C) but for membrane potential. (E) The same as in (C) but for rheobase. (F) The same as in (C) but for maximal frequency. *p < 0.05, **p ≤ 0.01, and ***p ≤ 0.001. A separate observation in our study was that L2/3 VIP- and SST-INs displayed diverse spiking patterns in response to the somatic current injection in whole-cell patch-clamp recordings and that in fact these patterns were very similar in both interneuron types. In literature, the same firing patterns might have variety of terms (Kawaguchi and Kubota, 1996; Kawaguchi, 1997; Gibson et al., 1999; Beierlein et al., 2003; Goldberg et al., 2004; Wang et al., 2004; Ma et al., 2006; Prönneke et al., 2015, 2019; Liguz-Lecznar et al., 2016; Urban- Ciecko and Barth, 2016; Jiang et al., 2023). Frontiers in Cellular Neuroscience frontiersin.org References Beierlein, M., Gibson, J. R., and Connors, B. W. (2003). Two dynamically distinct inhibitory networks in layer 4 of the neocortex. J. Neurophysiol. 90, 2987–3000. doi: 10.1152/jn.00283.2003 Beierlein, M., Gibson, J. R., and Connors, B. W. (2003). Two dynamically distinct inhibitory networks in layer 4 of the neocortex. J. Neurophysiol. 90, 2987–3000. doi: 10.1152/jn.00283.2003 Chavas, J., and Marty, A. (2003). Coexistence of excitatory and inhibitory GABA synapses in the cerebellar interneuron network. J. Neurosci. 23, 2019–2031. doi: 10.1523/ JNEUROSCI.23-06-02019.2003 Belelli, D., Harrison, N. L., Maguire, J., Macdonald, R. L., Walker, M. C., and Cope, D. W. (2009). Extrasynaptic GABAA receptors: form, pharmacology, and function. J. Neurosci. 29, 12757–12763. doi: 10.1523/JNEUROSCI.3340-09.2009 Belelli, D., Harrison, N. L., Maguire, J., Macdonald, R. L., Walker, M. C., and Cope, D. W. (2009). Extrasynaptic GABAA receptors: form, pharmacology, and function. J. Neurosci. 29, 12757–12763. doi: 10.1523/JNEUROSCI.3340-09.2009 Dobrzanski, G., Lukomska, A., Zakrzewska, R., Posluszny, A., Kanigowski, D., Urban-Ciecko, J., et al. (2022). Learning-induced plasticity in the barrel cortex is disrupted by inhibition of layer 4 somatostatin-containing interneurons. Biochim. Biophys. Acta 1869:119146. doi: 10.1016/j.bbamcr.2021.119146 Dobrzanski, G., Lukomska, A., Zakrzewska, R., Posluszny, A., Kanigowski, D., Urban-Ciecko, J., et al. (2022). Learning-induced plasticity in the barrel cortex is disrupted by inhibition of layer 4 somatostatin-containing interneurons. Biochim. Biophys. Acta 1869:119146. doi: 10.1016/j.bbamcr.2021.119146 Bracci, E., and Panzeri, S. (2006). Excitatory GABAergic effects in striatal projection neurons. J. Neurophysiol. 95, 1285–1290. doi: 10.1152/jn.00598.2005 Bracci, E., and Panzeri, S. (2006). Excitatory GABAergic effects in striatal projection neurons. J. Neurophysiol. 95, 1285–1290. doi: 10.1152/jn.00598.2005 Donato, C., Balduino Victorino, D., Cabezas, C., Aguirre, A., Lourenço, J., Potier, M.- C., et al. (2023). Pharmacological signature and target specificity of inhibitory circuits formed by martinotti cells in the mouse barrel cortex. J. Neurosci. 43, 14–27. doi: 10.1523/JNEUROSCI.1661-21.2022 Donato, C., Balduino Victorino, D., Cabezas, C., Aguirre, A., Lourenço, J., Potier, M.- C., et al. (2023). Pharmacological signature and target specificity of inhibitory circuits formed by martinotti cells in the mouse barrel cortex. J. Neurosci. 43, 14–27. doi: 10.1523/JNEUROSCI.1661-21.2022 Bright, D. P., and Smart, T. G. (2013). Methods for recording and measuring tonic GABAA receptor-mediated inhibition. Front. Neural Circ. 7:193. doi: 10.3389/ fncir.2013.00193 Drasbek, K. R., and Jensen, K. (2006). THIP, a hypnotic and antinociceptive drug, enhances an extrasynaptic GABAA receptor-mediated conductance in mouse neocortex. Cereb. Cortex 16, 1134–1141. doi: 10.1093/cercor/bhj055 Bryson, A., Hatch, R. J., Zandt, B.-J., Rossert, C., Berkovic, S. F., Reid, C. A., et al. Data availability statement All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author. Funding The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the National Science Centre, Poland (2020/39/B/ NZ4/01462 to JU-C). Supplementary material The animal study was approved by Polish Ministry of Environment. The study was conducted in accordance with the local legislation and institutional requirements. The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncel.2023.1270219/ full#supplementary-material Conflict of interest Taken together, our data indicate that tonic GABAAR-mediated inhibition modulates neocortical networks in an interneuron type- specific manner. In overall, the differential sensitivity of specific neurons to GABA modulation may fine-tune the balance of excitation and inhibition in the cortical networks. Further studies are required to fully understand the complex role of tonic inhibition on neuronal network function under physiological and pathological conditions. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Discussion In the somatosensory cortex, the majority of SST-INs show so called classical accommodating (c-AC, Wang et al., 2004) spiking responses to current injection. This expression might be analogous to other terms such as regular spiking (RS) non-pyramidal (RSNP, Kawaguchi and Kubota, 1996; Kawaguchi, 1997) or low-threshold spiking in other studies (LTS, Gibson et al., 1999; Goldberg et  al., 2004). In general, LTS interneurons are characterized by the relatively high input resistance, display accommodating pattern in response to depolarized currents and also generate rebound spike bursts following somatic hyperpolarization (Kawaguchi and Kubota, 1996; Goldberg et al., 2004). It has been revealed that not every SST-IN shows this phenomenon (Goldberg et al., 2004; Ma et al., 2006). In our experiments, SST-INs more often displayed LTS firing in the internal solution with the low [Cl−] concentration than in the high [Cl−] solution. As in other studies (Kawaguchi and Kubota, 1996; Wang et al., 2004; Ma et al., 2006), a small subgroup of SST-INs had a non-accommodating firing which was analogous to FS responses characteristic for PV (parvalbumin) interneurons. The same as for SST-INs, L2/3 VIP-INs might have firing patterns with regular spiking (adapting firing) as well as bursting adapting, bursting non-adapting or irregular spiking (Lee et al., 2010; Miyoshi et al., 2010; Prönneke et al., 2015, 2019; Jiang et al., 2023). In our experiments, a subset of VIP-INs exhibited high input resistance and displayed the classical LTS pattern with the rebound spikes. Similarly to SST-INs, LTS firing was observed more frequently in the internal solution with the low [Cl−] concentration. Taking into account that L2/3 SST- and VIP-INs fire with very similar patterns, careful consideration should be given to studies using the firing phenotype as the only category to determine an interneuron type, since this population might include interneurons expressing different molecular markers. Now, thanks to a variety of transgenic mouse lines we  can combine molecular and electrophysiological 10 frontiersin.org Bogaj et al. 10.3389/fncel.2023.1270219 Author contributions properties to study specific interneuron types. On top of that, recent investigations using single cell-PCR and multimodal classification methods differentiate several subgroups within VIP- and SST-INs (Gouwens et al., 2020; Donato et al., 2023; Hostetler et al., 2023; Jiang et al., 2023). Unfortunately, much of this diversity remains inaccessible for in-depth study due to lack of genetic targeting tools. KB: Formal Analysis, Investigation, Writing – original draft, Writing – review & editing. RK: Formal Analysis, Investigation, Writing – review & editing. JU-C: Formal Analysis, Investigation, Writing – review & editing, Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft. In conclusion, previous work has shown that ambient GABA was not sufficient to regulate L2/3 SST-IN excitability through GABABRs (Kanigowski et al., 2023). Here we demonstrate that GABAARs also do not regulate SST-IN firing, indicating that these interneurons are mainly immune to ambient GABA. In contrast, L2/3 VIP-INs are modulated by tonic GABAAR inhibition, as we report for the first time. Our results suggest that under the condition of ambient GABA, the differential sensitivity of VIP- and SST-INs to tonic GABAAR inhibition will favor SST-IN activity over VIP-INs. In this condition, SST-INs can generate powerful GABAergic inhibition thanks to their intrinsic excitability that is immune to both GABAA and GABABRs (Kanigowski et al., 2023). Frontiers in Cellular Neuroscience References Neuron 56, 763–770. doi: 10.1016/j.neuron.2007.11.002 Oliva, A. A., Jiang, M., Lam, T., Smith, K. L., and Swann, J. W. (2000). 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English
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New Hofmann-like spin crossover compound with 3,5-lutidine
Hyperfine interactions
2,013
cc-by
4,219
Hyperfine Interact (2014) 226:27–34 DOI 10.1007/s10751-013-0939-x Hyperfine Interact (2014) 226:27–34 DOI 10.1007/s10751-013-0939-x Proceedings of the 32nd International Conference on the Applications of the Mössbauer Effect (ICAME 2013) held in Opatija, Croatia, 1–6 September 2013. T. Kitazawa Research Centre for Materials with Integrated Properties, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan Keywords 57Fe Mössbauer spectroscopy · Coordination polymer · Spin crossover T. Kitazawa (B) · M. Takahashi Department of Chemistry, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan e-mail: kitazawa@chem.sci.toho-u.ac.jp 1 Introduction The spin crossover (SCO) phenomenon is found in first-row transition-metal com- plexes of d4-d7 configuration, the largest number of examples being available for octahedral iron(II). External stimuli, such as temperature, light, or pressure, can be used to switch between these states. SCO research areas continue to develop with the coming of new SCO materials, associated with theories and prospective applications [1]. Coordination polymer frameworks built up by self-assembly constitute a most useful and efficient process for building up nano-scale supramacromolecular ar- chitectures with unique network topologies and potentially interesting magnetic properties. Cyanometalates are useful building blocks for various dimensional co- ordination polymeric networks as transition metal templates. Special attention is currently paid to coordination polymer iron(II) SCO compounds acting in coopera- tive behaviour since they can manifest sensory and memory functions. The Hofmann pyridine coordination polymer Fe(py)2Ni(CN)4 1 shows iron(II) SCO behaviour, as revealed by 57Fe Mössbauer spectroscopy and the SQUID technique [2]. Pressure tuning Raman spectroscopy of 1 was reported [3] and an isotope effect of 1 was found [4]. An emission Mössbauer spectroscopic study of 57Co-labelled analogous 57Co(py)2Ni(CN)4 demonstrated nuclear-decay-induced excited spin state trapping (NIESST) [5]. The effect on the spin crossover of Cl and CH3 replacements located at different positions on the pyridine ring was also studied [6, 7]. The related spin crossover coordination compounds have been developed [8–18]. Some of them contain 3D pillared Hofmann-type-organic frameworks [10–16]. We previously reported two kinds of 2D coordination polymer iron(II) spin crossover complexes containing 3,5-lutidine with formula Fe(3,5-lutidine)2Ni(CN)4· [n(H2O)m(3,5-lutidine)],whose host framework is similar to that of 1 [17]. The type I material obtained from the pH 5.2 solution is Fe(3,5-lutidine)2Ni(CN)4·[0.8(H2O)] 2a, in which water molecules coordinate to iron(II) ions and/or are accommo- dated with the host Fe(3,5-lutidine)2Ni(CN)4framework. The type II material ob- tained from the pH 8.5 solution is Fe(3,5-lutidine)2Ni(CN)4·[0.5(H2O)(3,5-lutidine)] 2b, in which 3,5-lutidine molecules act both as ligands coordinating to octahe- dral Fe(II) atoms and as guest molecules accommodated with the host Fe(3,5- lutidine)2Ni(CN)4framework. Their SCO properties studied by temperature depen- dent 57Fe Mössbauer spectroscopy indicate incomplete SCO behaviour [17]. Some- times the SCO behaviour depends on the preparation methods and/or preparation conditions. We report here the preparation of the new type III material of Hofmann-like 3,5-lutidine spin crossover coordination compound with formula Fe(3,5-lutidine)2Ni(CN)4·2[(H2O)(3,5-lutidine)] 2c. We also prepared the Hofmann-like 3,5-dichloropyridine coordination compound Fe(3,5- dichloropyridine)2Ni(CN)4·2[(3,5-dichloropyridine)(H2O)] 2d to compare to 2c. New Hofmann-like spin crossover compound with 3,5-lutidine Takafumi Kitazawa · Miwa Takahashi Published online: 7 November 2013 © Springer Science+Business Media Dordrecht 2013 Published online: 7 November 2013 © Springer Science+Business Media Dordrecht 2013 Abstract A new type III of 3,5-lutidine spin crossover coordination compound with formula Fe(3,5-lutidine)2Ni(CN)4·2[(H2O)(3,5-lutidine)] 2c has been obtained. The ratio of the high spin state (HS) iron (II) changing to the low spin state (LS) iron (II) in 2c is higher than that of type I and type II 3,5-lutidine coordination polymer 2a and 2b previously reported. 57Fe Mössbauer spectra of 2c show two different doublets which correspond to HS1 (inner doublet lines) and HS2 (outer doublet lines). The intensity of the HS1 doublet decreases on cooling to 80 K while the intensity of another component, the LS singlet, increases. The 90 % of the HS1 doublet change to the LS singlet is probably due to suitable environments of octahedral iron (II) ions coordinated by four nitrogen atoms of cyano groups and two nitrogen atoms of 3,5-lutidine ligands. We also prepared the Hofmann-like 3,5- dichloropyridine coordination compound Fe(3,5-dichloropyridine)2Ni(CN)4·2[(3,5- dichloropyridine)(H2O)] 2d to compare it with 2c. 57Fe Mössbauer spectra of 2d show that 2d is not a spin crossover coordination compound. T. Kitazawa (B) · M. Takahashi Department of Chemistry, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan e-mail: kitazawa@chem.sci.toho-u.ac.jp T. Kitazawa, M. Takahashi 28 2 Experimental Although we previously prepared 2a and 2b using direct contact methods with the aqueous solution containing Mohr’s salt (Fe(NH4)2(SO4)2·6H2O) and potas- sium tetracyanonickelate(II) to neat 3,5-lutidine [17], we prepared the new type III Fe(3,5-lutidine)2Ni(CN)4·2[(H2O)(3,5-lutidine)] 2c using by vial-in-vial slow diffusion methods with aqueous solutions and 3,5-lutidine vapors. 3.14 g (8.80 mmol) of Mohr’s salt (Fe(NH4)2(SO4)2·6H2O) and 2.12 g (8.80 mmol) of potassium tetra- cyanonickelate(II) (K2[Ni(CN)4]) were added into 120 ml of water, and then a light blue precipitate formed immediately. The last one was dissolved by adjusting the pH 5.2 of an aqueous solution, using 4.79 g of citric acid and 2.2 ml of 1,3- diaminopropane. The aqueous solution was located in a small vial. The small vial was put in a large vial with 5 ml of 3,5-lutidine. After standing for a few days at room temperature (RT), yellow solid particles 2c were formed at the interface between the organic and aqueous phases or/and at the bottom of the aqueous phase. Fe(3,5- dichloropyridine)2Ni(CN)4· 2[(3,5-dichloropyridine)(H2O)] 2d was obtained by a similar method for 2c. Unfortunately single crystals suitable for X-ray determination have not yet been obtained. The materials were identified by 57Fe Mössbauer spec- troscopy, elemental analysis, powder X-ray diffraction, and infrared spectroscopy. The elemental analysis for C, H, and N was carried out with a Perkin-Elmer Model 2400. 57Fe Mössbauer spectra were obtained using an Austin Science S-600 spectrom- eter in connection with an EG & G Ortec Model 5500 multichannel analyzer. The temperature dependence of the spectra in the range of 80–295 K were measured by keeping the sample in a gas-flow type cryostat (Oxford Instrument CF1104) and the source was kept at RT. The temperature of the sample was controlled by a DTC2 digital temperature controller from Oxford Instruments. A platinum resistor was used to measure the temperature. The spectra were computer-fitted to Lorentzian lines. The values of the isomer shift are given relative to an α-iron foil at RT. 1 Introduction 57Fe Mössbauer data indicate that 2c is spin crossover compound, while 2d does not have SCO behaviour. New Hofmann-like spin crossover compound New Hofmann-like spin crossover compound 29 Table 1 Types of Fe(3,5-lutidine)2Ni(CN)4·[n (H2O) m(3,5-lutidine)] Guest ratio Abb. Preparation condition SCO behaviour Type I n = 0.8, m = 0 2a pH = 5.2 (direct method) incomplete (36 %) Type II n = 0.5, m = 1 2b pH = 8.5 (direct method) incomplete (27 %) Type III n = 2, m = 2 2c pH = 5.2 (vapor method) Almost (90 %) complete Type I, Type II ref. [17]; Type III [This work] 3 Results and discussion C, 56.24; H, 5.90; N, 16.40 %), in which 3,5-lutidine molecules act both as ligand coordinating to octahedral Fe(II) atoms and as guest molecules accommodated with the ma- jority of the host Fe(3,5-lutidine)2Ni(CN)4framework. The material 2d by using the vial-in-vial slow diffusion method is Fe(3,5-dichloropyridine)2Ni(CN)4·2[(3,5- dichloropyridine)(H2O)] (Found C, 33.19; H, 3.21; N, 13.15 %. Calcd. C, 34.02; H, 1.89; N, 13.23 %), in which 3,5-dichloropyridine molecules act both as ligand coordinating to octahedral Fe(II) atoms and as guest molecules accommodated with the host Fe(3,5-dichloropyridine)2Ni(CN)4framework. Fe(NH4)2(SO4)2·6H2O and K2[Ni(CN)4], and preparation methods with or with- out using the vial-in-vial slow diffusion method. Thematerial 2c obtained by us- ing the vial-in-vial slow diffusion method is Fe(3,5-lutidine)2Ni(CN)4·2[(H2O)(3,5- lutidine)] (Found C, 55.89; H, 5.49; N, 16.28 %. Calcd. C, 56.24; H, 5.90; N, 16.40 %), in which 3,5-lutidine molecules act both as ligand coordinating to octahedral Fe(II) atoms and as guest molecules accommodated with the ma- jority of the host Fe(3,5-lutidine)2Ni(CN)4framework. The material 2d by using the vial-in-vial slow diffusion method is Fe(3,5-dichloropyridine)2Ni(CN)4·2[(3,5- dichloropyridine)(H2O)] (Found C, 33.19; H, 3.21; N, 13.15 %. Calcd. C, 34.02; H, 1.89; N, 13.23 %), in which 3,5-dichloropyridine molecules act both as ligand coordinating to octahedral Fe(II) atoms and as guest molecules accommodated with the host Fe(3,5-dichloropyridine)2Ni(CN)4framework. 57Fe Mössbauer spectra of material 2c and 2d performed at 80 K and RT are shown in Figs. 1 and 2. 57Fe Mössbauer parameters for 2c and 2d are listed in Tables 2 and 3. Spectra of 2c indicate SCO behavior with 90 % inner HS sites changing to LS sites, while the percentage of HS changing to LS (90 %) in 2c is higher than those of the type I and the type II 3,5-lutidine coordination polymer 2a (36 %) and 2b (27 %) previously reported [17]. Spectra of 2d do not show SCO behaviour. 57Fe Mössbauer spectra parameters for 2d (δ = 1.11(2), EQ = 1.68(2) mm s−1 for RT and δ = 1.22(2), EQ = 2.19(2) mm s−1 for 80 K) indicate that iron(II) sites at both 80 K and RT are high spin states. 57Fe Mössbauer spectra of material 2c and 2d performed at 80 K and RT are shown in Figs. 1 and 2. 57Fe Mössbauer parameters for 2c and 2d are listed in Tables 2 and 3. 3 Results and discussion We prepared type III of 3,5-lutidine materials with the majority of two- dimensional layered framework consisting of Fe(3,5-lutidine)2Ni(CN)4. The for- mation of the three materials type I, II and III in Table 1 depends on the pH of the mother aqueous solution containing equimolar amounts of 30 T. Kitazawa, M. Takahashi Fig. 1 57Fe Mössbauer spectra of spin crossover coordination polymer Fe(3,5-lutidine)2Ni(CN)4· 2[(H2O)(3,5-lutidine)] 2c at RT and 80 K 100.0 99.5 99.0 98.5 98.0 97.5 Relative Transmission (%) 4 3 2 1 0 -1 -2 -3 Velocity (mm / sec.) 100 98 96 94 92 90 RT 80K Fe(NH4)2(SO4)2·6H2O and K2[Ni(CN)4], and preparation methods with or with- out using the vial-in-vial slow diffusion method. Thematerial 2c obtained by us- ing the vial-in-vial slow diffusion method is Fe(3,5-lutidine)2Ni(CN)4·2[(H2O)(3,5- lutidine)] (Found C, 55.89; H, 5.49; N, 16.28 %. Calcd. C, 56.24; H, 5.90; N, 16.40 %), in which 3,5-lutidine molecules act both as ligand coordinating to octahedral Fe(II) atoms and as guest molecules accommodated with the ma- jority of the host Fe(3,5-lutidine)2Ni(CN)4framework. The material 2d by using the vial-in-vial slow diffusion method is Fe(3,5-dichloropyridine)2Ni(CN)4·2[(3,5- dichloropyridine)(H2O)] (Found C, 33.19; H, 3.21; N, 13.15 %. Calcd. C, 34.02; H, 1.89; N, 13.23 %), in which 3,5-dichloropyridine molecules act both as ligand coordinating to octahedral Fe(II) atoms and as guest molecules accommodated with the host Fe(3,5-dichloropyridine)2Ni(CN)4framework. 57F M b f i l 2 d 2d f d 80 K d RT h 30 T. Kitazawa, M. Takahashi Fig. 1 57Fe Mössbauer spectra of spin crossover coordination polymer Fe(3,5-lutidine)2Ni(CN)4· 2[(H2O)(3,5-lutidine)] 2c at RT and 80 K 100.0 99.5 99.0 98.5 98.0 97.5 Relative Transmission (%) 4 3 2 1 0 -1 -2 -3 Velocity (mm / sec.) 100 98 96 94 92 90 RT 80K Fig. 1 57Fe Mössbauer spectra of spin crossover coordination polymer Fe(3,5-lutidine)2Ni(CN)4· 2[(H2O)(3,5-lutidine)] 2c at RT and 80 K 100.0 99.5 99.0 98.5 98.0 97.5 Relative Transmission (%) 4 3 2 1 0 -1 -2 -3 Velocity (mm / sec.) 100 98 96 94 92 90 RT 80K Fe(NH4)2(SO4)2·6H2O and K2[Ni(CN)4], and preparation methods with or with- out using the vial-in-vial slow diffusion method. Thematerial 2c obtained by us- ing the vial-in-vial slow diffusion method is Fe(3,5-lutidine)2Ni(CN)4·2[(H2O)(3,5- lutidine)] (Found C, 55.89; H, 5.49; N, 16.28 %. Calcd. New Hofmann-like spin crossover compound New Hofmann-like spin crossover compound 31 New Hofmann-like spin crossover compound 31 Fig. 2 57Fe Mössbauer spectra of Fe(3,5- dichloropyridine)2Ni(CN)4· 2[(3,5-dichloropyridine) (H2O)] 2d at RT and 80 K 100.0 99.5 99.0 98.5 98.0 97.5 97.0 Relative Transmission (%) -4 -2 0 2 4 Velocity (mm / sec.) 100 99 98 97 96 RT 80K Table 2 57Fe Mössbauer parameters of Fe(3,5-lutidine)2Ni(CN)4·2[(H2O)(3,5-lutidine)] 2c T K Site δ (mm s−1) EQ (mms−1) exp (mm s−1) A % RT HS1 1.02(2) 0.97(1) 0.34(2) 0.34(2) 67(2) HS2 1.06(2) 1.98(2) 0.41(2) 0.26(2) 33(2) 80 HS1 1.24(2) 1.21(1) 0.27(2) 7 (2) HS2 1.15(2) 2.63(2) 0.35(2) 0.52(3) 21(3) LS 0.45(2) 0.00(1) 0.57(3) 72(3) exp: The full experimental linewidth at half maximum height As shown in Fig. 1, the 57Fe Mössbauer spectrum of material 2c at RT shows two different doublets which correspond to the HS1(inner doublet lines: blue lines) and HS2(outer doublet lines: green lines) states. The intensity of the HS1 doublet decreases gradually on cooling to 80K while a new spectrum component, the LS singlet(red lines), increases in intensity. As listed in Table 2, the ratio of HS1 to HS2 at RT is 67(2): 33(2), and that of HS1 and HS2 to LS at 80 K is 7(2): 21(3): 72(3). The recoil-free fraction of HS1 is different of that of HS2 due to different octahedral iron(II) enviroments. Considering both the Mössbauer parameters of 2c (δ = 1.02(2), EQ = 0.97(1) mm s−1 at RT and δ = 1.24(2), EQ = 1.21(1) mm s−1 at 80 K) and the structural and Mössbauer data for Fe(py)2Ni(CN)4 [2], Fe(3-Cl-py)2Ni(CN)4 [6], Fig. 2 57Fe Mössbauer spectra of Fe(3,5- dichloropyridine)2Ni(CN)4· 2[(3,5-dichloropyridine) (H2O)] 2d at RT and 80 K 100.0 99.5 99.0 98.5 98.0 97.5 97.0 Relative Transmission (%) -4 -2 0 2 4 Velocity (mm / sec.) 100 99 98 97 96 RT 80K Fig. 3 Results and discussion Spectra of 2c indicate SCO behavior with 90 % inner HS sites changing to LS sites, while the percentage of HS changing to LS (90 %) in 2c is higher than those of the type I and the type II 3,5-lutidine coordination polymer 2a (36 %) and 2b (27 %) previously reported [17]. Spectra of 2d do not show SCO behaviour. 57Fe Mössbauer spectra parameters for 2d (δ = 1.11(2), EQ = 1.68(2) mm s−1 for RT and δ = 1.22(2), EQ = 2.19(2) mm s−1 for 80 K) indicate that iron(II) sites at both 80 K and RT are high spin states. New Hofmann-like spin crossover compound New Hofmann-like spin crossover compound 2 57Fe Mössbauer spectra of Fe(3,5- dichloropyridine)2Ni(CN)4· 2[(3,5-dichloropyridine) (H2O)] 2d at RT and 80 K 100.0 99.5 99.0 98.5 98.0 97.5 97.0 Relative Transmission (%) -4 -2 0 2 4 Velocity (mm / sec.) 100 99 98 97 96 RT 80K Relative Transmission (%) Table 2 57Fe Mössbauer parameters of Fe(3,5-lutidine)2Ni(CN)4·2[(H2O)(3,5-lutidine)] 2c T K Site δ (mm s−1) EQ (mms−1) exp (mm s−1) A % RT HS1 1.02(2) 0.97(1) 0.34(2) 0.34(2) 67(2) HS2 1.06(2) 1.98(2) 0.41(2) 0.26(2) 33(2) 80 HS1 1.24(2) 1.21(1) 0.27(2) 7 (2) HS2 1.15(2) 2.63(2) 0.35(2) 0.52(3) 21(3) LS 0.45(2) 0.00(1) 0.57(3) 72(3) exp: The full experimental linewidth at half maximum height exp: The full experimental linewidth at half maximum height As shown in Fig. 1, the 57Fe Mössbauer spectrum of material 2c at RT shows two different doublets which correspond to the HS1(inner doublet lines: blue lines) and HS2(outer doublet lines: green lines) states. The intensity of the HS1 doublet decreases gradually on cooling to 80K while a new spectrum component, the LS singlet(red lines), increases in intensity. As listed in Table 2, the ratio of HS1 to HS2 at RT is 67(2): 33(2), and that of HS1 and HS2 to LS at 80 K is 7(2): 21(3): 72(3). The recoil-free fraction of HS1 is different of that of HS2 due to different octahedral iron(II) enviroments. Considering both the Mössbauer parameters of 2c (δ = 1.02(2), EQ = 0.97(1) mm s−1 at RT and δ = 1.24(2), EQ = 1.21(1) mm s−1 at 80 K) and the structural and Mössbauer data for Fe(py)2Ni(CN)4 [2], Fe(3-Cl-py)2Ni(CN)4 [6], 32 Table 3 57Fe Mössbauer parameters of Fe(3,5-dichloropyridine)2Ni(CN)4· 2[(3,5- dichloropyridine)(H2O)] 2d T K Site δ (mm s−1) EQ (mm s−1) exp (mm s−1) A % RT HS 1.11(2) 1.68 (2) 0.39(2) 0.35(2) 100(2) 80 HS 1.22 (2) 2.19 (2) 0.47(2) 0.44(2) 100(2) exp: The full experimental linewidth at half maximum height Fe(3-Methyl-py)2Ni(CN)4 [7], and Fe(3-F-py)2Ni(CN)4 [8, 9], the iron(II) atoms in HS1 of 2c have six coordinations with four nitrogen atoms of cyano groups and two nitrogens of 3,5-lutidine. 90 % of HS1 sites change to the singlet LS state due to the suitable FeN6 environment for SCO behaviour. The Mössbauer parameters of LS at 80 K is δ = 0.45(2), EQ = 0.00(1) mm s−1 for 2c. New Hofmann-like spin crossover compound ( ) Q ( ) The minor HS2 parameters (δ = 1.06(2), EQ = 1.98(2) mm s−1 at RT and δ = 1.15(2), EQ = 2.63(2) mm s−1 at 80 K) with a large EQ value is probably due to the FeN5(OH2) or FeN4(OH2)2 environment. The FeN5O core consists of an octahedral iron(II) atom coordinated by four nitrogen atoms of the cyano group, one nitrogen atom of the 3,5-lutidine and one oxygen atom of the water ligand. The FeN4O2 core completes an octahedral iron(II) atom by coordinating two water molecules at the axial position. Taking into account the particle sizes of 2c, the HS2 doublet may be ascribed to the iron(II) units located at the nanocrystals edge as they must have coordinative defects, that is, coordinatively unsaturated sites or coordinated terminal water molecules and, consequently, cannot exhibit spin crossover [9]. [ ] In contrast to the HS1 site, the relative area of the HS2 site does not change over the temperature range measured, suggesting that the HS2 site does not participate in the SCO behaviour as would be expected from the suggested coordination envi- ronment. For material 2c with guest 3,5-lutidine species, another explanation about the outer high spin state sites may be possible such as π −π interactions between the ligands of 3,5-lutidine coordinated to iron(II) atoms and the guest molecules of 3,5-lutidine in 2D and 3D Hofmann-like coordination polymer compounds [10– 16]. While 2c has the 2D host framework, guest 3,5-lutidine molecules probably have π −π interactions with ligand 3,5-lutidine species. At RT, the Mössbauer spectrum is composed of two different doublets that can be attributed to iron(II) in the HS state. The low 0.97(1) mm s−1 value of EQ in HS1 indicates that local symmetry is lower than cubic and the ground orbital state is rather a doublet, which corresponds to an axial elongation of the octahedral environment around the iron(II) ion centre. Conversely, the large 1.98(2) mm s−1 value of EQ in HS2 indicates a local symmetry lower than cubic, but with a rather well-isolated ground orbital singlet, which corresponds to an axial compression of the octahedral structure. When decreasing the temperature to 80 K, the doublet with smaller quadrupole splitting HS1 was transformed into the LS state, while the second doublet HS2 was not affected by the spin crossover behaviour. New Hofmann-like spin crossover compound The large amount of the accommodated guest 3,5-lutidine molecules in the network involved in π −π stacking interactions with the coordinated 3,5-lutidine molecules in the type III decreases the electron density of the ligand molecule and thus explains the elongation of almost all the axial Fe(II)-N bonds, which represent the SCO-active Fe(II) ion centers [15]. By contrast, only a small amount of guest 3,5- New Hofmann-like spin crossover compound 33 lutidine molecules in the type I and II are included within the network; a proportion of iron(II) ion centers in which the coordinated 3,5-lutidine is not involved in π −π stacking interactions conserves an axial compression agreeing with the inactive iron(II) ion centers. Moreover, the important variation in the value of EQ of HS2 (1.98(2) −2.63(2) mm s−1) of the non-active HS doublet between RT and 80 K is due to a strongly distorted equatorial environment associated with a small energy gap between the dxy orbital and the thermally accessible dxz or dyz orbitals [15]. lutidine molecules in the type I and II are included within the network; a proportion of iron(II) ion centers in which the coordinated 3,5-lutidine is not involved in π −π stacking interactions conserves an axial compression agreeing with the inactive iron(II) ion centers. Moreover, the important variation in the value of EQ of HS2 (1.98(2) −2.63(2) mm s−1) of the non-active HS doublet between RT and 80 K is due to a strongly distorted equatorial environment associated with a small energy gap between the dxy orbital and the thermally accessible dxz or dyz orbitals [15]. y y Water molecules can act both as ligands coordinating to iron(II) ions and as guest molecules in the Fe(3,5-lutidine)2Ni(CN)4 host framework in 2c. Water molecules accommodated with the Fe(3,5-lutidine)2Ni(CN)4 2D framework may be associated with subtle coordination environments of iron(II) ions. The residual fractions of the high spin HS1 molecules in the low temperature range may be related to the packing mode of 3,5-lutidine in 2c. While a favorable π −π interaction between the 3,5- lutidine ligands of the host frameworks and the guest 3,5-lutidine molecules in 2c may be associated with a nature of spin transition which acts as 90 % HS1 transferring to LS, subtle environments around Fe(II)N6 coordinated by two pyridine ligands and four nitrogen atoms of cyano groups in 2c induce that 90 % of HS1 change to the LS singlet. 1. Gütlich, P., Goodwin, H.A. (eds.): Spin crossover in transition metal compounds. Top. Curr. Chem. 233, 234, 235. Springer Verlag, Berlin (2004) 2. Kitazawa, T., Gomi, Y., Takahashi, M., Takeda, M., Enomoto, M., Miyazaki, A., Enoki, T.: J. Mater. Chem. 6, 119 (1996) Acknowledgements This work was supported by MEXT(Ministry of Education, Culture, Sports, Science and Technology, Japan)-Supported Program for the Strategic Research Foundation at Private Universities 2012–2016. New Hofmann-like spin crossover compound We have obtained the new type III of 3,5-lutidine Hofmann spin crossover coordination compound 2c. The spin crossover behaviour of type III is different from that of type I and II. Contents of 3,5-lutidine and water molecules are linked to differences of SCO behaviours in 2a, 2b and 2c. Chemical pressure of 3,5-lutidine and water molecules are associated with different SCO rate of HS1 to LS in 2a, 2b and 2c. Guest water molecules in 2c are key to active SCO behaviour. Figures 1 and 2 also indicate that using 3,5-lutidine molecules may induce water molecules coordinating to iron(II) atoms. Probably water molecules in 2d may be guest molecules in the host framework Fe(3,5-dichloropyridine)2Ni(CN)4. Both 3-methyl-pyridine Hofmann complex Fe(3-Methyl-py)2Ni(CN)4 and 3- chloro-pyridine Hofmann complex Fe(3-Cl-py)2Ni(CN)4 act as incomplete spin crossover behaviour [6, 7]. The position-3 substituent of the pyridine ring may be associated with spin crossover behaviour in Fe(3-Cl-py)2Ni(CN)4, Fe(3-Methyl- py)2Ni(CN)4. Although the shape and the size of 3,5-lutidine are almost the same as those of 3,5-dichloropyridine, the 3,5-lutidine complex occurs in spin crossover while the 3,5-dichloropyridine complex does not. The effects of position-3,5 substituent to SCO behaviour are different from those of position-3. The existence of three kinds of 3,5-lutidine Hofmann-like spin crossover com- pounds may promise a new way of Hofmann-like spin crossover coordination com- pounds using linear [Au(CN)2]−ligands [16, 18, 19] and [Ag(CN)2]−ligands [20, 21]. References T. Kitazawa, M. Takahashi 34 Molnár, G., Kitazawa, T., Dubrovinsky, L., McGarvey, J.J., Bousseksou, A.: J. Phys.: Condens. Matter 16, S1129 (2004) ( ) 4. Hosoya, K., Kitazawa, T., Takahashi, M., Takeda, M., Meunier, J-F., Molnár, G., Bousseksou, A.: Phys. Chem. Chem. Phys. 5, 1682 (2003) y y , ( ) 5. Sato, T., Ambe, F., Kitazawa, T., Sano, H., Takeda, M.: Chem. Lett. 1287 (1997) 6. Kitazawa, T., Takahashi, M., Takahashi, M., Enomoto, M., Miyazaki, A., Enoki, T., Takeda, M.: J. Radio. Nucl. Chem. 239, 285 (1999) 7. Molnár, G., Guillon, T., Moussa, N.O., Rechignat, L., Kitazawa, T., Nardone, M., Bousseks A.: Chem. Phys. Lett. 423, 152 (2006) y ( ) 8. Martínez, V., Gaspar, A.B., Muñoz, M.C., Bukin, G.V., Levchenko, G., Real, J.A.: Chem. Eur. J. 15, 10960 (2009) ( ) 9. Martínez, V., Boldog, I., Gaspar, A.B., Ksenofontov, V., Bhattacharjee, A., Gütlich, P., Real, J.A.: Chem. Mater. 22, 4271 (2010) 10. Sciortino, N.F., Scherl-Gruenwald, K.R., Chastanet, G., Halder, G.J., Chapman, K.W., Létard, J-F., Kepert, C.J.: Angew. Chem. Int. Ed. 51, 10154 (2012) 11. Ohba, M., Yoneda, K., Agustí, G., Muñoz, M.C., Gaspar, A.B., Real, J.A., Yamasaki, M., Ando, H., Nakao, Y., Sakaki, S., Kitagawa, S.: Angew. Chem. Int. Ed. 48, 4767 (2009) 12. Southon, P.D., Liu, L., Fellows, E.A., Price, D.J., Halder, G.J., Chapman, K.W., Moubaraki, Murray, K.S., Létard, J-F., Kepert, C.J.: J. Am. Chem. Soc. 131, 10998 (2009) 13. Muñoz-Lara, F.J., Gaspar, A.B., Aravena, D., Ruiz, E., Muñoz, M.C., Ohba, M., Ohtani, Kitagawa, S., Real, J.A.: Chem. Commun. 48, 4686 (2012) g ( ) 14. Muñoz-Lara, F.J., Gaspar, A.B., Muñoz, M.C., Arai, M., Kitagawa, S., Ohba, M., Real, J.A.: Chem. Eur. J. 18, 8013 (2012) ( ) 15. Bartual-Murgui, C., Salmon, L., Akou, A., Ortega-Villar, N.A., Shepherd, H.J., Muñoz, M. C., Molnár, G., Real, J.A., Bousseksou, A.: Chem. Eur. J. 18, 507 (2012) 16. Yoshida, K., Akaboshi, D., Kawasaki, T., Saito, T., Kitazawa, T.: Polyhedron (2013). doi: 10.1016/j.poly.2013.05.003 j p y 17. Kitazawa, T., Takahashi, M., Kawasaki, T.: Hyperfine Interact. 218, 133 (2013) 18. Yoshida, K., Kosone, T., Kanadani, C., Saito, T., Kitazawa, T.: Polyhedron 30, 3066 (20 K., Kosone, T., Kanadani, C., Saito, T., Kitazawa 19. Kosone, T., Tomori, I., Kanadani, C., Saito, T., Mochida, T., Kitazawa, T.: Dalton Trans. 39, 1719 (2010) 20. Rodríguez-Velamazán, J.A., Carbonera, C., Castro, M., Palacios, E., Kitazawa, T., Letard, J- Burriel R.: Chem. Eur. J. 16, 8785 (2010) 21. References Rodríguez-Velamazán, J.A., Castro, M., Palacios, E., Burriel R., Kitazawa, T., Kawasaki, T.: J. Phys. Chem. B 111, 1256 (2007)
https://openalex.org/W2117037636
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English
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Characterizing the Diverse Mutational Pathways Associated with R5-Tropic Maraviroc Resistance: HIV-1 That Uses the Drug-Bound CCR5 Coreceptor
Journal of virology
2,015
cc-by
17,727
ABSTRACT Entry inhibitors represent a potent class of antiretroviral drugs that target a host cell protein, CCR5, an HIV-1 entry coreceptor, and not viral protein. Lack of sensitivity can occur due to preexisting virus that uses the CXCR4 coreceptor, while true resistance occurs through viral adaptation to use a drug-bound CCR5 coreceptor. To understand this R5 resistance pathway, we analyzed >500 envelope protein sequences and phenotypes from viruses of 20 patients from the clinical trials MOTIVATE 1 and 2, in which treatment-experi- enced patients received maraviroc plus optimized background therapy. The resistant viral population was phylogenetically distinct and associated with a genetic bottleneck in each patient, consistent with de novo emergence of resistance. Recombination analysis showed that the C2-V3-C3 region tends to genotypically correspond to the recombinant’s phenotype, indicating its primary importance in con- ferring resistance. Between patients, there was a notable lack of commonality in the specific sites conferring resistance, confirming the unusual nature of R5-tropic resistance. We used coevolutionary and positive-selection analyses to characterize the genotypic determi- nants of resistance and found that (i) there are complicated covariation networks, indicating frequent coevolutionary/compensatory changes in the context of protein structure; (ii) covarying sites under positive selection are enriched in resistant viruses; (iii) CD4 bind- ing sites form part of a unique covariation network independent of the V3 loop; and (iv) the covariation network formed between the V3 loop and other regions of gp120 and gp41 intersects sites involved in glycosylation and protein secretion. These results demonstrate that while envelope sequence mutations are the key to conferring maraviroc resistance, the specific changes involved are context de- pendent and thus inherently unpredictable. on November 24, 2017 by SWETS SUBSCRIPTION SERVIC p://jvi.asm.org/ vember 24, 2017 by SWETS SUBSCRIPTION SERVICE Characterizing the Diverse Mutational Pathways Associated with R5- Tropic Maraviroc Resistance: HIV-1 That Uses the Drug-Bound CCR5 Coreceptor on November 24, 2017 b http://jvi.asm.org/ Downloaded from on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from ertag,a,c Conor J. Meehan,d,e Grace P. McCormack,d Simon A. Travers,d,f Charles Craig,g,h Mike Westby,h R b t a Xiaowei Jiang,a,b Felix Feyertag,a,c Conor J. Meehan,d,e Grace P. McCormack,d Simon A. Travers,d,f Charles Craig,g,h Mike Westby,h Marilyn Lewis,g,h David L. Robertsona Computational and Evolutionary Biology, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdoma; Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdomb; Department of Biology, University of Nevada, Reno, Nevada, USAc; School of Natural Sciences, National University of Ireland Galway, Galway, Irelandd; Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgiume; South African National Bioinformatics Institute, South African MRC Bioinformatics Unit, University of the Western Cape, Bellville, South Africaf; The Research Network, Sandwich, United Kingdomg; Pfizer R&D, Sandwich, United Kingdomh November 2015 Volume 89 Number 22 IMPORTANCE 7 by SWETS SUBSCRIPTION SERVICE The entry inhibitor drug maraviroc makes the cell coreceptor CCR5 unavailable for use by HIV-1 and is now used in combination an- tiretroviral therapy. Treatment failure with drug-resistant virus is particularly interesting because it tends to be rare, with lack of sensi- tivity usually associated with the presence of CXCR4-using virus (CXCR4 is the main alternative coreceptor HIV-1 uses, in addition to CD4). We analyzed envelope sequences from HIV-1, obtained from 20 patients who enrolled in maraviroc clinical trials and experi- enced treatment failure, without detection of CXCR4-using virus. Evolutionary analysis was employed to identify molecular changes that confer maraviroc resistance. We found that in these individuals, resistant viruses form a distinct population that evolved once and was successful as a result of drug pressure. Further evolutionary analysis placed the complex network of interdependent mutational changes into functional groups that help explain the impediments to the emergence of maraviroc-associated R5 drug resistance. H uman immunodeficiency virus type 1 (HIV-1), the caus- ative agent of human AIDS, uses two main immune cell surface proteins, CD4 and a chemokine coreceptor, to enter cells. Pairs of viral envelope proteins (the heavily asparagine [N]-glycosylated gp120-gp41 heterodimers) form trimeric complexes anchored on the virus surface through gp41, and these facilitate entry into cells (1). CD4 receptor binding initi- ates virus-cell membrane interaction, and the coreceptors CCR5 (C-C chemokine receptor type 5) and CXCR4 (C-X-C chemokine receptor type 4) further facilitate viral entry into cells. HIV-1 that exclusively uses CCR5 is termed R5 tropic, whereas HIV-1 that exclusively uses CXCR4 is termed X4 tropic; viruses that use both coreceptors are termed dual or mixed tropic (R5X4) (2). Members of the CCR5 antagonist drug class, also known as HIV-1 entry inhibitors, such as mara- H Received 1 June 2015 Accepted 28 August 2015 Accepted manuscript posted online 2 September 2015 Citation Jiang X, Feyertag F, Meehan C, McCormack G, Travers SA, Craig C, Westby M, Lewis M, Robertson DL. 2015. Characterizing the diverse mutational pathways associated with R5-tropic maraviroc resistance: HIV-1 that uses the drug-bound CCR5 coreceptor. J Virol 89:11457–11472. doi:10.1128/JVI.01384-15. Editor: F. Kirchhoff Address correspondence to David L. Robertson, david.robertson@manchester.ac.uk. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JVI.01384-15. Copyright © 2015, Jiang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. IMPORTANCE Received 1 June 2015 Accepted 28 August 2015 Accepted manuscript posted online 2 September 2015 Citation Jiang X, Feyertag F, Meehan C, McCormack G, Travers SA, Craig C, Westby M, Lewis M, Robertson DL. 2015. Characterizing the diverse mutational pathways associated with R5-tropic maraviroc resistance: HIV-1 that uses the drug-bound CCR5 coreceptor. J Virol 89:11457–11472. doi:10.1128/JVI.01384-15. Editor: F. Kirchhoff Address correspondence to David L. Robertson, david.robertson@manchester.ac.uk. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JVI.01384-15. Copyright © 2015, Jiang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. November 2015 Volume 89 Number 22 Journal of Virology jvi.asm.org 11457 jvi.asm.org Jiang et al. viroc, are negative allosteric modulators and prevent HIV-1 from entering the host cell (3). screening who had received three classes of drugs and/or were infected with virus resistant to two drug classes. A number of these patients sub- sequently experienced therapy failure with an R5-tropic viral population. Virus from these patients was assessed using the PhenoSense Entry Assay. The HIV-1 envelope coding sequence was amplified from virus samples by PCR and ligated into a pCXAS expression vector to create an envelope expression vector library. Virus particles were produced by transfecting HEK293 cells with the purified envelope expression vector library and an HIV-1 genomic vector lacking the envelope-encoding region and con- taining a firefly luciferase gene. The ability of the pseudoviruses to infect U87 CD4 cells overexpressing CCR5 in the presence or absence of vari- ous concentrations of inhibitor was assessed by measuring luciferase-gen- erated relative light units (RLU). If increasing concentrations of maravi- roc failed to give 100% inhibition and a plateau was observed, then the maximal percentages of inhibition (MPI) were estimated visually from the inhibition curve (7). MPI values of less than 95% were considered resis- tant (7). Resistant samples were further analyzed by clonal analysis. One hundred to 200 individual env clones from each sample were prescreened for viability and tropism in single-well cultures of CCR5- or CXCR4- expressing cells. A number of viable clones from 20 patients were selected for further phenotypic and genotypic analysis. For most drugs, a defined set of point mutations in viral genes are associated with resistance to a specific drug (4). Lack of sensi- tivity to maraviroc can arise in two ways. IMPORTANCE In the first mode, sup- pression of maraviroc-sensitive R5 viruses revealed preexisting CXCR4-using variants (5, 6). As maraviroc has no direct impact on strains using the CXCR4 coreceptor, patients are routinely screened for the presence of CXCR4-using viruses prior to treat- ment (tropism testing). The aim is to distinguish a viral popula- tion that is R5 from a viral population harboring either X4 or R5X4 variants. In the second mode, true resistance arises, where R5-tropic viruses adapt to use the maraviroc-bound CCR5 core- ceptor (7, 8). Previous studies have documented that R5-tropic resistance to maraviroc is associated with point mutations in vari- able region 3 (the V3 loop) of gp120 (9, 10). However, no shared (and therefore predictable) set of sites of amino acid changes that conferred resistance in different patients have been reported. In addition, recent evidence indicated that R5 populations, as a con- sequence of mutations outside the V3 loop either in gp120 or in gp41, can also develop resistance (11, 12). Given the structural heterogeneity and the highly N-glycosylated nature of HIV-1’s envelope, such that specific compensatory/coevolutionary changes are required to maintain protein stability and full func- tionality under drug selective pressure (13–18), it is not surprising that maraviroc-associated resistance is more diverse than that to other drug classes. on November 24, 2017 b http://jvi.asm.org/ Downloaded from on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from on November 24, 2017 by SWETS SUBSCRIPTION SER http://jvi.asm.org/ Full-length clonal envelope sequences were obtained before treatment and at one or more on-treatment time points from patients with viral loads of 500 copies/ml. An average of 15 sensitive and 12 resistant clonal sequences were obtained per patient. In addition to optimized back- ground treatment, 17 patients (1 to 17) were given maraviroc, and 3 pa- tients (18 to 20) were from the placebo-treated arm. All samples were confirmed R5 tropic using the original Monogram Trofile assay. RLU values for CCR5 entry efficiency were retrieved from the assay for each clone. vember 24, 2017 by SWETS SUBSCRIPTION SERVICE In this study, we investigated the emergence of resistance to maraviroc associated with use of the drug-bound CCR5 receptor by R5-tropic viruses. IMPORTANCE We analyzed full envelope sequences cloned from viruses obtained from 20 patients (17 maraviroc treated and 3 treated with placebo) enrolled in the MOTIVATE 1 and 2 clin- ical trials (19), a sample of those who failed without evidence of a CXCR4-using virus population out of a total of 1,049 patients. Experimental phenotypic characterization using the Monogram PhenoSense Entry assay was performed on these sequences to es- tablish whether they were associated with a sensitive or resistant phenotype. This determined whether they were or were not able to enter the cell when maraviroc was bound to the CCR5 receptor, i.e., were susceptible or resistant, respectively. Analyzing these data, we confirmed the unusual nature of R5-tropic maraviroc resistance, and by performing evolutionary and structural analy- ses, we found complicated coevolutionary dynamics in maravi- roc-sensitive and -resistant viruses. Specifically, we observed that there were strong coevolutionary changes between sites involved in CD4 binding, V3, sites in the potential N-linked glycosylation motifs (PNGMs) (N-X-S/T-X, where X repre- sents any amino acid except proline [20]), and the signal pep- tide. These changes were associated with the important physi- cochemical properties of the covarying amino acids: hydrophobicity, molecular weight, and structure and/or func- tion of the envelope protein. Collectively, our results explain the unpredictable nature of the evolutionary changes associ- ated with the emergence of resistance to maraviroc in HIV-1’s envelope protein. An initial examination of the relationship between gp160 sequence identity and RLU scores, regardless of drug susceptibility, revealed that identical sequences varied in their CCR5 RLU values by up to 500,000 RLU. In addition, V3 loops that were identical between clones were asso- ciated with RLU values that differed by up to 3 million RLU. This suggests that the individual amino acid sequence of the V3 loop or the full gp160 does not have a direct correlation with the efficiency value for CCR5 usage (represented by the RLU). Despite this discrepancy, all the sequences in this study represent clones from CCR5-using viruses (see Fig. S1 in the supplemental material). by SWETS SUBSCRIPTION SERVICE The MPI score was not directly related to the individual amino acid sequence of either the full gp160 or the V3 loop, with identical sequences exhibiting different MPI scores (data not shown). IMPORTANCE To account for poten- tial conflicts in sequence analysis resulting from these ambiguities, we used the pooled MPI scores (calculated by averaging the MPI scores for all the clones sampled from a patient) to group sensitive and resistant se- quences, as they clustered together in all cases in the phylogenetic analysis. Due to a lack of scores of pooled resistant MPI, patient 17 was excluded from the coevolutionary analysis, because an imbalance of sequence num- bers might hamper this type of analysis when comparing the results be- tween sensitive and resistant viruses (21). For the between-patient analy- sis, this resulted in 182 and 181 sequences for sensitive and resistant viruses, respectively, in the group of patients receiving maraviroc, while in the placebo arm, it resulted in 34 sequences for the two time points (before and after treatment). Sequence alignment. Amino acid sequences were aligned using MUSCLE (22), and this alignment was used as a template for aligning nucleotide sequences with respect to the reading frame. Alignments were checked manually using Jalview (23), and Jalview was used to calculate the consensus sequences for between-patient and within-patient alignments (23). Due to alignment ambiguity, portions of the gp160 sequence align- ments were removed. These positions corresponded to (HXB2 amino acid numbering) 132 to 149, 151 to 153, 186 to 190, 352 to 355, 396 to 413, and 460 to 465. Sequence alignments are available upon request. MATERIALS AND METHODS Data. In the clinical studies MOTIVATE 1 and 2, 1,049 patients were enrolled and screened for R5-tropic virus. They were treatment-experi- enced patients with an HIV-1 RNA level of at least 5,000 copies/ml at November 2015 Volume 89 Number 22 Journal of Virology 11458 jvi.asm.org R5-Tropic Maraviroc Resistance motifs (N-X-S/T-X). In the between-patient coevolutionary analysis, consensus sequences of sensitive and resistant viruses were used for the prediction. In the within-patient analysis, consensus sequences from in- dividual patients were used for the prediction. To understand the signif- icance of observed glycosylation sites in covariation networks, we used 2 tests with the following equations: motifs (N-X-S/T-X). In the between-patient coevolutionary analysis, consensus sequences of sensitive and resistant viruses were used for the prediction. In the within-patient analysis, consensus sequences from in- dividual patients were used for the prediction. To understand the signif- icance of observed glycosylation sites in covariation networks, we used 2 tests with the following equations: Recombination analysis. Recombination plays an important role in HIV evolution, which can also lead to obscure results in phylogenetic and downstream analyses (24). To account for this, we performed recombi- nation analysis using Recco software, which employs an implementation of a dynamic programming algorithm for detecting likely parental se- quences and breakpoints from within an alignment by finding a path that minimizes costs based on mutations and putative recombination (25). For each patient, sequences were pooled, and 1,000-replicate permuta- tions were performed to assess the reliability of recombination between individual strains within a patient. This provides P value support for re- combination for an alignment, as well as for individual breakpoints. The nucleotide sequence alignment for each patient was analyzed individually, and sequences that were identified as being likely recombinants (P  0.05), were further investigated. Of particular interest were recombinant sequences that had resistant and sensitive parental sequences (see Re- sults). RESULTS Inference of evolutionary history and recombination analysis. We inferred evolutionary relationships from the clonal sequence data sampled before and after treatment (depicted for patient 4 in Fig. 1; see Fig. S2A and B in the supplemental material for all patients’ viral phylogenies). In all 17 maraviroc-treated patients, the overwhelming majority of resistant viruses (second and sub- sequent time points) form a monophyletic cluster emerging from the sensitive population (the first time point). Acquisition of R5- tropic maraviroc resistance is thus clearly associated with the emergence of resistant variants from the existing susceptible pop- ulation as opposed to the presence of preexisting (divergent) vari- ants, as is the case with the emergence of CXCR4-using viruses (5, 6). This de novo emergence of resistant virus was confirmed by Bayesian skyline analysis, which detected a distinct drop, by up to a factor of 10, in the genetic diversity, indicating a bottleneck in the size of the viral population between the time points, confirmed by reduced HIV-1 RNA concentrations (Fig. 1 shows an example; see Fig. S2A and B in the supplemental material for all maraviroc- treated patients’ analyses). Interestingly, a rebound in the genetic diversity to preresistance levels (again confirmed by viral loads) was observed in patients 1, 3, 4, 5, 6, 8, 9, 10, 11, and 13 after resistance developed (see Fig. S2A and B in the supplemental material). In the three patients from the placebo arm of the MOTIVATE trials, we found no significant shift of viral genetic diversity between time points, except for patient 20, who had a very low CD4 cell count at the starting time point (see Fig. S2C in the supplemental material). As no maraviroc was present, this g p q Coevolutionary analysis. To understand the nonindependent evolu- tion of amino acid sites in the envelope protein with and without drug selection, we used the coevolutionary method, CAPS, to detect sites un- dergoing coevolutionary changes using protein sequences (29–31). This method uses a strategy to identify structurally and/or functionally impor- tant covarying sites (inferred to be coevolving) that can be explained by compensatory physicochemical changes (with negative or positive corre- lations) (29–31). First, the method normalizes sequences by Poisson dis- tance in the alignment to minimize the effects of phylogenetic coevolu- tion. MATERIALS AND METHODS fe  n N (1) Ei  feNi (2) X2  (Oi  Ei) Ei (3) fe  n N (1) Ei  feNi (2) X2  (Oi  Ei) Ei (3) (1) on November 24, 2017 b http://jvi.asm.org/ Downloaded from on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from (2) (3) In equation 1, fe represents the expected frequency of the number of N-linked glycosylation sites/motifs, n denotes the predicted number of N-linked glycosylation sites/motifs in the envelope proteins, and N de- notes the total number of sites in the alignment/consensus sequence. In equation 2, Ei represents the expected number of N-linked glycosylation sites/motifs in a given number of covarying sites (Ni), and Oi denotes the observed number of N-linked glycosylation sites/motifs predicted in a given covariation network. For a given network i, the 2 test was per- formed with equation 3 and 1 degree of freedom (at a 95% significance level). Due to the small number of observed glycosylation sites present in some cases, we also used Fisher exact tests. These recombinant sequences involving sensitive and resistant viruses were removed from the phylogenetic and positive-selection analyses. A resistant virus from patient 10 (sequence number 18) that clustered with sensitive viruses in the initial phylogenetic analysis (not shown) was ana- lyzed with RDP (26) and found to exhibit evidence of recombination and so was also removed. on November 24, 2017 by SWETS SUBSCRIPTION SERV http://jvi.asm.org/ Molecular phylogenetic analysis. BEAST v1.8.1 was used to construct dated trees and to infer the variance of the effective population size over time using Bayesian skyline plots (27, 28). Separate trees were constructed for each patient from nucleotide sequence alignments (excluding the re- combinant sequences detected as described above), using an HKY model of nucleotide substitution with gamma-distributed rate heterogeneity, a proportion of invariant sites, an uncorrelated lognormal relaxed molecu- lar clock, and a Bayesian skyline coalescent tree prior with tip heights set to the number of days since the trial began. BEAST XML files were created using BEAUTi v1.8.1, and chains were run for 108 states and inspected using Tracer v1.6. Stepwise maximum clade credibility phylogenetic trees were created from the BEAST MCMC tree log file with a burn-in of 10% using TreeAnnotator v1.8.1, and stepwise Bayesian skyline plots were gen- erated using Tracer v1.6. The BEAST XML files are available upon request. Positive-selection analysis. MATERIALS AND METHODS We used HyPhy to detect positively se- lected sites (with posterior probability P  0.95) in sensitive and resistant viruses by comparing four models: M1 (neutral), M2 (selection), M7 (beta), and M8 (beta and ) (35, 36). HyPhy used the aligned codon sequences and the corresponding maximum-likelihood (ML) phyloge- netic tree. RAxML MPI was used to construct the ML tree with 100 boot- straps (37). We used ProtTest (with PhyML as its key component) to select the best-fit amino acid substitution model for the alignment (38, 39). ovember 24, 2017 by SWETS SUBSCRIPTION SERVICE Nucleotide sequence accession numbers. Sequences were deposited in GenBank under accession numbers KT452084 to KT452622. November 2015 Volume 89 Number 22 RESULTS (A) Bayesian skyline plot showing a sharp drop (blue line) in genetic diversity as therapy starts, with a rebound occurring as drug resistance develops, which is consistent with the viral-load plot of the patient (red line). The light-blue lines represent the 95% highest posterior density (HPD) confidence intervals. (B) Phylogenetic tree for patient 4 created with BEAST using a Bayesian skyline coalescent. The tree tips are calibrated by days into therapy, with the R5-sensitive strains sampled at day 1 in green and the R5-resistant strains sampled at day 57 in red. Patient and sequence numbers are shown at the tip nodes, as well as sampling day and pooled MPI, with the sequence MPI shown in parentheses. The internal nodes show the ancestral time points in days. on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from A p j g A on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from 20.0 Patient 4-20 (57/77/68) Patient 4-13 (57/7775) Patient 4-12 (1/100/100) Patient 4-16 (57/77/76) Patient 4-7 (1/100/100) Patient 4-18 (57/77/81) Patient 4-2 (1/100/100) Patient 4-23 (57/77/85) Patient 4-10 (1/100/100) Patient 4-17 (57/77/82) Patient 4-21 (57/77/87) Patient 4-14 (57/77/70) Patient 4-11 (1/100/100) Patient 4-6 (1/100/100) Patient 4-3 (1/100/100) Patient 4-4 (1/100/100) Patient 4-9 (1/100/100) Patient 4-24 (57/77/81) Patient 4-19 (57/77/72) Patient 4-15 (57/77/67) Patient 4-1 (1/100/100) Patient 4-8 (1/100/100) Patient 4-22 (57/77/96) Patient 4-5 (1/100/100) 56.43 18.36 17.31 25.95 57.52 106.98 117.09 22.97 21.38 23.96 15.73 109.33 136.5 132.69 139.22 162.25 119.57 146.32 29.9 110.58 20.44 20.69 57.72 B Day 57 Resistant Cluster Day 1 Sensitive Cluster FIG 1 Evolutionary analysis. Patient 4 is presented as a representative example. Phylogenetic trees and Bayesian skyline plots for patients 1 to 16 and placebo arm patients 18 to 20 are presented in Fig. S2A, B, and C in the supplemental material. (A) Bayesian skyline plot showing a sharp drop (blue line) in genetic diversity as therapy starts, with a rebound occurring as drug resistance develops, which is consistent with the viral-load plot of the patient (red line). The light-blue lines represent the 95% highest posterior density (HPD) confidence intervals. (B) Phylogenetic tree for patient 4 created with BEAST using a Bayesian skyline coalescent. The tree tips are calibrated by days into therapy, with the R5-sensitive strains sampled at day 1 in green and the R5-resistant strains sampled at day 57 in red. RESULTS Second, it identifies statistically significant pairs of covarying sites by comparing a calculated BLOSUM62 score correlation coefficient to a ran- domly generated distribution (30, 31), where we use a high correlation coefficient cutoff (  0.9) for initial filtering, a small alpha value (0.001), and 1 million random samples for the statistical test to minimize false positives. Third, it continues to look for covarying pairs by two important physicochemical properties in the results: hydrophobicity and molecular weight. The degree of connectivity in the resulting networks was measured by calculating the connection coefficient as 2e/(n  [n  1]), where e and n correspond to the number of edges and nodes in the network, respec- tively. Finally, we visualized and analyzed the covariation networks with all the information associated with nodes and edges in Cytoscape (version 2.8.3) (32, 33). The reference HIV-1 variant HXB2 was used for domain mapping (http://www.uniprot.org/uniprot/P04578). DOG (version 2.0) was used to illustrate HXB2 domain structures for covarying sites in the figures (34). WETS SUBSCRIPTION SERVICE Prediction of potential N-linked glycosylation sites. The NetNGlyc 1.0 server was used to predict potential N-linked glycosylation sites and motifs (http://www.cbs.dtu.dk/services/NetNGlyc/). The default cutoff value 0.5 was used for considering potential N-linked glycosylation sites/ jvi.asm.org 11459 November 2015 Volume 89 Number 22 Journal of Virology 11459 jvi.asm.org Jiang et al. 20.0 Patient 4-20 (57/77/68) Patient 4-13 (57/7775) Patient 4-12 (1/100/100) Patient 4-16 (57/77/76) Patient 4-7 (1/100/100) Patient 4-18 (57/77/81) Patient 4-2 (1/100/100) Patient 4-23 (57/77/85) Patient 4-10 (1/100/100) Patient 4-17 (57/77/82) Patient 4-21 (57/77/87) Patient 4-14 (57/77/70) Patient 4-11 (1/100/100) Patient 4-6 (1/100/100) Patient 4-3 (1/100/100) Patient 4-4 (1/100/100) Patient 4-9 (1/100/100) Patient 4-24 (57/77/81) Patient 4-19 (57/77/72) Patient 4-15 (57/77/67) Patient 4-1 (1/100/100) Patient 4-8 (1/100/100) Patient 4-22 (57/77/96) Patient 4-5 (1/100/100) 56.43 18.36 17.31 25.95 57.52 106.98 117.09 22.97 21.38 23.96 15.73 109.33 136.5 132.69 139.22 162.25 119.57 146.32 29.9 110.58 20.44 20.69 57.72 A B Day 57 Resistant Cluster Day 1 Sensitive Cluster FIG 1 Evolutionary analysis. Patient 4 is presented as a representative example. Phylogenetic trees and Bayesian skyline plots for patients 1 to 16 and placebo arm patients 18 to 20 are presented in Fig. S2A, B, and C in the supplemental material. RESULTS Patient and sequence numbers are shown at the tip nodes, as well as sampling day and pooled MPI, with the sequence MPI shown in parentheses. The internal nodes show the ancestral time points in days. 11460 jvi.asm.org November 2015 Volume 89 Number 22 Journal of Virology B B 20.0 Patient 4-20 (57/77/68) Patient 4-13 (57/7775) Patient 4-12 (1/100/100) Patient 4-16 (57/77/76) Patient 4-7 (1/100/100) Patient 4-18 (57/77/81) Patient 4-2 (1/100/100) Patient 4-23 (57/77/85) Patient 4-10 (1/100/100) Patient 4-17 (57/77/82) Patient 4-21 (57/77/87) Patient 4-14 (57/77/70) Patient 4-11 (1/100/100) Patient 4-6 (1/100/100) Patient 4-3 (1/100/100) Patient 4-4 (1/100/100) Patient 4-9 (1/100/100) Patient 4-24 (57/77/81) Patient 4-19 (57/77/72) Patient 4-15 (57/77/67) Patient 4-1 (1/100/100) Patient 4-8 (1/100/100) Patient 4-22 (57/77/96) Patient 4-5 (1/100/100) 56.43 18.36 17.31 25.95 57.52 106.98 117.09 22.97 21.38 23.96 15.73 109.33 136.5 132.69 139.22 162.25 119.57 146.32 29.9 110.58 20.44 20.69 57.72 B Day 57 Resistant Cluster Day 1 Sensitive Cluster November 24, 2017 by SWETS SUBSCRIPTION SERVICE Day 57 Resistant Cluster FIG 1 Evolutionary analysis. Patient 4 is presented as a representative example. Phylogenetic trees and Bayesian skyline plots for patients 1 to 16 and placebo arm patients 18 to 20 are presented in Fig. S2A, B, and C in the supplemental material. (A) Bayesian skyline plot showing a sharp drop (blue line) in genetic diversity as therapy starts, with a rebound occurring as drug resistance develops, which is consistent with the viral-load plot of the patient (red line). The light-blue lines represent the 95% highest posterior density (HPD) confidence intervals. (B) Phylogenetic tree for patient 4 created with BEAST using a Bayesian skyline coalescent. The tree tips are calibrated by days into therapy, with the R5-sensitive strains sampled at day 1 in green and the R5-resistant strains sampled at day 57 in red. Patient and sequence numbers are shown at the tip nodes, as well as sampling day and pooled MPI, with the sequence MPI shown in parentheses. The internal nodes show the ancestral time points in days. FIG 1 Evolutionary analysis. Patient 4 is presented as a representative example. Phylogenetic trees and Bayesian skyline plots for patients 1 to 16 and placebo arm patients 18 to 20 are presented in Fig. S2A, B, and C in the supplemental material. November 2015 Volume 89 Number 22 11460 jvi.asm.org RESULTS (A) Bayesian skyline plot showing a sharp drop (blue line) in genetic diversity as therapy starts, with a rebound occurring as drug resistance develops, which is consistent with the viral-load plot of the patient (red line). The light-blue lines represent the 95% highest posterior density (HPD) confidence intervals. (B) Phylogenetic tree for patient 4 created with BEAST using a Bayesian skyline coalescent. The tree tips are calibrated by days into therapy, with the R5-sensitive strains sampled at day 1 in green and the R5-resistant strains sampled at day 57 in red. Patient and sequence numbers are shown at the tip nodes, as well as sampling day and pooled MPI, with the sequence MPI shown in parentheses. The internal nodes show the ancestral time points in days. 11460 jvi.asm.org Journal of Virology November 2015 Volume 89 Number 22 Journal of Virology Journal of Virology 11460 jvi.asm.org 11460 jvi.asm.org R5-Tropic Maraviroc Resistance material). In one patient (patient 1), the signal peptide was in- volved in within-patient coevolution (see Fig. S3 in the supple- mental material). Analysis of the sensitive and resistant viruses of individual patients indicated that there was no statistically signif- icant difference between the sensitive and resistant viruses in terms of numbers of N-linked glycosylation sites (paired t test, df 15, Pr(|T|  |t|) 0.81). However, the location and number of glycosylation sites between sensitive and resistant viruses changed within some patients (patients 2, 5, 9, 11, 13, and 16). All the patients had their own unique covariation networks, indicat- ing a patient-dependent coevolutionary profile of the viral popu- lation. In the placebo arm (patients 18, 19, and 20), there were no covarying sites involved in the V3 loop (see Fig. S3 in the supple- mental material). drop in genetic diversity in patient 20 was presumably associated with the efficacy of the background treatment. Collectively, this indicates that the resistance to CCR5 antagonist maraviroc in the R5 viral population evolves de novo, and unlike CXCR4-using virus, the associated resistance is not due to an already present minority population of resistant virus that becomes dominant under drug selective pressure. Note that, while we cannot discount a resistant variant being present by chance prior to treatment, our results are consistent, unlike CXCR4-associated resistance, with no preexisting resistant population of any significance. RESULTS This suggests that the resistance mutations that enable the virus to use maraviroc-bound CCR5 are not independent and are con- strained by the need to maintain the structure/function of the envelope protein. To identify between-patient patterns of coevolution, we next combined sensitive and resistant sequences from all 16 patients identified by their population dynamics (Fig. 2; see Fig. S2A and B in the supplemental material). The general pattern observed in individual patients was consistent with the global pattern seen between patients. Interestingly, considering covarying sites asso- ciated with hydrophobicity and molecular weight covariation, we found that both (sensitive and resistant) covariation networks were enriched with the same number of sites associated with po- tential PNGMs (15 sites in the sensitive network [2 51.80, P  0.001; Fisher exact test, P  0.001] and 15 sites in the resistant network [2 55.81, P  0.001; Fisher exact test, P  0.001]). vember 24, 2017 by SWETS SUBSCRIPTION SERVICE by SWETS SUBSCRIPTION SERVICE In the sensitive R5 viruses, we identified a covariation network consisting of 130 amino acid sites forming 1,413 covarying pairs by BLOSUM score covariation (Table 1). This covariation net- work can be further refined by hydrophobicity and molecular weight covariation. One hundred three sites forming 295 covary- ing pairs (with a connection coefficient of 0.056) can be associated with hydrophobicity covariation, whereas 108 sites forming 319 covarying pairs (connection coefficient, 0.055) can be associated with molecular weight covariation (Table 1 and Fig. 3A). There were 81 sites forming 131 covarying pairs (connection coefficient, 0.040) that can be associated with both hydrophobicity and mo- lecular weight covariation (Table 1 and Fig. 3A). Furthermore, there were 120 sites forming 483 covarying pairs (connection co- efficient, 0.067) associated with hydrophobicity and/or molecular weight (Table 1 and Fig. 3A). Coevolution networks within and between patients. To in- vestigate potential coevolutionary/compensatory changes in Env, we identified structurally and/or functionally important covary- ing sites in both gp120 and gp41, which form complicated cova- riation networks in both sensitive and resistant R5 viruses. In the coevolution analysis, we focused on covarying sites that could be associated with hydrophobicity and/or molecular weight covaria- tion, as these two factors are among the most important for ex- plaining amino acid contributions to protein structural stability (46). We first analyzed data from individual patients and looked for common patterns that might be shared between patient data sets. RESULTS on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from on November 24, 2017 http://jvi.asm.org/ Downloaded from Recco identified 31 and 13 recombinant viruses that were as- sociated with phenotypically sensitive or resistant viruses, respec- tively (Fig. 2A). Comparing these, we delimited the regions of the envelope that confer the respective phenotype by identifying the regions that showed the highest conservation in either the sensi- tive or resistant recombinants: V1 to C3 in sensitive and C2 to C3 in resistant sequences (Fig. 2B). This indicates that the Env C2 to C3 regions, including in particular the V3 loop region, have an influence on resistance, i.e., the relevant amino acids must be pres- ent in these regions of the recombinant to confer the respective phenotype. This recombination analysis result, coupled with the high number of mutations observed in V2 and V3 and in the C3 N terminus, indicates that structural shifts in these areas are most likely involved in HIV-1’s use of drug-bound CCR5. To further understand the evolution of maraviroc resistance in individual patients, we analyzed the sensitive and resistant se- quences separately. Although we detected covarying sites in only two patients (patients 10 and 13), the results revealed two striking observations: the identified covarying sites were all in the vicinity of PNGMs, and the changes in hydrophobicity of these covarying pairs were negatively correlated in the sensitive viruses, while in the resistant viruses, they were positively correlated. Note that molecular weight changes were negatively correlated in both groups (see Table S7 in the supplemental material). on November 24, 2017 by SWETS SUBSCRIPTION SERVIC p://jvi.asm.org/ Mutations in both V2 and V3 have been previously implicated in coreceptor binding (40–45). As the C3 region is structurally proximal to the V3 loop, interactions between these domains may be important for coreceptor recognition or binding. However, unlike other cases of HIV-1 drug resistance, the sequence posi- tions in Env associated with R5-tropic resistance differed consid- erably between patients. Indeed, no identifiable shared sites (or specific residue changes) were found to be consistently involved in resistance emergence across all patients. Though sites within V3 were found to be divergent based upon drug susceptibility in most patients, few of these sites overlapped across multiple patients. November 2015 Volume 89 Number 22 jvi.asm.org RESULTS To do this, we identified covarying sites within each of the patient data sets, combining sequences from the two time points: before treatment (sensitive) and after treatment (resistant). Eighteen pa- tients had detectable covariation networks (see Fig. S3 in the sup- plemental material); the exceptions were patients 4 and 8. In the 18 covariation networks, there were covarying sites in both gp120 and gp41, except for three patients (5, 9, and 10). We observed seven patients (1, 2, 5, 9, 15, 16, and 17) with V3 loop involvement in their covariation networks (see Fig. S3 in the supplemental In the resistant R5 viruses, we identified a covariation network with 125 amino acid sites forming 1,400 covarying pairs in terms of BLOSUM score covariation (Table 1). After considering hydro- phobicity and molecular weight, we found 99 sites forming 305 covarying pairs (connection coefficient, 0.063) with hydrophobic- ity covariation, and for molecular weight covariation, we found 106 sites forming 326 covarying pairs (connection coefficient, 0.058) (Table 1 and Fig. 3B). RESULTS Combining both hydrophobicity and molecular weight, we identified 77 sites forming 147 covarying Journal of Virology 11461 jvi.asm.org Patient 1, Sequence 1 (Day 0, Sensitive) Patient 1, Sequence 26 (Day 252, Resistant) Patient 2, Sequence 2 (Day 1, Sensitive) Patient 2, Sequence 6 (Day 1, Sensitive) Patient 3, Sequence 11 (Day 1, Sensitive) Patient 3, Sequence 12 (Day 1, Sensitive) Patient 3, Sequence 21 (Day 172, Resistant) Patient 4, Sequence 3 (Day 1, Sensitive) Patient 4, Sequence 4 (Day 1, Sensitive) Patient 5, Sequence 4 (Day 1, Resistant) Patient 5, Sequence 9 (Day 1, Resistant) Patient 10, Sequence 3 (Day 1, Sensitive) Patient 11, Sequence 8 (Day 1, Sensitive) Patient 12, Sequence 22 (Day 86, Resistant) Patient 12, Sequence 23 (Day 86, Resistant) Patient 12, Sequence 26 (Day 114, Resistant) Patient 12, Sequence 27 (Day 114, Sensitive*) Patient 12, Sequence 30 (Day 114, Resistant) Patient 12, Sequence 34 (Day 114, Sensitive*) Patient 12, Sequence 36 (Day 114, Resistant) Patient 13, Sequence 2 (Day 1, Sensitive) Patient 13, Sequence 3 (Day 1, Sensitive) Patient 13, Sequence 5 (Day 1, Sensitive) Patient 13, Sequence 8 (Day 1, Sensitive) Patient 13, Sequence 9 (Day 1, Sensitive) Patient 13, Sequence 20 (Day 113, Resistant) Patient 13, Sequence 21 (Day 113, Resistant) Patient 14, Sequence 1 (Day 1, Sensitive) Patient 14, Sequence 11 (Day 1, Sensitive) Patient 15, Sequence 4 (Day 1, Sensitive) Patient 15, Sequence 7 (Day 1, Sensitive) Patient 16, Sequence 1 (Day 1, Sensitive) Patient 16, Sequence 14 (Day 87, Sensitive) Patient 16, Sequence 18 (Day 87, Sensitive) Patient 16, Sequence 19 (Day 87, Sensitive) Patient 16, Sequence 24 (Day 87, Sensitive) Patient 16, Sequence 33 (Day 353, Resistant) Patient 16, Sequence 34 (Day 353, Resistant) Patient 17, Sequence 7 (Day 1, Sensitive) Patient 17, Sequence 8 (Day 1, Sensitive) Patient 17, Sequence 9 (Day 1, Sensitive) Patient 17, Sequence 12 (Day 1, Sensitive) Patient 17, Sequence 24 (Day 41, Sensitive) Patient 17, Sequence 26 (Day 117, Sensitive) 1 2568 1 96 Signal peptide 96 390 C1 468 V1 588 V2 885 C2 990 V3 1152 C3 1254 V4 1380 C4 1413 V5 1533 C5 1596 FP 1725 1776 ImSup 1896 2001 Coil 2133 2145 EC sig 2562 DL rep 0.3 0.4 0.5 0.6 0.7 0.8 0 1000 2000 Envelope nucleotide position Conservation Sensitive Resistant Signal Peptide C1 V1 V2 C2 V3 C3 V4 C4 V5 C5 gp41 A B ng of recombination breakpoints for sensitive-resistant recombinants. RESULTS (B) Plot indicating the conservation of sensitive (green) and resistant (red) regions m org November 2015 Volume Journal of Virology 1 2568 1 96 gnal peptide 96 390 C1 468 V1 588 V2 885 C2 990 V3 1152 C3 1254 V4 1380 C4 1413 V5 1533 C5 1596 FP 1725 1776 ImSup 1896 2001 Coil 2133 2145 EC sig 2562 DL rep Patient 1, Sequence 1 (Day 0, Sensitive) Patient 1, Sequence 26 (Day 252, Resistant) Patient 2, Sequence 2 (Day 1, Sensitive) Patient 2, Sequence 6 (Day 1, Sensitive) Patient 3, Sequence 11 (Day 1, Sensitive) Patient 3, Sequence 12 (Day 1, Sensitive) Patient 3, Sequence 21 (Day 172, Resistant) Patient 4, Sequence 3 (Day 1, Sensitive) Patient 4, Sequence 4 (Day 1, Sensitive) Patient 5, Sequence 4 (Day 1, Resistant) Patient 5, Sequence 9 (Day 1, Resistant) Patient 10, Sequence 3 (Day 1, Sensitive) Patient 11, Sequence 8 (Day 1, Sensitive) Patient 12, Sequence 22 (Day 86, Resistant) Patient 12, Sequence 23 (Day 86, Resistant) Patient 12, Sequence 26 (Day 114, Resistant) Patient 12, Sequence 27 (Day 114, Sensitive*) Patient 12, Sequence 30 (Day 114, Resistant) Patient 12, Sequence 34 (Day 114, Sensitive*) Patient 12, Sequence 36 (Day 114, Resistant) Patient 13, Sequence 2 (Day 1, Sensitive) Patient 13, Sequence 3 (Day 1, Sensitive) Patient 13, Sequence 5 (Day 1, Sensitive) Patient 13, Sequence 8 (Day 1, Sensitive) Patient 13, Sequence 9 (Day 1, Sensitive) Patient 13, Sequence 20 (Day 113, Resistant) Patient 13, Sequence 21 (Day 113, Resistant) Patient 14, Sequence 1 (Day 1, Sensitive) Patient 14, Sequence 11 (Day 1, Sensitive) Patient 15, Sequence 4 (Day 1, Sensitive) Patient 15, Sequence 7 (Day 1, Sensitive) Patient 16, Sequence 1 (Day 1, Sensitive) Patient 16, Sequence 14 (Day 87, Sensitive) Patient 16, Sequence 18 (Day 87, Sensitive) Patient 16, Sequence 19 (Day 87, Sensitive) Patient 16, Sequence 24 (Day 87, Sensitive) Patient 16, Sequence 33 (Day 353, Resistant) Patient 16, Sequence 34 (Day 353, Resistant) Patient 17, Sequence 7 (Day 1, Sensitive) Patient 17, Sequence 8 (Day 1, Sensitive) Patient 17, Sequence 9 (Day 1, Sensitive) Patient 17, Sequence 12 (Day 1, Sensitive) Patient 17, Sequence 24 (Day 41, Sensitive) Patient 17, Sequence 26 (Day 117, Sensitive) A A on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from 1 2568 1 96 96 390 588 885 990 11521254 1380 1533 1725 ImSup 1896 Coil 2145 EC sig DL rep 0.3 0.4 0.5 0.6 0.7 0.8 0 1000 2000 Envelope nucleotide position Conservation Sensitive Resistant Signal Peptide C1 V1 V2 C2 V3 C3 V4 C4 V5 C5 gp41 B FIG 2 Mapping of recombination breakpoints for sensitive-resistant recombinants. RESULTS (A) Intrapatient recombination between sensitive and resi using Recco. The sequenced regions are mapped out relative to the HXB2 reference sequence, with a domain map showing the relat e listed, the patient name and sequence number are indicated, as well as the day in therapy and R5 resistance, shown in parenth arental sequence, with red indicating R5-resistant sequences and green indicating R5-sensitive sequences, separated by probable brea k. *, sensitive according to unpooled MPI score. Journal of Virology RESULTS The molecular weight covariation was also positively corre- lated (Spearman’s  0.86, P  0.0001). Moreover, in the placebo arm (patients 18, 19, and 20), both hydrophobicity and molecular weight covariation were positively correlated between the two time points (Spearman’s  0.82, P  0.0001, and Spearman’s  0.88, P  0.0001, respectively). on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from This finding of similar numbers of sites covarying in the sensi- tive and resistant viral data sets indicates that compensatory, i.e., structurally linked, changes that maintain various functionalities are a normal part of envelope evolution. Envelope-mediated drug resistance is thus a specific evolutionary path among possible evo- lutionary trajectories. To confirm the importance of the identified covarying sites, we performed positive-selection analysis (after re- moving the detected recombinants mentioned above). In the sen- sitive R5 viruses, we identified 45 sites under positive selection, and 33 of these sites exhibited detectable coevolution (2 102.08, P  0.00001), i.e., 17 sites in gp120 and 16 sites in gp41 (see Tables S1 and S2 in the supplemental material). Conversely, in the resistant R5 viruses, we identified 69 sites under positive a The numbers of covarying sites and pairs for sensitive and resistant networks are summarized. Four types of networks were compared: covariation by BLOSUM62 score covariation, covariation by hydrophobicity and/or molecular weight covariation. pairs (connection coefficient, 0.050) (Table 1 and Fig. 3B). There were 114 sites forming 484 covarying pairs associated with hydro- phobicity and/or molecular weight covariation (connection coef- ficient, 0.075). RESULTS (A) Intrapatient recombination between sensitive and resistant sequences was detected using Recco. The sequenced regions are mapped out relative to the HXB2 reference sequence, with a domain map showing the relative regions. For each sequence listed, the patient name and sequence number are indicated, as well as the day in therapy and R5 resistance, shown in parentheses. The colors indicate the parental sequence, with red indicating R5-resistant sequences and green indicating R5-sensitive sequences, separated by probable breakpoint regions, shown in black. *, sensitive according to unpooled MPI score. (B) Plot indicating the conservation of sensitive (green) and resistant (red) regions in recombinant sequences. 0.3 0.4 0.5 0.6 0.7 0.8 0 1000 2000 Envelope nucleotide position Conservation Sensitive Resistant Signal Peptide C1 V1 V2 C2 V3 C3 V4 C4 V5 C5 gp41 B B Conservation Envelope nucleotide position FIG 2 Mapping of recombination breakpoints for sensitive-resistant recombinants. (A) Intrapatient recombination between sensitive and resistant sequences was detected using Recco. The sequenced regions are mapped out relative to the HXB2 reference sequence, with a domain map showing the relative regions. For each sequence listed, the patient name and sequence number are indicated, as well as the day in therapy and R5 resistance, shown in parentheses. The colors indicate the parental sequence, with red indicating R5-resistant sequences and green indicating R5-sensitive sequences, separated by probable breakpoint regions, shown in black. *, sensitive according to unpooled MPI score. (B) Plot indicating the conservation of sensitive (green) and resistant (red) regions in recombinant sequences. 11462 jvi.asm.org Journal of Virology November 2015 Volume 89 Number 22 Journal of Virology Journal of Virology 11462 jvi.asm.org R5-Tropic Maraviroc Resistance TABLE 1 Summary of the sensitive and resistant covariation networksa Type Covarying sites (nodes) Covarying pairs (edges) Sensitive Resistant Sensitive Resistant BLOSUM62 130 125 1,413 1,400 Hydrophobicity 103 99 295 305 Molecular wt 108 106 319 326 Hydrophobicity and molecular wt 81 77 131 147 Hydrophobicity/molecular wt/hydrophobicity and molecular wt 120 114 483 484 a The numbers of covarying sites and pairs for sensitive and resistant networks are summarized. Four types of networks were compared: covariation by BLOSUM62 score covariation, covariation by hydrophobicity and/or molecular weight covariation. TABLE 1 Summary of the sensitive and resistant covariation networksa 0.0001) in the shared covariation network between sensitive and resistant sequences from all 20 patients (between-patient analy- sis). RESULTS Moreover, we found that there was positive corre- lation of hydrophobicity covariation (Spearman’s  0.71, P  A B gp41_c V2 gp41_d V3 V4 Signal peptide Immunosuppressive region V1 C3 gp41_b gp41_a Coiled coil C5 C4 CD4 binding C1 V5 C2 Fusion peptide C1 gp41_a V2 V2 CD4 binding C5 C4 C3 C2 C1 V5 V4 V3 V2 V1 Signal peptide Immunosuppressive region gp41_c gp41_a gp41_b Fusion peptide Coiled coil Signal peptide Signal peptide gp41_d Coevolution by both hydrophobicity and molecular weight p<0.001 Coevolution by molecular weight p<0.001 Coevolution by hydrophobicity p<0.001 gp120 gp41 gp41 positively selected site p<0.05 gp120 positively selected site p<0.05 Maraviroc Sensitive R5 (p<0.001) Maraviroc Resistant R5 (p<0.001) C 1 Signal peptide 32 C1 130 V1 156 V2 196 C2 295 V3 330 384 V4 418 C4 460 471 511 C5 gp120 gp41 511 532 Fusion peptide 575 a 575 592 632 b 667 711 c 715 d 854 856 Di-leucine repeat Endocytosis signal V5 Immunosuppressive region Coiled coil 364 374 C3 CD4 binding FIG 3 Covariation networks of gp120 and gp41 in sensitive and resistant R5 viruses. (A and B) Covariation networks of HIV-1 envelope protein associated with hydrophobicity and/or molecular weight covariation for maraviroc-sensitive (A) and -resistant (B) CCR5 viruses. (C) Covarying amino acid sites are organized and labeled by protein domains in gp120 (orange circles) and gp41 (blue circles). The circle sizes indicate the relative numbers of interactions in the covariation networks. Covarying sites are linked by colored lines, which indicate the physicochemical properties of covariation (see the key below and Table 1 for a summary). Sites under positive selection are shown as hexagons. Information regarding positive selection, protein domain, and glycosylation can be found in Tables S1 and S3 in the supplemental material for covarying sites in sensitive and resistant viruses, respectively. RESULTS The other 15 sites from gp41 were located in the coiled-coil region (636N; 644S, under positive selection; 658Q; 662E; and 659E), the fusion peptide (514G), gp41_b (620E, under positive selection, and 621Q, under positive selection), and gp41_d (734E; 746I; 750N; 754A; 792A, under positive selection; 804S, under positive selec- tion; and 817A) (Fig. 4). Interestingly, we found that in the sensi- tive R5 viruses, two (364S and 365S) of the three CD4 binding sites and one site (363Q) from C3 were located in the same potential N-linked glycosylation motif (N-X-S/T-X) (Table 2) (2 2.51, P  0.05; Fisher exact test, P  0.05). Moreover, there were two sites (365S in the CD4 binding region and 817A in the gp41_d region) from the resistant R5 viruses located in different PNGMs (Table 3) (2 1.79, P  0.05; Fisher exact test, P  0.05). Only the two CD4 binding sites (365S and 373T) from the resistant viruses were under positive selection, and the covariation network from the resistant viruses was more highly connected than the network from the sensitive viruses (Fig. 4A), which indicates an important role of CD4 binding in the evolution of maraviroc re- sistance. In summary, we have identified three (2 8, P 0.00468) (Table 2) and 10 (2 37.06, P  0.00001) (Table 3) sites under positive selection in sensitive and resistant covariation networks, respectively. selection, and 51 of these sites exhibited detectable coevolution (2 264.57, P  0.00001), with 27 sites in gp120 and 24 sites in gp41 (see Tables S3 and S4 in the supplemental material). Inter- estingly, we observed that positively selected sites were enriched for PNGM motifs in both sensitive (6 sites; 2 22.75, P  0.001; Fisher exact test, P  0.001) and resistant (8 sites; 2 24.69, P  0.001; Fisher exact test, P  0.001) viruses. To further validate our observations, we analyzed viruses from the three placebo-treated patients (18, 19, and 20) by combining their sequences from the first and second time points. We ob- served similar numbers of covarying sites before and after treat- ment—45 and 52 sites, respectively—which formed 256 and 795 covarying pairs and had connection coefficients of 0.259 and 0.599, respectively. RESULTS First, we compared three covariation networks between sensitive and resistant R5 viruses in the group of patients who received maraviroc, which centered on CD4 binding, CCR5 binding (V3 loop), and the secretory pathway (signal peptide dependent, which plays an important role in con- trolling the expression level of envelope protein on the virion). Second, we analyzed the results from the placebo arm to further validate our results. by SWETS SUBSCRIPTION SERVICE In the resistant R5 viruses, we observed 9 covarying sites from the V3 loop, which coevolved with 20 sites from gp120 and 16 sites from gp41; this network had a connection coefficient of 0.048 (Fig. 5B and Table 5). Among the 20 covarying sites from gp120, we identified 14 in the conserved regions of gp120 (specifically C2, C3, and C4) and sites in V2, V4, and V5. Among the 16 covarying sites in gp41, there were 5 sites in the coiled-coil region (Fig. 5B and Table 5). Of the 44 total covarying sites among the resistant R5 viruses, we identified 20 sites under positive selection (P  0.05) (Fig. 5B and Table 5). Interestingly, there were two positively se- lected sites in the V3 loop, which were located on opposite sides in the tip of the V3 loop (Fig. 5C and Table 5). Coevolution between CD4 binding sites and other regions of gp120 and gp41 reveals novel mutational pathways leading to maraviroc resistance. CD4 binding allows HIV-1 gp120 to bind to CCR5/CXCR4 and enter the host cell, which is a critical step in initiating the whole HIV infectious life cycle. In the sensitive R5 viruses, we found that 3 sites (364S, 365S, and 373T) in the CD4 binding region coevolved with 9 (5 in gp120 and 4 in gp41) sites forming a network with a connection coefficient of 0.288, while in the resistant viruses, we found that 2 sites (365S and 373T, with both under positive selection) from the CD4 binding region co- evolved with 19 (4 in gp120 and 15 in gp41) sites forming a net- work with a connection coefficient of 0.409 (Fig. 4). In the sensi- tive R5 viruses, the five sites from gp120 were located in C2 (238P), C3 (363Q, under positive selection), C4 (442Q, under positive selection), C5 (502K), and V2 (173Y) (Fig. 4A). RESULTS on November 24, 2017 by SWETS SUBSCRIPTION SERVICE g/ A gp41_c V2 gp41_d V3 V4 Signal peptide Immunosuppressive region V1 C3 gp41_b gp41_a Coiled coil C5 C4 CD4 binding C1 V5 C2 Fusion peptide Maraviroc Sensitive R5 (p<0.001) C 120 41 B C1 gp41_a V2 V2 CD4 binding C5 C4 C3 C2 C1 V5 V4 V3 V2 V1 Signal peptide Immunosuppressive region gp41_c gp41_a gp41_b Fusion peptide Coiled coil Signal peptide Signal peptide gp41_d Maraviroc Resistant R5 (p<0.001) A B A Maraviroc Sensitive R5 (p<0.001) Maraviroc Resistant R5 (p<0.001) Maraviroc Resistant R5 (p<0.001) on November 24, 2017 by SWETS SUBSCRIPTION SERVICE Coevolution b p<0.001 Coevolution b p<0.001 Coevolution b p<0.001 1 Signal peptide 32 C1 130 V1 156 V2 196 C2 295 V3 330 384 V4 418 C4 460 471 511 C5 511 532 Fusion peptide 575 a 575 592 632 b 667 711 c 715 d 854 856 Di-leucine repeat Endocytosis signal V5 Immunosuppressive region Coiled coil 364 374 C3 CD4 binding Di-leucine repeat FIG 3 Covariation networks of gp120 and gp41 in sensitive and resistant R5 viruses. (A and B) Covariation networks of HIV-1 envelope protein associated with hydrophobicity and/or molecular weight covariation for maraviroc-sensitive (A) and -resistant (B) CCR5 viruses. (C) Covarying amino acid sites are organized and labeled by protein domains in gp120 (orange circles) and gp41 (blue circles). The circle sizes indicate the relative numbers of interactions in the covariation networks. Covarying sites are linked by colored lines, which indicate the physicochemical properties of covariation (see the key below and Table 1 for a summary). Sites under positive selection are shown as hexagons. Information regarding positive selection, protein domain, and glycosylation can be found in Tables S1 and S3 in the supplemental material for covarying sites in sensitive and resistant viruses, respectively. November 2015 Volume 89 Number 22 Journal of Virology Journal of Virology Journal of Virology jvi.asm.org 11463 November 2015 Volume 89 Number 22 jvi.asm.org 1 Jiang et al. under positive selection) (Fig. 4A). In the resistant R5 viruses, the four sites from gp120 were located in C1 (99D), C4 (444R, under positive selection), V4 (415T), and V5 (471G) (Fig. 4B). RESULTS This increase of covarying pairs was in contrast to the group of patients receiving maraviroc because, interest- ingly, the trend of change for the number of covarying pairs was higher in the placebo arm (Table 1), indicating stronger structural and/or functional constraints acting on the maraviroc-resistant populations and consistent with a genetic bottleneck in the emer- gence of R5-tropic resistance. After considering hydrophobicity and/or molecular weight, in addition to covarying sites identified by BLOSUM62 score covariation, we still observed similar num- bers of covarying sites (45 and 50 sites), which formed 238 (see Fig. S4 and Table S5 in the supplemental material) and 702 (see Fig. S5 and Table S6 in the supplemental material) covarying pairs having connection coefficients of 0.240 and 0.573, respectively. Intrigu- ingly, we observed only a few PNGMs (three and six PNGMs before and after treatment [placebo arm], 2 3.13, P  0.05; Fisher exact test, P  0.05 and 2 16.99, P  0.001; Fisher exact test, P 0.002, respectively) involved in the two covariation net- works (see Tables S5 and S6 in the supplemental material), which suggests a potential role of N-linked glycosylation in the evolution of R5 maraviroc resistance. on November 24, 2017 b http://jvi.asm.org/ Downloaded from on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from on November 24, 2017 by SWETS SUBSCRIPTION SERV http://jvi.asm.org/ Coevolution between the V3 loop and other regions of gp120 and gp41 is enriched with sites in the N-linked glycosylation motifs. Among the sensitive R5 viruses, we identified 7 covarying sites in the V3 loop that coevolved with 19 sites in gp120 and 16 sites in gp41; this network had a connection coefficient of 0.051 (Fig. 5A and Table 4). Among the 19 covarying sites in gp120, sites were identified in C1 to C5 and V2, with sites in C3, C4, and V2 detected to be under positive selection (P  0.05) (Fig. 5A and Table 4). Among the 16 covarying sites in gp41, there were 6 sites from the coiled-coil region (1 positively selected site) and 10 sites from the rest of gp41, with 3 positively selected sites identified (Fig. 5A and Table 4). ovember 24, 2017 by SWETS SUBSCRIPTION SERVICE Next, to understand the mutational pathways that may lead to the evolution of maraviroc resistance, we focused on the key steps that HIV-1 requires to enter host cells. November 2015 Volume 89 Number 22 RESULTS Information regarding positive selection, protein domain, and glycosylation can be found in Tables 2 and 3 for covarying sites in sensitive and resistant viruses, respectively. on November 24, 2017 by SWETS http://jvi.asm.org/ Downloaded from CD4 binding on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from CD4 binding 442Q 520L 364S 363Q 502K CD4 binding on November 24, 2017 by SWETS SUBSCRIPTION SERVIC tp://jvi.asm.org/ November 24, 2017 by SWETS SUBSCRIPTION SERVICE 1 Signal peptide 32 C1 130 V1 156 V2 196 C2 295 V3 330 384 V4 418 C4 460 471 511 C5 gp120 gp41 511 532 Fusion peptide 575 a 575 592 632 b 667 711 c 715 d 854 856 Di-leucine repeat Endocytosis signal V5 Immunosuppressive region Coiled coil CD4 binding C3 FIG 4 Covariation networks of CD4 binding sites. (A and B) Shown are covariation networks of the CD4 binding sites associated with hydrophobicity and/or molecular weight for sensitive (A) and resistant (B) R5 viruses. Covarying sites are mapped to the gp120 protein crystal structure. Sites under positive selection are shown as hexagons. Covarying sites are linked by colored lines indicating the nature of their covariation. The sizes of the circles indicate the relative numbers of interactions in the covariation networks (Fig. 3). Information regarding positive selection, protein domain, and glycosylation can be found in Tables 2 and 3 for covarying sites in sensitive and resistant viruses, respectively. by SWETS SUBSCRIPTION SERVICE (Fig. 5B and Table 5). This indicates that all except two covarying sites in the conserved region of gp120 were associated with PNGMs (site 290T was predicted to be in a PNGM with a score, 0.49, below the threshold of 0.5) that coevolved with sites in the V3 loop. Similarly, in the V3 loop, we observed four sites (300N, 305K, 308R, and 309I) located in the vicinity of the PNGM. We found that covarying sites (357K and 335R) located in the PNGM also coevolved with site 12R in the signal peptide (Fig. 6B). In summary, we have identified 14 (2 57.41, P  0.00001) (Table 4) and 20 (2 66.97, P  0.00001) (Table 5) positively selected sites in sensitive and resistant covariation networks, respectively. pensatory changes between the V3 loop and other protein re- gions are also important for developing maraviroc R5 drug resistance. RESULTS The other four sites from gp41 were located in the coiled-coil region (662E and 658Q), the fusion peptide (520L), and the gp41_b region (620E, To further understand the evolution of drug resistance, we compared the numbers of covarying pairs established by the same sites in the V3 loop between sensitive and resistant viruses and found that two sites (315R and 319T) had dramatic changes in the number of linked changes between sensitive and resistant viruses. The interactions changed from 13 to 3 for 315R (Fig. 5A and Table 2) and 2 to 13 for 319T (Fig. 5B and Table 5) in sensitive and resistant networks, respectively. This suggests 319T may be important for R5 resistance. However, among the 13 interactions 319T established, 11 were under positive selec- tion (Fig. 5B and Table 5), which indicates that linked/com- 11464 jvi.asm.org Journal of Virology November 2015 Volume 89 Number 22 R5-Tropic Maraviroc Resistance A B Maraviroc Resistant R5 (p<0.001) Maraviroc Sensitive R5 (p<0.001) Maraviroc Sensitive R5 (p<0.001) 373T 365S 662E 620E 173Y 238P 658Q 442Q 520L 364S 363Q 502K 471G 621Q 804S 514G 415T 373T 365S 734E 750N 662E 754A 620E 746I 792A 636N 659E 644S 658Q 817A 444R 99D Maraviroc Sensitive R5 (p<0.001) A Maraviroc Resistant R5 (p<0.001) B Coevolution by both hydrophobicity and molecular weight p<0.001 Coevolution by molecular weight p<0.001 Coevolution by hydrophobicity p<0.001 gp120 gp41 gp41 positively selected site p<0.05 gp120 positively selected site p<0.05 CD4 binding CD4 binding CD4 binding CD4 binding CD4 binding 1 Signal peptide 32 C1 130 V1 156 V2 196 C2 295 V3 330 384 V4 418 C4 460 471 511 C5 gp120 gp41 511 532 Fusion peptide 575 a 575 592 632 b 667 711 c 715 d 854 856 Di-leucine repeat Endocytosis signal V5 Immunosuppressive region Coiled coil CD4 binding C3 FIG 4 Covariation networks of CD4 binding sites. (A and B) Shown are covariation networks of the CD4 binding sites associated with hydrophobicity and/or molecular weight for sensitive (A) and resistant (B) R5 viruses. Covarying sites are mapped to the gp120 protein crystal structure. Sites under positive selection are shown as hexagons. Covarying sites are linked by colored lines indicating the nature of their covariation. The sizes of the circles indicate the relative numbers of interactions in the covariation networks (Fig. 3). RESULTS In the resistant R5 viruses, we observed that covarying sites located in the PNGM from the V3 loop covariation network were enriched (Table 5 and Fig. 5B) (10 sites; 2 74.64, P  0.001). However, the covarying sites from the sensitive virus were also enriched (Table 4 and Fig. 5A) (6 sites; 2 25.09, P  0.001; Fisher exact test, P  0.001). Further inspection of these covarying sites in the resistant viruses located in the PNGM indicated that 7 of the 10 sites were located in gp120 (Table 5). Moreover, four sites were located in a conserved region of gp120 (C3), and three of these were detected to be under positive selection. There were three sites located in variable regions (1 in V3 and two in V4). Covarying sites located in the PNGM were also enriched in the conserved region (Table 5) (2 40.45, P  0.001; Fisher exact test, P  0.001). Besides the four sites located in the PNGM, we found there were seven covarying sites located in the vicinity of the PNGM (within five sites upstream or downstream of the N-X-S/ T-X motif), with the exception of site 440S in C4 and 490K in C5 Coevolution between the signal peptide and other regions of the envelope protein is enriched with sites in the N-linked gly- cosylation motifs in the resistant viruses. Site 12R in the resistant viruses, but not in the sensitive viruses, was also under positive selection, suggesting the signal peptide plays a role in maraviroc resistance. Recently, mutational effects at position 12 in the signal peptide have been experimentally studied and shown to modulate the expression level of the envelope protein on the virion and viral infectivity (47). Some early studies also found that the signal pep- November 2015 Volume 89 Number 22 Journal of Virology jvi.asm.org 11465 11465 jvi.asm.org Jiang et al. on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from on November 24, 2017 by SWETS SUBSCRIPTION SERVIC p://jvi.asm.org/ f The sites are in the same N-X-S/T-X motif. vember 24, 2017 by SWETS SUBSCRIPTION SERVICE tide can control glycosylation and HIV-1’s use of the secretory pathway (48, 49). To understand the association between the signal peptide and maraviroc resistance, we analyzed the subnetwork formed be- tween the signal peptide and other regions of the envelope protein. There were 3 and 12 covarying sites in the sensitive and resistant viruses, respectively; these networks had connection coefficients of 0.667 and 0.136 for sensitive and resistant networks, respec- tively (Fig. 6 and Tables 6 and 7). We found that the covariation network of the signal peptide in resistant viruses was enriched with sites in a PNGM (Table 7) (2 10.32, P  0.01; Fisher exact test, P 0.036). Interestingly, site 12R was involved only in the covariation network of resistant viruses (Tables 6 and 7), which was also positively selected (P  0.01). Strikingly, as reported above, in the resistant viruses, 12R coevolved with sites 357K and 335R, which were located in the PNGM (Fig. 6B and Table 7). Moreover, all other sites that were not in the signal peptide also coevolved with sites from the V3 loop (Fig. 5B and 6B). Among these sites, 85V, 275V, 360I, and 444R in conserved regions of gp120 and site 644S from the coiled-coil region of gp41 were lo- cated in the vicinity of the PNGM (Table 7). We found that sites 85V, 335R, 360I, 444R, 644S, and 792A were also under positive selection (Table 7) (P  0.05). These results from combining the resistance data indicate that the signal peptide indeed plays a role in HIV-1 N-linked glycosylation and the structure and/or func- tion of the V3 loop, which can in turn have a role in the evolution of maraviroc resistance. by SWETS SUBSCRIPTION SERVICE P  0.05) and the signal peptide (2 1.56, P  0.05; Fisher exact test, P  0.05) covariation networks before placebo treatment. After placebo treatment, we observed five and four PNGMs in the V3 loop (2 20.26, P  0.001; Fisher exact test, P 0.002) and the signal peptide (2 9.39, P  0.01; Fisher exact test, P 0.019) networks (see Tables S5 and S6 in the supplemental mate- rial). 11466 jvi.asm.org November 2015 Volume 89 Number 22 RESULTS TABLE 2 Covarying sites in the CD4 binding network for sensitive R5 viruses viruses HXB2a Degreeb Selectionc Sited Domain Protein N-Gly motife 173Y 13 Null 151 V2 gp120 238P 15 Null 211 C2 gp120 3 363Q 17 0.998267 331 C3 gp120 N-X-S/T-Xf 364S 4 Null 332 CD4 binding gp120 N-X-S/T-Xf 365S 4 Null 333 CD4 binding gp120 N-X-S/T-Xf 373T 1 Null 341 CD4 binding gp120 442Q 21 0.999411 392 C4 gp120 2 502K 7 Null 446 C5 gp120 520L 4 Null 464 Fusion peptide gp41 620E 24 1 564 gp41_b gp41 658Q 5 Null 602 Coiled coil gp41 662E 22 Null 606 Coiled coil gp41 a HXB2 numbering is used as a reference. b Degree, number of covarying pairs with the site in the BLOSUM62 covariation network. c Selection indicates the results of positive-selection analysis (at 95% posterior probability); null indicates no positive selection. d Site indicates the position of the covarying site in the alignment. e N-Gly motif, the position of the covarying site in the N-linked glycosylation motif N-X-S/T-X. The position is highlighted in boldface and underlined, and positive/negative numbers indicate the position of the covarying site relative to its closest N-X-S/T-X motif in the sequence. Negative () means the covarying site is located on the left side of the motif, while positive () means the site is located on the right side of the motif. f The sites are in the same N-X-S/T-X motif. on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from Nevertheless, we observed two PNGMs in both the V3 loop (2 4.55, P  0.05; Fisher exact test, Journal of Virology 11466 jvi.asm.org November 2015 Volume 89 Number 22 R5-Tropic Maraviroc Resistance Maraviroc Sensitive R5 (p<0.001) V3 loop V3 loop V3 loop V3 loop V3 loop Maraviroc Resistant R5 (p<0.001) V3 loop V3 loop V3 loop V3 loop V3 loop V3 loop V3 loop Coevolution by both hydrophobicity and molecular weight p<0.001 Coevolution by molecular weight p<0.001 Coevolution by hydrophobicity p<0.001 gp120 gp41 gp41 positively selected site p<0.05 gp120 positively selected site p<0.05 A B 362K 319T 347S 80N 322K 34L 620E 333I 644S 444R 360I 343K 746I 467I 316A 336A 836A 779T 750N 651N 315R 170Q 340N 648E 636N 659E 734E 297T 300N 165I 238P 195S 490K 804S 754A 440S 346A 668S 640S 619L 309I 350R 315R 347S 195S 818T 173Y 636N 316A 351E 340N 658Q 394T 754A 183P 321G 290T 297T 275V 300N 700A 651N 335R 440S 471G 621Q 309I 360I 389Q 336A 734E 305K 308R 706N 357K 618S 350R 746I 612A 319T 641L 640S 554N 337K 362K 620E 490K V3 loop V3 loop V3 loop C 1 Signal peptide 32 C1 130 V1 156 V2 196 C2 295 V3 330 384 V4 418 C4 460 471 511 C5 gp120 gp41 511 532 Fusion peptide 575 a 575 592 632 b 667 711 c 715 d 854 856 Di-leucine repeat Endocytosis signal V5 Immunosuppressive region Coiled coil 364 374 C3 CD4 binding Tip Stem Base 297T 300N 305K 308R 309I 315R 316A 319T gp120 V3 Loop 321G 322K FIG 5 C i ti t k f th V3 l (A d B) C i ti t k f th HIV 1 l t i V3 l i t d ith h d h bi it d/ on November 24, 20 http://jvi.asm.org/ Downloaded from Maraviroc Sensitive R5 (p<0.001) V3 loop V3 loop V3 loop V3 loop V3 loop Maraviroc Resistant R5 (p<0.001) V3 loop V3 loop V3 loop V3 loop V3 loop V3 loop V3 loop Coevolution by both hydrophobicity and molecular weight p<0.001 Coevolution by molecular weight p<0.001 Coevolution by hydrophobicity p<0.001 gp120 gp41 gp41 positively selected site p<0.05 gp120 positively selected site p<0.05 A B 362K 319T 347S 80N 322K 34L 620E 333I 644S 444R 360I 343K 746I 467I 316A 336A 836A 779T 750N 651N 315R 170Q 340N 648E 636N 659E 734E 297T 300N 165I 238P 195S 490K 804S 754A 440S 346A 668S 640S 619L 309I 350R 315R 347S 195S 818T 173Y 636N 316A 351E 340N 658Q 394T 754A 183P 321G 290T 297T 275V 300N 700A 651N 335R 440S 471G 621Q 309I 360I 389Q 336A 734E 305K 308R 706N 357K 618S 350R 746I 612A 319T 641L 640S 554N 337K 362K 620E 490K V3 loop V3 loop V3 loop C 1 Signal peptide 32 C1 130 V1 156 V2 196 C2 295 V3 330 384 V4 418 C4 460 471 511 C5 gp120 gp41 511 532 Fusion peptide 575 a 575 592 632 b 667 711 c 715 d 854 856 Di-leucine repeat Endocytosis signal V5 Immunosuppressive region Coiled coil 364 374 C3 CD4 binding Tip Stem Base 297T 300N 305K 308R 309I 315R 316A 319T gp120 V3 Loop 321G 322K FIG 5 Covariation networks of the V3 loop. on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from (A and B) Covariation networks of the HIV-1 envelope protein V3 loop associated with hydrophobicity and/or molecular weight for sensitive (A) and resistant (B) R5 viruses. (C) Covarying sites in the V3 loop mapped to the gp120 protein crystal structure. Sites under positive selection are shown as hexagons. Covarying sites are linked by colored lines indicating the nature of their covariation. The sizes of the circles indicate the relative numbers of interactions in the covariation networks (Fig. 3). Information regarding positive selection, protein domain, and glycosylation can be found in Tables 2 and 3 for covarying sites in sensitive and resistant viruses, respectively. A Maraviroc Sensitive R5 (p<0.001) Maraviroc Sensitive R5 (p<0.001) Maraviroc Resistant R5 (p<0.001) B on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from November 24, 2017 by SWETS SUBSCRIPTION SERVICE FIG 5 Covariation networks of the V3 loop. (A and B) Covariation networks of the HIV-1 envelope protein V3 loop associated with hydrophobicity and/or molecular weight for sensitive (A) and resistant (B) R5 viruses. (C) Covarying sites in the V3 loop mapped to the gp120 protein crystal structure. Sites under positive selection are shown as hexagons. Covarying sites are linked by colored lines indicating the nature of their covariation. The sizes of the circles indicate the relative numbers of interactions in the covariation networks (Fig. 3). Information regarding positive selection, protein domain, and glycosylation can be found in Tables 2 and 3 for covarying sites in sensitive and resistant viruses, respectively. by SWETS SUBSCRIPTION SERVICE works and in individual patients, in particular, the covariation networks of CD4 binding, the V3 loop, and the signal peptide (not observed in the sensitive viruses), indicating its importance in R5 resistance evolution (53). Interestingly, we identified three sites (514, 515, and 520) located in the gp41 fusion peptide that may contribute to resistance, which are very close to three sites (516, 518, and 519) identified previously in another CCR5 antagonist, vicriviroc (54). Intriguingly, site 514 covaries with sites in the CD4 binding region in the resistant virus, but none of the three sites we identified covaries with any site in the V3 loop and signal peptide. This suggests the fusion peptide can contribute to developing CCR5 antagonist resistance in general, which is V3 independent. on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from Moreover, there was a unique CD4 binding covariation network independent of the V3 loop but dependent on the gp41 fusion peptide contributing to the evolution of R5 maraviroc resistance, which is consistent with a previous report that there might be unique CD4 binding-dependent mutational pathways leading to the emergence of maraviroc resistance without the requirement for V3 loop mutations (12), possibly by binding more strongly to the CD4 coreceptor through coevolutionary changes in non-V3 regions and the fusion peptide. This suggests a potential treatment and 365S, also positively selected covarying sites) visualized in Fig. S7 in the supplemental material. Intriguingly, when we mapped the sites to the complex, all the sites (excluding one, 92N), con- tributed to functionality: CD4 binding (350R, 279N, 467I, 471G, 365S, and 373T), N-linked glycosylation (279N and 365S), and CCR5 binding (316A) (see Fig. S7 in the supplemental material). DISCUSSION November 2015 Volume 89 Number 22 on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from This evidence supports the view that N-linked glycosylation is important for V3 loop function (50) and that the signal peptide is crucial for both normal evolution of HIV-1 and entry inhibitor resistance (47, 51). In summary, we found no sites under positive selection in the sensitive covariation network (Table 6), whereas there were seven positively selected sites in the resistant covaria- tion network (Table 7). Finally, to understand how positively selected sites unique to maraviroc-resistant virus can contribute to resistance evolution, we constructed a covarying network of positively selected sites unique to resistant virus (see Fig. S6 and Table S8 in the supple- mental material). Interestingly, key sites unique to resistant virus, as reported above (including CD4 binding, V3 loop, and signal peptide regions; N-linked glycans; the gp41 fusion peptide; and other gp41 regions) were found in this network (see Table S8 in the supplemental material). To understand the functional impli- cations of these unique sites, we mapped them into a hypothetical structural complex, CD4-GP120-CCR5, with two PNGMs (279D To validate the patterns observed from the V3 loop and the signal peptide covariation networks, we compared the results with those from the placebo arm. We found that these patterns were not observed in the placebo arm. DISCUSSION In this study, we detected significant patterns of genetic change in the HIV-1 envelope that confer R5-tropic maraviroc resistance. We found that resistant sequences formed a new and distinct clus- ter, which was associated with a bottleneck occurring after therapy started, consistent with the resistant population most likely emerging de novo, i.e., as a result of the selective pressure provided by the drug, which leads to significantly more amino acid sites under positive selection and higher connection coefficients of the resistant virus covariation networks. Note that while we cannot discount a preexisting variant being resistant by chance and emerging under drug pressure, as reported in another CCR5 an- tagonist, aplaviroc (APL) (52), our results support the absence of any preexisting virus population of any significance. We found that gp41 was involved in all the covariation net- jvi.asm.org 11467 Journal of Virology November 2015 Volume 89 Number 22 11467 jvi.asm.org Jiang et al. strategy that uses both entry and fusion inhibitors (such as enfu- virtide [55]) to minimize the chance for emergence of V3-inde- tance. DISCUSSION Collectively, these results demonstrate that while envelope TABLE 4 Covarying sites in the V3 loop network for sensitive R5 viruses HXB2 sitea Degreeb Selectionc Domaind Protein Site N-Gly motife 34L 12 Null C1 gp120 33 80N 5 Null C1 gp120 79 238P 15 Null C2 gp120 211 3 333I 2 Null C3 gp120 305 N-X-S/T-X 336A 4 0.999804 C3 gp120 308 1 340N 1 0.988433 C3 gp120 312 N-X-S/T-X 343K 5 0.981324 C3 gp120 315 1 346A 4 Null C3 gp120 318 4 347S 2 1 C3 gp120 319 5 350R 1 Null C3 gp120 322 2 360I 5 0.999305 C3 gp120 328 1 362K 17 0.997297 C3 gp120 330 N-X-S/T-X 440S 13 0.997212 C4 gp120 390 444R 15 0.999696 C4 gp120 394 4 490K 14 Null C5 gp120 434 165I 4 Null V2 gp120 143 2 170Q 14 0.962951 V2 gp120 148 195S 9 Null V2 gp120 168 2 297T 3 Null V3 gp120 270 N-X-S/T-X 300N 2 Null V3 gp120 273 2 309I 15 Null V3 gp120 282 4 315R 13 Null V3 gp120 286 316A 9 Null V3 gp120 287 319T 2 Null V3 gp120 290 322K 1 Null V3 gp120 293 467I 3 Null V5 gp120 411 636N 24 Null Coiled coil gp41 580 1 640S 12 Null Coiled coil gp41 584 N-X-S/T-X 644S 18 0.997303 Coiled coil gp41 588 4 648E 26 Null Coiled coil gp41 592 651N 19 Null Coiled coil gp41 595 659E 19 Null Coiled coil gp41 603 619L 6 0.999707 gp41_b gp41 563 N-X-S/T-X 620E 24 1 gp41_b gp41 564 1 668S 9 Null gp41_c gp41 612 734E 19 Null gp41_d gp41 678 746I 30 Null gp41_d gp41 690 750N 9 Null gp41_d gp41 694 754A 20 Null gp41_d gp41 698 779T 19 Null gp41_d gp41 723 804S 18 0.999353 gp41_d gp41 748 836A 13 1 gp41_d gp41 780 a HXB2 numbering is used as a reference. b Degree, number of covarying pairs with the site in the BLOSUM62 covariation network. c Selection indicates the results of positive-selection analysis (at 95% posterior probability); null indicates no positive selection. d Site indicates the position of the covarying site in the alignment. e N-Gly motif, the position of the covarying site in the N-linked glycosylation motif N-X-S/T-X. DISCUSSION The position is highlighted in boldface and underlined, and positive/negative numbers indicate the position of the covarying site relative to its closest N-X-S/T-X motif in the sequence. Negative () means the covarying site is located on the left side of the motif, while positive () means the site is located on the right side of the motif. TABLE 5 Covarying sites in the V3 loop network for resistant R5 viruses HXB2 sitea Degreeb Selectionc Domaind Protein Site N-Gly motife 275V 10 Null C2 gp120 248 1 290T 15 Null C2 gp120 263 335R 5 0.98715 C3 gp120 307 N-X-S/T-X 336A 13 0.999229 C3 gp120 308 1 337K 1 0.99455 C3 gp120 309 2 340N 2 0.99799 C3 gp120 312 N-X-S/T-X 347S 3 1 C3 gp120 319 5 350R 3 0.974134 C3 gp120 322 2 351E 16 Null C3 gp120 323 1 357K 25 Null C3 gp120 325 N-X-S/T-X 360I 6 0.999998 C3 gp120 328 1 362K 4 0.988469 C3 gp120 330 N-X-S/T-X 440S 9 0.974215 C4 gp120 390 490K 6 Null C5 gp120 434 173Y 7 Null V2 gp120 151 183P 10 0.963713 V2 gp120 161 195S 6 Null V2 gp120 168 297T 2 Null V3 gp120 270 N-X-S/T-X 300N 1 Null V3 gp120 273 2 305K 2 Null V3 gp120 278 1 308R 7 0.999995 V3 gp120 281 4 309I 17 Null V3 gp120 282 5 315R 3 Null V3 gp120 286 316A 3 0.954869 V3 gp120 287 319T 13 Null V3 gp120 290 321G 1 Null V3 gp120 292 389Q 16 Null V4 gp120 357 N-X-S/T-X 394T 5 Null V4 gp120 362 N-X-S/T-X 471G 14 0.972233 V5 gp120 415 636N 22 0.971159 Coiled coil gp41 580 1 640S 15 0.999965 Coiled coil gp41 584 N-X-S/T-X 641L 3 0.994541 Coiled coil gp41 585 1 651N 10 0.964727 Coiled coil gp41 595 658Q 10 Null Coiled coil gp41 602 554N 12 Null gp41_a gp41 498 612A 19 1 gp41_b gp41 556 618S 19 Null gp41_b gp41 562 N-X-S/T-X 620E 16 0.999999 gp41_b gp41 564 1 621Q 23 0.999999 gp41_b gp41 565 2 700A 18 Null gp41_c gp41 644 706N 15 Null gp41_c gp41 650 734E 26 Null gp41_d gp41 678 746I 24 Null gp41_d gp41 690 754A 27 Null gp41_d gp41 698 818T 18 Null gp41_d gp41 762 N-X-S/T-X a HXB2 numbering is used as a reference. 11468 jvi.asm.org DISCUSSION b Degree, number of covarying pairs with the site in the BLOSUM62 covariation network. c Selection indicates the results of positive-selection analysis (at 95% posterior probability); null indicates no positive selection. d Site indicates the position of the covarying site in the alignment. e N-Gly motif, the position of the covarying site in the N-linked glycosylation motif N-X-S/T-X. The position is highlighted in boldface and underlined, and positive/negative numbers indicate the position of the covarying site relative to its closest N-X-S/T-X motif in the sequence. Negative () means the covarying site is located on the left side of the motif, while positive () means the site is located on the right side of the motif. on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from TABLE 4 Covarying sites in the V3 loop network for sensitive R5 viruses TABLE 5 Covarying sites in the V3 loop network for resistant R5 viruses on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from Maraviroc Resistant R5 (p<0.001) Signal Peptide Signal Peptide Signal Peptide 792A 29S 24M 444R 360I 31T 85V 644S 12R 357K 335R 275V Coevolution by both hydrophobicity and molecular weight p<0.001 Coevolution by molecular weight p<0.001 Coevolution by hydrophobicity p<0.001 gp120 gp41 gp41 positively selected site p<0.05 gp120 positively selected site p<0.05 B peat B Maraviroc Resistant R5 (p<0.001) Maraviroc Sensitive R5 (p<0.001) A 24M on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from on November 24, 2017 by SWETS SUBSCRIPTION SERVIC tp://jvi.asm.org/ FIG 6 Covariation networks of the signal peptide. (A and B) Shown are covariation networks of the HIV-1 envelope protein signal peptide associated with hydrophobicity and/or molecular weight for sensitive (A) and resistant (B) R5 viruses. Sites under positive selection are shown as hexagons. Covarying sites are linked by colored lines indicating the nature of their covariation. The sizes of the circles indicate the relative numbers of interactions in the covariation networks (Fig. 3). Information regarding positive selection, protein domain, and glycosylation can be found in Tables 4 and 5 for covarying sites in sensitive and resistant viruses, respectively. November 24, 2017 by SWETS SUBSCRIPTION SERVICE three-dimensional conformation, which stabilizes the protein and is vital for cell entry by the virus (17, 50, 57–60). It also acts as a “glycan shield” to protect the virion from neutralizing antibodies (56, 61). Moreover, our novel results suggest that coevolutionary changes between the signal peptide, N-linked glycosylation, and other functional domains of gp120/gp41 (especially the V3 loop) individual infection and thus are difficult to predict. This is a consequence of the malleable nature of HIV-1’s envelope, which results in distinct constraints, so that different mutations are re- quired to confer resistance in different infections. Therefore, ap- propriate coevolutionary/compensatory changes of important amino acid residues are vital for the maintenance of functional viral proteins. HIV-1’s envelope, as a consequence, presents itself as a good drug target (in terms of a low propensity to evolve resistance) accounting for the uncommon observation of maravi- roc resistance in R5-tropic virus on failure in clinical trials. on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from 2017 by SWETS SUBSCRIPTION SERVICE TABLE 7 Covarying sites in the signal peptide network for resistant R5 i TABLE 7 Covarying sites in the signal peptide network for resistant R5 viruses HXB2 sitea Degreeb Selectionc Domaind Protein Site N-Gly motife 85V 1 0.997736 C1 gp120 84 3 275V 10 Null C2 gp120 248 1 335R 5 0.98715 C3 gp120 307 N-X-S/T-X 357K 25 Null C3 gp120 325 N-X-S/T-X 360I 6 0.999998 C3 gp120 328 1 444R 15 0.999052 C4 gp120 394 4 12R 2 0.995959 Signal peptide gp120 12 24 M 6 Null Signal peptide gp120 23 29S 1 Null Signal peptide gp120 28 31T 1 Null Signal peptide gp120 30 644S 10 0.999716 Coiled coil gp41 588 4 792A 18 0.999976 gp41_d gp41 736 a HXB2 numbering is used as a reference. b Degree, number of covarying pairs with the site in the BLOSUM62 covariation network. c Selection indicates the results of positive-selection analysis (at 95% posterior probability); null indicates no positive selection. d Site indicates the position of the covarying site in the alignment. e N-Gly motif, the position of the covarying site in the N-linked glycosylation motif N-X-S/T-X. The position is highlighted in boldface and underlined, and positive/negative numbers indicate the position of the covarying site relative to its closest N-X-S/T-X motif in the sequence. Negative () means the covarying site is located on the left side of the motif, while positive () means the site is located on the right side of the motif. What is crucial to appreciate is that HIV-1 envelope glycosyla- tion and protein folding are not independent, and this subjects Env to additional structural constraints that impact potential drug resistance pathways. The heavy glycosylation of gp120 (about 55% of its molecular mass is contributed by N-linked glycans [56]) facilitates folding of the Env polypeptide chain into its correct TABLE 6 Covarying sites in the signal peptide network for sensitive R5 viruses HXB2 sitea Degreeb Selectionc Domaind Protein Site N-Gly motife 16R 2 Null Signal peptide gp120 15 194T 7 Null V2 gp120 167 3 161I 2 Null V2 gp120 139 a HXB2 numbering is used as a reference. b Degree, number of covarying pairs with the site in the BLOSUM62 covariation network. c Selection indicates the results of positive-selection analysis (at 95% posterior probability); null indicates no positive selection. d Site indicates the position of the covarying site in the alignment. on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from November 24, 2017 by SWETS SUBSCRIPTION SERVICE strategy that uses both entry and fusion inhibitors (such as enfu- virtide [55]) to minimize the chance for emergence of V3-inde- pendent CCR5 antagonist resistance. tance. Collectively, these results demonstrate that while envelope sequence mutations do confer R5-tropic maraviroc resistance, the specific changes involved are largely context dependent, i.e., they are dependent on the genetic variation present in the patient’s tance. Collectively, these results demonstrate that while envelope sequence mutations do confer R5-tropic maraviroc resistance, the specific changes involved are largely context dependent, i.e., they are dependent on the genetic variation present in the patient’s N-linked glycosylation and signal peptide involvement further constrained the emergence of envelope-associated drug resis- November 2015 Volume 89 Number 22 11468 jvi.asm.org 11468 jvi.asm.org Journal of Virology R5-Tropic Maraviroc Resistance Maraviroc Sensitive R5 (p<0.001) Maraviroc Resistant R5 (p<0.001) Signal Peptide Signal Peptide Signal Peptide 792A 29S 24M 444R 360I 31T 85V 644S 12R 357K 335R 275V Signal Peptide 194T 16R 161I Coevolution by both hydrophobicity and molecular weight p<0.001 Coevolution by molecular weight p<0.001 Coevolution by hydrophobicity p<0.001 gp120 gp41 gp41 positively selected site p<0.05 gp120 positively selected site p<0.05 A B 1 Signal peptide 32 C1 130 V1 156 V2 196 C2 295 V3 330 384 V4 418 C4 460 471 511 C5 gp120 gp41 511 532 Fusion peptide 575 a 575 592 632 b 667 711 c 715 d 854 856 Di-leucine repeat Endocytosis signal V5 Immunosuppressive region Coiled coil 364 374 C3 CD4 binding FIG 6 Covariation networks of the signal peptide. (A and B) Shown are covariation networks of the HIV-1 envelope protein signal peptide associated with hydrophobicity and/or molecular weight for sensitive (A) and resistant (B) R5 viruses. Sites under positive selection are shown as hexagons. Covarying sites are linked by colored lines indicating the nature of their covariation. The sizes of the circles indicate the relative numbers of interactions in the covariation networks (Fig. 3). Information regarding positive selection, protein domain, and glycosylation can be found in Tables 4 and 5 for covarying sites in sensitive and resistant viruses, respectively. November 2015 Volume 89 Number 22 REFERENCES First, in order to be secreted through the host Sec pathway, the viral signal peptide has to pres- ent a configuration similar to that of a typical host secreted protein (66). Therefore, the viral signal peptide does not deviate from the basic configuration of a eukaryotic signal peptide (67). What is unique to HIV-1 is a higher negative charge in the N-terminal region of the signal peptide, which is suggested to play a role in gp120 secretion efficiency (48). Second, virus-specific changes in the signal peptide are required throughout its life cycle (47). Third, the HIV signal peptide also determines gp120 glycosyla- tion, which is critical for envelope structure and/or function. 4. Clavel F, Hance AJ. 2004. HIV drug resistance. N Engl J Med 350:1023– 1035. http://dx.doi.org/10.1056/NEJMra025195. on November 24, 2017 by SWETS SUBSCRIPTION SERVIC tp://jvi.asm.org/ p g 5. Westby M, Lewis M, Whitcomb J, Youle M, Pozniak AL, James IT, Jenkins TM, Perros M, van der Ryst E. 2006. Emergence of CXCR4-using human immunodeficiency virus type 1 (HIV-1) variants in a minority of HIV-1- infected patients following treatment with the CCR5 antagonist maraviroc is from a pretreatment CXCR4-using virus reservoir. 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The signal peptide of the HIV-1 envelope protein has been shown to determine the expression level of the glycosylated Env on the virion (47) and potentially determines vaccine efficacy (63). As the expression levels of the host coreceptors CCR5 and/or CXCR4 also fluctuate between cell lines (64), dynamic Env expres- sion on the virion surface regulated by the signal peptide may confer great selective advantage on HIV populations (53). This can also be true for using the maraviroc-bound CCR5 for cell entry of R5-tropic viruses (65). The signal peptide plays a vital role in protein subcellular localization and function. The mutational effects of key amino acid substitutions in both the general secre- tion (Sec) pathway and the signal peptide have been extensively studied in bacteria. These studies found that mutations in both the secretion system and the signal peptide can affect protein secre- tion (62). More specifically, changes of charged residues in the signal peptide of the N-terminal end can increase or decrease the secretion rates of the underlying proteins (49). HIV envelope pro- tein is secreted through the host Sec pathway and N-glycosylated cotranslationally (57). Moreover, the viral envelope signal peptide has to satisfy several constraints of the Sec pathway and the viral life cycle, which is only possible through extensive coevolutionary changes or compensatory mutations. on November 24, 2017 by SWETS SUBSCRIPTION SERVICE http://jvi.asm.org/ Downloaded from e N-Gly motif, the position of the covarying site in the N-linked glycosylation motif N-X-S/T-X. The negative number indicates the position of the covarying site relative to its closest N-X-S/T-X motif in the sequence. Negative () means the covarying site is located on the left side of the motif. TABLE 6 Covarying sites in the signal peptide network for sensitive R5 i jvi.asm.org 11469 Journal of Virology November 2015 Volume 89 Number 22 Journal of Virology jvi.asm.org Jiang et al. modulate envelope protein expression efficiency and glycosyla- tion patterns. Note that expression differences associated with variants can also change without signal peptide involvement. These changes help the virus adapt to the host environment under “normal” immune pressure and during the emergence of drug resistance, such as we have investigated here. Although it is not clear how the signal peptide affects N-linked glycosylation, previ- ous research has shown that oligosaccharides (glycans) are added to the translocating peptide cotranslationally, suggesting the sig- nal peptide may control the glycosylation process by interacting with amino acids flanking the N-linked glycosylation sites/motifs in the envelope protein (57, 62). 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